[From the U.S. Government Printing Office, www.gpo.gov]



	Shellfish Microbiology: A literature review including 
	conclusions and recommendation to address current and future
	research needs.

	A report prepared for the Shellfish Enhancement Task Force
	by Howard Kator

	Microbiology Program
	Virginia Institute of Marine Science
	School of Marine Science
	The College of William and Mary
	Gloucester Point, Virginia 23062

	This study was funded in part by the Virginia Coastal Resources Management Program
	with funds from the National Oceanic and Atmospheric Administration under Section 306
	of the Coastal Zone Management Act of 1972 as amended. Grant No. NA90AA-H-
	CZ796.

	Council on the Environment 						Commonwealth of Virginia









							VIRGINIA
						COASTAL RESOURCES
					   MANAGEMENT PROGRAM


QR
118
.K38
1990					Coastal Resources Management Program links state programs to manage coastal resources.
					n's coastal boundary includes the 29 counties and 15 cities within Tidewater Virginia. The
					coordinated and monitored by the Virginia Council on the Environment.















                                                                             
                                         This report was  funded in part, by the Virgina Coastal Resources  
                                         Management Program through Coastal Zone Management Act grant
                                         funds provided by the National Oceanic and Atmospheric Administration.
                                         The Virginia Coastal Resources Management Program is managed and
                                         coordinated by the Virginia Council on the Environment.


                                         Virginia, Council on the Environment,
                                         202 N. Ninth Street, Suite 900
                                         Richmond
                                                    VA   232019
                                         (804) 786-4580440.
 











                   Shellfish Microbiology: A literature review including
                   conclusions and recommendations to address current and future
                   research needs.


                   A report prepared for the Shellfish Enhancement Task Fo=
0                  by Howard Kator

                   Microbiology Program
                   Virginia Institute of Marine Science
                   School of Marine Science
                   -Me College of William and Mary
                   Gloucester Point, Virginia 23062



                   This study was funded in part by the Virginia Coastal Resources Management Program
                   with funds from the National Oceanic and Atmospheric Administration under Section 306
                   of the Coastal Zone Management Act of 1972 as amended. Grant No. NA90AA-H-
                   CZ796.
                   Council on the Environment                            Commonwealth of Virginia





                                                   U - S - DEPARTMENT OF COMMERCE NOAA
                                                   COASTAL SERVICES CENTER
                                                   2234 SOUTH HOBSON AVENUE
                                                  CHARLESTON      SC 29405-24 13




        :z                                             Property of CSC Library








                        Highlights and Summary of Literature Review:

 "44
                        1. The coliforni and fecal coliform indicators werechosen as indicators of sanitary water
                        quality on the basis of their presence in feces and sewage.


                        2. The current NSSP (National Shellfish Sanitation Program) shellfish growing area
                        'standard', expressed in terms of either coliforms or fecal coliforms, is based on a
                        hypothetical relationship between the densities of coliforms and Salmonella spp. in a
                        homogeneous sewage treatment plant effluent and an assumption concerning pathogen
                        infectivity. The U. S. Public Health Service research report which established the
                        standard" recognized its arbitrary nature and recommended confirmation of its validity.
                        This has not been done.


                        3. Importantly, the U. S. Public Health Service report also noted t  hat the standard was
                        only applicable to point sources of sewage pollution. This is because assumptions
                        regarding pathogen occurrence and ratios of pathogens to indicators only hold in sources
                        from large populations- The presence of multiple small potential sources in nonpoint
                        growing areas was recognized and the Public Health Service report suggested sole reliance
                        on the numerical standard in these growing areas was not likely to provide effective public
                        health protection. The importance of the sanitary survey and experienced judgement of an
                        informed regulator were suggested as mechanisms to deal with such growing areas.


                        4. Over the past 20-25 years deficiencies with coliform and fecal coliform indicators have
                        been noted. Departures from characteristics of an'idear indicator have been described.
                        These include prolonged persistence under favorable conditions in estuarine waters, poor
                        persistence under other conditions, lack of source specificity, problems with recovery
                        methodologies related to sublethal stress and culturability, the poor precision of current
                        MPN based methods, lack of epidemiological information relating indicator numbers to
                        risk, presence of viruses (and other pathogens) in approved waters, lack of parity between
                        bacterial indicators and viruses coupled with observations that viruses are probably the
                        most important etiological agents of shellfish-borne illness in recent years, etc.


                        5. As a consequence of Item 4., efforts have been directed toward finding and verifying
                        'better' indicators of fecal contamination. This has resulted in evaluations of fecal
                        st@ reptococci, enterococci, various anaerobic bacteria, clostridia, viruses and direct detection
                        of pathogens. Some of these have the potential to differentiate human from nonhuman








                        sources. The concept of multiple indicators, including those based on chemicals, has also
                        been suggested but inadequately evaluated.


                        6. Because human enteric viruses (Norwalk agent, hepatitis) appear to be the most
                        important pathogenic agents in point source impacted growing areas, a number of
                        investigators have proposed bacterial viruses or coliphages as candidate indicators. These
 0
                        viruses, which are not pathogenic to humans, offer significant advantages in terms of cost
                        and ease of analysis. In particular, one group of coliphages, the 'male-specific'FRNA
                        coliphages, has been identified as a promising candidate indicator. The chlorine resistance
                        characteristics of this group axe similar to hepatitis virus and Norwalk agent and may be as
                        resistant under environmental conditions. The National Indicator Study is now funding
                        research to examine aspects of male-specific coliphages and B=erotdesfivgffis
                        bacteriophage as indicators of fecal contamination in growing areas.


                        7. Preliminary research reports suggest male-specific bacteriophages may be useful as
                        indicators of sewage contamination. However, its utility as an indicator in nonpoint source
                        impacted growing areas remains equivocal and must be subjected to field verification.
 0                      There is also a need to evaluate its utility as an indicator of human fecal pollution. Ideally,
                        indicators should be sought which allow for differentiation of human from animal fecal
                        pollution. Similarly, bacterial hosts used to enumerate these phages must be tested in
                        natural waters.


                        8. The literature describing relationships between land usage and microbial pollution is
                        rather inconclusive, especially concerning relationships between indicators and pathogens
                        in runoff. Fecal coliforms are not specific as to source of fecal contamination and therefore
 0                      cannot yet differentiate human from animal pollution in runoff, except perhaps by
                        differences in concentrations when obviously different land usages are compared. A
                        number of investigators have concluded the public health significance of cofiform
                        organisms in runoff remains unclear. Consequently, continued use of these
 0                      microorganisms to assess water quality and public health risk remains an equivocal
                        assumption. A priority research issue should be development of BMPs coupled with
                        methods to effectively evaluate BW strategies in term of concentrations and annual
                        outputs of human-specific fecal indicator microorganisms in runoff and receiving waters
 0                      used for shellfish harvesting.








                        Specific research recommendations:


                        1. Support the National Indicator Study and other studies to identify and evaluate new
                        indicators (focus on new indicators, rapid methods, verification via epidemiological study).


                        2. Support reduction of point source effluents to estuaries and u'riprove effluent quality.
                        Current studies indicate densities of male-specific phages are not significantly affacted by,
                        chlorination, dechlorination. Their presencein sewage effluents suggests current
                        disinfection methods are ineffective as far as viruses are concerned. Approaches to
 0'                     improve effluent quality by existing methods or exploration of new methods including
                        ultrafiltration or moving effluents to ocean outfalls (as New Jersey) is needed. Evaluate
                        use of male-specific phages released in sewage treatment plant effluents to model pollutant
                        fields in receiving waters. Such information may be useful to address the issue of buffer
                        zone phmment.
 0

                        3. Continue to support efforts to identify and validate new indicators in nonpoint source
                        areas. Reliance on a fecal coliform numerical standard to protect public health remains
                        equivocal. Public health would be better served by improving the scope and degreee of
 0
                        coverage obtained with the watershed or shoreline survey, minimizing development based
                        on, use of on site sewage disposal in areas where conditions are marginal for their
                        installation. Bringing central sewer systems to areas where septic systems are
 0                      inappropriate, and supporting in general efforts to improve the quality of surface runoff and
                        groundwater. As long as the fecal coliform. indicator continues to be used, the latter are
                        sources of these orgamisms as well as nutrients that may facilitate their persistence.

 0                      4. In conjunction with Item 3., a priority research need is to perform studies to evaluate
                        BW strategies in terms of concentrations and annual outputs of human-specific fecal
                        indicator microorganisms (and pathogens) in runoff and shellfish growing waters. Such
                        studies would necessarily include evaluation of indicators in the most likely sources of fecal
 0                      contamination present@ i.e., animals and septic effluents, and their persistence and transport
                        in estuaries. The effectiveness of BMP strategies to limit the impact of septic systems on
                        sanitary water quality would be an especially important issue to evaluate.


 0






 0







                         INDICATORS OF THE SANITARY QUALITY OF SHELLFISH
                         GROWING WATERS



                         Microbiological Indicators of Fecal Pollution for Use in
                         Shellfish Growing Waters - General Comments



                         Historically, recognition of feces as a source of waterborne disease transmitted by the fecal-
                         oral route occurred in Europein the mid- to late 18Ws. In the early 190(Ys it was
                         generally recognized that microotganisms were more sensitive indicatDrs of fecal
                         contamination than chemical parameters and that direct detection of pathogens to assess
                         water quality was fundamentally limited in usefulness because of difficulties associated
0                        with detection of multiple pathogen  s and their unpredictable occurrence. Use of surrogate
                         microorganisms was considered as a reasonable means to detect the presence of fecal
                         contamination in receiving waters. This review is generally restricted to indicators found
                         exclusively in feces, sewage or septage that have applicability to shellfish waters.
                         Microorganisms found in sewage or septage and which can grow saprophytically in natural
                         waters are not included. Examples of such microorganisms are Aeromonas hydrophila,
                         KlebsieM pneunioniae, and Pseudomonas aeruginosa.


                         Although bacteria have been and are now the approved fecal indicator organism, use of
                         fungi, and specifically the yeasts, should be briefly mentioned as they have seen limited
                         advocacy as candidate indicators of fecal contamination. Simard (1971) discussed the use
                         of "pink yeasts" as a possible indicator of fecal pollution, presenting various observations
                         associating densities of yeasts with sewage pollution. On the basis of in vitro experiments
                         which demonstrated the superior survival of Candida affikans compared with selected
                         bacterial pathogens exposed to seawater, Jamieson et al. (1976) suggested this organism
                         might be a useful indicator of fecal pollution in estuarine waters. Buck (1977) in a review
                         of C albicans as a candidate indicator of health hazards, cited deficiencies in information
                         concerning its survival characteristics in natural receiving waters and the lack of rapid and
                         selective enumeration methods. A "Standard Test Method" is now available based on
                         membrane filtration and a selective medium designated mCA (ASTM 1987). Cabelli
                         (1979) eliminated Candida albicans from serious consideration as a health effects indicator
                         because of its inconsistent presence in feces and low densities. Hood (1983) failed to
                         observe a relationship between levels of the yeast Rhodotorula rubra in fi-esh Gulf of


                                                                 1









                      Mexico oysters and clams with the classification of the harvest waters, but observed that
                      populations increased during storage suggesting possible utility as an indicator of product
 0
                      quality-



                      Issues T    hat Affect Indicator Usefulness


                      The coliform group of indicator bacteria has traditionally been the basis for the
                      microbiological growing area standard for shellfish waters since the early 1900s. Since
                      that time researchers have identified critical:deficiencies in its use as an indicator of fecal
                      contamination.or potential health risk in aquatic systems (eg.,, Berg 1978; Cabelli 1978b;
                      Dutka 1973; Shear and Gottlieb 1980). Responses to some of these criticisms are evident
                      in a measured progression to adopt the most fecal-specific coliforin organisms as approved
                      indicators. Thus, the fecal coliform. has supplanted the total coliform group, and
                      Fschenchia colt may replace the fecal coliform. Recent studies challenge the fiindamental
                      assumption that R coft is the most efficient predictor of enteric disease risk in marine
                      waters (Cabelli et al. 1983). Other workers have identified processes whose effects on
0                     indicator densities seriously question the validity of the coliform, group as the basis for the
                      growing water standard. Differences in resistance to disinfection and persistence
                      characteristics of bacterial indicators compared to the most prevalent viral agents causing
                      shellfish-associated disease continues to focus attention on the adequacy of bacterial
                      indicators to protect public health.


                      As a result, -interest in other indicators of fecal pollution has intensified, although
                      information concerning the survival characteristics and recovery methodologies of alternate
                      indicators is for the most part restricted to freshwater environments. In Section 9010 A.
                      entitled "General Discussion" of Standard Methods (APHA 1989), the statement is made
                      with regard to pollution in %tidal estuaries and other bodies of Wine water. " that "In the
                      following sections, applications of specific techniques to saline water are not discussed
                      because the methods used for fresh waters also can be used satisfactorily with saline
                      waters." Contrary to Standard Methods (APRA 1989) there is no a priori basis. to conclude
                      that enumeration methods for freshwater will be effective in shellfish growing waters. In
                      fact, much of the literature suggests this assumption is unwarranted.






                                                              2








                         The Indicator Concept.. Use of microbiological water standards to minimize the
                         transmission of enteric disease through consumption of raw molluscan shellfish has been a
                         rather successful public health strategy, eliminating major outbreaks of gastroenteritis
                         caused by salmonellae, The indicator concept, first elaborated for drinking water where
                         contamination was assessed on the basis of an operat ional response using a multiple-tube
                         enumeration procedure, was  later applied to the waters of Raritan Bay in the. early 1900s
                         following incidents of typhoid associated with consumption of raw clams (Kehr et al.
                         194 1). A target density of total coliforms was derived based on the dilution of a Large point
                         source of domestic sewage with a sufficient volume of water to yield a theoretical final ratio
                         of indicator to bacterial pathogen. Dilution was anticipated to lower the pathogen density to
                         a value yielding an unknown but significantly reduced potential public health risk. Use of a
                         water-based rather than shellfish-based standard was supported by coliform data showing
                         that bivalves (ie., hard clams) grown in water at or below that standard indicator level
                         would not bioconcentrate pathogens to densities exceeding a presumed minimum infective
                         dosage.. Adoption of a surrogate (indicator) for the pathogen Salmonella typhi was
                         expedient for reasons that included the absence of an accurate and selective method for the
                         recovery of this organism, and, the inability to predict its, occurrence in sewage effluent.
                         Moreover, Kehr et al. (1941) recognized the standard as an arbitrary index that does not
                         index a pre*termined level of risk, requiring verification through epidemiological
                         investigation. "It is believed, therefore, that the most favorable method of reducing the
                         danger of infection from the ingestion of raw hard clams is through the adoption of an
                         arbitrary standard that would reduce to a satisfactory degree the estimated coliform. content
                         of the annual production of Raritan Bay hard clams" (Kehr et al. 194 1, p. 94).


                         A number of basic assumptions were implicit to the indicator concept. It was assumed that
                         the index or standard was applicable only to a diluted waste effluent, that there existed a
                         constant density ratio of indicator to the pathogen of concern (ie., S. typid) in the effluent,
                         and that the ratio was conserved in the environment, at least within the immediate vicinity
                         of a discharge (Kehr et al. 194 1). However, theauthors, recognized that if pollution  -
                         sources departed from these conditions, reliance on the bacteriological standard to reflect
                         health risk was unwarranted. Analysis, of contributing sources, the interdiction of
                         judgement, and skepticism concerning the ability of a numerical standard value to offer
                         unequivocal health protection were aspects of their thinking.


                         Many authors (Bonde 1977; Cabelli 1978; Berg 1978; Wheater et al. 1980) have listed
                         those characteristics that an ideal indicator should possess. Most have agreed that no single


                                                                3








                         indicator is "ideal" or can be universal applied. Indicators possess unique characteristics
                         whose applicability depends on the question b    eing asked and the specific environment
                         involved4 Thus, an indicator diat is not very resistant to chlorination (a poor indicator of
                         disinfection) would be inappropriate for sewage effluents but could be perfectly adequate in
                         a nonpoint source polluted estuary. Very persistent indicators such as Clostridium
                         perfiftens spores or coprostanol may not be appropriate in a nonpoint source area but can
                         be useful as indices of sewage plume dispersion or transport of particulate-bound material.


                         One paradigm of an indicator in diellfishmaters would be to provide a relationship to index
0                        health risL However, epiderruolog:    ical studies to establish predictive relationships are
                         lac1drig and the standards now used can reflect the presence of fecal contamination and
                         disease-causing "potential". As noted, this usage is based on an assumed quantitative
                         relationship between coliform bacteria 9nd an enteric pathogen in diluted sewage from a
0                        large population.. However, in nonpoint impacted estuarine and marine receiving waters,
                         the constancy of this association may not hold because of variation inherent in small
                         multiple sources, which are by nature intermittent, stormwater runoff, the interaction of
                         both indicators and pathogens in the environment, and fecal pollution from nonhuman

                         sources.


                         Indicators may also serve to provide information concerning source or "age" of pollution in
                         nonpoint impacted watersheds. An indicator can be used as an investigatory tool to provide
                         information relevant to fecal source, i.e., location within a watershed, human versus animal
                         sources or animal type, and the age of pollution or its "freshness." Indicators inappropriate
                         as indices of health risk may be useful for this purpose.



                         Persistence. Sanitary engineers and microbiologists have expended considerable effort
                         to identify environmental factors and processes that affect densities of enteric bacteria and
                         their recovery. Much of the early literature oncoliform survival suggested that -dieoff- (or
                         "decay") was the only functional response of coliforms exposed to marine or estuarine
                         environments. This was primarily attributed to the "bactericidal" property of seawater
                         (Ketchum et al. 1952). Immediate decreases in concentrations of coliform organisms
                         discharged from a sewage treatment plant can result from physical dilution &mugh
                         turbulent transport and mixing, processes that presumably occur over time penods of
                         minutes or hours. At some distance from the discharge where the effluent becomes
                         dynamically passive, and in addition to.  dilution, light, temperature, bacterivory,








                        antagonism, inhibition, sedimentation and autecological responses become important
                        factors affecting indicator fate.


                        Use of the term "dieoff" to describe changes in indicato r densities over time is
                        inappropriate. The apparent reduction of recoverable counts ftom estuarine waters is not
                        only the result of "true" cell death, i.e.9 cells, that become non-viable, but is also a function
                        Of-


                        L. Enumeration methodology
                        2. Physiological adaptation to an adverse environment
                        3. Complex intemcdons of physical, biological, and chemical processes


                        The net effect of these processes will be'a function of their degree of interaction, relative
                        dominance andthe unique characteristics of each environment.

                        Initially, dilution was considered the major physical factor affecting the densities of
                        indicator bacteria, i.e., indicator bacteria were treated as "quasi" conservative elements.
                        We now recognize that the approved coliform. indicator for shellfish growing areas does not
                        behave as a conservative elenient@ and can exhibit changes in density that are functions of
                        .(I) physiological responses to a variety of physical and chemical properties of the
                        environment that may be expressed as loss of viability, change in culturability, persistence
                        or aftergrowth, and (2) complex interactions between the indicator and components of the
                        microbiota. In estuaries where entrainment and flushing characteristics increase contact
                        times between indicator organisms and the environment these are important processes
                        affecting indicator densities.



                        Nonfecal origin. Other deficiencies of coliform indicators include new evidence for the
                        extraenteral origins of some species that comprise the fecal coliform. group (Lopez-Torres et
                        al. 1987), the capacity of organisms defined as fecal coliforms to persist or even grow in
                        aquatic environments under favorable tempwature and nutrient regimes (Santo Domingo et
                        al. 1989; Hazen 1988; Knittel. et al. 1977; Rhodes and Kator 1988; Verstraete and Voets,
                        1976), the inability of the indicator to differentiate vertebrate source, the effect of
                        environmental exposure on recoverability (Rhodes et al. 1983; Xu et al. 1982), and the
                        recognition that allochthonous bacterial indicators are prey to microbial grazers (Anderson
                        et al. 1983; McCambridge and McMeekin 1979, 1980; Rhodes and Kator 1988).


                                                               5









                         if these criticisms promote uncertainty about the validity of the fecal coliform indicator,
                         they must also diminish confidence in its use as a numeric standard applicable to all
                         shellfish growing waters. Arguably, other bacterial indicators may be found that offer
                         advantages in terms of source specificity, determination of source age, or recovery
                         methodology. However, considering the important influence of the environment on
                         bacterial indicator fate and recovery, and the inherent regional and temporal variations that
                         characterize dynamic estuarine environments, it is difficult to find credible the proposition
                         that a standard based on a bacterial indicator can be "universally applied."



                         Recoverabiffity. Another important concept concerning the fate and culturability of
                         enteric organisms following environmental exposure was described by Xu et al. (1982).
0                        These workers noted a differential between numbers of viable cells recovered from
                         seawater using a direct measure of viability -and the viable count obtained using
                         nonselective maditional culture methods. It was demonstrated that a significant proportion
                         of cells starved in seawater rapidly enter a "nonrecoverable" stage, ie., do not grow in
0                        standard media, but remain viable as shown by a direct viable count assay (Kogure et al.
                         1979). The phenomenon of noriculturability has been observed by other workers (Lopez-
                         Torres et al. 1987; Munro et al. 1987; Roth et al. 1988; Martinez et al. 1989; Desmonts et
                         al. 1990,; Garcia-Lam et al. 199 1). Culturable cell counts drop significantly within 2-4
0                        days exposure. At these times culturable counts are 20% or less of total direct viable
                         counts. Although significant numbers of culturable cells may remain (depending on the
                         initial cell density), these responses suggest the "true" numbers of indicator organisms
                         could be significantly underestimated because of the low culturable densities fbund in
0                        approved shellfish waters. Garcia-lan et al. (1991) employed the thymidine-labeling
                         method of Servais et al. (1985) to demonstrate that actual mortality rates of E Cok S.
                         typhimuiium and S. faecium (E fbecium) in natural seawater were an order of magnitude
                         lower than mortality rates measured by culturable counts. Ibis method involves measuring
0                        loss of radioactivity ftom the trichloroacetic acid-insoluble ftwtion (containing the DNA
                         from intact cells) after addition of [3H]thymidine-labeled cells to seawater. E faecium
                         exhibited the lowest rate of disappearance as a culturable count. Desmonts et al. (1990)
                         combined the direct viable count (DVQ procedure of Kogure et al. (1979) with an indirect
0                        fluorescent-antibody (IFA) for Salmonella spp. Salmonella spp. were detected in all
                         samples of raw domestic and chlorinated wastewaters by I]FA-DVC at comparatively high
                         densities, even when culturable cells were not found. Of the IFA detected cells in







                      chlorinated wast'ewater, 5-31.5% were viable by DVC. The public health significance of
 0                    these studies rema *in to be, evaluated but the relatively high counts of salmonellae observed
                      in disinfected. effluents should produce an, examination of the disinfection procm Grimes
                      and Colwell (1996) demonstrated that cellIs of an enterotoxigenic strain of E COU became
                      nonculturable on a BM-synthetic seawater medium after 13 hours exposure to ocean water
 0                    and remained vindent based o n retentionof plasrmds coding for virulence genes and
                      subsequent rabbit ileal loop assays A detailed discussion of the viable but nonculturable
                      phenomenon can be found in Grimes et al. (1986) and Roszak and Colwell (1987).
                      Recognition of. this physiological adaptation to starvation has done much-to stimulate
                      rethiril@ing the fate of allochthonous, bacteria in marine and estuarine environments as well
                      as the efficacy of traditional enumeration methods. Entry of a significant proportion of an
                      enteric indicator population during exposure to seawater into a non-recoverable stage would
                      seriously question the validity of bacterial indicators.

 0
                      However, numerous reports also demonstrate coliforms (generally E coli ) can persist for
                      extended periods. Awong et al. (1990) found both wild-type and genetically engineered
                      strains of E. coli survived for periods of 10-30. days,.albeit with gradual reductions in
 0                    culturable densities, in filtered and nonfiltered lalm water. Flint (1987) reported that E coli
                      survived in sterile river water for 260 days in the absence of predators and that direct and
                      cultural counts were similar over the first 40 days of exposure. Fiskdal et al. (1989)
                      observed both rapid and slow declines in culturable counts of E coli in filtered seawater.
 0                    Extended culturability (ca. 5 months) of E coli in seawater was reported by Munro et AL
                      (1989). Munro et al. (1989) suggested that genetic differences in osmoregulatory
                      capabilities, acting in response to the effects of test cell preft-eatment and exposure
                      conditions, are responsible for differences in survivaland culturability.

 0
                      We are uncertain about the causes of variation concerning entry into a nonculturable state.
                      Some evidence suggest these may be related to properties of test strains, inocula
                      preparation methods (e.g., Gauthier et al. 199 1) or to water quality properties such as the
 0                    concentration or type of organic matter. Because of the importance of this phenomenon in
                      terms of indicator selection, its consequences on culturable enumeration methods, and
                      recent reports that document its occurrence, a rigorous and standardized evaluation of
                      factors that control entry of cells into the nonculturable state is desirable.

 0

                      Sources of Indicators to Shellfish Growing Waters


 0                                                          7









                        Feces. Since the early work of Escherich G 885) who identified Bacillus coli (now
 .0
                        Fschetichia colt) as a dominant bacterium in feces, considerable effort has been expended
                        to identify the dominant bacteria asso ciated with the gastrointestinal tracts of humans and
                        warm-blooded animals. Microorganisms now used as indicators of fecal contamination are
                        necessarily (but not exclusively) inhabitants of the alimentary tracts of humans and warm-
                        blooded animals and are not numerically dominant components of the microbiota, (Savage
                        1977). The bacterial composition of mammalian feces, which can vary with host species,
                        age, diet, and geographic location (Savage 1977; Feachem et al. 1983), is dominated by
                        obligate anaerobic bacteria belonging to major taxa that include Bacteroides,
                        Bifidobactenum, Closm&un4 and EubacteriunL         Commensal facultative anaerobes include
                        the lactobacilli, Enterobacteriacae, and various cocci belonging to Enterococcus and
                        Streptococcus. Concentrations of these -fecal organisms range ftom 105- 1011 organisms/g
                        in the 150 g/day feces produced by humans in industrial societies (Feachem et al. 1983). It
                        is estimated that up to 40% of feces is composed of microbial cells (Savage 1972).
                        Considering that obligate anaerobes are numerically dominant in human feces (Holdeman et
                        al. 1976; Moore and Holdeman 1974), the use of aerobic recovery methods and facultative
 0                      anaerobes as fecal indicators may appear somewhat paradoxical. There is little doubt this
                        reflects the latter's ea of recovery from aquatic environments compared to the somewhat
                        rigorous procedures necessary for recovery of obligate anaerobes and the presumption of
                        poor survival in oxygen-containing environments. Human and animal feces also may
 0                      harbor bacteriophages that utilize bacteria as hosts. Bacteriophages that use E colf as a
                        host, e.g., somatic coliphages, F-specific coliphages and Bacteroidesftagilis phages will
                        be discussed in subsequent sections.

 9                      Domestic and feral animals are important potential sources of fecal contamination in urban
                        and rural areas (Feachem et al. 1983; Geldreich 1972). The microbial composition of
                        domestic animal feces has been examined to catalog dominant genera (Barnes 1986), to
                        compare ratios of common indicators to those in humans (Geldreich and Kenner 1969),
 9                      and to identify indicator microorganisms unique to animals (e.g., Cooper and Ramadan
                        1955; Geldreich and Kenner 1969; Wheater et al. 1979). Similar data for feral warm-
                        blooded animals living adjacent to or in shellfish growing waters (e.g, harbor seals,
                        Calambolddis et al. 1989; sea lion, Oppenheimer and Kelly 1952) are rare. Information
                        relating the effects of animal presence on indicator and pathogen levels in adjacent estuarine
                        receiving waters in the absence of gross'pollution is needed.



                                                                8









                       Wastewatem Anthropogenic sources of fecal organisms to shellfish growing areas
                       include discharges of treated municipal sewage, discharges of partially treated or raw
                       sewage that occur owing to mechanical failure, combined sewer systems, falling septic
                       systems, and vessel discharges. Bypassing, the release of untreated sewage, during periods
                       of high rainfall, is not uncommon. Levels of bacterial indicators in effluents from a.
                       properly functioning sewage treatment plant using disinfection will be considerably reduced
                       compared with those in the influent (Miescier and Cabelli 1982). However, disinfection is
                       not a stoichiornetric process and its effectiveness varies with waste composition, influent
                       volume, target organism, and flow. Recent unpublished studies,based on male-specific
                       coliphages, suggest chlorination of sewage effluents may not be removing the more resistant
                       viruses (H. Kator and M. Rhodes, unpublished data; M. Sobsey, unpublished data; W.
                       Watldns and S. Rippey; unpublished data, NETSU). Other sources include agricultural
                       and stonn water runoff, which can transport fecal wastes from humans, and domestic and
                       feral animals to shellfish growing areas. Resuspension and transport of contaminated
                       sediments may also be a source to growing waters. Discharges from commercial and
                       recreational vessels into marina waters, tributaries, and growing areas have been cited as
                       sources of fecal contamination (Faust 1982).



                       Nonpoint Source Runoff. Much of the literature concerning indicators in shellfish
                       growing waters seems to have developed around the concept that fecal pollution is
                       primarily derived from large point sources of sewage or river discharges. These rivers may
                       integrate multiple point sources and typify conditions encountered in some high ly urbanized
                       northeastern. coastal cities. Of municipal sewage facilities discharging into marine and
                       estuarine waters, 42% are located in the northeast and these account for 60% of the total
                       volume discharged (Shigenaka and Price 1988). Diffuse or nonpoint pollution is
                       recognized as a major source of fecal contamination to all types of receiving waters
                       (Geldreich et al. 1968; Gilliland and Baxter-Potter 1987; Faust 1976). Indeed, in the mid-
                       Atlantic and Southeast regions, significant proportions of the total shellfish growing
                       acreage are closed to direct harvesting presumably because of runoff from rural,
                       agricultural, and wildlife sources (Leonard et aL 1989). Improperly functioning septic
                       systems are implicated as important sources of fecal contamination. It is estimated that
                       approximately 25-30% of households in the United States use on-site septic tank systems
                       for treatment and disposal of wastewaters (Chen, 1988; Snowdon and Cliver, 1989;
                       Reneau et al. 1989). The resulting per capita rate of discharge to the groundwater may


                                                              9









                         exceed 10 billion I per day of wastewater and there is reason to suspect that at least 50% of
                         septic systems are operating marginally (Canter and Knox, 1985). Specific issues of
                         concern are the degree of treatment afforded by such systems, which is minimal and does
                         not inactivate viruses, and the potential migration of effluent to estuarine waters because of
                         inadequate dminfields, poor soil characteristics, or leaching through subsurface ground
 0                       water that can be affected by tidal fluctuations. A common path of septic contamination is
                         through transport by runoff of leachate that has broken through the soil surface because of
                         malfunctioning systems, owing to improper size, poor maintenance, inappropriate soils or
                         water tables are common causes of this. In a study of the bacteriological quality of a
 0                       recreational lake, Hendry and Toth (982) found elevated levels of indicator bacteria along
                         the lakeshore were associated with malfunctioning or flooded home septic systems. Lower
                         bacterial densities (below water, quality target levels) were found adjacent to homes with
                         effective systems.  Chen (1988) found wastewater effluents from septic systems located on
                         the shores of New York lakes contaminated the lakes with nutrients and fecal coliforms,
                         through infilitration by groundwater. Such contamination, which has been associated with
                         waterborne disease in fivshwaters (Vaughn and Undry, 1983; Powelson et al., 1990),
                         may also represent a path of transport of indicator bacteria and pathogens to adjacent
                         estuarine waters. These and other concerns are especially relevant to issues of
                         development@ where residential construction of homes using traditional septic systems may
                         have to be curtailed and communities adopt central sanitary sewage systems. Resolution of
                         these issues awaits the application of i        and imaginative experimental approaches that
                                                               rigorous
                         facilitate tracing this contamination.


                         The public health significance of coliform levels in growing areas contaminated by diffuse
                         sources remains unclear. The paradigm of a diluted sewage effluent does not apply in this
0                        scenario, because sources and ratios of indicators to pathogens are expected to vary
                         considerably and less predictably than in large sources of domestic sewage. Fecal coliform.
                         densities that exceed the growing area standard are not uncommon in Chesapeake Bay and
                         other estuaries lacking identifiable point sources of human fecal pollution. Although direct
9                        evidence is lacking, it is believed conditions in estuaries can promote indicator survival
                         (Erkenbrecker 198 1). Contributing factors include inorganic and organic nutrient loading,
                         high suspended solids, elevated temperatures, the presence of fine grained organic rich
                         sediment and poor tidal flushing. Indeed, the need to identify and validate indicator
0                        systems to assess the sanitary quality of growing areas impacted by nonpoint sources may
                         be the greatest challenge to sanitariansa.nd shellfish microbiologists since the adoption of
                         the coliform growing area standard. An evaluation of indicators to index health risk in non-


                                                                10








                        point source impacted areas must also consider the effects of microbial food webs and the
 0                      estuarine environmenton the removal and enumeration of allochthonous, bacteria, the
                        possible extraenteral origin of indicators, and contamination derived from multiple sources
                        including wild and domestic animals. A paucity of data to evaluate these concerns has
                        engendered criticism of the cun  ent indicator and its validity as a public health standard in
                        these environments. However, it appears reasonable in.these environments to determine if
                        indicator data are consistent with the results of shoreline surveys.


 0                      Land Use-Effects On Sanitary Quality Of Receiving Waters.

                        Many studies have identified urban and rural stormwater runoff as a source of large
                        numbers of indicator bacteria and variable levels of pathogens (eg., Davis et al. 1977;
 0                      Ofivieri 1980). In general, many observers have observed the water quality of stormwaters
                        often exceed water quality standards based on total or fecal coliform. bactem Undetected
                        sewage sysytern failures, wild and, domestic animal feces, poor sanitation and perhaps
                        garbage are all contributing sources. In rural areas, runoff ftorn agricultural land has been
                        implicated as a major source of microbial pollution to surface waters (Gilliland and Baxter-
                        Potter 1987; Glendening 1985). Despite the importance of these sources qualitative and
                        quantitative assessments of actual contributions and controlling mechanisms are lacking.
                        Without such information it is difficult to evaluate the effectiveness of management
                        strategies to reduce bacterial pollution, and the absence of real time or rapid methods to
                        measure microbial contamination in runoff and receiving waters remains a significant
                        limitation.


                        Burge and Parr (1980) reviewed the kinds of pathogenic and indicator microorganisms
                        present in sewage and animal wastes, modes of transport to receiving waters, and aspects
                        of indicator fate. One important conclusion was that viruses and bacteria vary greatly in
                        their adsorptive interactions with soils. Ihese interactions are complicated by physical and
                        chemical processes related to specific land use, soil characteristics and water saturation,
                        current and antecedent precipitation, temperature, age of fecal deposits, proximity to
                        receiving waters, etc. Microbial responses (i.e., migration of microorganisms) are difficult
                        to predict because of an imperfect understanding of these many interacting factors (Baxter-
                        Potter and Gilliland 1988). For example, Faust (1976) found no or poor correlation
                        between fecal coliforms discharged in runoff with nutrients, suspended sediment, or
                        rainfall. Meiman and Kunkle (1967) claimed bacterial indicators in runoff provided a bettez








                        indication of land use Ow turbidity or suspended solids. Colfiorm, fecal coliform and
                        fecal streptococci densities in runoff from nongrazed and partially-grazed lands were clearly
                        different Gillilan d and Baxter-Potter (1987) developed a geographically-based method to
                        predict the degree of bacterial pollution as a function of agricultural land use. Actual field
                        data confirmed previous observations that fecal coliform densities in surface runoff
                        consistently exceed surface water quality standards and that similm PC densities can arise
                        from land use as different as a feed lot and a corn field (Gilliland and Baxter-Potter 1987).
                        Faust and Goff (1977) compared annual fecal colfform discharge rates by monitoring water
                        flow from seven watersheds that included mixtures of forest, old field, cultivated cropland,
                        pasture, residential, and wetlands * Although fecalcoliform. counts were higher overall in
                        drainage from pasture land, fecal coliform contributions expressed as fecal coliforms, per
                        ha-year were similar for all basins. Doran and I= (1979) compared. the microbiological
                        quality of runoff from a cow-calf pastureland and a control or utigrazed land. Fecal
                        coliform counts were 5-10 times higher in runoff from the grazed land but fecal
                        streptococci were elevated in the control area, presumably because of feral animals.
                        Streptococcus boWs was detected in streams close to the grazed area and its use to
                        differentiate domestic from feral animal pollution was suggested. Boyer and Perry (1987)
                        observed significant increases in fecal colifiorm densities in runoff from reclaimed mine
                        land after cattle were paswred. Elevated densities were observed for several months after
                        cattle removal and fecal colifornis survived overwintering in manure at temperatures as low
                        as -300C. The authors suggested that lacking information describing the comparative
                        survival of fecal coliforms and microorganisms pathogenic to humans inder these
                        conditions, the presence of fecal coliforms in runoff "may not be indicative of a potential
                        health threat- Elliott and Ellis (1977) observed indicator organisms in animal wastes of
                        various types were not always reliable determinants of pathogen presence. Burge and Parr
                        (1980) concluded that despite the ability to differentiate runoff from lands gazed by
                        livestock from that which supports  only feral animals, the public health significance of fecal
                        coliform indicator densities remains unlmown. Gilliland and Baxter-Potter 1987) stated
                        that attempts to judge the efficacy of mitigation activities using these indicators to measure
                        public health risk remains equivocal. Geldreich (1981) suggested direct detection of
                        pathogens in nonpoint pollution might be necessary to assess public health concerns. Elliot
                        and Ellis (1977) discussed the information required to adequately assess the health hazards
                        associated with the presence of enteric pathogens in the environment. Rather than applying
                        the coricept of zero-tolerance, public health decisions should be based on knowledge
                        concerning the effects of environmental exposure on pathogen virtilence and survival, host
                        susceptibility, and historical incidence of disease attributed to suspect pathogens and routes


                                                               12








                        of transmission. That few managers apply this thinking is a reflection of the paucity of
                        information required, willingness to take risks, and the conservative nature of public health
                        regulation.


                        Various states.have developed management strategies to reduce non-pomt pollution. These
                        include notification.of sewage treatment facility failure and effective warning mechanisms,
                        BMPs to deal with animal wastes usually developed in cooperation with local soil and
                        water conservation personnel, identification of malfunctioning on-site sewage disposal
                        systems, correction and followup. There are very few data available relating BMPs to
                        microbial quality of runoff or receiving waters. Doyle et al. (1975).found forest buffer
                        strips (ca. 8 in wide) are an effective barrier against microbial stream pollution from
                        manured fields. Levels of fecal coliforms and fecal streptococci were reduced to
                        background levels during transport across forested buffer zones. Clausen and Meals
                        (1989) considered the effectiveness of various BMPs on measures of water quality that
                        included criteria for nutrients, suspended. soilds, and fecal coliforms. Measured against the
                        fecal coliform water quality criterion (200 FC/100 ml) for recreational waters), a vegetated
                        filter strip did not reduce densities below this level in runoff However, use of BMPs did
                        result in overall reductions of pollutant mass output and this measure is considered as
                        important as those based only on pollutant concentration. Clearly, new BMPs and methods
                        to effectively evaluate BMP strategies in term of concentrations and annual outputs of
                        microorganisms in runoff and receiving waters should be a priority research issue.



                        Environmental Factors Affecting the                     Fate of Allochthonous
                        Indicators of Fecal Contamination


                        Effects of various environmental parameters on indicator survival have been examined by
                        many investigators but it is often difficult to unequivocally assess their "true" significance.
                        Concerns arise with studies based on the use of in vitro experi ments, laboratory-adapted
                        strains, artificial or filtered menstrua, and exposure devices incapable of preventing external
                        contamination or containment of test strains (Roper and Marshall 1979; Anderson et al.
                        1983). Given the probability that the microbiota. can be a major factor affecting bacterial
                        numbers, it seems unwise to generalize based on experimental designs that exclude this
                        component. This is not to discredit the   value of in vitro studies, which are useful to
                        establish basic principles, but ecological hypotheses should be tested under conditions that
                        duplicate open or natural systems as closely as possible. Another concern focuses on


                                                                13








                        methods used for preparation of test cells. A majo    rity of survival studies involved
                        procedures for preparation of test cells known to result in sublethal stress or be
                        physiologically debilitating. Culture age, growth conditions, and laboratory manipulations
                        are important contributing factors contributing to sublethal injury. Use of cells pregrown in
                        rich media and harvested during the exponential phase of growth, cold-shocking, and
                        harvesting by repeated centrifugation in unfavorable solutions are known to compromise
                        physiological indices, increase sublethal stress, reduce adenylate levels, reduce enzyme
                        activities, produce changes in membrane integrity leading to leakage of cell constituents,
                        and render cells sensitive to fi-ee radicals or heavy metals (Postgate 1967; Strange 1976;
                        Anderson et al. 1979; Granai and Sjogren 198 1; Rhodes et al. 1983). Failure to recognize
                        these factors as sources of experiniental bias has led to incorrect  inferences of causality.
                        SimAu reservations apply to using laboratory adapted strains because these cells may
                        exhibit survival characteristics that are "atypical" compared with "fresh" fecal isolates
                        (Anderson et al. 1979).


                        In view of the importance accorded test cell preparation, a relevant hypothesis that should
                        be tested concerns the effect of indicator origin (exposure prehistory) on its fate Are there
                        differences in the survival of indicator cells prepared under laboratory conditions and cells
                        derived from "natural" sources such as sewage treatment plant (STP) effluent, septic tank
                        effluent, agricultural or stormwater runofP Ile STP or septic system provides a nutrient
                        rich environment, at times maintained at a higher temperature than t    he receiving waters, and
                        exposes cells to varying degrees of toxic materials or disinfectant-mediated injury. Could it
                        be diat indicator bacteria from diffuse natural sources are physiologically adapted to adverse
                        nutrient concentrations, intermittent lack of water, unfavorable temperatures, etc? Such
                        adaptation could indicator persistence and recoverability compared with laboratory-grown
                        cells.


                        Finally, it is difficult to see how investigations that treat the effects of environmental factors
                        asif they were independent operators can lead tD developing basic principles of indicator
                        cell survival. Treatments that isolate single variables will only provide information
                        concerning the potential role of that variable. In the environment survival will be affected
                        by the interaction of that factor with perhaps more dominant processes. lAboratory studies
                        must be augmented by admittedly more complex and variable in situ exposure studies that
                        integrate physicochemical and biological factors. Conversely, in situ experiments must be
                        carefully interpreted because of the potential for undetected processes that may affect test
                        cell densities. Examples include breaching of exposure devices by autochthonous


                                                                 14








                       organisms owing to design or structural damage (Roper and Marshall 1979; Anderson et al.
                       1983) and physical penetration through the membrane "pores7 by bacterivorous
                       nannoflagellates (Cynar et al. 1985) or other procaryotes (U and Dicide 1985). Chambers
                       may exhibit highly variable "bottle" effects because of nutrients contributed by fouling
                       communities on chamber surfaces, blooms caused by exclusion of autochthonous
                       organisms or predators, and attenuation of incident light. Finally, estuaries are unique
                       dynamic sy stems, characterized by temporal and spatial heterogeneity. Generalizations
                       based onexperiments performed in Puget Sound may not be applicable to Chesapeake Bay
                       or Gulf of Mexico growing waters.


                       The following sections summarize important aspects of the effects of physical, chemical
                       and biological factors on indicator survival, primarily under estuarine and marine
                       conditions.



                       Physical and Chemical Factors

0                      Temperature. At first glance the    literature dealing with the role of temperature on the in
                       situ survival of E coli in saline waters appears equivocal. Investigators have reported both
                       positive (Anderson et al. 1983; Rhodes and Kator 1988) and negative (Faust et al. 1975;
                       Vasconcelos and Swartz 1976; Lessard and Sieburth 1983) correlations of temperature and
0                      survival. This confusion can be resolved if it is understood that temperature has both
                       indirect and direct effects on indicator fate. It can have a direct effect on bacterial activity,
                       so that under appropriate conditions multiplication may occur at warmer seasonal
                       temperatures. E coli cells prepared under conditions to minimize stress initially exhibit
0                      multiplication (large negative values of the mortality rate coefficient, k) in membrane
                       filtered water (0.2 p m) at elevated temperatures in Chesapeake Bay (Rhodes and Kator
                       1988). Maximum negative values of k correspond to increases in viable counts of
                       approximately 1.0 to 1.5 log units. Although it may be thought that low environmental
0                      temperatures (<IOOC) would reduce coliform metabolic activity based on Q values, thereby
                       favoring persistence, low environmental temperatures do not appear to have this effect as
                       claimed by some authors. T"he indirect effects of low environmental temperature will be
                       addressed in the context of sublethal stress but exposure of bacterial cells to low
0                      temperatures is known to compromise cell envelope integrity and physiological indices
                       (Strange 1976). Indeed, positive values of k were obtained at temperatures below 10C in
                       filtered estuarine water (Rhodes and Kator 1988).


0                                                              15






 3




                       An indirect and highly significant relationshipbetween temperature and indicator recovery
                       and enumeration is the effect of temperature on the development of sublethal stress.
                       Sublethal stress reflects the inability of a microorganism to be cultured in a medium because
                       of prior injury, hiapairment or damage that resulted from exposure to unfavorable
                       environmental conditions. Sublethally stressed cells are particularly sensitive to recovery
                       methodologies that use selective temperatures, lack resuscitative protocols or use inhibitory
                       substances to enhance media specificity and selectivity (Hackney et al. 1979). We have
                       quantified the development of sublethal stress in E coli as a function of both temperature
                       and salinity, (Anderson et al. 1979; Rhodes et al. 1983). Sublethal stress and mortality are
                       inversely related to temperature. At temperatures below 100C transiently acute or
                       progressive development of sublethal stress is detectable using a variety of techniques.
                       These include differential counts on selective vs. non-selective recovery media and an assay
0                      technique (Anderson et al. 1979) based on the observation that sublethally stressed cells
                       require a longer period of time to produce an electrochernically-induced potential difference
                       compared with non-stressed cells [electrochemical detection time (ED71)]. In practical
                       terms, sublethally stressed cells may not be detected using enumeration methods that
0                      incorporate selective procedures. The effect of exposure to estuarine water on enumeration
                       efficiency using experimental and approved recovery methods (Rhodes et al. 1983) causes
                       progressive stress over time even with favorable environmental temperatures. Selective
                       methods, such as the direct M-FC procedure, an approved method (APHA 1989), exhibit
0                      poor enumeration efficiency. The rosolic acid in this medium significantly reduces the
                       recoverability of chlorine-injured fecal coliforms from sewage (Presswood and Strong
                       1978). Although resuscitation procedures in non-selective media have been employed to
                       diminish the impact of selective enumeration, such measures may not be completely
                       effective.


                       Sublethal stress is an important phenomenon that must be considered if conventional
                       selective enumeration procedures are used for the recovery of indicator cells. Results from
                       survival studies with K coli where viable counts were not "corrected" for the effects of
                       sublethal stress tend to overestimate mortality (underestimate cell densities), with the error
                       being most significant at temperatures below 10*C. As a result, the responses of cells to
                       physical par-ameters or treatments where sublethal injury was unrecognized must be
                       interpreted with caution because the observed mortality may have been incorrectly attributed
                       to another variable and not the enumeration process. These concerns reinforce the notion



                                                            16








                        that sublethal stress is an important factor affecting indicator choice when viable recovery
                        methods are used and.observed cell densities are to have regulatory significance.


                        Temperature-induced sublethal stress also rendered cells sensitive to other stressors.
                        Mackey and Derrick (1986) presented evidence that cold-shock sensitized E colt to very
 0                      low concentrations of hydrogen peroxide and as a consequence reduced recovery of viable
                        cells on an organic-rich medium. Postgate (1967) cautioned against chilling of samples and
                        cultures as a practice that promoted the death of stressed cells. Jackson (1974) observed
                        loss of viability of Staphylococcus aureus and increased sensitivity to a selective medium at
                        .50C and noted F. coft became more sensitive to violet-red-bile agar exposed at tins
                        temperature. The implications of these observations apparently remain unheeded because
                        Standard Methods (APHA 1989) suggests icing of microbiological samples if they cannot
                        be processed within I hour. A systematic examination of sample storage parameters to
                        opitimize recovery under a variety of seasonal temperature regimes should be part of the
                        indicator evaluation process.


                        indirect effects of temperature on coliforin fate arise from the influence of seasonal
                        temperature on the densities, composition and activities of the indigenous microbiota
                        (Verstraete and Voets 1976; Anderson et al. 1983; Rhodes and Kator 1988). The role of
                        the microbiota on indicator persistence and survival has been until recently a topic of
                        considerable speculation. Thus, although the activities of antagonistic substances, parasitic
                        and lytic. microorganisms, and protozoans were recognized as potentially important factors
                        controlling indicator abundance (Mitchell and Yankofsky 1969; Mitchell 1972; Mitchell and
                        Chamberlin 1975; Drake and Tsuchiya 1976), it is only recently that microbial ecologists
                        have identified bactenvory as important carbon and energy pathways (Wright and Coffin
 0                      1984). The importan   ce of bacteria in coastal waters as food for heterotrophic
                        microflagellates has been demonstrated (Fenchel 1982; Anderson and Fenchel 1985).
                        Using diffusion chambers, Awong et aL (1990) observed that densities of genetically
                        engineered strains of F. coli were significantly reduced in nonsterile lake water compared
 0                      with cells in filter-sterilized water. Gonzalez et al. (1990) observed bacterivory of F. coft
                        and E faecalis by natural assemblages of estuarine flagellates and ciliates incubated in
                        Whirl-pak bags. Both predators groups exhibited grazing preference for large bacterial
                        cells but ciliates ingested E faecas at a higher rate thaii E coft. Studies with diffusion
 9                      chambers, perhaps somewhat artifactual in terms of community development and enhanced
                        encounters between predator and prey, have revealed the potential influence of the
                        microbiota on E coli survival in estuarine water (Rhodes and Kator 1988). Generally,


 0                                                             17










                       times of maximum decline in nonfiltered water coincided with maximum densities of
                       heterotrophic flagellates or other predators (Rhodes and Kator 1988). Although predation
                       and other microbially-mediated removal processes, and the "intrinsic" growth responses of
                       enteric bacteria were direct functions of temperature, the net combined effect of increased
                       temperature was indicator removal. It is evident that failure to recognize both direct and
                       indirect effects of temperature has lead to the erroneous conclusion that elevated
                       temperature, per se, does not favor coliform survival.



                       Salinity. Efforts to determine the effect of salinity on indicatoi survival,constitutes a
                       small and somewhat inconclusive literature (Carlucci and Pramer 1960; Faust et al. 1975;
                       Orlob 1956; Vasconcelos and Swartz 1976). Results reported, which are generally for E
                       coh, range from no. effect of salinity on E coli viability to reduced survival with increasing
                       salinity. This literature remains problematic for many of the same reasons noted for
                       temperature studies. The combination of all-or-none viable counting methods with factors
                       known to be stressors, e.g., selective recovery methods, artificial seawater with possible
                       trace toxicants, harsh inocula preparation methods, contributed to the apparent
                       disappearance of test cells. These problems were avoided with techniques that measured
                       graded responses and used cell preparation techniques that minimi@ed injury (Anderson et
                       al. 1979). Graded responses included EDT in selective and non-selective media, 0-
                       galactosidase specific activity, and growth rate. Increased salinity was accompanied by
                       decreased recovery of viable cells, an increase in sublethal stress expressed both in terms of
                       larger EDT values and as the difference in cell recovery on selective versus non-selective
                       media, and as a marked reduction in 0-galactosidase specific activity (Anderson et al.
                       1979). Cells exposed to 30 psu seawater for 2-9 days exhibited an apparent mortality
                       about 9 times greater when recovered *in EC medium than the same cells recovered in TSB.
                       The idea that exposure of E coli to saline water yields cells with altered physiological
                       properties, thereby requiring modified enumeration methods, has been suggested. Dawe
                       and Penrose (1978) observed that salinity-induced debility in coliforms is reduced by
                       incorporation of seawater into the recovery medium. Gauthier et al. (1987) increased
                       recovery of E coli cells adapted to seawater by including sodium chloride in an
                       enumeration medium, although the degree of response was strain and time dependent.
                       Munro et al. (1987) observed that E coli starved in seawater manifested a variety of
                       physiological and structural responses including loss of 0-galactosidase activity, increased
0
                       activity of other enzymes, and altered sensitivities to antibiotics, phages and heavy metals.
                       Increased enzyme activity toward 4-methylumbelliferyl heptanoate observed during


                                                             18








                         starvation of E coh in seawater was presumably a response to low nutrient conditions
                         (Fiksdal et al. 1989). E coli cell envelope composition and functional characteristics are
                         different in ce Ils grown in an estuarine water-based medium compared with those grown in
                         a standard medium (Chai 1983).. The effect of high osmolarity in E.coli is to repress
                         synthesis of OmpF porin protein, a protective mechanism to reduce cell permeability to
 0                       naturally occurring detergents, i.e., bile salts (Nikaido, and Vaara 1987). 'This adaptative
                         process retains membrane permeability, albeit reduced, to nutrients with molecular weights
                         of 100-200. An indirect consequence of reduced membrane permeability under
                         hyperosm6lar conditions could be to alter significantly or reduce the uptake of molecules
                         used in direct cell viability assays such as the tetrazolium salts. Gauthier et al. (1991)
                         discussed the significance of cell preparation methods on E coli survival in seawater.
                         Procedures causing loss of intracellular K' and glutarnate affect the ability of cells to
                         regulate osmotic pressure and reduce their resistance and survival in seawater. Restoration
                         of intracellular K' and glutamate restores regulation of cellular osmotic pressure and
                         enhances survival in seawater. In summary, the studies mentioned support an emerging
                         hypothesis that survival of E coh in seawater is an active process involving physiological
                         adaptation to an adverse environment. Recovery of adapted cells may be optimized using a
                         resuscitative environment that allows cells to readapt to conditions of lower osmolarity.


                         Strange (1976) in his monograph on stress noted that osmotic shock causes loss of cell
                         viability, reduces active transport of solutes, and releases a variety of metabolites and
                         enzymes. Roth et al. (1988) demonstrated diat a large proportion of E coli cells exposed
                         to hyperosmotic conditions are not recoverable on a non-selective medium. However, if
                         the cells are osmotically upshocked in the presence of betaine (N, N, N-trimethylglycine, a
                         naturally occurring and ubiquitous nitrogen-containing compound produced by
                         microorganisms, animals and plants), the cells remain culturable. Betaine accelerates
                         uptake of ATP and reduces intracellular ATP that accumulates in osmotically upshocked
                         cells and facilitates protein synthesis. Conjugative transfer of a plasmid between donor and
                         recipient E coli cells was also enhanced by glycine bewine in autoclaved sediments
                         (Breittmayer and Gauthier 1990). Roth et al. (1988) developed a resuscitation method
                         using a medium incorporating betaine, chloramphenicol (to prevent changes in cell density
                         during resuscitation), ammonium and glucose as nitrogen and carbon sources. At this
                         writing we are unaware if this method has been evaluated with estuarine samples.






                                                                 19








                       Adsorption and Sedimentation. The effect of estuarine particulates on the persistence
                       of autochthonous bacteria continues to be an interesting area of research. Rubentschik et
                       al. (1936) suggested that adsorption of E coli to particulates with subsequent deposition in
                       sediments is an important aspect of self-purification in salt lakes. Weiss (195 1)
                       investigated F. coli adsorption on river and estuarine silts and concluded that adsorption
                       enhances bacterial sedimentation rate and is a function of particle type and size. Although
                       flo6culation of silts increase in seawater, the adsorptive capacity of silts toward bacteria
                       decreases with increased salinity and the bacteria desorb. Milne et al. (1986) concluded
                       that removal of fecal coliforms from estuarine water by deposition was directly related to
                       the concentration of naturally-occurring suspended solids.     A similar relationship was not
                       observed in seawater and attributed to the differences in the depositional behavior of
                       suspended solids in the two systems. Roper and Marshall (1974, 1979) observed that E
                       coli adsorb to sediments at high electrolyte concentrations and desorb below a critical
 0                     concentration. They hypothesized that in low electrolyte (freshwater) systems ele@trostatic
                       forces allow bacteria to exist as stable colloidal dispersions. As salinity is increased,
                       flocculation and sedimentation of bacteria and particles will occur. This straightforward
                       model can be complicated by organics present on the microorganisms "surface." Thus,
 0                     adsorbed bacteria can adhere very strongly to sediment particles owing to production of
                       organic exopolymers (Marshall, 1985). Viruses also adsorb to estuarine sediments.
                       Labelle and Gerba (1979) observed >99% adsorption of various enteric viruses to sediment
                       at salinities of 1-35 psu. Alterations in salinity and pH produce small but variable effects
 0                     on adsorption and desorption of most virus types examined. Because enteric viruses did
                       not readily desorb they concluded that vn-al transport would be dependent on particle
                       resuspension and transport.

 0                     Based on the above observations it is not surprising that densities of fecal indicator bacteria
                       (Erkenbrecker, 198 1; Shiaris et al., 1987) and enteric viruses (LaBelle et al., 1980, Rao et
                       al., 1984) are elevated in estuarine sediments compared to overlying waters. Sediments
                       from an urban shellfish growing area in the Chesapeake Bay contained fecal colifbrms at
 40                    densiti es two orders of magnitude larger than in the water column (Erkenbrecker 198 1).
                       Variable but generally smaller values of the ratio of fecal coliform densities in sediment to
                       water were observed in a small subestuary subject to nonpoint pollution (Kator and Rhodes
                       1989). LaBelle et al. (1980) found no correlation between densities of bacterial indicators
 0                     and virus in estuarine waters. There was a positive correlation, between the numbers of
                       viruses and fecal coliforms in sediments.




                                                               20









                         In vitro and in situ experiments (Gerba and McLeod 1976; Roper and Marshall 1974, 1979;
 .0                      Peresz-Rosas and Hazen 1988) have shown the positive effect of marine or estuarine
                         sediments on E coli persistence. Similarly, adsorption to estuarine sediments enhances
                         survival of enteroviruses (Smith et al. 1978; Toranzo et al. 1982) and bacteriophage T7
                         (Bitton and Marshall 1974). Protective effects are attributed primarily to accumulation of
                         nutrients on particle surfaces and reduced predation (Roper and Marshall 1978) and
 0
                         antibiosis. As previously mentioned, Roth et al. (1988) reported the organic compound
                         betaine minimizes osmotic stress and restores the cu'lwrability of E coil exposed to
                         seawater. Le Rudulier and Bouillard (1983) have shb*h that betaine concentrations as low
                         as I mM eliminate osmotic stress in X pneumoniae, S. typhimuhum          and E coli over a
                         range of high NaCI concentrations (0.65- 1.0 M). There was no evidence that betaine is
                         utilized as a growth substrate in K pneumoniae. Betaine is a ubiquitous and relatively
                         abundant compound in benthic organisms and is an important substrate in fermentation
 0                       pathways coupled to methanogenesis in marine sediments (King 1984; King 1988;
                         Heijthuijsen and Hansen 1989). The apparent enrichment and persistence of E coft (or
                         other enteric bacteria) in sediments may be augmented by the osmotolerance afforded by
                         betaine or other osmolytes (Munro et al. 1989; Gauthier and Le Rudulier, 1990; Ghoul et
 0                       al. 1990). The effects of anaerobic sediments, which are sources of inorganic nutrients and
                         small molecular weight fermentation products, on indicator persistence is obviously a
                         research issue requiring further study.

 0                       The accumulation and enhanced survival of sewage microorganisms in sediments have
                         been employed as a justification for questioning the validity of using a water quality
                         criterion as the basis for classification of shellfish growing waters (Shiaris et al. 1987).
                         Sediments provide a more integrated recent history of fecal pollution than overlying waters
 0                       that reflect transient pollution events. Relationships between indicators in sediment and
                         river water were examined in a study by Matson et al. (1978). Sediment deposition,
                         resuspension, and transport were identified as processes that significantly affect densities
                         of indicators (and enteric pathogens) in overlying waters (Matson et al. 1978; LaBelle et al.
 0                       1980;      enbrecker 1981  ). Thus, the absence o  f microorganisms from the water column
                         may not reflect a diminished health hazard. Metcalf et al. (1973) noted that fecal coliform
                         concentrations and the likelihood of isolating salmonellae in the water column were
                         functions of tidal stage, suggesting an association between tidal currents and the
 0                       resuspension and transport of particulate maUnal. Particle-associated pathogen transport is
                         emphasized by greater viral accumulation in shellfish exposed to resuspended sediment as



 0                                                               21








                          compared to those placed in a system containing undisturbed sediment (Landry et al.
                          1983).



                          Ught. Direct lethal effects of light on enteric bacteria in seawater have been demonstrated
                          under in situ (Gameson and Saxon 1%7; Gameson and Gould 1975; Bellair et al. 1977;
                          Fujioka et al. 1.981) and in vitro experimental treatments (Gameson and Gould 1975;
                          Fujioka, et al. 1981; Kapuscinski and Mitchell 1981; McCarnbridge and McMeekin 1981;
                          Fujioka and Siwak 1987; Coniax et al. 1990. Tartera et al. (1988) compared the
                          sensitivities of bacterial and viral indicators to UV light under in vitro conditions.
                          Coliphage f2 and a Bacteroidesfragilts bactenophage are more resistant than E coli and E
                          fbecalu. Male-specific coliphages are significantly more resistant to UV radiation than
                          indicator bacteria (Havelaar 1986; 19n. The overall importance of these studies to the
                          mortality of indicator organism  s in shellfish growing waters remains somewhat equivocal.
                          This is because estuaries where shellfish are produced, for example in the eastern United
                          States and Gulf of Mexico, are highly turbid and can be dominated by complex microbial
                          food webs. The importance of these factors resides in the attenuating capacity of
                          suspended and dissolved material toward lethal wavelengths of light and the light-
                          stimulating effect on components of the microbiota that can lead to enhanced bacterial
                          mortality. Attenuation of UV-B (280-320 nm) light may occur in the uppermost layer of
                          the water column although the degree of attenuation in coastal waters is influenced by local
                          properties that affect light absorption such as dissolved organic matter, chlorophyll
                          concentration and particulate load (Calkins 1982). Interactive effects of light and the
                          autochthonous microbiota have been demonstrated in fresh and estuarine waters under in
                          vitro conditions (McCambridge and McMeekin 198 1; Barcina. et al. 1989). A series of in
                          situ experiments were performed in the Chesapeake Bay using diffusion chambers specially
                          modified to maximize light penetration and employing light and dark, filtered and
                          nonfiltered treatments (Rhodes and Kator 1990). Compared with cells suspended at 1.0
                          cm below the water surface, mortality from sunlight was essentially insignificant at 25.0
                          cm. except during periods (fall, winter) of minimal light attenuation. Cells at 1.0 cm also
                          exhibited significant sublethal stress. During the warm seasons significantly greater
                          mortality occurred in the presence of light and the microbiota than with either alone.
                          Enhanced mortality of the combined treatment can be attributed to stimulation of predation,
-0                        by sunlight, light-dependent release of antagonistic substances, or formation of
                          photochernically induced toxicants. Therefore, in turbid shellfish growing waters sunlight-



                                                                  22








                        induced injury and mortality result from direct and ind:trect effects whose relative influence
                        will be a function of local conditions.




                        Organic Compounds and Nutrients. In a review of factors affecting the survival of
                        enteric microorganisnis in marine and estuarine environments, Mitchell and Chamberlin
                        (1975, p. 24 1) concluded a section on "Nutrient deficiencies" as follows: "Iliese results
                        coupled with laboratory findings tend to suggest a significant role for nutrients in
                        determining survival of enteric bacteria in seawater". Until recently the literature did not
 0                      refute this conclusion, but did little to amplify it or providedata supporting this hypothesis.
                        Fortunately, this area has been the focus of a number of research groups such as Lopez-
                        Torres, et al. (1987), who describe a positive statistical association between nutrient
                        concentrations and persistence of E coli.' E coli starved in seawater manifest a variety of
 0                      physiological and structural responses including changes in enzyme activity, and altered
                        sensitivities to antibiotics, phages and heavy metals (Munro et al. 1987). Fiksdal et al.
                        (1989) concluded increased hydrolase activity toward 4-methylumbelliferyl heptanoate by
                        E colt starved in seawater is an adaptation to nutrient limitation. Studies are still needed to
                        evaluate the direct effects of inorganic nutrients and organic carbon on enteric survival,
                        especially in nutrient-rich shellfish growing areas that are not well flushed, where high
                        productivity associated with detrital food webs, allochthonous inputs, and organically
                        enriched fine sediments may provide nutrient conditions favoring indicator persistence.



                        Toxic Compounds. Although the lethality of disinfectants toward indicator bacteria is
                        well described, the effects of toxicants present in shellfish growing waters and sediment on
                        these microorganism   s is. a rather. impoverished area of research. This is somewhat
                        surprising considering that many estuarine areas are polluted by high levels of toxic heavy
                        metals, xenobiotics and other materials. Runoff from agricultural and urban areas contains
                        a variety of pesticides of varied toxicity and persistence characteristics. Elevated
                        concentrations of zinc have been found in coastal sediments impacted by sewage discharges
                        (Bruland et al. 1974). Effluents from kraft pulp mill plants may elicit a broad range of
                        toxic effects (Sodergren 1989). Oil shale process waters are lethal to coliforms and S.
                        faecalis under in vitro exposure conditions (Adams and Farrier 1982). Jones and Cobet
                        (1975) noted the toxicity of naturally occurring heavy metals in Caribbean seawater to
                        enteric bacteria. Widespread use of organotin compounds as antifouling paints has been
                        recognized as contributing to high tributyltin concentrations in estuarine waters and


                                                                23








                        sediments frequently exposed to vessels. Pettibone and Cooney (1986) concluded that
 -0                     organotin compounds are not acutely toxic to E coli and E faecalis isolates at naturally-
                        occurring concentrations but act as stressors. Chai (1993) noted that F. coli grown in a
                        medium containing estuarine water manifested changes (adaptations) in cell envelope
                        Composition, changes that afforded decreased sensitivity to bacteriophage infection and
                        colicins but also rendered cells more sensitive to heavy metals and detergents. In a series
 0                      of experiments to evaluate th e effects of 10 MM Zn2+ on. bacteria and selected coliphages,
                        the toxicity of zinc to E coli was increased in the presence of .0. 1 M or higher sodium
                        chloride (Babich and Stotzky 1978). In vitro exposure of E coli to estuarine water
                        containing various toxic chemicals alters cell en velope protein composition, detectability of
                        plasmids, and affects carbohydrate and amino acid metabolism (Palmer et al. 1984).
                        Creosote contamination of estuarine sediments is detrimental to microbial communities as
                        reflected in reduced secondary production and biomass (Koepfler and Kator 1986).
                        Although definitive information is lacicing, sediments may be sources of chemical and
                        biological stressors toward indicator bacteria although it is likely the degree of effect will be
                        site specific.


 0                      Biologic-al Factors

                        Biological factors that affect indicator fate have been discussed or mentioned in other
                        sections. A complete survey of this literature is beyond the scope of this review.
                        Biological interactions between indicators (bacteria or viruses) and components of the
                        microbiota, are complex and definitive in situ rate measurements describing interactive
                        processes are lacldng. Processes effecting removal through predation, parasitism or lytic
                        activity (e.g. Berk et al. 1976; Roper and Marshall 1977, 1978; Enzinger and Cooper 1976;
                        Anderson et al. 1983; Rhodes and Kator 1988) or antiblosis (Sieburth and Pratt 1962;
                        Moebus 1972; Aubert et al. 1975; Toranzo et al. 1982; Girones et al. 1989), and
                        competition for nutrients (Jannasch 1966) have been described. A need exists to determine
                        the relative importance of biological interactions compared with other processes that affect
                        indicator survival or removal. Because biological processes in estuaries are highly variable
                        in temporal and spatial dimensions, studies to assess these effects must be performed with
                        sufficient replication and seasonal coverage to ensure the collection of representative data.
                        Technical impediments to in situ experimentation that require resolution are uncontrolled
                        contamination of exposure chambers by autochthonous microorganisms, inadequate sample
                        volumes, and the need to unequivocally differentiate test cells from the autochthonous cells.


                                                             24







                        Garcia-Lara et al. (1991) proposed use of [3]41thymidine-labeled cells to evaluate the
                        overall contribution of grazing and lytic processes on indicator mortality. Analytical
                        approaches combining rapid direct viable counting methods with those permitting
                        unequivocal identification of test organisms (e.g., fluorescent antibody, gene probes, etc.,
                        Roszak and Colwell 1987) may also provide solutions.

 0

                        INDICATORS., THEIR DETECTION AND ENUMERATION



                        Traditional Indicators for Water


                        Coliforms. Historically coliforms have evolved to become approved indicators of
                        sanitary water quality for drinldng, surface and estuarine receiving waters. During this
 0                      period, methods for their recovery and enumeration have been under contimial scrutiny,
                        yielding refinements in selectivity, specificity, and efficiency characteristics. Over the last
                        several decades the effects of environmental exposure on coliform recovery have been
 0                      recognized and repair procedures evaluated. The 17th edition of Standard Methods (1989)
                        recognizes injury as a factor affecting recovery of colifiorm indicator bacteria, suggesting a
                        variety of steps to enhance recovery.


                        Although the total cohforin group can be found in most current compilations of approved
                        microbiological methods, the overwhelming thrust of literature dealing with indicators,
                        relationships between indicators and pathogens and the epidermological verification of
                        indicators is rather clear in authenticating the extrafecal origins and growth potentials of
 0                      members of this group, the superior specificity of the fecal coliform as an indicator of
                        feces, and its lack of correlation with pathogens and health effects. Based on these facts
                        one must conclude that although the group remains an accepted indicator and standard for
                        drinldng water, its continued application to shellfish growing waters is not justified.

 0
                        Although as previously noted the validity of total coliforms as an indicator of fecal
                        contarnination in natural shellfish waters is equivocal, use of the total coliform group to
                        classify shellfish growing waters is still recognized by the National Shellfish Sanitation
 0                      Program (1990) and therefore methods for its recovery are included in this review.
                        Approved methods (APRA, 1989) for enumeration of the coliform group of bacteria from
                        shellfish waters include multiple-tube fermentation and membrane filtration techniques..


 0                                                            25








                        Each method has its unique characteristics and advantages. The multiple-tube fermentation
                        method (MPN) is -comparatively simple to perform but mechanically repetitious, and using
                        3 or 5 tubes per dilution (most commonly used) provides a relatively imprecise estimate of
                        the density of coliforms in a given sample. However, the NPN method does allow for
                        processing samples that contain relatively high amounts of suspended particulates,
                        chlorine, or other toxic chemicals compared with membrane filtration.


                        The total coliform group consists of all aerobic and facultative anaerobic, gram negative,
                        non-spore-forming rod-shaped bacteria that ferment lactose with gas and acid formation
                        within 48 h at 350C (APHA, 1989). Approved methods call for incubation first in lauryl
                        sulfate trptose broth, although lactose broth may be used, followed by transfer of tubes
                        producing gas to brilliant green lactose bile broth, etc. Approved MW-based methods do
                        suffer from interferences caused by the presence of microorganisms that inhibit gas
                        production or are antagonistic to coliforms (Evans et al. 198 1). Coliform masking can be
                        intensified with sublethally stressed cells and was demonstrated with seawater samples
                        (Olson 1978). Some coliforms may be anaerogenic and therefore tubes with growth
                        should be confirmed through additional and time consuming testing (Olson 1978; Evans et
 0                      al. 1981). Another source of interference are organisms such as Aeromonas spp. which
                        produce gas in standard media or can overgrow coliforms. These effects can lead to
                        underestimation of coliform densities or failure to detect their presence.


                        Total coliforms may also be enumerated using a membrane filter approach (Geldreich
                        1981). The latest edition of Standard Methods (APHA, 1989) offers an approved total
                        coliform membrane method (using either M-Endo or LES Endo agars) and also presents a
                        method for recovery of injured total coliforni bacteria as well as an abbreviated discussion
                        of stress. Avila et al. (1989) recommended M-Endo as the most suitable medium for
                        recovery of coliforms from seawater by membrane filtration. The membrane method offers
                        better reproducibility, sensitivity and precision than the MPN approach (Clark et al. 195 1).
                        However, the obvious advantage of sample concentration leads to problems associated with
                        high turbidity, injury owing to adsorption of chlorine or other toxics by the filter, and
                        concentration of noncoliform bacteria. Interference caused by the physical presence of
                        background microorganisms or even the production of coliform-specific bacteriocin-like
                        substances has been considered (Means and Olson 198 1; Burlingham et al. 1984). The
                        volume filtered in saline waters may be restricted because of high particulate concentrations
                        which interfere with colony formation and counting. Geldreich (1981) and Hartman et al.
                        (1966) consider basic problems associated with membrane filtration-based methods.


                                                              26











                        Fecal Coliform. The fecal coliform, group, the most frequently used indicator of fecal
                        contamination to classify shellfish growing waters, replaced the total coliform group as a
                        more specific indicator of fecal pollution in natural waters. However, it also possesses
                        characteristics that have engendered criticism of the total coliform group. The fecal
                        caliform group is operationally defined (APHA 1985) and includes genera that are not
                        restricted to fecal habitats and may grow in saline receiving waters. The standard
                        procedure for enumerating fecal coliforms does not differentially resolve component
                        genera. By extension, if the numeric value of the total coliform standard was not based on
                        a quantitative assessment of health risk, then the same must be said of the fecal coliform
                        standar d, the latter derived by correlative analysis of paired total and fecal coliform. data
                        from growing areas (Hunt and Springer'1974). The discrete value chosen also reflects a
 0                      methodological bias dictated by the constraints of the MPN distribution table. Compared
                        with the enterococci, the fecal coliform indicator (and E colt) was considered an inferior
                        indicator of sewage pollution because of large reductions in its density that occur during
                        sewage treatment, its greater sensitivity to chlorination, and its higher rate of dieoff
                        (Miescier and Cabelli 1982). F. coli has been shown capable of persistence for periods of
                        extended duration, or even aftergrowth in estuarine waters. It is noteworthy that shellfish
                        harvested from approved growing areas in the Gulf of Mexico during periods of seasonally
                        warm temperatures may be rejected because fecal coliforms levels in the meats exceeded the
                        market guideline owing to multiplication of fecal coliforms after harvesting (FDA 1984).
                        Recent reports note how its entry into a nonculWrable but viable physiological state, its
                        physiological adaptation to seawater, and the effects of sublethal stress can lead to
                        significant underestimation of its numbers in seawater. Sublethal stress can have a
                        significant effect on bacterial recovery at environmental temperatures below 100C, in waters
                        of high salinity, in the presence of toxic chemicals, or due to light-induced damaged.


                        An alternative to the APHA MPN method (APHA 1985) is the approved 24 h modified A- I
                        MPN method (Hunt and Springer 1978; APHA 1985) method. For better precision a
                        membrane filter direct counting method based on the use of mFC agar is approved for use
                        (APHA 1989) but is subject to the same limitations discussed for the total coliform
                        membrane filter method. Use of rosolic acid may be inhibitory to injured coliforms and
                        may be omitted (Geldreich 1981). Pagel et al. (1982) found recovery of fecal coliforms on
                        mT'EC agar, a medium designed for selective recovery of E coh, is superior in overall
                        performance characteristics to mFC. A alternate filter-based approach is the hydrophobic


                                                               27








                       grid membrane filter (e. g., Sharpe 198 1) coupled with an appropriate selective media such
                       as described by Entis and Boleszczuk (1990). These filters accommodate an increased
                       range of counts, i. e., 3 orders of magnitude compared with ordinary membrane filters, and
                       provide considerably better precision than the MPN method. Waxed grid lines used to
                       divide the membrane into hundreds of independent compartments or cells, confine colony
                       growth, prevent spreading and inhibition by background bacteria.



                       Unapproved or Emergent Methods for Enumeration of Coliforms and Fecal
                       Coliforms. Methods discussed in this section have been developed to enumerate the
                       Larget microorganisms within a significantly shorter time interval than required for
                       approved APHA WN methods (APHA 1989, "HA 1985). The methods can be
                       arbitrarily classified into approaches thavare modifications of the existing enumerationr
                       procedures but use different media or procedures, those that combine different media with a
                       variety of techniques to detect activity or densities of viable cells, and those that are based
                       on direct detection of cellular nucleic acids. Additional variations may be obtained by
                       combining these th= categories. A recent review by Boardman et al. (1989) discusses
                       new methods for detection of coliform bacteria and other indicators found in marine waters.


                       Kamplema  cher et al. (1976) compared the existing APHA WN enrichment method for
                       detection of total coliforms with three enrichment media used in Europe and also compared
                       direct recovery of E coft from these same media. One medium, formate glutamate, was
                       found to be result in higher recoveries and significantly fewer false positives after 48 h than
                       LST broth.


                       A variety of rapid enumeration methods for coliforms and fecal coliforms are referred to in
                       Standard Methods as Special Techniques (APHA 1989). A table of methods with
                       appropriate refaences is provided (APHA 1989) and include radiometric, enzyme assay,
                       electrochemical, impedance, gas chromatographic, colorimetric and potentiometric assays.
                       Many of these techniques are generally considered inappropriate for routine enumeration of
                       fecal coliforms in water samples. Only methods based on the colorimetric enzyme assays
                       and 14C-labeled substrates are recommended by the APHA (1989). All these methods are
                       calibrated against standard curves obtained by viable count methods. This can lead to
                       problems of interpretation and large errors because the responses obtained with
                       environmentally stressed and substrate non-responsive cells complicates interpretation of
                       standard curves.



                                                           28






 0




                       Berg and Fiksdal (1988) evaluated three fluorogenic substrates, 4-methylumbelliferyl-O-D-
                       galactoside, 4-methylumbelliferyl-heptanoate, and 4-methylumbelhferyl-O-D-glucurx)mde
                       for a rapid method to detect total and fecal coliforms based on enzyme activity. Samples of
                       river water, potable water, or sewage effluents were filtered through 0.45 pin membrane
 0                     filters, the filters incubated in a nutrient broth containing a fluorogenic substrate, lactose,
                       sodium lauryl sulfate, and buffer at temperatures selective for each coliform group. Initial
                       rates of substrate hydrolysis, measured over time by fluorescence, were related to initial
                       coliform densities. Linearity of fluorescence over the first hour of incubation was very
                       good. Of the substrates examined 4-methylumbelhferyl-O-D-galactoside gave the best
 0
                       response time, providing a positive result in 15 minutes. Incorporating this substrate in M
                       7h FC agar (APHA 1989) provided a direct method for enumeration of fecal coliforms
                       within 6 hours.




                       Eschetichia coh. This organism is considered the dominant fecal coliform in human and
                       animal &ces and is generally considered an indisputable indicator of fecal contamination
 0                     from warm blooded animals. Its thermotolerance facilitates its separation from most other
                       members of the coliform group using selective and differential methods. Replacement of
                       the fecal coliform indicator with E colt has been considered because of thermotolerant-fecal
                       coliforms whose presence in aquatic environments is not necessarily related to fecal
 0                     contamination. Klebsiella is a ubiquitous FC-positive organism that can be found in
                       aquatic environments and is associated with plant materials (Bagley and Seidler 1977),
                       industrial effluents (Huntley, Jones and Cabelli 1976), and degraded water quality
                       (Vlassoff 1977). The validity of the approved fecal coliform method has been questioned
 0                     because of FC-positive klebsiellae and estuarine "fecal coliform mimicldng" bacteria that
                       proliferate in shellfish harvested from approved growing areas in the Gulf of Mexico and
                       other areas during seasons of maximum water temperature (Miescier et al. 1985). Hood et
                       al. 1983) reported KZebsiella sppwere abundant in Gulf of Mexico oysters and clams
                       during the months from April through October. A similar phenomenon may also occur to a
 0
                       lesser extent in the water column. Another criticism focuses on the multiple species
                       composition of the fecal coliform group. It is reasonable to assume that the development of
                       rapid and direct enumeration methodologies could be simplified if the target was a single.
 0                     species. For these and other reasons R coli has been suggested as a more specific
                       indicator of fecal contamination in estuarine waters.




 0                                                          29








                        However, we should ask if the information available is sufficiently compelling to support
                        this conclusion. As noted for fecal coliforms, E coli does not fulfd the requirements of an
                        ideal indicator for various reasons despite the alleged exclusivity of its fecal origin.
                        Besides its persistence and aftergrowth capabilities, its biomass is significantly impacted by
                        the nab i1ral microbiota. In contrast, we would not anticipate that human enteric viral
                        pathogens would enter the microbial food web and be removed through similar predator-
                        prey or other microbiallyv-mediated interactions. This hypothesis implies a fundamental
                        difference between viral and bacterial indicator removal mechanisms. A selective removal
                        process of this Idnd may contribute to viral isolations without detecting indicator bacteria.
                        Therefore, although E coli may be a useful indicator downstream from definitive or large,
                        stable point sources of fecal contamination, its value as a quantitative standard in shellfish
                        growing waters impacted by diffuse sources appears equivocal. This conclusion stands
                        despite anticipated approaches to -fine-tune- the specificity, selectivity and accuracy of
                        traditional or'state-of-the-art" enumeration methods because the deficiencies noted owe
                        their origin to ecological phenomena.


                        The APHA approved WN method using lauryl sulphate tryptose or lactose broth followed
                        by EC broth is effective but suffers from the poor analytical precision because it is a
                        statistical estimate of the true population density and is time consuming. The most-
                        probable number based A- 1 method was developed by Andrews and Presnell (1972) and
                        subsequently modified as A-I-M (Andrews et al. 1978) as a one step enumeration
                        procedure. The modified method incorporates an initial low temperature (35'Cï¿½0.5*) 3
                        hour resuscitation period followed by incubation at 44.5*Cï¿½0.2* in a waterbath for 21ï¿½2
                        hours (APHA 1984). Cook (1981) developed a temperature control mechanism to
                        automatically program an incubator for the required resuscitation and incubation periods at
                        the appropriate temperatures. E coli may be enumerated by direct count using the EPA
                        approved mTEC procedure (USEPA 1985). Water samples are filtered through 0.45pm
                        membrane filters, placed on mTEC medium, incubated for 2 h at 35'C to allow for
                        resuscitation of stressed or injured cells, and incubated at 44.5. After 22h the filter is
                        removed and placed on a pad saturated with urea. Target colonies are urease-positive and
                        turn yellow to yellow-brown.



                        Nonapproved or Emergent Enumeration Methods for Eschefichia coli. Rapid
                        methods for detection of E coli in water based on hydrolysis of various fluorogenic or
                        chromogenic conjugated substrates have been developed in recent years and few have been


                                                             30








                       evaluated in shellfish waters.. A review of several methods through 1986 can be found in
                       Hartman et al. (1986). Several assays are based on the ability of F. coli to produce
                       catabolic enzymes in response to particular substrates that are conjugated to a fluorogenic or
                       chromogen moiety through a O-D glycosidic linkage. Hydrolysis of the glycosidic bond
                       releases the fluorogen or chromogen which can be detected by fluorescence or
                       spectrophotometry. Kilian and Bfilow (1976) evaluated a variety of nitrophenyl-conjugated
                       enzyme substrates for rapid detection of bacterial glycosidases in representative genera
                       including Escherichia spp. A widely used fluorogenic substrate is 4-methylumbelliferyl-0-
                       D-glucuronide (MUG) which has been shown to be hydrolyzed by E colt and some
                       Salnwnella and Shigella spp. (Kilian and Bfdow 1976). However, Dahlen and Lmde
                       (1973) found glucuronidase activity in sorneDmeroides spp. and Corynebactefium spp.
                       Basic assumptions concerning these methods include: (1) that the ability to hydrolyze the
                       substrate is uniformly present in the target species, (2) that this ability can be expressed
                       under a given set of cultural conditions , (3) interferences caused indirectly (overgrowth) or
                       directly (false positives) by the presence of other organisms can be minimized to an
                       acceptable level, and (4) that the sample matrix itself does not autofluorescence or contain
                       enzymes that will hydrolyze the assay substrate. Most reports reveal that only a small
                       proportion of E cob are O-glucuronidase negative and cannot hydrolyze this substrate
                       (Freier and Hartman 1987; Adams et al. 1990). However, reports by Chang et al. (1989)
                       and Lum and Chang (1990) suggest the incidence of glucuronidase-negative strains is
                       much higher thaii the aforementioned reports suggest and caution against use of MUG
                       hydrolysis as a singular method to detect E coli. MPN, plate, and microtiter based
                       methods employing 4-methylumbelhferyl-f@-D-glur-uromde (MUG) have been described by
                       (Feng and Hartman 1982). Freier and Hartman (1987) developed two membrane filtration
                       media containing MUG and inhibitors to gram-positive bacteria for simultaneous recovery
                       of both total coliforms and E coli within 24 h. The method, which incorporates a 35*C
                       incubation temperature, does not require a resuscitation step and effectively recovers the
                       target organisms from sewage and surface freshwater. There are no reports of these media
                       applied to shellfish waters. Ley et al. (1988) synthesized the glucuronide, indoxyl-O-D-
                       glucuronide, as a less expensive analog to MUG and chromogenic compounds. Watkins et
                       al. (1988) examined the chromogenic glucuronide BCIG (5-bromo-4-chloro-3-indoxyl-p-
                       D-glucuronide) for the specific, differential identification of E coli from wastewaters and
                       shellfish using direct enumeration methods on agar media. Adams et al. (1990) described a
                       colorimetric method for enumeration of E coli based on hydrolysis of the conjugate p-
                       nitrophenol-o-D-glucuronide in a liquid freshwater medium at 37* and 44*C. The method
                       is based on a graded response whereby the time required for spectrophotometric detection


                                                              31








                         of the chromogen is inversely related to the F. coli cell density. Relationships between log
                         cell density and time to detect dye is established on the basis of a series of standard curves.
                         Interference, demonstrated in the presence of highnumbers of competing microorganisms
                         such as Klebsiella spp. and FAterobacter spp., resulted in overestimations of the "true" cell
                         densities. It is probable that sublethal stress caused by hyperosmolarity, temperature
                         stress, sunlight, starvation, etc. would also cause departures from predicted densities.


                         The comparative advantages of direct counting methods compared to enumeration methods
                         based on cultivation of target cells are widely recognized (e.g., Daley 1979; Atlas 1982).
                         Specific issues concerning indicator enumeration based on viable recovery methods have
                         been reviewed by Roszak and Colwell (1987). Many studies demonstrating
                         nonculturability have used F. coli, Salmonella spp. or VibHo spp. as target organisms. A
                         variety of direct counting procedures to distinguish viable from non-viable cells have been
                         developed. Representative approaches include cell elongation (Kogure et al. (1979),
                         measurements of elect= transport (Zimmerman et al. 1978), autoradiography (Tabor and
                         Neihof 1982), use fluorescent antibodies (Desmonts et al. 1990), measuring the change in
                         activity over time of 3[H]-thymidine-labeled DNA recovered from cells exposed to seawater
                         (Servais et al. 1985), and recombinant methods such as gene probes and PCR-based
                         amplification of target sequences (Steffan and Atlas, 1988). The APHA recognizes use of
                         fluorescent-antibodies as a valid method for direct detection of microorganisms (APHA
                         1989). Singh et al. (1989) demonstrated the advantages of combined automated image
                         analysis with the direct viable counting method of Kogure et al. (1979) for bacterial
                         enumeration. Advantages include rapidity and improved counting efficiency. Although this
                         method was used under ideal conditions, e.g., pure cultures of E coli grown in laboratory
                         media, the concentration of nalidixic acid used was observed to significantly affect direct
                         viable counts, the effect generally being to decrease counts with increased antibiotic
                         concentration. Furthermore, the exposure time and concentration of nalidixic acid required
                         to maximize the counts of pure cultures of E coli, S. typhimufium, P. aeruginosa, Y
                         enterocolitica, and V cholerae varied over almost a ten-fold range. This observation
                         emphasizes the importance of prior knowledge of the effects of nalidixic acid concentration,
                         assay incubation time, and substrate on enumeration of a target indicator or segment of a
                         microbial community in natural samples by direct viable count.


                         Thus far, methods using recombinant DNA/RNA techniques applied to environmental
                         microbiology have included direct detection through hybndization of the target nucleic acid
                         sequence using naturally occurring organisms in an environmental sample or biological


                                                                32








                        matrix, or (2) detection by colony hydri dization after cultivation of the target
                        microorganism on a particular medium. Although the first method has been limited by
                        amount of DNA or RNA required to hybridize with a probe, use of cell concentration
                        techniques (Somerville et al. 1989 and
                                                                 new PCR methods (Atlas and Bej 1990; Bej et al.
                        1990) offer improved sensitivity- Atlas and Bej (1990) claim a sensitivity of 1-10 fg of
                        genomic DNA winch, is equivalent to only 1-5 R coli cells in a I - 100 ml water sample.
                        Issues of concern with the viable approach include those problems associated with viable
                        recovery methods, e.g., interference and overgrowth of the target organisms, poor
                        selectivity and specificity, sublethal stress, nonculturability, etc. Amy and Hiatt (1989)
                        illustrated several of these problems using a DNA chromosomal gene probe against colony
                        blots prepared from water samples containing indigenous microbiota and target R coh.
                        Optimum conditions for detection of target cells were the absence of a competing
                        microbiota and use of selective pressure; conditions that are probably unrealistic with
                        natural samples. Knight et al. (1990) used a commercially available DNA probe for direct
                        detection of Salmonella spp. from estuarine water samples Sterivex filter units Wpore
                        Corporation, Bedford, MA) were used to concentrate and recover cells from water
                        samples. Similar methods could be applied to recovery of indicator bacteria. The probe
                        hybridization procedure, which can be accomplished in 2 days, detected Salmonella spp.
                        DNA in direct extracts of cell concentrates and in samples negative for cultured cells
                        following addition of medium to the filter unit . At this time it is not possible to determine
                        with sufficient accuracy the number of cells, viable or nonviable, corresponding to a given
                        yield of probe-bound DNA. Sayler and Layton (1990) recently reviewed environmental
                        applications of nucleic acid probe techniques for detection of rmicroorganisms.



                        Fecal Streptococci. 1he term "fecal streptococci" has been used to describe a group of
                        taxonomically diverse streptococci that are Gram-positive, caWase negative, nonspore-
                        forming, facultative anaerobes, associated with the gastrointestinal tracts of humans and
                        animals. Functionally, the fecal streptococci were variously divided into groups and
                        subgroups on the basis of serology and physiological characteristics. Thus, the
                        serologically-defined group D streptococci of the fecal streptococci (Clausen, Green, and
                        Litsky 1977), was divided into an "enterococcal" subgroup (S. faecalis, S. faecium, S.
                        aWum, and S. durans) and a "nonenterococcal" subgroup (S. boWs and S. equinus). As
                        noted, the later term is curiously inappropriate and has contributed to confusion regarding
                        the meaning of the term "enterococci" (Deibel and Hartman 1984).



                                                               33








                        Recent studies have indicated that although the "nonenterococcal" species Of the genus
                        Streptococcus shared the group-D antigen and a fecal habitat, these similarities did not
                        reflect underlying taxonomic relationships. Comparative analysis of oligonucleotides, from
                        16S-rRNA of representative streptococci (Ludwig et al. 1985), and nucleic acid
                        hybridization studies, have led to creation of a new genus, Enterococcus, (Schleifer and
                        Kilpper-Balz 1984), that includes members of the enterococcal group as well as other
                        species not associated with humans. Tbe genus Emerv    coccus now includes 9 valid species
                        and proposals for the inclusion of 3 newly discovered species have been made (Facklarn
                        and Collins 1989). @ S. boWs and S. equinus, were taxonomically distinct and not
                        considered valid members of this genus owing to differences in physiology and metabolic
                        characteristics (Bergey's Manual of Determ@ive Bacteriology 1986).


                        Although far from being nurnerically-dominant in human feces, constituting only about
                        0. 1 % or less of the gut microbiota. (based on cell densities) (Holdeman et al. 1976), this
                        group has been considered as an important indicator of fecal pollution for a considerable
                        period of time. This is attributed to its association with feces, the alleged differences in
                        numbers of coliforms to numbers of fecal streptococci in human and animal feces
                        (Geldreich and Kenner 1969), and observations suggesting that the enterococcal group
                        does not manifest many of the negative characteristics associated with the coliform group,
                        one of the most important being its reported inability to grow in seawater (Slanetz and
                        Bartley 1965). As a group, the complex nutritional requirements of these organisms are
                        generally considered to preclude extraenteral growth (Mundt 1982).


                        Use of the ratio of fecal coliforms to fecal streptococci has been accorded considerable
                        importance in the literature and much discussion has concerned its validity as an index
                        capable of revealing the nature of a pollution source, i.e., human versus animal fecal
                        contarnination (Geldreich and Kenner 1969; Feachem 1975). Values of the ratio equal to
                        4.0 or more are assumed to reflect human pollution; values less than 4.0 animal (Geldreich
                        and Kenner 1969). This is because there are proportionately more fecal streptococci in the
                        feces of animals than man. A number of investigators have presented data that critically
                        questions the validity of this concept in freshwater environments, including accepted values
                        of the ratio in feces (McFeters et al. 1974; Wheater, Mara, and Oragui 1979). Values of the
                        ratio in feces from a variety of animals and man varied considerably and certain animal
                        species exhibited mean values essentially similar to human sources (Kjellander 1960;
                        Wheater, Mara, and Oragui 1979). Furthermore, the ratio did not remain stable in
                        receiving waters and was difficult to interpret in systems with multiple sources of fecal


                                                             34








                        pollution. Significantly less attention has focused on the survival characteristics of fecal
                        StreptOCOCCi in marine and estuarine waters (Slanetz; and Bartley 1965; Vasconcelos and
                        Swartz 1976; Lessard and Sieburth 1983). These studies have yielded contradictory results
                        so that a definitive conclusion regarding use of the ratio in these waters is not possible.
                        However, in an estuarine environment, differential dieoff of fecal coliforms and fecal
                        streptococci and the species comprising these groups, and the effects of biological and
                        physicallchernical factors, will likely contribute to variations in the ratio suggesting it would
                        be difficult to deduce the origin of pollution based solely on its value. Furthermore, it is
                        difficult to see how the recommended criteria for use of the fecal coliform/fecal
                        streptococcus ratio (Geldreich and Kenner 1969), no doubt derived with defined point
                        sources in mind, can be met in growing areas polluted by multiple, diffuse sources of fecal
                        contamination. Finally, as will be noted, enumeration of fecal streptococci in estuarine
                        waters is very method sensitive and contributes a source of variation that must be
                        minimized if the ratio is to be viewed as a meaningful parameter (Brodsky and Schiemann
                        1976). Attention to reduce variability is also important because these organisms occur at
                        significantly lower derisities than fecal coliforms. Fortunately, the newest edition of
                        Standard Methods (APHA 1989) cautions against the-use of the ratio to differentiate
                        pollution source.


                        Although certain genera (e.g. E faecalis, E faecium) of the fecal streptococci have been
                        considered specific to feces from human or other warm-blooded animals, phenotypically-
                        similar strains and biotypes, can be isolated from other environmental sources (Mundt 1982;
                        Clausen et al. 1977; Beaudoin and Litsky 198 1). Strains and biotypes of E faecalis and of
                        E faecium can be isolated from a variety of plant materials9 reptiles and insects (Mundt et
                        al. 1958; Mundt 1963; Geldreich et al. 1964; Geldreich and Kenner 1969). Moreover,
                        Mundt et al. (1962) reported growth of E faecalis biotypes, on plants and vegetables. In
                        comparison, the distribution and viability of S. boWs and S. equinus in extr-aenteral
                        environments appears restricted, exhibiting significantly reduced survival in aquatic habitats
                        (Slanetz; and Bartley 1965; Geldreich and Kenner 1969; Wheater et al. 1979). The,
                        usefulness of this group as an indicator of fecal pollution in shellfish growing waters
                        appears to depend on development of specific enumeration methods that distinguish
                        between strains of fecal versus non-fecal origin-


                        A large variety of media h ave been developed for the recovery of fecal streptococci
                        (Brezensid 1973; Yoshpe-Purer 1989). Methods have been based on most-probable-
                        number techniques, direct pour plate or membrane filtration techniques, some of which


                                                                35








                        incorporate resuscitative steps to minimize sublethal stress. Comparisons of the various
                        media and methods can be found in references such as Switzer and Evans (1974), Clausen
                        et al. (1977), Pagel and Hardy (1980)9 Voltera et al. (1985) and Yosphe-Purer (1989). It
                        is evident from this literature there exist considerable differences *in recovery efficiency,
                        specificity and selectivity among the different methods, and that each method requires some
                        degree of confirmatory testing. Use of this group as an indicator in marine and estuarine
                        shellfish waters requires improved methods, specifically the seleovity and specificity of
                        media, coupled with an improved understanding of factors affecting the survival of this
                        group. Methods evaluation should be performed in shellfish growing areas, with attention
                        to identifying typical fecal streptococcal species recovered and those microorganisms
                        indigenous to shellfish growing waters that produce false positives. E&wntially similar
                        recommendations with respect to methods were articulated quite a few years ago by
                        Kiellander (1960) and Hartmanet al. (1966). 1hus, until recently KF was considered the
                        medium of choice in the 15th edition of Standard Methods (APHA 1985), although
                        anecodotal reports by many investigators and most recently Yosphe-Purer (1989)
                        suggested that KF medium is not sufficiently selective to apply to marine waters. The 16th
                        edition of Standard Me thods (APHA 1989) now recommends use of azide-dextrose broth
                        with confirmation on Pfizer selective enterococcus agar (PSE) for MPN determinations and
                        m Enterococcus agar for membrane filtration assay.


                        Other considerations aside, use of this group as a fecal indicator necessitates adoption of a
                        common functional definition of fecal streptococci (as with fecal colfforms), that is based
                        on a formalized and unique method for recovery of specific enterococcal and streptococcal
                        species associated with feces. This must be viewed as. a challenging but formidable task.



                        Enterococci. The term "enterococci," with its connotation of fecal origin, is
                        "understood" by some workers to exclude those fecal streptoccocal species (S. equinus and
                        S. boWs) associated almost exclusively with animal gastrointestinal tracts. This usage is
                        misleading because the term implies streptococci sharing a fecal habitat Diebel and
                        Hartman (1984) consider the terms group-D streptococci and enterococci the same.
                        Creation of the genus Enterococcus spp. (Schliefer and Kilpper-Balz 1984) (whose
                        constituents are "enterococci"?), which contains several non-human species (Facklarn and
                        Collins 1989) but excludes S. equinus and S. boWs, does little to resolve this semantic
                        confusion.,




6                                                            36








                      Functionally, the enterococci are defined on the basis of selective recovery obtained using
                      one of several methods developed for this purpose (e.g., Slanetz and Bartley 1965;
                      Isenberg et al.. 1970; IYAoust and Litsky 1975; Levin et al. 1,975). The range of species
                      selected varies with each method, some being more selective for the classic "enterococcal"
                      (F-fibecalis, E faecium) group organisms than others. In reviewing this literature the
                      reader must be wary of the operational nature of the results and current taxonomic
                      relationships.


                      Tlm most recent use of enterococci in marine waters is by Cabelli et al. (1983). mE
                      medium (as modified by Levin et al. (1975) and Dufour (1980; who substituted indoxyl-13-
                      D-glucoside for esculin in the primary medium) was u  sed in polluted marine and estuarine
                      waters to derive a statistical relationship between indicator density and the incidence of
                      swimming-associated gastrointestinal disease. Cabelli et al. (1983) concluded enterococcal
                      densities were better predictors of health risk than fecal coliforms or E coft. Prospective
                      epidemiological studies to evaluate the enterococci and other indicators as predictors of
                      enteric disease associated with consumption of raw shellfish were recently conducted and
                      the results being evaluated (A. Dufour, USEPA, personnel communication). Enteroccoci
                      are now recommended as the indicators of choice for recreational waters by the
                      Environmental Protection Ageny (1986).


                      Validation of the enterococci as an indicator of public health risk in shellfish growing areas
                      will require improved recovery methods. The range of selectivity and specificity
                      characteristics of methods for enumeration of enterococci has been mentioned. Current
                      recovery methods require confirmatory testing of presumptive isolates (Encksen and
                      Dufour 1086), increasing the time and cost of analysis. Although the mE-based method
                      has been applied to marine and e stuarine waters (Cabelli et al. 1983), its utility in nonpoint
                      source impacted shellfish growing areas is undetermined. Similarly, enterococcal presence
                      in stormwater ninoff (Geldreich et al. 1968; Pagel and Hardy 1980) and differences
                      concerning its persistence in estuarine and marine waters (Lessard and Sieburth 1983;
                      Slanetz and Bartley 1965; Vasconcelos, and Swartz 1976) demonstrate a need for
                      distribution and survival studies. Research requirements include : (1) improving methods
                      for recovery of stressed cells, (2) improving specificity for various Enterococci spp. and
                      Streptococci spp., (3) reducing the incidence of false-positives, which can vary with
                      temperature, season, and geographic region, and (4) minimizing background growth. The
                      relative occurrence and densities of non-fecal biotypes of Efecaelis and Ejbedum in
                      nonpoint source and stormwater runoff impacted areas should be determined. Rapid


                                                           37









                       methods for confirmation of selected enterococcal species, based on serological or
                       biochemical characteristics, could significantly improve its candidacy as an indicator in
                       shellfish growing waters. Until recently there have been few reports of direct methods for
                       enumeration of this group or species identification, e.g.,'fluorescent antibody-based
                       methods (Abshire and Guthrie 1971; Pugsley and Evison 1975). Bosley et al. (1983)
                       describe a useful and rapid (4 hours) identification method to separate enterococci. from
                       group D nonenterococci based on hydrolysis of. L-pyrrolidonyl-0-naplithamide (PYR). A
                       colony hybridization method, employing oligonucleotide probes synthesized for specific
                       sequences of 23S rRNA of selected. enterococci,, was used to successfully detect and
                       identify R faecalis, E faecium, and F. aWum in mixed culture (Betzl et al. 1990).



                       Alternate Microbiological Indicators for Water


                       Bacteroides fragiUs group. Although the obligate anaerobic bacteria are the dominant
                       constituents of the human fecal microbiota (Moore and Holdeman 1974), attempts to utilize
                       these organisms as indicators of fecal pollution have-been limite(L This may be attributed
0                      to the need for rigorous anaerobic cultural and enumeration procedures and the assumption
                       that anaerobes do not persist under extraenteral conditions.


                       Bacteroides spp. are nonsporu.1ating obligately anaerobic gram-negative bacteria considered
0                      the dominant genus in human feces (Holdeman et al. 1976; Salyers 1984). Like the
                       bifidobacteria, Bacteroides spp. is an important candidate indicator of fecal pollution as its
                       major habitat is restricted to the gastrointestinal tracts of humans and warm-blooded
                       animals. As a group, Bacteroides, may be more tolerant of oxygen than the bifidobacteria-,
0                      some Bacteroides spp. produce catalase and R ftagifts and R distasonis synthesize
                       superoxide dismutase (Salyers 1984).


                       Until recently, the term Bacteroidesfiragilis group (BFG), was used to describe dominant
                       bacteroides found in the human colon that included R ftagilis and its subspecies. The
                       subspecies were subsequently accorded species rank on the basis of DNA homology
                       studies (Salyers 1984) and dominant species now include R vulgatus, B. ftagilis, B.
                       thetaimaomicron, and R distasonis. The term BFG still appears in the current literature, an
                       although taxonomically incorrect, has utility as a simple phrase referring to the dominant
                       human colonic bacteroides species. B. vulgatus, B. thetaiotaomicron, and B. distasonis.
                       are numerically dominant (1010 cells/gdry weight feces) and A vulgatus that can constitute


0                                                            38








                       > 10% (as cell count) of the culturable fecal microbiota (Holdeman et al. 1976). R fragifis9
 Am                    which is more aerotolerant than B. vtdgatus, occurs at densities similar to E coli. The
                       observation that Bacteroides species other than those found in humans dominate in
                       ruminants (Macy 1981) supports the use of BFG species as a human-specific indicator.
                       Macy (1981) provides a summary of host-related species composition and methods for
                       recovery and cultivation of non-pathogenic Bacteroides SM.


                       Although Bacteroides spp. and Rjr*agi&, specifically, are medically significant and can be
                       isolated from a variety of clinical specimens and blood samples (Goldstein and Citron
                       1988), there is little in formation describing the occurrence of nonpathogenic bacteroides in
                       natural waters. Post et al. (1967) first suggested use of the BFG group as an indicator of
                       fecal pollution. Allsopand Stickler (1985) evaluated BFG as a fecal indicator based on its
                       presence in sewage, various freshwater, and marine environments. Organisms belonging
                       to this group were readily isolated downstream from waters impacted by sewage. Because
                       Bacteroides spp. disappeared faster from these waters than E coft, Allsop and Stickler
                       (1985) concluded the ratio of BFG to R coft counts reflects "aging" of a fecal pollutant and
                       also proximity to the source. The presence of BFG organisms in natural waters was
                       attributed to human fecal pollution because analysis of feces from a variety of domestic
                       animals and feral birds revealed that BFG species occur at much lower densities (i.e., 105-
                       1010 fold lower) in common domestic farm animals (cattle, horses, pigs, chickens, and
                       sheep)(Allsop and Stickler 1985). Higher densities occur in domestic pets and seagulls but
                       these were still lower than levels in human feces. A variety of obligate anaerobes including
                       Bacteroides spp. were detected during the warmer months at a polluted river site (Daily et
                       al. 1981). These observations, admittedly limited, support the potential use of BFG as a
                       human-specific indicator in nonpoint source impacted shellfish growing areas.


                       General comments made concerning Bifidobactena spp. recovery methods also apply to
                       this group. Perhaps more work on identification methods has occurred with Bacteroides
                       spp. owing to its clinical importance as a pathogen and possible involvement in colon
                       cancer. Allsop and Stickler (1994) developed an improved medium for recovery of BFG
                       from natural waters. Penicillin is incorporated into the medium to inhibit clostridia and
                       membrane filtration (0.22 pm) is used for sample concentration. Application of this
                       medium to samples of sewage, and fresh and marine waters revealed interference by
0                      obligate and facultative anaerobes. Increasing the concentration of gentamycin improves
                       the selectivity of the medium but decreases recovery efficiency. A resuscitation period is
                       incorporated to minimize the effects of sublethal in ury. Serious consideration of the BFG


                                                             39








                         group requires a detailed assessment of this recovery method in shellfish growing waters,
0                        including its selectivity, validation of confirmatory steps required, as well as data
                         describing the effects of season on BFG occurrence, and persistence in estuarine waters.


                         Anaerobic bacteria vary in their sensitivity toward oxygen (Lmsch 1969). Ix)esch (1969)
                         classified anaerobes. into two groups: (1) those that do not exhibit growth on agar media if
                         pO2 levels exceed 0.5 % were called strict anaerobes, and (2) those that could grow at pO2
                         levels from 2 % through 8 % (mean, 3 %) were called moderate anaerobes. , R fragilis was
                         found among the most oxygen-tolerant of the latter group. Cells exposed to the atmosphere
                         on an agar surface survived for at least 8 hours with only a small reduction in colony
                         counts. T'his tolerance explains detection of this organism in environmental waters but also
                         suggests that modifications to sample storage and processing protocols that reduce oxygen
                         exposure could improve recovery and enumeration efficiency. Walden and Hentges (1975)
                         confirmed the moderate tolerance of R fivgilis to oxygen and demonstrated that oxygen,
                         not Eh, was adverse to anaerobic growth because anaerobes grew well under conditions of
                         positive, intermediate, and negative oxidation-reduction potentials in the absence of
                         oxygen. Onderdonk et al. (1976) confirmed the independence of Eh on growth but
0                        observed that under conditions of continuous culture oxygen concentrations from 10 to
                         100% were bacteriostatic and elicited a decline in metabolic activity.


                         Chromosomal DNA probes have been developed to speciate Bacteroides as an alternative
0                        to conventional phenotypic methods. Roberts et al. (1987) and Morotomi et al. (1988)
                         used whole cell dot blot assays that can correctly differentiate Bacteroides by species.
                         Kuritza and Salyers (19185) used a cloned DNA probe to enumerate B. vulgaw in feces
                         and concentrations measured were similar to viable counts. Although the probe was highly
0                        specific for a segment of the B. vulgatus genome, its use for enumeration of cells in
                         environmental samples unlikely because of its low sensitivity (ca. 107- 108 cells required).
                         A sample concentration procedure (eg., Somerville et al. 1989) that would eliminate
                         problems associated with particulates in estuarine water, i.e., clogged filters, and
9                        interfering overgrowth by the autochthonous microbiota (Amy and Hiatt 1989) will be
                         neede(L However, these probes should be valuable in supplanting or augmenting the
                         specialized and labor -intensive biochemical identification of presumptive isolates. Use of
                         R firagilis bacteriophage as an alternate indicator is discussed in the section on viral
                         indicators.






                                                                40








                      Bifidobacteria. Bifidobacterium spp. are obligately anaerobic, Gram-positive,
                      nonmotile bacteria that possess a characteristic pleomorphic and branching cell
                      morphology. They are potentially important candidate indicators of fecal pollution as the
                      habitat of many species appears restricted to the feces of adult humans and infants and
                      major groups of warm  blooded animals (Levin 1977; Cabelli 1978a; Bergey's Manual of
                      Systematic Bacteriology 1986)). In humans, bifidobacteria are a major component of the
                      intestinal microbiota occurring at densities greater than 1010 celIs/g dry feces and some
                      species may comprise more than 6% of the culturable microbiota (Holdeman et al. 1976).
                      A unique metabolic feature of tins genus is that glucose is metabolized exclusively and
                      characteristically through the fivctose-&phosphate shunt (bifid shunt) (Bezkorovainy
                      1989). The first reaction step is mediated by the enzyme fructose-6-phosphate
                      phosphoenolketolase (F6PPK). This enzyme can be detected using a colorimetric assay
                      and is a reliable phenotypic characteristic of the genus (Scardovi 198 1; Bergey's Manual of
                      Systematic Bacteriology 1986). Bifidobacteria can also utilize ammonium as a sole source
                      of nitrogen. The sensitivity of these organisms to oxygen varies with species and strains,
                      the presence of C02 allowing some species to tolerate exposure to air for several hours.


                      A potentially valuable property of this group relates to its ability to differentiate human from
0                       .mal fecal pollution based on "human" and "animal" strains (Levin 1977). Tanaka and
                      Mutai (1980) found that R adolescentis and R longum accounted for 74% of the
                      bifidobacterial strains isolated from human feces. Levin and Resnick (1981) were unable
                      to isolate bifidobacteria dominant in human feces (i.e., R longum , R adolescentis) from a
                      variety of domestic and wild animals except swine. Mara and Oragui (1983) confirmed the
                      low occurrence of these microorganisms in animal feces and developed a medium based on
                      sorbitol fermentation, which selects for bifidobacteria, derived from human sources. The
                      ability of human-specific strains to ferment sorbitol can vary and not all sorbitol-
                      fermenting bifidobacteria are derived from human feces (Bergey's Manual of Systematic
                      Bacteriology 1986). Nevertheless, the possibility of differentiating human from animal
                      fecal pollution should provide an impetus for the evaluation of this anaerobe.


                      Use of the bifidobacteria as an indicator of fecal contamination in shellfish growing waters
                      has been considered (Cabelli 1978a), but recent studies have focused primarily on its
                      occurrence and recovery from freshwater environments (Mara and Oragui 1983; Carrillo et
                      al. 1985; Munoa and Pares 1988). Information on the distribution of these bacteria, albeit
                      limited to natural and sewage contaminated freshwater, suggests its use as an indicator of



                                                           41








                      very recent fecal pollution is valid as the organisms appear incapable of extraenwal
                      growth.


                      Although obligate anaadbes, Gyllenberg et al. (1960) reported bifidobacteria survive as
                      well as E coli in freshwater. In contrast, Levin and Resnick (1981) observed B. longum
                      and R adolescends, exposed in vitro to fresh and marine water samples, are less persistent
0
                      than E coft. B. adokscentis populations decline considerably after 48 hours of in situ
                      exposure in tropical freshwaters (Carrillo et al. 1985). Under these same conditions E coli
                      densities remain constant or increased. Results of an in vitro experiment to compare
                      survival of R adolescentis in fresh estuarine water (Kator and Rhodes 1988) confirmed
                      these observations using samples of Chesapeake Bay water. R adolescentis cells were
                      incubated in flasks containing membrane filtered (0.2 gm) water at 60C and 250C. B.
                      adolescends persistence at 6*C was moderately better than E colt, but at higher
                      temperatures was significantly worse. In contrast, Levin and Resnick (1981) observed
0
                      considerably less persistence of bifidobacteria from sewage exposed in vitro to membrane-
                      filtered seawater (32 psu). Survival was also independent of temperature (at temperatures
                      of 40, 120 and 200C), with approximately 15 % of the initial density remaining after about 6
                      h. Individual species and strains varied in survival capability.


                      The generally low bifidobacterial densities found in receiving waters may be attributed in
                      part to poor recovery owing to oxygen toxicity and sublethal stress, factors that are
                      exacerbated by selective media and harsh recovery methods. Munoa and Pares (1988)
                      demonstrated the mability of Bifidobactenum spp. cells to produce colonies on a selective
                      medium was caused by sublethal injury following exposure to seawater. To minimize the
                      effect of sublethal strew on enumenation, a two-layer recovery procedure was designed that
                      incorporated plating and incubation of the sample on resuscitative medium (reinforced
                      clostridial agar). This was followed with an overlay of BIM-25, a selective medium
                      developed to improve the poor selectivity of YN-6 medium with environmental samples
                      (Levin and Resnick 198 1). Unfortunately, B., adolescentis did not grow as well on BIM-
                      25 as other Bifidobacterium spp. (Munoa. and Pares 1988). Improving recovery methods
                      for this group of microorganisms, especially human-specific species, should be an
                      objective of future research efforts. Beerens (1990) described a modified Columbia
                      (Pasteur Production, Bioservice, France) agar medium containing propionic acid that is
                      selective and enhances recovery of bifidobacteria. This and other recovery media should be
0
                      evaluated using resuscitative techniques and membrane filtration because of the need for
                      sample concentration. Despite the recognition that the bifidobacteria. are sensitive to


                                                           42








                       oxygen, use of prereduced media or anoxygen-fi-ee environment for recovery
                       manipulations has not been prescribed. Although Levin and Resnick (198 1) suggest
                       oxygen toxicity is not a concem with recovery of bifidobactei* sorbitol-fermenting strains
                       are catalase negative, and data such as those of Munoa and'Pares (1988) and Kator and
                       Rhodes (1988) suggest factors that exacerbate sublethal stress should be minimmA
                       Differences in the tolerance of bifidobacterial species or strains to oxygen (Bergey's
                       Manual of Systematic Bacteriology 1986) may target the choice of a indicator
                       bifidobacterial species. The role of particle association in protecting Bifidobacteyium spp.
                       and other anaerobes f1rom oxygen in the environment is undetermined. In situ exposure
                       studies, are needed to measure the persistence and recovery of selected bifidobacterial
                       species in estuarine waters and sediment.


                       If using indicators to identify sources of fecal pollution to nonpoint impacted estuarine
                       growing areas, the unequivocal fecal origin and limited survival properties of the
                       bifidobacteria are potentially valuable because their detection implies immediate fresh
                       sources. The appearance of bifidobacteria in feeder streams may be used to locate sources
                       of fecal pollution and to differentiate human from nonhuman sources. Although
                       discrimination of vertebrate source will require studies to verify the restricted host range of
                       the sorbitol-fermenting bifidobacteria species composition in feces of domestic animals,
                       humans, and septic tank effluents, the ability to differentiate human from animal pollution
                       could aid management strategies directed toward reduction of pollutant sources. Finally,
                       various bacteriophages, including those lytic to BacteroidesfiragiM Qofre et al. 1986;
                       Tartera and Jofre 1987), are being considered as human-specific indicators of fecal
                       pollution. Similarly, if bacteriophages lytic to bifidobacteria occur in feces or sewage,
                       these could be used as human-specific viral indicators of fecal contamination.



                       Clostyidium perpingens. C per
                                                           .ftingeru is an anaerobic, Gram-positive, spore
                       forming rod whose presence in receiving waters has been linked to contamina.tion by feces
                       and wastewaters (Cabelh 1977a; Bisson and Cabelli 1980). Bisson and Cabelli (1980)
                       developed a membrane filtration method for C perfnngens recovery applicable to saline
                       waters based on a highly selective medium (mCP). The composition of this medium was
                       later modified to reduce the concentration of a costly component without compromising'its
                       selectivity (Armon and Payment 1988). Sartory (1986) compared recovery of C

                       per
                          .fiingens on mCP and egg yolk-free tryptose-sulphite-cycloserine (TSC) agar as
                       membrane-based tests. For a variety of sample types, which included polluted river water,


                                                              43








                        egg yolk-free TSC was found as selective as mCP and more efficient Egg yolk-free TSC
                        was recommended because of its ease of preparation, lower cost, and availability of a
                        simple confirmation scheme.


                        Bisson and Cabelli (1980) concluded that C perfiingens has value as an indicator of
                        chlorination efficiency and the presence of unchlorinated sources of fecal contamination,
                        and as a "c onservation tracer" delineating the areal impact and transport of wastewater
                        effluents. Moreover, detection of vegetative cells in the environment reflects fresh and
                        untreated fecal matter because the persistence of vegetative cells is very short (Bisson and
                        Cabelli 1980). Fujioka and Shizumura (1985) confirmed the utility of C. perfiingens to
                        detect wastewater discharge into fresh water streams. However, there is doubt C.
                        perfiingens would be a useful indicator in nonpoint impacted shellfish growing areas
                        because (1) it is so persistent that it may be difficult to index to current pollution conditions,
                        (2) it is widely distributed in soils and sediments, and (3) it is carried into growing areas
                        from extraenteral sources by stormwater runoff and transport of suspended sediment
                        (Matches and Liston 1974, Smith 1975).



                        Rhodococcus coprophilus. Rhodococcus coprophilus is an aerobic nocardioform
                        actinomycete proposed as an indicator of domestic faun animal fecal pollution (Rowbotharn
                        and Cross 1977b; Mara and Oragui 1981; Oragui and Mara 1983). As a fecal indicator this
                        organism is unique because it is associated with the feces of domestic grazing farm animals
                        but is notconsidered an active component of the rumen microbiota (Rowbothan and Cross
                        1977b). Rowbotharn and Cross (I 977b) noted that numbers (colony forming unitstgram
                        of R coprophilus in pasture grass and in dung collected from cattle fed this pasture grass
                        were similar, implying no multiplication occurs during passage through the animals. In
                        contrast, significant increases in densities of R coprophilus that occur during incubation of
                        fresh dung at a moderate temperature (ca. 20*C) with water, confirms the coprophilic
                        habitat of this organism. Consequently, this organism is commonly found in pasture grass
                        and soils grazed by cattle and other domestic farm animals. Studies have shown that R
                        coprophilus is absent from human feces but consistently found in feces of cattle, sheep,
                        pigs, horses, donkeys, farm-raised poultry and sporadically in dog and seagull feces (Mara
                        and Oragui 1981).


                        R coprophilus can survive both dessication and temperatures of 2*@-4*C in dung for periods
                        of up to 6 weeks (Rowbotham and Cross 1977b). Goodfellow and Williams (1983)


                                                               44








                       concluded that the coccal stage of R coprophilus, carried by runoff into either fi-esh or salt
                       water habitats, does not grow but remains viable. R coprophilus persists in vitro for 17
                       weeks in nonfiltered fi-eshwater incubated at 5*, 20* and 30*C, whereas E coft and fecal
                       streptococd disappeared within 5 weeks (Oragui and Mara, 1983). These investigators
                       suggested these and other properties of the organism could be used (in conjunction with
                       other bacterial indicators) to determine the temporal characteristics, location and source of
                       fecal contamination to fivshwater systems. Information concerning the distribution and
                       persistence of this organism in estuarine waters are insufficient to evaluate its utility as an
                       indicator in shellfish growing waters.   In an experiment using a recovery medium (NW)
                       devised by Mara and Oragui 198 1), Kator and Rhodes (1988) compared its persistence to
                       F. coil over a 30 day period in 0.2 pm filtered estuarine water incubated at 60 and 250C.
                       Although R. coprophilus appeared to multiply more slowly than E coli, its persistence in
                       water over the range of salinities and temperatures tested appeared generally better than E
                       coh, andit manifested less initial aftergrowth at 250C. Whether the observed density
                       increases were due to growth or an artifact of the organisnfs fi-agmentable nocardioform
                       morphology (see methodological considerations below) must be addressed in future
                       studies. Similarly, the persistence of this organism in marine and estuarine sediments has
                       not been examined, although it is readily isolated from marine and estuarine sediments
                       (Goodfellow and Williams 1983; Attwell and Colwell 1984; Goodfellow and Haynes 1984;
                       Kator and Rhodes 1989). Isolation of R coprophilus in a small subestuary of the York
                       River, Virginia, was more fi-equent and the densities higher in water and sediment adjacent
                       to a livestock farm compared to locations lacking this activity (Kator and Rhodes 1989).
                       Studies of R coproplulus persistence in estuarine water and sediment, related particularly
                       to seasonal temperature, are needed to assess its value as an index of current pollution
                       conditions.


                       Disadvantages now associated with use of this indicator are primarily limitations inherent in
                       the current enumeratio  n procedure (Mara and Oragui 1981). Only small volumes (0.2 ml)
                       of sample can be spread plated onto MM3 agar, currently the medium of choice. Direct
                       spread plating limits detection to 1 cfu/ml (eg., five replicate plates containing 0.2 ml
                       each). A recovery procedure is needed for concentrating R coprophilus ftom water using
                       MEN43 or other suitable medium for expressing characteristic colony morphology. The
                       combination of membrane filtration (Pisano et al. 1986) with the novel selective method of
                       Hirsch and Christensen (1983) could be evaluated for enumeration of R coprophilus.
                       Hirsh and Christensen (1983) described a "selective" method for recovery of actinomycetes
                       that eliminates bacterial contamination. This is based on the ability of actinomycetes to


                                                               45









                      Penetrate the "pores" of a membrane filter on a growth medium, whereas bacteria remain
 0                    on the filter surface- After an incubation period the filter is discarded and only
                      actinomycetes that penetrate the filter form colonies. Another limitation noted by Mara and
                      Oragui (1981) is the long incubation period (17-18 days) required fbr development of
                      characteristic stellate colonies having both substrate hyphae and bright orange central
 0                    Papillae (Rowbotham. and Cross 1977a). The prolonged incubation is a consequence Of (1)
                      the low organic content of. the medium, (2) the presence of inhibitors used to suppress
                      bacterial growth, (3) the photDchromogemc nature of color production, and (4) the time
                      required to visibly develop the characteristic colony morphology. Biochemical
 0                    confirmation Of nOcardiOfOrms to the generic level is very time-consuming and
                      identification on the basis of colonW morphology alone is neither reliable nor valid
                      (Goodfellow and Williams 1983; Bergey's Manual of Systematic Bacteriology 1986).
                      Rapid and direct methods for species vedification (and enumeration) are needed. An
                      indirect method to detect or identify R coprophilus using actinophage appears doubtful
                      because of the broad cross-reactivities of actmophage to both Nocardia and RWococcus
                      (Prauser 1984).


                      Precise enumeration of R coprophilus is complicated by the inherent molphogenetic
                      nature of nocardioforms, and particularly for R coprophilus , which forms extensively
                      branched hyphae and coccoid elements that are connected in chains of varying length.
                      Thus, a given colony-forming unit in an water or sediment sample can originate from a
                      variety of cell configurations. Rowbotham and Cross (1977a) observed that a typical
                      colony enumerated from dung is derived from single or multiple coccoid elements, the latter
                      being the most prevalent form. The complicating effect of R coprophilus morphology on
                      enumeration is neither a desirable indicator characteristic nor conducive to analytical
 0                    precision.


                      Streptococcus bovis. S. boWs may be the dominant fecal streptococcus in warm
                      blooded animals (Kjellander 1960; Wheater et al. 1979). Use of S. boWs as a specific
                      indicator of animal fecal pollution was first suggested by Cooper and Ramadan (1955).
                      Since their report few investigators have evaluated its use as an indicator of animal fecal
                      pollution in freshwater and none that we know of in marine and estuarine waters. Wheater
                      et al. (1979) claim the proportion of S. boWs to total fecal streptococci was largest in
                      ruminants but note the important role of diet and geographic location on this ratio
                      However, the association of S. bovis with ruminants is not unique as it has been be found


                                                           46








                         in the feces of dogs, cats, various birds, horses and pigs (Kenner et al. 1960; Clausen et al.
                         1977). Osawa and Mtsuoka (1990) isolated S. boWs biotype 1 (mannitol-fermenting) in
 0                       the feces of koalas on a selective medium. Geldreich and Kenner (1969), Clausen et al.
                         (1977) and Wheater et al. (1979) were unable to isolate S. boWs from human feces.
                         Oragui and Mara (1981) drew siniffiar conclusions but did isolate S. boWs from sewage
                         effluent. This contradiction was resolved when they located an abattoir that discharging
 0
                         into the sewage treatment facility. However, other workers have isolated S. boWs from the
                         feces of healthy humans (Kjellander 1960; Switzer and Evans 1974). Kjellander (1960)
                         isolated S. boWs from about 15 % of humans examined; Dalton et al. (1986) observed a
                         fecal carriage rate in healthy humans of 10-16%. About 1.0% of the group D strepwocci
 0
                         recovered by Abshire (1977) from human feces during an evaluation of a new presumptive
                         medium for this group were S. boWs. Osawa and Mitsuoka (1990) indicate mannitol
                         fermenting S. boWs (biotype 1) are commo     nly isolated from ruminants whereas mannitol
                         nonfermenting strains (biotype II) are more fi-equent in human infections. Some workers
                         suggest these contrastitig results are due in part to dietary variation and regional effects.
                         Another explanation is that investigators used methods with different recovery efficiencies,
                         selectivity and specificity characteristic& Based on the literature the hypothesis that S.
 0                       bovis is a unique indicator of animal fecal pollution remains equivocal.

                         Greater concurrence exists concerning the survival of S. boWs in natural waters compared
                         with other enteric aerobic cocci. S. boWs mortality in freshwater is much greater than E
 0                       fibecalis or E coli (Geldreich and Kenner, 1969; McFeters et al. 1974; Clausen et al. 1977;
                         Wheater et al. 1979). Results of an in vitro exposure experiment using an isolate of S.
                         boms, exposed to filtered feeder stream or estuarine water at 60C and 250C, confirmed its
                         inability to persist, especially at the higher temperature (Kator and Rhodes 1988). As
 0                       stated earlier, poor survival may be viewed as a positive attribute of this indicator because
                         detection of S. boWs implies fecal pollution of very recent origin. Conversely, its poor
                         survival violates the requirement that an ideal indicator be at least as persistent as enteric
                         pathogens.
 0                       Although S. boWs can be isolated with other Enterococcus spp. and Streptococcus spp. on
                         some of the media used for recovery of these groups (e.g., Switzer and Evans 1974), only
                         one medium is specifically designed for its isolation (Oragui and Mara 198 1). Littel and
 0                       Hartman (1983) described a selective medium for the fecal streptococci that differentiates S.
                         boWs from other streptococci and enterococci based on hydrolysis of a fluorogenic
                         substrate and a colorimetric indicator of starch hydrolysis, amylose azure, The ability of S.


 0                                                               47






0



                       bovis to ferment starch has been considered an important phenotypic characteristic
                       separating it from Enterococcus spp. and other fecal suvptococci. Membrane-bovis agar
                       (m-BA) (Oragui and Mara 198 1) is a selective medium for S. boWs whose specificity is
                       based on the ability of S. boWs under anaerobic conditions to utilize NH4+ as the sole
                       source of nitrogen and the absence of a requirement for exogenous, vitamins. A
                       resuscitation step to minimize the effect of temperatwe stress is incorporated m-BA is
                       more specific and more efficient for recovery,of S. bovis ftorn freshwater and sewage than
                       KF. Subsequently, Oragui and Mara (1984) described a modified m-BA medium (called
                       mm-BA) containing less sodium azide because of reports noting its inhibitory effect on S.
                       bo-Ws strains from different geographical regions. Using m-BA Oragui and Mara (1981)
                       reported that more than 65% of the fecal streptococci in animal feces were S. bovis.
                       Moreover, a proportion (10%) of typical isolates later confirming as S. boWs failed to
                       ferment starch. Isolates lacking this characteristic could lead to underestimation of S. bovis
                       densities using the medium of Littel and Hartman (1983). A significant proportion of
                       isolates we confirmed as S. boWs that were recovered on mm.-BA from fteshwater feeder
                       streams and shellfish-growing waters in a subestuary of Chesapeake Bay did not hydrolyze
                       starch (Kator and Rhodes 199 1).


                       Our experience using this medium with samples from freshwater feeder streams and
                       estuarine shellfish growing areas revealed departures from selectivity and specificity
                       characteristics reported by Oragui and Mara (1981; 1984). Thus, in the analysis of
                       approximately 400 mm-BA isolates, a significant proportion (65.1 %) of "typical yellow
                       colonies", i.e., presumptive S. bows, were confirmed as E faecium, S. salivarius and
                       non-group D streptococci (Kator and Rhodes 199 1). This figure is considerably higher
                       than the 1.3% false-positive rate observed by Oragui and Mara (1981). Conversely, in
                       terms of false- negatives 8.9% of the atypical, non-yellow colonies were confirmed as S.
                       boWs. E faecium was frequently recovered on m-BA, a point subsequently noted by Mara
                       and Oragui (1984). Although S. boWs has potential as a source-specific and time-sensitive
                       indicator, and the mm-BA method has the advantage of sample concentration, use in
                       shellfish waters requires an evaluation of the medium's recovery efficiency, specificity and
                       selectivity over seasons and in different geographic regions. Also needed is development
                       and validation of rapid screening methods for confirmation of presumptive S. bows.


                       Osawa and Mitsuoka (1990) reported a selective medium for recovery of S. bovis based on
                       treatment of brain heart infusion agar with tannin and addition of colistin-oxolinic acid to
                       inhibit enterobacteria, in feces. Although the medium selectively recovers and differentiates


 0                                                            48









                      S. bovis biotypes, I and 11 present in fresh koala feces, it has not been evaluated with other
                      animal feces, applicability to membrane concentration, and is not particularly effective
                      recovering S. boWs in pure culture.


 0                    BacteriOPhages- Occurrences of enteric viral disease attributed to shellfish consumption
                      (Richards, 1985) have stimulated research for an appropriate viral indicator. Inadequacies
                      of the current bacterial standard have been further emoiasized by the lack of parity in
                      survival characteristics of fecal coliforms and enteric viruses (Feachern et al. 1983) and the
                      detection of enterovirus in shellfish from approved growing waters. (Goyal et al. 1979;
                      Vaughn et al. 1979; Ellender et al. 1980). Richards (1985) has advocated the direct use of
                      enterovirus as indicators, in particular poliovirus because it is easily cultivated and
                      prevalent in sewage due to universal vaccination. Although cost, method and time
                      constraints currently preclude routine detection of enteric viruses, such analyses could be
                      used to assess potential health risk in growing areas lacking identifiable sources of human
                      fecal pollution and closed to direct harvesting because of elevated indicator densities.


                      Compared with the costs and limits of direct viral detection, bacteriophage indicators are
                      appealing due to their resistance to disinfection (Keswick et al. 1985) and physical factors
                      that eliminate bacterial *indicators (Debartolomeis 1988), their ease of detection, low
                      analytical cost, and short assay periods. Justification for use of coliphages, as indicators of
                      fecal and sewage pollution or as simulants of human enteric viruses in estuarine and marine
                      receiving waters (Borrego et al. 1987, 1990; Cornax 199 1; OKeefe and Green 1989;
                      Grabow et al. 1984; Kott et al. 1974; Kott 1981; Scarpino 1975) has been reviewed by
                      Gerba (1987) and IAWPRC Study Group on Health Related Water Microbiology (1991).
                      Gerba (1987) expressed concern over the paucity of basic data describing ratios of specific
                      coliphages to viral pathogens and the occurrence, persistence, and seasonal stability of
                      coliphages in shellfish growing waters. The following studies illustrate the basis of his
                      concern. Vaughn and Metcalf (1975) recovered enterovirus from shellfish growing waters
                      free of coliphages. Seeley and Primrose (1980) described a subpopulation of coliphages
                      apparently capable of replication at temperatures found in freshwater environments In situ
                      replication of somatic coliphages in estuarine water has been reported (Vaughn and Metcalf,
                      1975). It is evident that studies of coliphage ecology in marine and estuarine waters are
                      necessary to evaluate its utility as an indicator.





                                                            49








                     Use of male-specific RNA coliphages as indicators of sewage pollution has been discussed
                     (Havelaar and Hogeboom 1984; Furuse 1987; Havelaar and van Olphen 1989). The
                     gastrointestinal tract of warm blooded animals and domestic sewage are major habitats fbr
                     these viruses. (Furuse et al. 1978). FRNA coliphages share some properties with human
                     enteroviruses such as type of nucleic acid, structure and size, although these similarities do
                     not necessarily translate to similar functional properties in saline environments or shellfish.
                     FRNA phage may also be source specific. Furuse et al. (19178) isolated and classified
                     FRNA phages in domesti     c waste from various countries in South and East Asia into four
                     major serological groups (groups I, H, 1H and IV). The relative proportion of FRNA
                     phages. in domestic waste and sewage ranges from 10-90% of total coliphages (Furuse et
                     al. 1978; Osawa et al. 1981b). Most FRNA'phages from countries other than mainland
                     Japan belonged to group HI and those in mainland domestic waste were in group 11.
                     Dominance of phage groups in Korea was intermediate between Japan and Southeast Asia,
                     i.e., groups R and III were equally prevalent (Osawa et al. 198 lb). Differences in
                     geographic temperature and its effect on viral replication was hypothesized responsible for
                     the distributions observed (Osawa et al. 1981b; Snowdon and Cliver 1969). From a global
                     perspective, fewer FRNA phages were detected in sewage from Central and South
                     American countries than in Asian sources (Furuse et al. 1978). Additional studies (Osawa
                     et al. 1981a) showed FRNA phages belonging to group I were only detected in feces or
                     gastrointestinal contents of mammals (domestic farm and feral zoo animals) other than
                     humans. FRNA phages isolated from pigs belonged to groups I and 11, and those from
                     humans groups II and Hl. Phages belonging to group IR were exclusive to humans.
                     However, in a study of domestic sewage from treatment plants in Japan (Furuse et al.
                     1981), FRNA phages belonging to groups I, H and Il were found. Occurrence of group I
                     phages, albeit at low frequencies compared to those in H and IH, was attributed to inputs of
                     sewage derived from animal sources such as slaughterhouse wastes. Basic studies
                     concerning the ecology and occurrence of FRNA phages in sewage and feces in the United
                     States are lacIdng. Poppell (1979) recovered FRNA coliphages at low frequency (2%)
                     from human feces and consistently from all parts of municipal sewage collection systems in
                     the Northeast United States. The validity of FRNA serological groupings to differentiate
                     human and animal fecal contamination should be examined, and, if corroborated, could be
                     useful in nonpoint impacted growing areas.


                     Two coliphage assay systems have been recently developed that are selective for F-specific
                     phages. Previous coliphage assays required labor intensive screening to identify RNase
                     sensitive phage to avoid counting somatic'and filamentous DNA phages. Debartolorneis


                                                            50








                      (1988) developed and applied a F-specific sensitive host strain to estuarine and marine
0                     waters. The assay uses a male E coli host mutant (called Famp) which is also resistant to
                      lysis by DNA somatic phages. RNase.has to be incorporated in a parallel assay to
                      distinguish between FRNA and FDNA filamentous coliphages. Asecond F-specific
                      coliphage assay was developed by Havelaar and Hogeboorn (1984) through addition of an
                      R coli plasmid coding for sex pilus production to a F- Salmonella typhimurium host strain
                      (WG49). S. 13V&mufium was chosen as the host to avoid interference by somatic
                      coliphages which are abundant in sewage. Theoretically, plaques observed on this host
                      would be attributed primarily to FRNA phage because somatic salmonellae and FDNA
                      filamentous phages occur at much lower densities in sewage. This assay has been
                      extensively applied to studies of feces and wastewaters (Havelaar et al. 1984; Havelaar and
                      Nieuwstad 1985; Havelaar et al. 1986) but not to marine or estuarine waters. Recently we
                      compared densities of fecal coliforms and phages lytic against F1 S. ophimufium WG49
                      from samples collected along a salinity gradient in an estuary subject to nonpoint pollution
                      (Rhodes and Kator, in press). Verified FRNA phages were infrequently recovered from
                      100 ml samples of feeder streams or estuarine water with fecal coliform. densities
                      significantly above the growing area standard (range = <2-7900 FC 100 ml-1). Moreover,
                      higher levels of phages were detected in estuarine sediments compared with feeder stream
                      sediments. Although fecal coliform densities in freshwater feeder stream sediments were
                      one to four orders of magnitude larger than phage densities, densities of phages and fecal
                      coliforms in estuarine sediments were similar. This pattern of phage distribution was
                      inconsistent with the fecal coliform data which identified the feeder streams as sources of
                      fecal pollution.


                      Our experience confirms the critical unportance of the bacterial host strain to the coliphage
                      assay (Sinton and Ching 1987; Havelaar and Hogeboorn 1983; Seeley and Primrose 1982).
                      Host strains vary as to the accessibility, specificity and location of phage receptor sites.
                      Evaluation of coliphage indicator systems must be conducted and verified using field
                      samples collected to obtain seasonal coverage. Seeley and Primrose (1982) considered
                      coliphages inappropriate indicators because of the likelihood of interfering phages. For
                      example, Vaughn and Metcalf (1975) found the abundance of coliphages, in shellfish
                      growing waters varied over a three year period as a function of the E coli host employed.
                      In the study previously mentioned, Rhodes and Kator (in press) randomly picked plaques
                      on WG49 for confirmation as male-specific phages. Of the total number of plaques
                      produced on S. typhimurium WG49,99% (293 of 294) were produced by RNW-resistant
                      phages. Phages purified from these plaques were lytic against the female parent S.








                      typhimurium WG45, were not lytic against a male strain of E coli, and were lytic against
                      environmental Salnwnella spp. isolates. The susceptibility of the S. typhimurium WG49
                      host strain to MS2 was routinely retested and confirmed. A s* a result, not only is the
                      sanitary significance of these data is uncertain, but the application of this particular assay
                      host to nonpoint source impacted growing areas may be inappropr  iate.


                      If FRNA phages are to be used as fecal indicators more must be known about their ecology
                      and fate. A possible limitation concerns their comparative occurrence in feces and sewage;
                      FRNA phages are an infi-equent component of human (and some animal) feces (Havelw et
                      al. 1986; Furuse 1987; Havel= 1987) and occur at densities significantly higher in
                      sewage. Their abundance in sewage treatment plants may be the result of extraenteral
                      multiplication at ambient temperatures on hosts that formed pili at temperatures above 30*C
                      (Havehw et al. 1986; Havelaar 1987; Havelaar and Pot-Hogeboom 1988). However,
                      Poppell (1979) calculated the low occurrence of FRNA phages found in humans was
                      sufficient to account for the densities found in sewage because the numbers of phages
                      contributed by those individuals was very high. Therefore, the extent to which FRNA
                      phages can infect and lyse enteric bacteria in wastewater facilities, septic systems and
                      growing area environments is an issue of concern because such multiplication would alter
                      the ratios of indicator to bacterial and viral pathogens. Thus, although FRNA (and FDNA)
                      phages may serve as indicators of wastewater and sewage contamination, their use as
                      indicators of fecal contamination or a predictor of health risk in shellfish growing waters
                      requires carefid.analysis. Unless it can be demonstrated that male-specific phages occur at
                      reasonably high densities in septic leachate, these phages may be a poor indicator of human
                      fecal contamination in non-point impacted shellfish growing areas On the other hand,
                      FRNA phages may have potential use as an indicator of animal contamination because its is
                      significantly more abundant in the feces of certain domestic farm animals (Havelaar and
                      Pot-Hogeboorn 1988; Havel= et al. 1986).


                      Another issue of concern is the persistence of bacteriophages in estuarine environments and
                      shellfish. Borrego and Romero (1985) found selected coliphages persisted for extended
                      periods, i.e., hundreds of days in sterilized seawater, but their numbers decreased rapidly
                      in natural nonfiltered seawater. Mitchell and Jannasch (1969) reported that filtered natural
                      seawater (using either 0.45 pm or 0.22 prn membrane filters) is strongly antiviral toward
                      coliphage 4@X-174, producing a decrease of viral titer from 1012/ml to 103/ml in 6 days.
                      To assess the validity of bacteriophages as indicators of viruses, studies are needed to



                                                           52








                        examine the survival of target phages under various seasonal conditions in natural waters,
                        sediments, and commercial species of shellfish.


                        Quantitative assessment of F-specific phages in estuarme waters may require processing
                        sample volumes of 100 ml or larger and the use of concentration methods. Cornax et al.
                        (199 1) concluded (perhaps prematurely) that male-specific coliphages and R firagilis
                        bacteriophages are inappropriate indicators in seawater because of their low densities in
                        sample volumes of only 0. 1 to 1.0 ml assayed by double-layer-agar method. Grabow and
                        Coubrough (1986) used a single-agar-layer method to assay 100 ml sample volumes
                        although this is time consuming and costly. Methods for phage concentration have relied
                        primarily on concentration techniques developed for human enteric viruses. However,
                        there is no a priori basis to assume these methods are appropriate or equally effective for
                        bacteriophages (Borrego et al. 199 1). Seeley and Primrose (1982) reviewed a variety of
                        viral concentration procedures and considered their applicability to phage concentration.
                        Positively charged filters have been used successfully to concentrate coliphages from
                        potable and freshwater systems (Logan et al. 1980; Singh and Gerba 1983). Although
                        positively-charged filters are considered effective for-concentrating enterovirus from
                        estuarine waters (Kilgen and Cole 1983)@ 'Havelaar (1986) reported they were ineffective
                        for recovery of coliphage from artificial seawater. Concentration methods for enteric
                        viruses from marine waters have relied primarily on the use of electronegative microporous
                        filters (Sobsey 1987). Debartolomeis (1988) evaluated a method for recovery and
                        concentration of FRNA bacteriophages modified after the procedure of Purdy et al. (1984,
                        1985) for recovery of Bacillus spp. bacteriophage from water samples. The procedure
                        involves adsorption of bacteriophages to host cells added to a water sample, concentrating
                        the infected cells by centrifugation or membrane filtration, and plaque assay of the
                        concentrate by agar overlay. Debartolomeis (1988) obtained mean recoveries of 103.5 and
                        87.4 percent@ respectively, for naturally-occurring F-specific phages in seawater and river
                        water when compared against the direct overlay method. Using the same approach with
                        marine samples spiked with Bacillus spp. phages, Purdy et al. (1984, 1985) obtained
                        anomalous results. Gerba et al. (1978) described a successful protocol for concentration of
                        poliovirus from seawater using pleated membrane filters in conjunction with aluminum
                        chloride flocculation. Isbister et al. (1983) incorporated 2,3,5-triphenyltetrazolium chloride
                        into a single layer coliphage assay procedure to improve plaque visualization. Bor  rego et
                        al. (1991) evaluated a variety of electronegative and chemically-produced elechqvsitive
                        filters for recovery of coliphages from nonsaline water containing diluted sewage effluent
                        A protracted "drop-by-drop" elution technique with 3 % beef extract and positive pressure


                                                              53








                       gave superior recovery of coliphages adsorbed to diatomaceDus. earth filters treated with
                       cationic polymers. Application of these and other methods for enumeration of F-specific
0                      and other phages requit-es ffirther evaluation before being considered for routine use in
                       shellfish growing waters.


                       Sobsey et al. (1990) recently described a simple membrane filter-based method for
                       concentration of bacteriophages from drinking and surface water. This method may have
                       applicability to shellfish waters provided that particulate loading on the filter is not an
                       uncontrolled source of variation to ova-Al recovery. Briefly, M902 is added to a water
                       sample, which is then vacuum filtered through a 0.45 pm membrane filter, and the filter
                       placed face down on an agar surface inoculated with the assay host. Adsorbed phages
                       subsequently desorb to produce plaques in this agar layer and a tetrazolium dye is used to
                       improve detection. Particulates were shown to reduce plaque numbers, possibly by
                       competitively inhibiting phage adsorption, suggesting this method will have to be carefidly
                       evaluated in estuarine waters which are generally characterized by high particulate loads.
                       Methods for the recovery of coliphages, from a variety of representative estuarine and
                       marine sediments must be developed and rigorously verified. In a study of this type,
                       Armon and Cabelli (1988) compared the effectiveness of various eluants tD release Q phage
                       experimentally adsorbed to purified clay minerals and those in natural sediment. Efficient
                       recovery of phages, from shellfish will be needed because the numbers are likely to be low
                       because only small volumes of homogenized samples can be accommodated by direct
                       plating methods. Brodisch et al (1986) enumerated coliphages, in mussel homogenates, by
                       direct plaque assay in a medium augmented by antibiotics to suppress background. A
                       similar approach should be evaluated for different species of commercial shellfish in this
                       country.


                       Use of direct pour plate assay methods for phage enumeration generally requires steps to
                       reduce or suppress the growth of background microbiota, depending on sample type.
                       Incorporation of antibiotics, decontamination with chloroform (Tartera and Jofre 1987;
                       Osawa et al. 1981a), membrane filtration (Tartera, and Jofre 1987), and selective media
                       (Kennedy et al. 1985) have been used for this purpose. Adams (1959) used chloroform to
                       lyse background cells to optimize coliphage recovery. Osawa et al.(1981a) diluted sewage
                       samples with 5 ml of PG medium and treated with chloroform (5 %, vtv) to kill bacteria.
                       Kennedy et al. (1985) compared chloroform (5%, v/v) with other methods including
                       selective media to reduce background for recovery of coliphages from sewage and lake
                       waters. Following an exposure interval after mixing of I hour to chloroform, samples


                                                            54








                        were removed from the aqueous phase for phage enumeration. Chloroform eliminated the
                        background but also reduced plaque numbers on most media examined and use of selective
                        media was recommended. Tartera, and Jofre (1987) found that although chloroform did not
                        inactivate R ftagilis phages, it also did not suppress growth of anaerobic sporeformers,
                        which sporulated under anaerobic conditions. Potassium sorbate (0.05 %) and a lowered
                        pH (5.7) were used to eliminate spore germination. Cornax et al. (1990) compared use of
                        chloroform (20%, v/v), antibiotics, and membrane filtration for removal of the background
                        during recovery of Rfiragilis bactenophages; ftorn sewage and sewage-contaminated
                        seawater. Membrane filters (0.45 prn and 0.22 pore nitrocellulose) were used either
                        untreated or treated with 10 ml of 3% beef extract (pH 9.5). Recovery was statistically
                        better in terms of phage numbers and suppression of background growth using 0.45 prn
                        filters treated with beef extract . It was hypothesized that treatment with beef extract
                        combined with the larger pore size membranes is the most effective because most bacterial
                        cells were retained and yet phages were able to pass unadsorbed through the nonbinding
                        filter matrix. Ile combined use of membrane filtration with incorporation of antibiotics
                        was re commended to increase ova-all assay selectivity.


                        In contrast to coliphage, bacteriophages active against the anaerobe Bacteroidesfiragilis
                        demonstrate a high degree of host strain specificity and appear to lack activity against other
                        species of Bacteroides spp. (Booth et al. 1979; Cooper et al. 1984; Keller and Traub 1974;
                        Kory and Both 1986; Tartera and Jofre 1987; Tartera. et al. 1989). As a potential indicator
                        of fecal contamination, R fragilis phages, were detected exclusively in human feces and
                        sewage and appear to reflect the dominance of the Bacteroidesfragilis host in human feces
                        (Booth et al. 1979; Cooper et al. 1984; Salyers 1984; Tartera and Jofre 1987; Tartera, et aL
                        1989). Phages lytic to the most efficient host strain examined, Bfiragilis HSP40, were
                        recovered only from environmental areas subjected to sewage and never detected in
                        nonpolluted areas or those occupied exclusively by feral animals (Jofre et al. 1986; Tartera
                        and Jofre 1987). These observations, coupled with the apparent inability of B. ftagifis
                        phage to multiply in freshwater, seawater or sediment habitats (Jofre et aL 1986; Tartera et
                        al. 1989), suggest this phage is a promising subject for verification as an indicator of
                        human fecal pollution. The low isolation frequency of HSP40 bacteriophages in humans,
                        Le.,:@10%, suggests the need for sampleconcentration and an assessment of its occurrence
                        in waters and shellfish in growing areas contaminated by nonpoint pollution.


                        The occurrence of phages, active against alternate bacterial indicators such as B.
                        adolescentis or R breve, S. boWs, and R coprophilus appears to have received received


                                                               55








                        scant attention. Phage assay systems could provide an alternative detection method to
                        viable enumeration procedures which are    unsatisfactory for reasons previously discussed.
                        Enumeration of phages active against human specific sorbitol-fermenting bifidobacteria
                        could provide an assay system similar to that proposed for R fragilis (Jofre et al. 1986).
                        As noted, crom-reactivities between Nocardia spp. and Rhodococcus spp. phages may
                        preclude development of a Rhodococcus-assay system.


                        Although validation of a viral indicator will depend upon establishing a statistically
                        significant relationship between densities of a given ph4e and target enteric pathogen or
                        risk of enteric disease, such information will at first be very difficult and expensive to
                        obtain. Therefore, candidate viral phage indicators must be carefully evaluated before these
                        studies are begun to understand their ecology and to confirm their target specificities with
                        samples collected from regionally-characteristic growing areas over a variety of seasonal
                        conditions.




                        Enumeration of Indicators in Shellfish


                        The object of this section is not an exhaustive coverage of a very large and (sometimes
                        redundant) literature concerning methods of recovery of indicators from shellfish. Rather,
                        its purpose is to highlight areas we believe warrant attention or are of current interest.
                        Specifically, all methods should be considered with regard to factors that affect indicator
                        recovery discussed in previous sections and the overall need to improve analytical

                        precision.




                        Methods for Shellfish Preparation. Procedures for preparation of shellfish for
                        enumeration of microorganisms vary with the microorganism being sought and the
                        detection method. The first step of the currently approved method (APHA 1985) for
                        detection of coliform organisms is an initial breakup and dilution of a sample consisting of
                        10-12 whole animals to release and uniformly distribute microorganisms throughout a
                        homogeneous suspension. However, Al-Jebouri and Trollope (1981) found processing
                        only parts of the digestive system (through excision of the "stomach and intestine") of
                        mussels yielded improved sensitivity of bacterial numbers compared with the total animal
                        homogenate method. In contrast, Metcalf et al. (1980) recovered more virus from whole
                        shellfish than from selected tissues which included hepatopancreas from oysters and


                                                               56








                      hepatopancreas and siphons for clams. Mechanical breakup of the tissue is normally
                      performed by homogenization (low-speed blending or high-speed homogenization) or
                      stomaching. Stomaching, which seems to be preferred for many foods, has only been
                      evaluated for shellfish by a few investigators and it has not been rigorously tested.
                      Trollope (1984) compared counts of lactose-fermenting bacteria from mussels (M
                      edulis) after stomaching and mechanical blending. Average counts from stomached tissue
                      wer e about 2x higher than mechanically blended tissue. Andrews et al. (1978), in a
                      comparison of stomaching and blending using a variety of foods, fbund stomaching to
                      recover higher aerobic plate counts (APQ from oysters. A smaller volume model was
                      judged more efficient than a larger one for recovery of APC in oysters. Stomaching is
                      intrinsically a uniform and "clean" method because the sample is encased in a plastic bag,
                      preventing aerosolization and heating because the sample is repetitively struck with a
                      paddle. However, its effectiveness for release of naturally-polluting bacteria and viruses
                      from different species of edible shellfish requires evaluation. It has been claimed for
                      example, that one problem with stomaching shellfish is bag puncturing because shucked
                      shellfish occasionally contain large bits of shell debris (A. P. Dufour, personal
                      communication).


                      Because of better precision, methods that utilize membrane filtration for direct estimates of
                      target indicators in shellfish may be preferable to MPN-based or direct pour-plate methods.
                      Direct pour-plate methods are limited by small plant volume, the potential for heat-shocking
                      stressed cells, and background interference. The potential accuracy of direct counting
                      methods using membrane filtration for epifluorescence microscopy or culturable counts is
                      influenced by requirements to: (1) optimize release microorganisms from the food matrix,
                      (2) minimize clogging of the filter by reducing the size s of food particles, and (3) to prevent
                      adsorption of food on and within the filter. Food particles that remain on the filter are a
                      potential substrate for competing organisms and may obscure colony development of target
                      microorganisms. An approach to achieve these goals is to treat shellfish with hydrolytic
                      enzymes. Hydrolysis must breakdown the food matrix without reducing the target
                      indicator population, especially if sublethally stressed cells are present. Trypsin and other
                      enzymes have been used to effectively digest a variety of food types (Entis et al. 1982) for
                      microbiological analysis using the hydrophobic grid membrane filter (0.45gm) method.
                      Enzyme digestion has also been coupled with the direct epifluorescent filter technique
                      (DEFI) for estimation of total microbial numbers in meat and poultry (Shaw et al. 1987).
                      Rodrigues and Kroll (1988) proposed use of DEFr for rapid enumeration of coliforins by
                      counting microcolonies growing on selective media. DEFr methods are amenable to


                                                           57








                       automated counting by image analysis and use of fluorescent antibodies for added
 46                    specificity. Although homogenized clams and oysters have been treated with trypsin for
                       enumeration of E coli and enterococci by membrane filtration (A. P. Dufour, personal
                       communication), an evaluation of the usefulness of enzymatic digestion for the
                       microbiological analysis of shellfish is needed.


                       Whatever methods used for release of microorganisms, different species of shellfish are
                       unique in terms of d= overall structure and organization, physiology, and accumulation
                       strategies. Given the same natural exposure conditions, Manila clams (Tapesjoponica)
                       consistently accumulate colifornis and fecal coliforms, at higher concentrations than oysters
 46                    (Crassostrea gigas) (Vasconcelos et al. (1969). Viral infection experiments with
                       Crassostrea gigas suggest viruses are absorbed intracellula rly, reflecting a different
                       mechanism of sequestering than indicator bacteria (Hay and Scott 1986). Power and
                       Collins (1990) found similar uptake pattems of E coli and an iscohedral coliphage in
                       various tissues of the mussel Mytilus eduhs. Oysters are considered more easily
                       homogenized than hard clams. Metcalf et al. (1980) observed significant differences in the
                       recovery efficiency of spiked viruses as a function of shellfish species. Use of identical
                       procedures for hard and soft-shelled clams resulted in lower viral recoveries from the latter.
                       Additional extraction steps were necessary to release virus from soft-shell clams. In view
                       of the national interest in alternate indicators and direct counting methods it seems prudent
                       to optimize recovery methods as a function of target indicator and shellfish species.
                       Commercially important filter-feeding shellfish include oysters (Crassostrea Wrginica,
                       Crassosma gigas, Crussostrea lunda), hard-shell clams (Mercenand mercenana,
                       Mercenafia campechiensis), soft-shell clams (Mya arenaria), Pacific little neck cLvn (Tapes
                       japonica), and mussels (Mywhs edulis).



                       Approved Methods for Indicators



                       Total colifornis and fecal coliforms. One approved method for examining the
                       bacterial quality of shellfish is the multiple tube fermentation test (APHA 1985). Used in
                       either 3- or 5-tube most-probable-number (MPN) configurations, the method offers poor
                       precision, providing an estimate of the "true" population and large confidence intervals.
                       The largest sample portion generally inoculated is 10 ml of a 1:10 diluted homogenate
                       which is equal to I gram. of shellfish meats. Media used are the same as for water, lactose


 46                                                         58









                       or lauryl sulfate tryptose broths for the presumptive test, followed by EC broth and brilliant
                       green bile broth (BGB). Total analytical time for presumptive and confirmed tests is 72
                       hours for fecaland 96 hours for total colifixms. Confirmation that fecal cohforms are E
                       coli with this method takes 10 days. Tbe validity of th e approved fecal coliform MPN
                       test, and particularly its use to assess product quality in terms of the recommended market
                       guideline of 230 FC/100 grams meats, has been questioned for shellfish from the Gulf of
                       Mexico. Counts exceeding this guideline were attributed to EC-positive klebsiellae and
                       estuarine "fecal coliform. mimicking" bacteria which proliferate during seasons of maximum
                       water temperature (INfiescier et al. 1985). Hood et al. (1983) reported Mebsie& spp. were
                       abundant in Gulf of Mexico oysters and clams during the months from April through
                       October.


                       There is only one appro ved pour plate method to detect and measure densities of fecal or
                       elevated temperature coliforms in shellfish (hard and soft clams) meats (APHA 1985). It
                       offers a short analysis time (24 hours) compared with the MPN, the improved precision of
                       a direct count, but is restricted to the shellfish mentioned and less sensitive in that only I
                       gram of shellfish meats can be processed per plate. In addition to the important issue of
                       recovery of sublethally-stressed cells (Yoovidhya and Fleet 1981), other problems
                       associated with this method are reports of significant interferences caused by background
                       growth with shellfish from the Gulf of Mexico and variations in dye lot (John Miescer,
                       FDA, personal communication).



                       Nonapproved and Emergent Methods


                       A succinct discussion of rapid methods, media evaluated, and international methods for
                       recovery of fecal coliforms and E. coli from shellfish meats, is available in the
                       Compendium of Methods for the Microbiological Examination of Foods (APHA 1984).
                       The following sections focus on recent methods not covered in the Compendium.



                       Coliforms. Detection methods based on hydrophobic grid-membrane filters offer
                       significant advantages in enumeration of indicators in foods (Sharpe et al. 1979). These
                       include larger numerical operating range, improved precision, sensitivity, reduced
                       background interference, and removal of iiihibitory substances and substrates that may
                       support growth of background species. These are very desirable characteristics and in


                                                           59@









                      combination with appropriate media could prove valuable wi th shellfish if a reliable and
                      reproducible method can be developed for the initial filtration step. Entis and Boleszczuk
                      (1990) evaluated a hydrophobic grid membrane filter method for enumeration of total
                      coliforms and E coli within 24 hours. The method compared favorably with a MUG-
                      based 3-tube MPN. 'Me method has been used for scallops and shrimp, digested with
                      trypsin, but not bivalve shellfish.




                      Eschetichia coli. As noted
                                                   previously the fluorogen, 4-methylumbelliferyl-0-
                      glucuronide (MUG), which has found application for enumeration of E coli in water, has
                      also been examined for similar use in foods and seafoods. The initial work of Feng and
                      Hartman (1982) was followed by Alvarez (1984) and Moberg (1985). Moberg (1985)
                      examined factors such as specificity, sensitivity, and optimum MUG concentration in IST
                      (lauryl sulfate tryptose broth)-MUG to detect R coft in a variety of food and dairy samples.
                      The ILST-MUG method had a lower false-positive rate and detected more E.coli in non-
                      seafoods thaii the standard APHA method. False-positives were attributed primarily to
                      staphylococci. Coliforms such as E cloacae and K pneumoydae did not produce
                      detectable fluorescence from MUG in I.ST. Alvarez (1984) compared recovery of E coli
                      from a variety of fresh and frozen seafoods (including bivalve shellfish). MUG was used
                      directly in lactose broth, in Violet Red Bile agar (VRBA) as an overlay, and in M-Endo
                      broth with membrane filters. All assays were performed within 24 hours. The high
                      specificity of MUG for E coli was verified by confirming fluorescent tubes or colonies in
                      EC broth, strealdng on EMB, and with traditional confirmatory tests. The lowest false-
                      positive rates (EC positive, no fluorescence) and highest recoveries were found using
                      lactose broth-MUG.   This latter observation was attributed to the nonselectivity of lactose
                      broth toward stressed cells, an observation mirroring earlier findings of Feng and Hartman
                      (1982) who recovered heat-stressed cells on LST-MUG. Koburger and Miller (1985)
                      applied MUG-IST to enumeration of E coli in oysters. They found incorporation of
                      MUG into LST was impractical because oysters possess endogenous glucuronidase activity
                      which yields false positives. Incorporation of MUG into EC broth eliminated this problem
                      although an additional 24-48 h is required. The 25 oyster samples tested yielded 127 gas-
                      positive EC tubes. Of this total 103 tubes were fluorescence-positive and only one was not
                      positive for E coli. Twenty-four tubes were both fluorescent-negative and E coli-
                      negative. However, Chang et al. (1989) suggest that the incidence of glucuronidase-
                      negative strains of E coli is is higher than commonly observed and may lead to
                      underestimations of E coli densities in shellfish or water samples. Frampton et al. (1988)


                                                           60








                       evaluated another glucuronidase substrate, 5-bromo-4-chloro-3-indolyl-o-D-glucuronide
                       (X-GLUC) as an alternative to MUG. This chromogenic substrate does, not require
                       illumination by near-UV to detect f@-glucuronidase activity and does not diffuse into agar
                       (as MUG) but remains within a colony. Disadvantages includes high cost, poor intensity
                       in liquid medium, and possible interference if additional color-based tests are performed.
                       Finally, as previously mentioned E coft has been enumerated from shellfish by combining
                       trypsin digestion with membrane filtration and incubation on mTEC (A. P. Dufour,
                       personal communication). Because of the advantages of direct count, this method should
                       be evaluated with regard to reducing the potential deleterious effects of hydrolysis on
                       recovery of stressed cells.


                       Recovery of other indicators such as the enterococci and bactenophages will require
                       development and validation of rapid and accurate methods. Methods currently available for
                       recovery of bacteriophages from shellfish meats are tedious, with recovery efficiencies that
                       may vary with species of, shellfish. Direct plating of hoinogenates using the conventional
                       double-layer-overlay procedure lacks sensitivity and is visually demanding to read.
                       Clearly, methods for recovery of viral indicators from- shellfish meats must be improved in
                       sensitivity, reliability, and ease of use if they are to become routine.



                       CHEMICAL INDICATORS

                       Coprostanol


                       Coprostanol (50(M-cholestan-30-ol) is formed by bacterial reduction of cholesterol and is
                       one of the principal sterols in the feces of man and other mammals (Murtaugh and Bunch
                       1967; Walker et al. 1982). Unique anaerobic cholesterol-reducing bacteria, possibly
                       Eubactefium spp., use cholesterol as an electron acceptor, reducing the 5,6-double bond of
                       the molecule to form coprostanol. (Sadzikowski et al. 1977). Individuals vary as to the
                       amount of coprostanol produced. Wilkins and Hackman (1974) reported that 25 % of a
                       human test group reduced less than 50% of fecal cholesterol to coprostanol. Coprostanol
                       can be the dominant sterol in domestic wastes.


                       Unlike microbial indicators which may be subject to chemical, biological, and physical
                       processes that influence their numbers and detection, coprostanol offers advantages of an
                       abiotic marker of sewage pollution. The areal distribution of coprostanol has been used to


                                                             61









                      delineate impacts of point sources such as sewage outfaRs or dumping areas (Kanazawa
                      and Teshima 1978; Hatcher et aL 1981; Boehm 1983; Pierce and Brown 1984; Brown and
                      Wade 1984; DOreth et al. 1986; Holm and Windsor 1986) but has not been applied to the
                      study of nonpoint pollution. Attempts to correlate densities of bacterial. indicators to
                      coprostanol. concentrations in estuaries subject to known inputs have yielded conflicting
                      results (Goodfellow et al. 1977; Churchland and Kan 1982; and Yde et al. 1982). Dutka et
                      aL(1988)app   lying a 'battery of biochemical, microbiological and bioassay tests" to
                      evaluate the water quality of the Saint John River and its basin, concluded that fecal sterols
                      were not a useful indicator of fecal pollution.


                      Concentrations of coprostanol. in estuarine (Holm and Windsor 1986) and marine
                      (Kanazawa and Teshima 1978) waters decrease with increasing distance from sources.
                      Brown and Wade (1984) demonstrated that coprostanol in sewage effluent is primarily
                      associated with particulate matter, which may be deposited immediately proximate to an
                      ouW or transported out of the areadepending on the dynamics affecting the distribution of
                      fine-grained sediments Increases of coprostanol concentrations with depth in estuarine
                      waters is attributed to settling of sewage-associated particulates and resuspension (Wade et
                      al. 1983). T'he distribution of coprostanol concentrations in waters adjacent to the
                      entrances of major estuaries from small- and large-scale physical processes and the
                      buoyancy characteristics of particulate matter. The hydrophobic nature of coprostanol.
                      causes it to be associated with particles whose transport subsequently affects its spatial
                      distribution.


                      Serious consideration of coprostanol. as an indicator in nonpoint impacted shellfish growing
                      areas remains hampered by the lack of data describing background levels in such
                      environments and possible origins from nonfecal sources. Laboratory studies using
                      estuarine water and sediment amended with radiolabeled cholesterol demonstrate its
                      conversion to coprostanol by microorganisms (Teshima and Kanazawa 1978). Nishimura
                      and Koyama (1977) suggested that stanols in recent sediments are derived from
                      phytoplankton and sterol conversion. Under anaerobic conditions 5P-isomers of stanols
                      are produced from autochthonous sedimentary organic matter (Nishimura 1982).
                      Tornabene et al. (1974), in a report describing sterols of the diatom, Nitzschia alba, noted
                      cholesterol has been identified in both blue-green and red algae. Cholesterol and other
                      sterols have been detected in both surface waters (Matthews and Smith 1968) and
                      sediments (Attaway and Parker 1970) from the Gulf of Mexico. Kanazawa and Teshima
                      (1971) found cholesterol was usually the dominant sterol in both suspended and dissolved


 ON                                                       62








                       fractions in Kagoshima Bay, Japan. Pocklington. et al. (1987) concluded that coprostanol
                       was an equivocal indicator of fecal pollution, identifying the marine biota as a probable
                       source because patterns of coprostanol occurrence in particulate matter were conjunctve
                       with those of various chemical indicators of primary production, including natural
                       phytosterols.


                       Qualitative and quantitative aspects of coprostanol degradation in estuarine systems remain
                       poorly understood. Coprostanol appears to persist, particularly under anaerobic
                       conditions, in both lacustrine (Nishimura and Koyama 1977) and marine (Bartlett 1987)
                       sediments. Coprostanol attributed to marine mammals (Venkatesan et al. 1986) dating to
                       3500 Bc. has been found in Antarctic sediments remote from anthropogenic inputs.
                       Recovery of 50-stanols from late Pleistocene sediments has also been reported (Nishimura
                       1982).


                       Observations that demonstrate the longevity of coprostanol as a geochernical marker raise
                       questions concerning its applicability as a quantitative indicator of health risk or as a marker
                       for growing areas affected by varied and intermittent pollution sources such storm or
                       agricultural runoff. Eganhouse et al. (1988) noted concentrations of coprostanol and linear
                       alkyl benzenes (the anionic surfactants found in household laundry detergents)) were rather
                       variable in samples of sewage sludge. This was attributed to differences in concentrations
                       of coprostanol in human feces and the differential effects of biosynthetic and degradative
                       processes that can take place in feces and during waste processing. Another argument
                       against the use of coprostanol is that although samples can be preserved for analysis at a
                       late date, the analysis remains technically detailed, lengthy, and costly. Alternate
                       approaches may result in shortened processing time. Wun et al. (1979) described a column
                       adsorption method using XAD-l resin (Rohm and Haas,     Inc., Philadelphia) for the rapid
                       extraction of both cholesterol and chlorophyll a from water samples. Hoskin and Bandler
                       (1987) described a rapid thin layer chromatographic method for detection of fecal
                       contamination through recovery of coprostanol from foods. Although this method would
                       be of limited value for analysis of water samples, it could possibly be adapted for use with
                       shellfish meats. Current improvements in solid phase packings, especially the silica-based
                       bonded pacldngs, could result in rapid and improved recovery procedures for detection of
                       cholesterol from shellfish waters or shellfish. Krahn et al. (1989) described a rapid, semi-
                       automated high performance liquid chromatograpluc (HPLC) method for separating
                       coprostanol from interfering compounds in sediment extracts. Finally, Eganhouse et al.
                       (1988) sounded a cautionary note concerning prior and future quantitative surveys of


 00                                                         63









                       coprostanol; unless its co-eluting , epicoprostanol, is clearly resolved, reported
                       concentrations have been and will be overestimated. Ova-all, the preponderance of
                       information suggests the use of coprostanol as an indicator of fecal pollution in shellfish
                       harvesting waters should be viewed as very low priority, because of multiple sources and
                       absence of data supporting its use to nonpoint polluted areas.



                       Hydrolytic Enzymes


                       Measur ing the activities of selected hydrolytic enzymes as indicators of fecal pollution has
                       been proposed. Lathard (1967; 1969) examined the relationship between urease activity
                       and the presence of sewage or sewage contamination in bottom sediments. Sldba and
                       Wainright (1982) evaluated urease activity in unpolluted and sewage-polluted beach sands
                       as an indicator of sewage contamination. @ Urease activity was relatively high in sands
                       adjacent to a sewage outfall and decreased with distance from the source. Urea. is a major
                       nitrogenous compound in urine, untreated or partially treated sewage, and its release
                       presumably elicits elevated urease activity in impacted sediments owing to its availability as
                       a bacterial substrate. Urease may also be present in the sewage source. Urease activity can
                       quantified by measuring evolved NH4' (Lenhard 1969) or loss of urea (Sadler 1989).
                       Either procedure is relatively uncomplicated and rapid compared with the standard fecal
                       coliform assay. Positive relationships between urease activity associated with known fecal
                       contamination (Lenhard 1967) and E coft densities (Sadler 1989) have been observed but
                       the data base is very limited. Because the urease assay measures a soluble constituent, it is
                       probable that its ability to reflect the behavior of free- or particle-associated bacterial or viral
                       pathogens in estuarine waters would be imperfect. Data are also needed that describe how
                       quickly sediment urease levels change in response to changing levels of sewage
                       contamination. Compared with coprostanol urease might be a simpler and less labor
                       intensive alternative to trace established sewage plumes or discharges.



                       Long-Chain Alkylbenzenes, Fluorescent Whitening Agents and
                       Sodium Tripolyphosphate


                       Certain chemicals found in commercial and domestic detergents and present in municipal
                       wastewaters and domestic sewage have been identified as waste-specific markers or
                       indicators of sewage contamination. Eganhouse (1986) reviewed the chemistry,


                                                            64








                       occurrence and fate of long-chain allcylbenzenes in various environments. Because of their
                       chemical stability, he concluded use of these compounds would be to delimit areal and
                       temporal impacts of plumes reflecting discharge of municipal sewage. Close et al. (1989)
                       detected septic contamination of groundwater by measuring fluorescent whitening agents
                       and sodium tripolyphosphate. These compounds, which are present in domestic
                       detergents, were detected in 17% of groundwater samples and significantly correlated with
                       densities of fecal colifams. The value of these indicator compounds to detect the presence
                       of malfunctioning septic systems in a watershed or transport of septic effluents through
                       subsurface infiltration to estuarine systems has not been evaluated. This will also require
                       determination of compound stability and extent of degradation by microorganisms in
                       natural systems.



                       PROTOZOA


                       Acanthamoeba


                       Free-living protozoans belonging to Acanthamoeba, a genus ubiquitous in soils and
                       freshwater environments, have been detected in sewage contaminated      sediments from
                       freshwater, estuarine and oceanic dump sites and outfalls (Sawyer et al. 1977; Sawyer
                       1980; Daggett et al 1982; 011alley et al. 1982; Sawyer et al. 1987). Generally, the
                       occurrenceof Acanthamoeba spp. has been associated with elevated levels of fecal bacteria
                       although sediments positive for Acanthantoeba spp., enteroviruses, coprostanol, PCBs,
                       and heavy metals were sometimes negative for bacterial indicators (Sawyer et al. 1987).
                       Sawyer et al. (1987) proposed Acanthamoeba as as a monitoring indicator based on
                       persistence of its cysts (for periods up to, 23 years), the pathogenicity of some strains, and
                       its association with sewage. These amoebae are widely distributed in nature. The
                        gnificance of their association with fecally polluted marine samples requires further study
                       to determine if in situ Acanthantoeba densities reflect utilization of bacteria growing in
                       dumpsite sewage sludge or the direct input of populations carried in the sludge (Sawyer et
                       al. 1982; Daggett et al. 1982). Perhaps, the most likely answer will be a combination of
                       both processes. Quantitative data regarding Acanthanweba      densities in human feces,
                       sewage, agricultural runoff and nonpoint impacted areas would be needed to assess its
                       applicability as an indicator in shellfish waters. The complexity and cost of this
                       undertaking, drawbacks associated with indicator organisms that form resistant cysts, the
                       lengthy incubation periods, and degree of expertise necessary to distinguish Acanthamoeba


                                                             65








                       spp. from other genera of free-living amoebae are concerns that do not supports its use as a
                       broadly based indicator of fecal pollution.



                       CONCLUDING COMMENTS


                       Some investigators (Berg 1978; Richards 1985)) have proposed direct detection of
                       microbial pathogens as an alternative to indicators, citing documented examples of
                       instances where current indicators failed to predict pathogen presence or pathogens were
                       detected in the absence of indicators. In our view, arguments to retain surrogate
                       microbiological indicators (Cabelli 1977b; James 1979) remain valid because of the
                       unpredictable occurrence of pathogens, variations in pathogen virulence, morbidity rates,
                       and minimum infective dosages within. a target population. Ultimately, of course
                       pathogens intrinsically lack the ability to predict risk. Arguments based on the lack of
                       accepted methods for direct and accurate routine detection of pathogens we less compelling.
                       Recent advances in biotechnology suggest the feasibility of dir-ect pathogen detection is
                       now reality. Experimental gene probes for hepatitis A virus (HAV) have been used to
                       detect HAV in polluted estuarine waters (Jiang et al. 1987; Metcalf and Jiang 1988) and a
                       gene probe for Norwalk agent is now being developed. A gene probe for Salmonella spp.
                       (GENE-TRAK Systems, 31 New York Avenue, Framingham, MA 01701) is commercially
                       available.


                       Emergent methods may revolutionize some aspects of microbiology that traditionally have
                       been associated with frustrating drudgery, elaborate and repetitive procedures, Le,
                       enumeration and phenotypic characterization of isolates. After more than half a century
                       using essentially unchanged cultivation methods for recovery of the coliform indicator
                       group, new methods for rapid detection are becoming available. Ilese may be based on
                       traditional cultivation methods, direct enummdon procedures, target phenotypic
                       characteristics such as constituitive enzymes or antigenic factors, or use recombinant
                       DNA/RNA technology such as PCR to target an oligonucleotide sequence with high
                       specificity. We welcome these new methods, anticipating their promise of ultimate
                       specificity and rapidity but feel compelled to offer the following cautionary remarks.


                       First the best detection systems will not eliminate the need to understand the ecology of an
                       indicator, its survival characteristics and the relationship between environmental exposure
                       and, the assay. For example, allochthonous bacteria exposed to marine and estuarine


                                                             66








                       environments lose their ability to grow on selective and conventional bacteriological media
                       (Barcina. et al. 1089; Roszak and Colwell 1987; Rhodes et al. 1983) and important
                       diagnostic characteristics may be altered or lost False-negatives or misidentification can be
                       a consequence of enumerative or identification schemes based on expression of a sensitive
                       phenotypic characteristic. E colt loses its ability to ferment lactose (Kasweck and
                       Fliernians 1978), O-galactosidase activity was reduced in cells exposed to seawater
                       (Anderson et al. 1979; Munro et al. 1987). Loss or alteration of plasmids encoding for
                       antibiotic and heavy metal resistance occurs in various Enterobacteriaceae during long-term
                       starvation (Chai 1983; Caldwell et al. 1989) and streptococcal (and enterococcal) isolates
                       from animal and environmental sources do not consistently yield valid reactions in
                       miniaturized biochemical testing systems (Molitoris et al. 1985; Rhodes and Kator
                       unpublished results). Chang et al. (1989) reported a significant proportion of E coli
                       isolated from human fecal samples were P-D-glucuronidase negative


                       Finally, it is conceivable that advances in sensitivity and accuracy of analytical methods
                       could lead to requests for growing area closures based on improved detection of pathogens
                       without considering issues such as pathogen infectivity, host susceptibility, historical
                       incidence of disease, and sanitary surveys. Such action could produce an untenable
                       situation for the shellfish industry and regulatory agencies. Detection of pathogens in
                       approved shellfish growing waters using new (or old) methods should not be construed as
                            -facie evidence of health risk. Cabelli's (1978b; 1979) arguments that standards must
                       be based on functional relationships, i.e., correlating rates of illness fro m prospective
                       epidemiologic. investigations with indicator densities, must be heeded.




















                                                            67


0








                     REFERENCES


                     Abshire, R. 0L 1977. Evaluation of a new presumptive medium for group D streptococci.
                     Ami. Environ. microbiol, 33: 1149-1155.


                     A2bshire, R., and Guthrie, 8K K. 1971. The use of fluorescent antibody techniues for
                     5:1089-1097.


                     Adams, M. H. 1959. bacteriophages. Interscience, New York, N. Y.


                     Adams, 2L C. and Farrier, D. S. 1982. The effect of some oil shale process waters upon
                     the viability of indicator, bacteria. J. Environ. ual. 11: 171-174.


                     Adams, M. R., Grubb, S. M., Hamer, A. and, Clifford, M. N. 1990. Colorimetric
                     enumeration of Escherichia coli based on 0-glucuronidase activity. A2N-1. Environ,
                     Microbiol, 56: 2021-2024.


                     AI-Jebouri, M. M. and Trollope, D. R. 1981. The Escherichia coli content of Myfilus
                     edulis from analysis of whole tissue or diges, tive tract. 1. -A-401. Bacteriol 51:135-142.


                     Allsop, K., and Stickler, D. J. 1984. The enumeration of Bacteroides ftagili group
                     organisms from sewage and natural waters. J. A 1. Bacteriol, 56:1524.
                                                                  


                     Allsop, K., and Stickler, D. J. 1985. An assessment of Bacteroides fimas group
                     organisms as indicators of human faecal pollution. J. 2A2W-1. Bacteriol. 58:95-99.


                     Alvarez, R. J. 1984. Use of 4f4luorogenic assays for the enumeration of Escherichia coli
                     from selected seafoods. J. Eod Sci. 49:1186-1232.


                     American Public Health Association. 1984. Comondium of Methods for
                     Microbiological Examination of Food& 2nd ed. American Public Health Association,
                     Washington, D. C.


                     American Public Health Association. 1985 Standard Methods for the Examination o
                     Water and Wastewater, 16th ed. American Public Health Association, Washington, D.C.




                                                        68
 







                     American Public Health Association. 1984. LWxgat= Ptocedures for the Examination of
                     Seaw= and Shellfish. 5th ed. Amen-can Public Health Association, Washington, D. C.


                     American Public Health Association. 1989. Standard Methods for the Examination o
                     Water and Wastewater, 17th ed. American Public Health Association, Washington, D.C.


                     Amy, P. S. and Hiatt, H. D. 1989. Survival and detection of bacteria in an aquatic
                     environment _A4W_l. Environ. Microbiol, 55:788-793.


                     Andersen, P., and Fenchel, T. 1985. Bacterivory by microheterotrophic flagellates in
                     seawater samples. Limnol. Oceanogr 30:198-202.


                     Anderson, 1. C., Rhodes, M., and Kator, H. 1979. Sublethal stress in Escherichi coii: a
                     function of salinity. -A-MI. Environ, Microbiol. 38:1147-1152.


                     Anderson, 1. C., Rhodes, M., and Kator, H. 1983. Seasonal variation in survival of
                     Eschetichia coli exposed in situ in membrane diffusion chambers containing filtered and
                     nonfiltered estuarine water. AWL Environ. Microbiol, 45 :1877-1883.


                     Andrews, W. H. and Presnell, M. W. 1972. Rapid recovery of Escherichi coli from
                     estuarine water. AML Microbiol. 23:521-523.


                     Andrews, W. H., Wilson, C. R., Poelma, P. L. , Romero, A., Rude, R. A., Duran, A.
                     P., McClure, D., and Gentile, D. E 1978. Usefulness of the stomacher in a
                     microbiological regulatory laboratory. AWL Environ. Microbiol. 35:89-93.


                     Armon, R., and Payment, P. 1988. A modified m-CP medium for enumerating
                     Clostridium pprffin&tn_s, from water samples. Can, J. Microbiol. 34:78-79.


                     Armon, R_, and Cabelli, V. J. 1988. Phage f2 desorption from clay in estuarine water
                     using nonionic detergents, beef extract, and chaotropic agents. Can. J. Microbiol.
                     34:1022-1024.


                     ASTM. 1987. Standard Test Method for enumeration of Candi albican in water. In:
                     ASTM Standards on Materials and Environmental Migm @ol y. American Society for
                     Testing and Materials, Philadelphia, pp. 113-118.


0                                                       69


0








                     Atlas, R. M. 1982. Enumeration and estimation of biomass of microbial components in
                     the biosphere. In: Bums R. G. and Slater J. H. (eds.) Experimental microbial ecology
                     Blackwell Scientific Publishers, Oxford, pp. 84-102.


                     Atlas, R. M., and Bej, A. K. 1990. Detecting bacterial8pathogens, in environmental water
 0                   samples by using PCR and gene probes. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J.,
                     andWhite, T. 2L (eds.) R Protocols: A- Guide to Methods and Applications . Academic
                     Press, San Diego, pp. 399-406.


                     Attaway, D., and Parker, P. 4L 1970. Sterols in recent marine sediments. Science 169:
                     674-675.


                     Attwell, R. W., and Colwell, R. R. 1984. Thermoactinomycetes as terrestrial indicators
                     for estuarine and marine waters. In: Ortiz-Or8f8iz, L, Bojalil, 0L F., and Yakoleff, V. (eds.)
                     Biolggical. biochemical and biomedical aspects of actinomycetes. Academic Press, New
                     York, pp. 441-452.
 0                   Aubert, M., Pesando, D., and Gauthier, M. G. 1975. Effects of antibiosis in a marine
                     environment. In: Garneson, 8L H. (ed.) Discharge of sewage from sea outfall . Pergarnon
                     Press, New York, pp. 191-197.
 0                   Avila, M. J., Morifigo, M. A., Comax, R., Romero, P., and Borrego, J. J. 1989.
                     Comparative study of coliform-enumeration media from seawater samples. J. Microbiol.
                     Meth     9:175-193.
 9                   Awong, J., Bitton,G., and Chaudhry, G. R. 1990. Microcosm for assessing survival of
                     genetically engineered microorganisms in auatic environments. 4&8Wl. Environ.
                     Microbiol. 56:977-983.
 0                   Ayres, P. A. 1977. Co0liphages in sewage and the marine environment. In: Skinner, F.
                     A., and Shewan, J. M. (eds.) Aquatic microbiology. Academic Press, London, pp. 275-
                     298.
 9                   Babich, H., and Stotzky, G. 1978. Toxicity of zinc to fungi, bacteria and coliphages:
                     influence of chloride ions. 72A4p44pl. Environ. Microbiol. 36: 906-914.


 0                                                        70
 

0








                      Bagley, S. T. and Seidler, R. J. 1977. Significance of fecal coliform-positive Klebsiel
                      Appl. Environ, Microbiol, 33:1141-1148.


                      Barcina, I., Aranaj., Iriberri, J., and Egea, 2L 1986. Influence of light and natural
                      microbiota of the Butron river4on 8R coli survival. A4ntonie van leeuwenhoek J. Microbiol.
                      52: 555-566.


                      Barcina, L, Gonzalez, J. M., Iriberri, J., and Egea, 4L 1989. Effect of visible light on
                      progressive dormancy of Escherichia eoli cells during the survival process in natural fresh
                      water. _AWL Environ. hfigrobiol. 55: 246-251.


                      Barnes, 8R L. 1986. Anaerobic bacteria of the normal intestinal microflora, of animals. In:
                      Barnes, 8R M., and Mead, G. C. (eds.) Anaerobic bacteria in habitats other than man
                      Blackwell Scientific Publications, Oxford, England, pp. 225-238.


                      Bartlet@ P. 1987. Degradation of coprosta4nol in an experimental system. Mar, Pollut.
                      Bull. 18: 27-29.


                      Baxter-Potter, W. R. and Gilliland, M. W. 1988. Bacterial pollution in runoff from
                      agricultural lands. J. Envir0on, 0ual. 17:27-34.


                      Beaudoin, 8R C., and Litsky, W. 1981. Fecal streptococci. In: Dutim, B. L(ed.)
                      Membrane filtration: a4p420h@cations, technigues. and bigblems. Marcel Dekker, New York,
                      pp. 77-118.
 0                    Beerens, H. 1990. An elective and selective isolation medium for Bifidobacterium spp.
                      Lett. A 1. Microbiol. 11: 155-157.
                            8M

 0                    Bej, A. K., Steffan, R. J., DiCesare, L, Haff, L., and Atlas, R. M. 1990. Detection of
                      coli8form bacteria in water by polymerase chain reaction and gene probes. AWL Environ.
                      Micro44biol. 56:307-314.


 0                    Bellair, J. T., Parr-Smith, G. A., and Wallis, L G. 1977. Significance of diurnal
                      variations in fecal coliform die-off rates in the design of ocean outfalls. J, Wat. Pollut.
                      Control     .49: 2022-2030.



 0                                                        71
 


                     Berg, G. 1978. The indicator system. In: G. Berg (ed.) Indicators of viruses in water
                     and food Ann Arbor Science Publishers Inc., Ann Arbor, Mich, pp. 1-13.


                     Bergey's Manual of Systematic Bacteriology. Vol. 1. 1984. Krieg, N. R., and Holt, J.
                     G. (eds.) Williams and Wilql2qdns, Baltimore.


                     Bergey's Manual of Systematic Bacteriology. Vol, 2. 1986. Sneath, H. A., Mair, P. H.
                     A., Sharpe, M. 8qR, and Holt, J. G. (edqs.) Williams and Wilkins, Baltimore.


                     Berk, S. G. , Colwell, R. R., and Small, N. E. B. 1976. A study of feeding responses to
                     bacterial prey by estuarine ciliates. Trans, Amer, Microscope. Soc. 95:514-520.


                     Betzl, D., LudwigW., and Schleifer, K. H. 1990. identification of Lactoccoci and
                     Enterococci by colony hydridization with 23S rRNA-targeted oligonucleotide probes.
                     Appl. Environ. Microbiol. 56:2927-2929.
                      


                     Bezkorovainy, A. 1989. Chapter 4. Nutrition and metabolism of bifidobacteria. In:
                     Bezkorovainy, A. and Miller-Catchpole, R. (ed.) Biochemistry and Physiology of
                     Bifidobacteri . CRC Press, Boca Raton, Florida, pp. 93-130.


                     Bisson, J. W., and Cabelli, V. J. 1980. Clostridium perfringens as a water pollution
                     indicator. J. Wat. Pollut. Control Fed 52: 241-248.


                     Bissonnette, G. K., Jezeski, J. J., McFeters, G. A., and Stuart,.D.G. 1975. Influence of
                     environmental stress on enumeration of indicator bacteria from natural water. Appl.
                     Microbiol. 29:186-194.


                     Bitton, G., and Mitchell, R. 1974. Effect of colloids on the survival of bacteriophages, in
                     seawater. Wat. Res. 8:227-229.


                     Boardman, G., McBrayer, T. R., and Kohlhepp, P. 1989. Detection and occurrence of
                     waterborne bacterial and viral pathogens. J. Wat. Pollut. Con. Fed. 61:1097-1109.






                                                         72








                      Boehm, P. D. 1983. Coupling of organic pollutants between the estuary and continental
                      shelf and the sediments and water column in the New York Bight Region. Can. J. Fish
                      Aquat. Sci, 40: 262-276.


                      Bonde, G. J. 1977. Bacterial indication of water pollution. In: Droop, M. R., and
                      Jannasch, H. W. (ed.) Advances in agUt
                      London, pp.273-364.                    Lic microbiology. Vol. 1. Academic Press,

                      Booth, S. J., Van Tamil, R. L., Johnson, J. L., and Willdns, T. D. 1979.
                      Bacteriophage s of Bacteroides. Rev. Infect. Dis. 1:325-336.


                      Borrego, J., and Romero, P. 1985. Coliphage survival in seawater. Water Res. 19:557-
                      562.


                      Borrego, J. J., Moriffigo, M. A., de Vicente, A., Cornax, R., and Romero, P. 1987.
                      Coliphages, as an indicator of fecal pollution in water. Its relationship with indicator and
                      pathogenic microorganisms. Wat. Res, 21:1473-1480.


                      Borrego, J. J., Comax, R., Moriffigo, M. A., Martinez-Manzanares, 11, and Romero, P.
                      1990. Coliphages as an indicator of fecal pollution in waten Their survival and productive
                      infectivity in natural aquatic environments. Wat Res. 24:111-116.
 41                   Borrego, J. J., Cornax, 1;L, Preston, D. R., Farrah, S. R., McElhaney, B., and Bitton, G.
                      199 1. Development and application of new positively charged filters for recovery of
                      bacteriophages fi-om water. &Wl. Environ. Microbiol. 57: 1218-1222.
 0                    Bosley, G. S., Facklarn, R. R., and Grossman, D. 1983. Rapid identification of
                      En           J. Clin. Mcrobiol. 18:1275-1277.


 0                    Breittmayer, V. A, and Gauthier, M. 1. 1990. Influence of glycine betaine on the transfer
                      of plasmid RN between Escheric      coli strains in marine sediments. Letters -Awl,
                      AEcrobiol. 10: 65-68.


 0                    Brezens1d, F. T. 1973. Fecal streptococci. In: Proceedings of the First Microbiology
                      Seminar on Standardization of Methods, San Francisco, California. U. S. Environmental
                      Protection Agency, Washington, D. C., pp. 47-68.


 0                                                        73










                      Brodisch, K. E. U., Idema, G. K., Coubrough, P., and Grabow, W. 0. K. 1986. The
                      recovery of enteric viruses and coliphages from shellfish. Water Sci, Technol. 18:157.


                      Brodsky, M. H., and Schiemann, D. A. 1976. Evaluation of Pfizer selective enterococcus
                      and KF media for the recovery of fecal streptococci from water by membrane filtration.
                      A
                        MI. Environ, Microbiol, 31: 695-699.


                      Brown, F- C., and Wade, T. L. 1984. Sedimentary coprostanol and hydrocarbon
                      distribution adjacent to a sewage outfall. Water Re 18:621-632.


                      Bruland, K. W., Bertine, K., Koide, M., and Goldberg., E D. 1974. History of heavy
                      metal pollution in southern California coastal zone. Environ. Sci. Tech, 8:425-432.


                      Buck, J. D. 1977. Can        albicans. In: Hoadley, A. W. and Dutka, B. J. (eds.)
                      Bacteriat indicatorOrAlth hazards associated with        American Society for Testing and
                      Materials, Philadelphia, pp. 139-147.

 0                    Burge, W. D. and Parr, J. F. 1980. Movement of pathogenic organisms fi-om. waste
                      applied to agricultural lands. In: Overcash, M. R. and Davidson, J. M. (eds.)
                      Environmental iwp= of noripgint source Wllu          Ann Arbor Science, Ann Arbor, pp.
                      107-124.


                      Burlingham, G. A., McElhaney, J., Bennett, M., and Pipes, W. 0. 1984. Bacterial
                      interference with coliform colony sheen production on membrane filters. AWL Environ,
                      Microbiol. 47:56-60.
 0

                      Busta, F. F. 1978. Introduction to injury and repair of microbial cells. Ady, AML
                      Microbiol. 23: 219-243.


 0
                      Cabelli, V. J. 1977a. Clostridium Rgddrig-ens as a water quality indicator. In: Hoadley.
                      A. W. and Dutka, B. J. (eds.) Bacterial indicators/health hazards associated with
                      Special Technical Publication 635, American Society for Testing and Materials,
 0                    Philadelphia, pp. 65-79.


 0                                                         74








                      C abelli, V. J. 1977b. indicators of recreational water quality. In: Hoadley, A. W. and
                      Dutka, B. J. (eds.) Bacterial Indicators/Health Hazards Associated with Water. Special
                      Technical Publication 635, American Society for Testing and Materials, Philadelphia, pp.
                      222-238.


                      Cabelli, V. J. 1978a. Obligate anaerobic bacterial indicators. In: Berg, 6. (ed.) Indicators
                      of Viruses in Water and         Ann Arbor Science Publishers, Ann Arbor, Michigan, pp.
                      171-200.


                      Cabelli, V. J. 1978b. New standards for enteric bacteria. In: Mitchell, R. (ed.) Water
                      Pollution Nficrobiology,-Yo-1.2. John Wiley & Sons, New York, pp. 233-271.


                      Cabelli, V. J. 1979. Evaluation of recreational water quality, the EPA approach. In:
                      James, A. and Evison, L (eds.) Biological Indicators of Water QgWily John Wiley &
 0                    Sons, Chichester, pp. 14-1 - 14-23.

                      Cabelli, V. J., Dufour, A. P., McCabe, L. J., and Levin, M. A. 1983. A marine
 0                    recreational water quality criterion consistent with indicator concepts and risk analysis. J.
                      Water Pollut. Control Ee,&_ 55:1306-314.


                      Calambolddis, J., McLaughlin, B. D., and Steiger, G. H. 1989. Bacterial contamination
 0                    related to harbor seals in Puget Sound, Washington. A final report to Jefferson County
                      and Washington Department of Ecology, Cascadia Research, Olympia, Washington.


                      Caldwell, B. A.., Ye, C., Griffiths, R. P., Moyer, C. L., and Morita, R. Y. 1989.
 0                    Plasmid expression and maintenance during long-term starvation- survival of bacteria in
                      well water. A01. Environ. Microbiol. 55:1860-1864.


                      Callcins, J. 1982. 'Me role of solar ultraviolet radiation in marine g&Mstems. Plenum
 0                    Press, New York.

                      Canter, L W., and Knox, R. C. 1985. Safic tank systems effects of ground
                      quality Lewis Publishers, Chelsea, Michigan.


                      Carlucci, A. F., and Pramer, D. 1960. An evaluation of factors affecting the survival of
                      Escherichi coli in seawater. H. Salinity pH, and nutrients. A @1. Microbiol. 8:247-250.
                                                                                     P



                                                           75










                     Carrillo, M., Estrada, E., and Hazen, T. C. 1985. Survival and enumeration of the fecal
                     indicators Bifidobacterium adolescentis, and         coh in a tropical rain forest
                     watershe(L AW-1. Environ. Microbiol 50:468-476.


                     Chai, T.-J. 1983. Characteristics of Escheric  coh grown in bay water as compared
                     with rich medium. AWL Environ. Microbiol 45:1316-1323.


                     Chang, G., Brill, J., and Lum, R. 1989. Proportion of P-D-glucuronidase-negative
                     Escheric   coli in human fecal samples. A 1. Environ. Microbiol 55:335-339.


                     Chen, M. 1988. Pollution of ground water by nutrients and fecal coliforms from
                     Ukeshore-septic tank systems. Water, Air and Soil Pollut. 37:407-417.


                     Churrhland, L M. and Kan, G. 1982. Variation in fecal pollution indicators through tidal
                     cycles in the Fraser River estuary. Can. J. Microbiol 28:239-247.


                     Clark, H. F., Geldreich, E. E., Jeter, H. L, and Kabler, P. W. 195 1. The membrane
                     filter in sanitary bacteriology. Public Health  66:951-977.


                     Clausen, J. C. and Meals, D. W., Jr. 1989. Water quality achievable with agricultural
                     best management practices. J. Soil WaL Cons. 44:593-596.


                     Clausen, E. M., Green, B. L., and Litsky, W. 1977. Fecal streptococci: indicators of
                     pollution. In: Hoadley, A. W., and Dutka, B. J. (eds.) Bacterial indicators/health hazards
                     associated with w   . Special Technical Publication 635, American Society for Testing
                     and Materials, Philadelphia, pp. 247-264.


                     Close, M. E., Hodgson, L. R., and Tod, G. 1989. Field evaluation of fluorescent
                     whitening agents and sodium tripolyphosphate as indicators of septic tank contamination in
                     domestic wells. New Zealand J. Mar, Freshwat. Res, 23: 563-568.


                     Colburn, K. G., Kaysner, C. A. , Abeyta , C. Jr., and Wekell, M. M. 1990. Listeria.
                     species in a California coast estuarine environment. AWL Environ, Microbiol. 56: 2007-
                     2011.





                                                         76








                     Collins', C. H., and Lyne, P. M. 1984. Microbiological methods. Fifth edition.
                     Butterworths, London.


                     Cook, D. W. 1981. Automatic incubator for use with modified A- I test for enumerating
                     fecal coliform bacteria in shellfish growing waters. 1. Ass oc. Off. Anal. Chem. 64:771 -
                     773.


                     Cooper, K. E, and Ramadan, F. M. 1955. Studies in the differentiation between human
                     and animal pollution by means of thecal streptococci. J. Gen. Microbiol 12:180-190.


                     Cooper, S. W., Szymczak, R G., Jacobus, N. V., and Tally, F. P. 1984. Differentiation
                     of ftcjmii@s ovatu and Bacteroi      thetaiotaomicron by means of bacteriophage. J.
                     Clin. Microbiol. 20:1122-1125.


                     Comax, R., Moriffigo, M. A., Romero, P., and Borrego, J. V 1990. Survival of
                     pathogenic microorganisms in seawater. Curr. Mimb-iol, 20:293-298.


                     Comax, R., Moriffigo, M. A., Balebona, M. C., Castro, D., and Borrego, J. J. 1991.
                     Significance of several bacteriophage groups as indicators of sewage pollution in marine
                     waters. Wat. Res, 25:673-678.


                     Cynar, F. J., Estep, K. W., and Sieburth, J. McN. 1985. The detection and
                     characterization of bacteria-sized protists in "protist-ftW filtrates and their potential impact
                     on experimental marine ecology. Microb, Ecol. 11:281-288.


                     IYAoust, R. A., and Litsky, W. 1975. Pfizer selective enterococcus agar overlay method
                     for the enumeration of fecal strWtococci by membrane filtration. -AM-1, Environ.
                     Microbiol. 29:584-589.


                     Daggett, P.-M., Sawyer, T. K., and Nerad, T. A. 1982. Distribution and possible
                     interrelationships of pathogenic and nonpathogenic Acanthamoeb from aquatic
                     environments. Nficrob, Ecol. 8:371-386.


                     Dahlen, G., & Linde, A. 1973. Screening plate method for detection of bacterial
                     glucuronidase. A-MI. Microbiol. 26: 863-866.



                                                         77








                      Daily, 0. P., Joseph, S. W., Gillmore, J. D., Colwell, R. R., and Seidler, R. J. 1981.
                      Identification, distribution and toxigenicity of obligate anaerobes in polluted waters. ARg Li.
                      Environ. Microbiol, 41:1074-1077.


                      Daley, R. J. 1979. Direct epifluorescence enumeration of native aquatic bacteria: uses,
                      limitations and comparative accuracy. In: Costerton, J. W., and Colwell, R. R.(eds.)
                      Native aQuatic bacteria: enumeration, activft and ecolM Special Technical Publication
                      695, American Society for Testing and Materials, Philadelphia, pp.29-45.


                      Dalton, H. P., Archer, G. L, Slifkin, M., Harris, R. C.,, Welshimer, H. J., Sosnowski,
                      K. M., Nottebart, H. C., Jr., Duma, R. J., Clark, R. B., Warren, N. G. , Kerkering, T.
                      M., Swenson, P. D., Ager, A. L., Jr., and May, R. G., Jr. 1986. Blood specimens. In:
                      Dalton, H. P., and Nottebart, H. C., Jr. (eds.) Injg&Ketive medical microbiology.
                      Churchill Livingstone, New York, pp. 28-29.


                      Davis@ E M., Casserly, D. M., and Moore, J. D. 1977. Bacterial relationships in
                      stormwaters. Wat. Resour. Bull. 13:895-905.          -


                      Dawe, L L., and Penrose, W. R. 1978. "Bactericid    al" property of seawater: death or
                      debilitation? _A@Ml, Environ. Microbiol. 35:829-833.


                      Debartolomeis., J. 1988. Enumeration of F male-specific bactetiophages from sewage
                      and fecally polluted waters. Ph.D. Dissertation, University of Rhode Island, Kingston,
                      Rhode Island.


                      Deibel, R. H., and Hartman, P. A. 1984. The Enterococci. In: Speck, M. L. (ed.)
                      QqmWndium. of methods for the microbiological examination of foods. American Public
                      Health Association, Washington, D. C., pp. 405-410.


                      Desmonts, C., Minet, J., Colwell, R., and Cormier, M. 1990. Fluorescent-antibody
                      method useful for detecting viable but nonculturable Salmonell spp. in chlorinated
                      wastewater. AW-1. Environ. Microbiol. 56: 1448-1452.


                      Doran, J. W. and Linn, D. M. 1979. Bacteriological quality of runoff water from
                      pastureland. Appl. Environ. Microbiol. 3,7:965-991.



                                                           78








                     Dowell, V. &, Jr., and Hawkins, T. M. 1968. LaboraW methods in anaerobic
                     bacteriology. U. S. Department of Health, Education, and Welfare. National
                     Communicable Disease Center, Atlanta, Georgia.


                     Doyle, R. C., Wolf, D. C., and Bezdicek, D. F. 1975. Effectiveness of forest buffer
                     strips in improving the water quality of manure polluted runoff In: Mm=g
                     Wastes. Proceedings 3rd International Sympgsium on Livestock           University of
                     Illinois, Urbanna-Champaign, April 21-24, 1975, pp.299-302.


                     Drake, J. F., and Tsuchiya, H. M. 1976. Predation on Escheri     gghLby CglMda
                     steinii. AM-1. Environ. Microbiol. 31: 870-874.


                     Dufour, A. P. 1980. A 24-hour membrane filter procedure for enumerating enterococci.
                     Abstracts of the 80th Annual Meeting of the American SgdM for MicrobiolQgy. Miami.
                          da. May. 1980.


                     Dufour, A. P., Strickland, E, R., and Cabelli, V. J. 1.981. Membrane filter method for
                     enumerating Escherichi coli. Ap4j Environ. Microbiol. 41: 1152-1158.


                     DOreth, S., Herrman, R., and Pecher, K. 1986. Tracing fecal pollution by coprostanol.
                     and intestinal bacteria in an ice-covered Finnish lake loaded with both industrial and
                     domestic sewage. Wat Air and Soil Pollut. 28: 131-149.


                     Dutka, B. J. 1973. Coliforms are an inadequate index of water quality. J. Environ. HIth.
                     36:39-46.


                     Dutka, B. J. 1979. Microbiological indicators, problems and potential of new microbial
                     indicators of water quality. In: James, A., and Evison, L (eds.) Biological indicators o
                     waterquali1y. John Wiley and Sons, Chichester, pp. 18-1 - 18-24.


                     Dutka, B. J., Jones, K., Kwan, K. K., Bailey, H., and McInnis, R. 1988. Use of
                     microbial and toxicant screening tests for priority site selection of degraded areas in water
                     bodies. Wat. Res, 22:503-510.


                     Eganhouse, R. P. 1986. Long-chain alkylbenzenes: their analytical chemistry,
                     environmental occurrence and fate. Inter. J. Environ. Anal. Chem. 26: 241-263.


                                                         79










                     Eganhouse,R. P., Olaguer, D.P., Gould, B. R., and Phinney, C. S. 1988. Useof
                     molecular markers for the detection of municipal sewage sludge at sea. Mar. Environ. Res
                     25:1-22.


                     Ellender, R. D., Mapp, J. B., Middlebrooks, B. L., Cook, D. W., and Cake, E W.
                     1980. Natural enterovirus and fecal coliform contamination of Gulf Coast oysters. J.
                     Food         .43:105-110.


                     Elliott, L F. and Ellis, J. R. 1977. Bacterial and viral pathogens associated with land
                     application of organic wastes. J. Environ. Qual. 6:245 -25 1.


                     Entis, P., Brodsky, M. H., and Sharpe, A. N. 1982. Effect of pre-filtration and enzyme
                     treatment on membrane filtration of foods. J. Food Protect, 45:8-11.


                     Entis, P. and Boleszczuk, P. 1990. Direct enumeration of coliforms and Escheri     coli
                     by hydrophobic grid membrane filter in 24 hours using MUG. J. Food. Protect. 53:948-
                     .952.


                     Enzinger, R. M., and Cooper, R. C.. 1976. Role of bacteria and protozoa in the removal
                     Of             coli from estuarine waters. &MI. Environ. Microbiol. 31:758-763.


                     Ericksen, T. H., and Dufour, A. P. 1986. Methods to identify waterborne pathogens and
                     indicator organisms. In: Craun, G. F. (ed.) Waterborne diseases in the United State .
                     CRC Press, Boca Raton, Florida, pp. 195-214.


                     Erkenbrecher, Jr., C. W. 198 1. Sediment bacterial indicators in an urban shellfishing
                     subestuary of the lower Chesapeake Bay. A4MI-. Environ. Microbiol. 42:484-492.


                     Evans, T. M., Warvick, C. E., Seidler, R. J., and LeChevallier, M. W. 1981. Failure of
                     the most-probable-number techniques to detect coliforms in drinking water and raw water
                     supplies. Appl. EAviron. Microbiol. 41:130-138.

                     Facklam, R. R., and Collins, M. D. 1989@. Identification of Enterococcus species isolated
                     from human infections by a conventional test scheme. J. Clin. Microbiol 27:731-734.



6                                                        80








                     Fauat, M. 1976. Coliform bacteria from diffuse sources as a factor in estuarine pollution.
                     Wat. Re . 10: 619-627.


                     Faust, M. 1982. Contribution of pleasure boats to fecal bacteria concentrations in the
                     Rhode River estuary, Maryland, U.S.A. Sci. Total Environ. 25: 255-262.


                     Faust, M. A., Aotaky, A. E, and Hargadon, M. L 1975. Effect of physical parameters
                     on the in situ survival of Escherichia gkhLMC-6 in an estuarine environment. App-1.
                     Microbiol. 30:800-806.


                     Faust, M. A. and Goff, N. M. 1977. Basin size, water flow and land-use effects on fecal
                     coliform pollution from a rural watershed. Watershed Res. Volume H:611-634.


                     Feachem, R. 1975. An improved role for faecal coliform to &ecal streptococci ratios in
                     the differentiation between human and non-human pollution sources. Water Res. 9:689-
                     690.


                     Feachem, R. G., Bradley, D. J., Garelick, H., and Mara, D. D. 1983, Sanitation and
                     disease. Health aspects of excreta and wastewater management. World Bank studies in
                     water &O-ly and sanitation. John Wiley and Sons, New York.


                     Fenchel, T. 1982. Ecology of heterotrophic microflagellates. H. Bioenergetics and
                     growth. Mar. Ecol. Pro&. S . 8:225-23 1.


                     Feng, P. C. S. and Hartman, P. A. 1982. Fluorogenic assays for immediate confirmation
                     Of            coli. AWL Environ. Microbiol. 43:1320-1329.


                     Fiksdal, L, Pommepuy, M., Derrien, A., and Cormier, M. 1989. Production of 4-
                     methylumbelliferyl heptanoate hydrolm by Esc          coli exposed to seawater. AM-1.
                     Environ. Microbiol. 55:2424-2427.


                     Flint, K. P. 1987. The long term survival of Escheric   coh in river water. J. AMI.
                     Bacteriol. 63:261-270.








                     Food and Drug Administration. 1984. Shellfish sanitation interpretation 34: Interpretation
                     of bacteriological market standards for sheRfish. Shellfish Sanitation Branch, Washington,
                     D. C., 12 pp.


                     Food and Drug Administration. 1987. Bacteriological Anal3djW Manual, 6th edition,
                     1994. Supplement 9/87, Association of Official Analytical Chemists, Arlington, Va.


                     Frampton, E. W., Restaino, L, and Blaszko, N. 1988. Evaluation of the f)-
                     glucuronidase. substrate 5-bromo-4-chloro-3-indolyl-P-D-glucuronide (X-GLUC) in a 24-
                     hour direct plating method for Escherich coh. J. Food          51:402-404.


                     Freier, T. A., and Hartman, P. A. 1987. Improved membrane filtration media for
                     enumeration of total coliforms, and Escheric ggh
                                                                     @ from sewage and surface waters. AwL
                     Environ. Microbiol. 53:1246-1250.


                     Fujioka, R. S. and Shizumura, L K. 1985. Clostridium pgrffingen : a reliable *indicator
                     of stream water quality. J. Water Pollut, Control Fed. 57:986-992.


                     Fujioka, R. S., and Siwak, E. B. 1987. Bactericidal properties of long ultraviolet and
                     visible wavelengths of sunlight. J. Amer. Water Works Assoc. 79:56.


                     Fujioka, R. S., Hashimoto, H. H., Siwak, E, B., and Young, R. H. F. 1981. Effect of
                     sunlight on survival of indicator bacteria in seawater. A
                                                                          MI. Environ. Microbiol. 41:690-
                     696.


                     Furuse, K. 1987. Distribution of coliphages in the environment: general considerations,.
                     In: Goyal, S. M., Gerba, C. P., and Bitton, G. (eds.) Phage ecology. John Wiley and
                     Sons, New York, pp. 87-124.


                     Furuse, K., Ando, A., Osawa, S., and Watanabe, 1. 1981. Distribution of ribonucleic
                     acid coliphages in raw sewage from treatment plants in Japan. AML Environ, Microbiol.
                     41: 1139-1143.


                     Furuse, K., Sakurai, T., Hirashima, A., Katsuki, M., Ando, A., and Watanabe, 1. 1978.
                     Distribution of ribonucleic acid coliphages in South and East Asia. AML Environ.
                     Mir-robiol, 35:995-1002.



                                                         82








                      Gameson, A. L k., and Gould, D. J. 1975. Effects of solar radiation on the mortality of
                      some terrestrial bacteria in sea water. In: Gameson, A. L H. (ed.) Disch=e- of sewa
                      from sea outfalls. Pergamon Press, Oxford, pp. 209-219.


                      Gameson, A. L H., and Saxon, J. R, 19.67. Field studies on effect of daylight on
                      mortality of coliform bacteria. Water    1:279-295.


                      Garcia-Lara, J., Menon, P., Servais, P., and Billen, G. 1991. Mortality of fecal bacteria
                      in seawater. AWL Environ. Microbiol, 57: 885-888.


                      Gauthier, M. J. and Le Rudulier, D. 1990. Survival in seawater of Escheric  coli cells
                      grown in marine sediments containing glycine betaine. AML Environ. Microbiol. 56:
                      2915-2918.


                      Gauthier, M. L, Munro, P. M., and Mohadjer, S. 1987. Influence of salts and sodium
                      chloride on the recovery of Escherichig coli ftorn seawater. Curr, Microbiol. 15:5-10.


                      Gauthier, M. J., Faluau, G. N., Le Rudulier, D., Clement, P.- L, and Combarro, M. C.
                      199 1. Intracellulair accumulation of potassium and glutamate specifically enhances survival
                      of Escherich coli in seawater. AWL Environ. Microbiol 57:272-276.


                      Qeldreich, R E 1972. Water-borne pathogens. In: Mitchell, R. (ed.) Water Pollution
                      Microb "lo y Wiley-Interscience, New York, pp. 207-24 1.


                      Geldreich, E. E 1978. Bacterial populations and indicator concepts in feces, sewage,
                      stormwater and solid wastes. In: Berg, G. (ed.) Indicators of Viruses in Water and Food.
                      Ann Arbor Science Publishers, Ann Arbor, Michigan, pp. 51-97.


                      Geldreich, E, R 198 1. Current status of microbiological water criteria. Am. Soc.
                      Microbiol, 47:23-27.


                      Geldreich, E, E 198 1. Membrane filter techniques for total coliform and fecal coliform
                      populations in water. In: Dutka, B. J. (eq) Membrane filtration: apWic-ations. techniques.
                      and prphlems. Marcel Dekker, New York, pp. 41-76.



                                                         83








                      Geldreich, E. E., and. Kenner, B. A. 1969. Concepts of faecal streptococci in stream
                      pollution. J. Wat, Pollut, Control Fe 41:R336-R352.


                      Geldreich, E. E., Kenner,, B. A., and Kabler, P. W. 1964. Occurrence of coliforms, fecal
                      coliforms, and streptococci on vegetation and insects. Ap@l. Nficrobiol. 12:63-69.


                      Geldreich, E. E. Best, L C., Kenner, B. A., and van Donsel, D. J. 1968. The
                      bacteriological aspects of stormwater pollution. J, Water Pollut. Control 40:1861-
                      1872.


                      Gerba, C. P. 1987. Phage as indicators of fecal pollution. In: Goyal,, S. M., Gerba, C.
                      P., and Bitton, G.(eds.) Phage EcoLogy. John Wiley and Sons, New York, pp. 197-209.


                      Gerba, C. P., and McLeod, J. S. 1976. Effect of sediments on the survival of Escherichi
                      coli in marine waters. AIWI. Environ. Mcrobiol. 32:114-120.


                      Gerba, C. P., Farrah, S. R., Goyal, S.M., Wallis,C. and Melnick, J. L 1978.
                      Concentration of enteroviruses from large volumes of tap water, treated sewage and
                      seawater. AWI, Environ, Microbiol. 35: 540-548.


                      Ghoul, M., Bernard, T., and Cormier, M. 1990. Evidence that Escherich coli
                      accumulates glycine betaine from marine sediments. AM-1. Environ. Microbiol. 56:551-
                      554.


                      Gilliland, M. W., and Baxter-Potter, W. 1987. A geographic information system to
                      predict non-point source pollution potential. Water Resources Bun. 23:281-29 1.


                      Girones, R., Jofre, J., and Bosch, A. 1989. Natural inactivation of enteric viruses in
                      seawater. J. Environ. QjW. 18:34-39.


                      Glendening, E. A. 1985. Bacterial water quality and shellfish harvesting. In:
                      Em=tives on Nonpgint Source Pollution, Proceedings Of a National Conference, hUy
                      19-22, 1985, Kansas CiV, pp. 447-454.


                      Goldstein, E. J. C. and Citron, D. M. 1988. Annual incidence, epidemiology, and
                      comparative in vitro susceptibility to cefoxitin, cefotetan, cefmetazole, and ceftizoxime of


                                                         84








                     recent community-acquired isolates of the Bacteroides kagilis group. I Clin. Microbiol.
                     26:2361-2366.


                     Gonzalez, JM., Sherr, E. B., and Sherr, B. F. 1990. Size-selective grazing on bacteria
                     by natural assemblages of estuarine flagellates and ciliates. AWL Environ. Microbiol. 56:
                     583-589.


                     Goodfellow, M. 1983. Ecology of actinomycetes. Ann. Rev. Microbiol. 37:189-216.


                     Goodfellow, M. and Haynes, J. A. 1984. Actinomycetes, in marine sediments. In: Ortiz-
                     Ortiz, L, Bojalil, L F., and Yakoleff, V. (eds.) Biologgical, biochemical and biomedical
                     aMeqts of actinomycetes Academic Press, New York pp. 453-472.


                     Goodfellow, M., and Williams, S. T. 1983. Ecology of actinomycetes. Ann. Rev.
                     Microbiol. 37:18-216.


                     Goodfellow, R. M., Cardoso, J., Eglinton, G., Dawson, J. P., and Best, G. A. 1977. A
                     faecal sterol survey in the Clyde Estuary. Mar. Pollut. Bull. 8:272-275.


                     Goyal, S. M., Gerba, C. P., and Melnick, J. L 1979. Human enteroviruses, in oysters
                     and their overlying waters. AWL Environ, Microbiol. 37:572-581.


                     Grabow, W. 0. K., Coubrough, P., Nupen, R M., and Bateman, B. W. 1984.
                     Evaluation of bacteriophages as indicators' of the virological quality of sewage-polluted
                     waters. Wat. SA 10:7-14.


                     Grabow, W. 0. K., and Coubrough, P. 1986. Practical direct plaque assay for
                     coliphages in 100-ml samples of drinicing water. AMI. Environ, Microbiol. 52:430-433.


                     Granai, C., RI, and Sjogren, P, R 1981.' In situ and laboratory studies of bacterial
                     survival using a microporous membrane sandwich. Avol. Environ. Microbiol. 41:190-
                     195.


                     Grimes, D. J. and Colwell, R. R. 1986. Viability and virulence of Escherichia coh
                     suspended by membrane chamber in semitropical ocean water. FEMS Mcrobiol. Let. 34:
                     161-165.



                                                         85










                     Grimes, D. J., Atwell, R. W., Brayton, P. R., Palmer, L. M., Rollins, D. M., Roszak, D.
                     B., Singleton, F. L, Tamplin, M. L, and Colwell, R. R. 1986. The fate of enteric
                     pathogenic bacteria in estuarine and ma mie environments. Microbiol. Sci, 3:324-329.


                     Gyllenberg, H., Niemela, S., and Sonnunen, T. 1960. Survival of bifid bacteria in water
                     as compared with that of coliform bacteria and enterococci. AWL Microbi . 8:20.


                     Hackney, C. R., Ray, B., and Speck, M. L 1979. Repair detection procedure for
                     enumeration of fecal coliforms and enterococci from seafoods and marine environments.
                     AML Environ. Microbiol. 37:947-953.


                     Hatcher, P. G., Berberian, G. A., Cantillo, A. Y., McGillivary, P. A., Hanson, P., and
                     West, R. H. 1981. Chemical and physical processes in a dispersing sewage sludge
                     plume. In: Ketchum, B. H., Kester, D. R., and Park, P. K. (eds.) Ocean DuMping o
                     Industrial Wastes. Plenum Press, New York, pp. 347-378.


                     Hartman, P. A., Reinbold,G. W., and Saraswat, D. S. 1966. Media and methods for
                     isolation and enumeration of the enterococci. In: Umbreit , W. W. (ed.) Advances in
                     Apph
                          Led Microbiology. Vol. 8. Academic Press, New York, pp. 253-289.


                     Havelaar, A. H. 1986. F-specific RNA bacteriophages as model viruses in water
                     treatment processes. Ph.D. Dissertation, University of Utrecht, The Netherlands.


                     Havelaar, A. H. 1987. Bacteriophages as model organisms in water treatment.
                     Microbiol. Sci. 4:362-364.


                     Havelaar, A. H. and Hogeboom, W. M. 1983. Factors affecting the enumeration of
                     coliphages in sewage and sewage-polluted waters. Antonie van Izeuwenhoek J.
                     Microbiol. 49:387-397.


                     Havelaar, A. H., and Hogeboom, W. M. 1984. A method for the enumeration of male-
                     specific bacteriophages in sewage. J. AWL Bacteriol. 56:439-447.






                                                         86







IML



                     Havelaar, A. H., and Nieuwstad, Th. J. 1985. Bacteriophages and faecal bacteria as
                     indicators of chlorination efficiency of biologically treated wastewater. J. Water Pollut.
                     Control-Fed. 57:1084-1088.


                     Havelaar, A. H. and Pbt-Hogeboom, W. M. 1988. F-specific RNA-bacteriophages as
                     model viruses in water hygiene: ecological aspects. Wat. Sci. Tech. 20:399-407.


                     Havelaar, A. H., and van Olphen, M. 1989. Water quality standards for bacteriophages?
                     In: Wheeler, D., Richardson, M. L, and Bridges, J. (ed.) Watershed-89. ne Future
                     Water QMjjV in            Pergamon Press, Oxford, pp. 357-366.


                     Havelaar, A. H., Hogeboom, W. M., and%Pot, R. 1984. F spedific RNA bacteriophages
                     in sewage: methodology and occurrence.         Sci, Tech. 17: 645-655.


                     Havelaar, A. H., Furuse, K., and Hogeboom, W. M. 1986. Bacteriophages and indicator
                     bacteria in human and animal faeces. J. AIV-1. Bacteriol. 60:255-262.


                     Hay, B. and Scott, P. 1986  Evidence for intracellular absorption of virus by the Pacific
                     oyster, Crassostrea gigas. New Zealand J. Mar, Freshwat. Res. 29:655-659.


                     Hazen, T. C. 1988. Fecal coliforms as indicators in tropical waters: a review. Toxicijy
                     Assessment: An International Journal. 3:461-477.


                     Hendry, G. S. and Toth, A. 1982. Some effects of land use on bacteriological water
                     quality in a recreational lake,. Wat. Res. 16:105-112.


                     Heijthuijsen, J. H. F. G., and Hansen, T. A. 1989. Betaine fermentation and oxidation
                     by marine Desulfuromonas strains. -A-MI,, Environ, Microbiol 55:965-969.

                     Hirsch, C. F. and Christensen, D. L 1983. Novel method for selective isolation of
                     actinomycetes. AWL Environ. Microbiol. 46:925-929.


                     Hobbie, J. E,, Daley, R. J., and Jasper, S. 1977. Use of nuclepore filters for counting
                     bacteria by fluorescence microscopy. -App-1. Environ. Microbiol. 33:1225-1228,.




                                                         87








                       Holdeman, L V., Good, I. J., and Moore, W. R C. 1976. Human fecal flora: variation
0                      in bacterial composition within individuals and a possible effect of emotional stress. AMI-.
                       Environ. Microbiol. 31:359-375.


                       Holm, S. E, and Windsor, J. G., Jr. 1986. Chemical monitoring of sewage effluents
                       using saturated hydrocarbons and coprostanol in estuarine waters. Oceans'86 Conference
                       Record: Science-Engineering-Adventure.               F-Skaiggiga Sympgsium 3:839-844.


                       Hood, M. A. 1983. Effects of harvesting waters and storage conditions on yeast
                       populations in shellfish. J. Food Prot. 46:105-108.


                       Hood, M. A., Ness, G. E, and Blake, N. J. 1983. Relationship among fecal coliforms,
                       Escherichi coli, and Sal          spp. in shellfish. AW-1. Environ. Microbiol, 45:122-126.


                       Hoskin, G. P., and Bandler, R. 1987. Identification of mammalian feces by coprostanol
                       thin layer chromatography: method development. J. Assoc, Off, Anal, Chem. 70:496-498.


                       Hunt, D. A., and Springer, J. 1974. Preliminary report on a comparison of total coliform
                       and fecal coliform. values in shellfish growing area waters and a proposal for a fecal
                       coliform area standard. In: Wilt, D. S. (ed.) Proceedings 8th National Shellfish Sanitation
                                   Food and Drug Administration, Shellfish Sanitation Branch, Washington, D.
                       C., pp. 97-104.


                       Hunt, D. A. and Springer, J. 1978. Comparison of two rapid test procedures with the
                       standard EC test for recovery of fecal coliform bacteria from shellfish-growing waters. L
                       Assoc, Off, Anal, Chem, 61:1317-1323.


                       Huntley, B. E., Jones, A. C., and Cabelh, V. J. 1976. Klebsiell densities in waters
                       receiving wood pulp effluents. J. Water Pollut. Control Fed, 48: 1766-1771.


                       IAWPRC Study Group on Health Related Microbiology. 199 1. Bacteriophages as model
                       viruses in water quality control. Wat. Res. 25:529-545.


                       Isbister, J. D., Simmons, J. A., Scott, W. M., and Kitchens, J. F. 1983. A simplified
                       method for coliphage detection in natural .. waters. Acta. M   iologica          32:197-
                       206.



                                                             88



                     Isenberg, H. D., Goldberg, D., and Sampson, J. 1970. Laboratory studies with a
                     selective enterococcus medium. Appl. Microbiol. 20:433-436.


                     Jackson, H. 1974. Loss of viability and metabolic injury of S2qWhqylococcu qaureus
                     resulting from storage at 5. J. Appl. Bacteriol, 37: 59-64.


                     James, A. 1979. The value of biological indicators in relation to other parameters of water
                     quality. In James, A., and Evison, L (eds.) Biological indicators of Water Quality. John
                     Wiley and Sons, Chichester, pp. 1-1 - 1-16.


                     Jamieson, W., Madri, P., and Claus, G. 1976. Survival of certain pathogenic
                     microorganisms in sea water. Hydrobiology 50: 117-121.


                     Jannasch, H. W. 1966. Competitive elimination of Enterobacteriaceae from seawater.
                     Appl. Microbiol. 16:1616-1618.
                   
                     Jiang, X., Estes, M. K., and Metcalf, T. G. 1987. Detection of hepatitis A virus by
                     hybridization with single-stranded RNA probes. Appl. Environ. Microbiol. 53:2487-
                     2495.


                     Jofre, J., Bosch, A., Lucena, F., Girones, R., and Tartera, C. 1986. Evaluation of
                     Bacteroides fragilis bacteriophages as indicators of the virological quality of water. Water
                     Sci. Technol. 18:167-177.
                               
                     Jones, G. E., and Cobet, A. B. 1975. Heavy metal ions as the principal bactericidal agent
                     in Caribbean sea water. In Gameson, A. L H. (ed.) Discharge of sewage, from
                     outfall . Pergamon Press, Oxford, England, pp. 199-207.


                     Kanazawa, A. and Teshima, S.   1971. Sterols of the suspended matters in sea water. J.
                     Oceanogr. Soc. Japan 27:207-212.


                     Kanazawa, A. and Teshima, S. 1978. The occurrence of coprostanol, an indicator of
                     faecal pollution, in sea water and sediments. Oceanol. A 1:39q-44.




                                                        89








                      Kapelmacher, E, H., lzussink, A. B., and van Noorle Jansen, L M. 1976. Comparative
                      studies of methods for the enumeration of coh aerogenes bacteria and E coli in suface
                      water. Wat. Res. 10:285-288.


                      Kapuscinski, R. B., and Mitchell, R. 1991. Solar radiation induces sublethal injury in
                      Esc
                                     I_in seawater. A 1. Environ. Microbiol. 41:670-674.


                      Kapuscinski, R. B., and Mitchell, P.- 1983. Sunlight-induced mortality of viruses and
                                  goli in coastal seawater. Environ, Sci. Technol. 17:1-6.


                      Kasweck, K. L, and Fliermans, C. B. 1978. Lactose variability of Escherichi coli in
                      thermally stressed reactor efflu6nt watersA
                                                                  "L Environ. Microbiol. 36-.739-746.


                      Kator, H. and Rhodes, M. W. 1988. EvtAfion of alternate microbial indicators of fecal
                      MIlution in a non:pQin1 source im9aqkd shellfish gmndng area, A Final Boo Submitted
                      to the Council on the Environme_nL Richmond. Virginia. Virginia Institute of Marine
                      Science, Special Report in Applied Marine Science and Ocean Engineering No. 297.


                      Kator, H. and Rhodes, M. W. 1989. Occurrence of indicators of fecal pollution in water
                      and sediment of a subestuary impacted by non-point source pollution. Submitted to
                      Virginia Department of Conservation and Historic Resources, Division of Soil
                      Conservation, Richmond, Virginia. Virginia Institute of Marine Science, Special Report in
                      Applied Marine Science and Ocean Engineering No. 303.


                      Kator, H. and Rhodes, M. R. 199 1. Indicators and alternate indicators of growing water
                      quality. In: Ward, D. R. and Hackney, C. R. (eds.) Microbiology of Marine
                      Products. Van Nostrand Reinhold, New York, pp. 135-196.


                      Kehr, R. W., Levine, B. S., Butterfield, C. T., and Miller, A. P. 194 1. A report on the
                      public health aspects of clamming in Raritan Bay. Public Health Service Report. Reissued
                      in June 1954 by Division of Sanitary Engineering Services, Public Health Services,
                      Department of Health, Education, and Welfare.


                      Kelland, L. R., Moss,S. H., and Davies, D. J. G. 1983. Damage to bacterial cell
                      membranes by UV radiation in sunlight. BioS         33: 334-335.



                                                           90






 0



                      Keller, R. and Traub, N. 1974. The characterization of Bacteroides ft=h bacteriophage
 0                    recovered from animal sera; observations on the nature of Bac      phage carrier
                      cultures. J. Gen. Virol, 24:179-189.


                      Kenard, R. P. and Valentine, R. S. 1974. Rapid determination of the presence of enteric
                      bacteria in water. AML Microbiol. 27: 484-487.


                      Kennedy, J.E. Jr., Bitton, G., and Oblinger, J. L. 1985. Comparison of selective media
                      for assay of coliphages in sewage effluent and lake water. -A 1. viron. Microbiol. 49:
                                                                                     En
                      33-36.


                      Kenner, B. A. 1978. Fecal streptococcal indicators. In: Berg, G. (ed.) Indicators of
                      viruses in water and food Ann Arbor Science Publishers, Ann Arbor, Mich., pp. 147-
                      169.


                      Kenner, B. A., Clark, H. F., and Kabler, P. . 1960. Fecal streptococci. H.
                      Quantification of streptococci in feces. Amer. J. Public Health. 50.1553-1559.


                      Keswick, B. H., Satterwhite, T. K., Johnson, P. C., DuPont, H. L, Secor, S. L.,
                      Bitsura, J. A., Gary, G. W., and Hoff, J. C. 1985. Inactivation of Norwalk virus in
                      drinking water by chlorine. AML Environ. Microbiol. 50:261-264.


                      Ketchum, B. H., Ayers, J. C., and Vaccaro, R. F. 1952. Processes contributing to the
                      decrease of colfform bacteria in a tidal estuary. Ecolog . 33:247-258.


                      Kilgen, M. B. and Cole, M. T. 1983. Recovery of pohovirus type I from artificial
                      seawater and natural estuarine water at varying salinities using electropositive filtem Abst.
                      Ann, MeeL Amer. Soc. Microbiol. 283.


                      Kilian, M. and Bfilow, P. 1976. Rapid diagnosis of Enterobacteriaceae 1. Detection of
                      bacterial glycosidases. Acta. Path. Microbiol, Scand. Sect. B. 84: 245-25 1.


                      King, G. M. 1984. Metabolism of trimethylarnine, choline, and glycine betaine by
                      sulfate-reducing and methanogenic bacteria in marine sediments. AML Environ.
                      Microbiol. 48:719-725.




                                                          91


                      King, G. M. 1988. Distribution and metabolism of quaternary amines in marine
                      sediments. In: Blackburn, T. H., and Sorenson, J. (eds.) Nitrogen cycling in the marine
                      environment. John Wiley & Sons, New York, pp. 143-73.

                      Kjellander, J. 1960. Enteric streptococqci as indicators of fecal contamination of water.
                      Acta Pathol. Et Microbiol. Scand. Suppl. 136.48:1-124.


                      Klein, D. A., and Wu, S. 1974. Stress: a factor considered in heterotrophic
                      microorganism enumeration from aquatic environments. Appl. Microbiol. 27:429-431.


                      Knight, I.T., Shults, S., Kaspar, C. W., and Colwell, R. R. 1990. Direct detection of
                      Salmonella spp. in estuaries by using a DNA probe. Appl. Environ. Microbiol.56:1059-
                      1066.

                      Knittel, M. D., Seidler, R. J., Eby, C., and Cabe, L. M. 1977. Colonization of the
                      botanical environment by Klebsiella isolates of pathogenic origin. Appl. Environ.
                      Microbiol 34:557-563.

                      Koburger, J. A. and Miller, M.L. 1985. Evaluation of a fluorogenic MPN procedure for
                      determining Escherichia coli in oysters. J.. Food. Prot. 48:244-245.

                      Koepfler, E. T., and Kator, H. I. 1986. Ecotoxicological effects of creosote
                      contamination on benthic microbial populations in an estuarine environment. Toxicity
                      Assessment: An International Quarterly. 1:465-485.


                      Kogure, K., Simidu, U., and Taga, N. 1979. A tentative direct microscopic method for
                      counting living bacteria. Can. J. Microbiol. 25:415-420.


                      Kory, M. M. and Booth, S. J. 1986. Characteristics of Bacteroides fragilis
                      bacteriophages, and comparison of their DNAs. Curr. Microbiol. 14:199q-203.


                      Kott, Y. 198 1. Viruses and bacteriophages. Science Total Environ. 18:13-23.


                      Kott, Y., Roze, N., Speber, S., and Betzer, N. 1974. Bacteriophages as viral pollution
                      indicators. Wat.Res. 8:165-171.

								
                                                           92






                      Krahn, M. M., Wigren, C. A., Moore, L K., and Brown, D. W.. 1989. High-
                      performance liquid chromatographic method for isolating coprostanol from sediment
                      extracts. J. Chrom. 481:263-273.


                      Kuritza, A. P., and Salyers, A. A. 1985. Use of a species-specific DNA hybridization
                      probe for enumerating Bacteroides vulgatu in human feces. A 1. Environ, Microbiol.
                      50:95.8-964.


                      LaBelle, R. L, and Gerba, C. P. 1979. Influence of pH, salinity, and organic matter on
                      the adsorption of enteric: viruses to estuarine sediment. _AWl. Environ. Microbio 38:93-
                      101.


                      LaBelle, R. Y., Gerba, C. P., Goyal, S. M., Melnick, J. L, Cech, I., and Bogdan, G. F.
                      1980. Relationships between environmental factors, bacterial indicators, and the
                      occurrence of enteric viruses in estuarine sediments.A
                                                                          Mi. Environ. Microbiol 39:588-
                      596.


                      Landry, E F., Vaughn, J. M., Vicale, T. J., and Mann, R. 1983. Accumulation of
                      sediment-associated viruses in shellfish. &Wl. Environ. Microbiol 45:238-247.


                      Le Rudulier, D., and Bouillard, L 1983. Glycine betaine, an osmotic effector in
                      Klebsi    pneurrion   and other members of the Enterobacteri      A_Wl. Environ.
                      Microbiol. 46:152-159.


                      Lenhard, G. 1967. Determination of protease activity in bottom deposits of sewage
                      stabilization ponds. Hydrobiolog 27:67-79.


                      Lenhard, G. 1969. Determination of urease activity in biological purification systems.
                      H_ ydr iglogia, 33:193-200.


                      Leonard, D. L, Broutman, M. A., and Harkness, K. E 1989. The quality of shellfish
                      growing waters on the east coast of the United States. National Oceanic and Atmospheric
                      Administration. Ocean Assessments Division. Rockville, Maryland, 45 pp.






                                                         93








                      Lessard, R J., and Sieburth, J. McN. 1983. Survival of natural sewage populations of
                      enteric bacteria in diffusion and batch chambers in the marine environment AW-1. Environ.
 0                    Mcrobiol. 45:950-959.


                      I"in, M. A. 1977. Bifidobacteria as. water quality indicators. In: Hoadley, A. W., and
                      Dutka, B. J. (eds.) Bacterial indicators/health hazards associated with w    Special
                      Technical Publication 635, American Society for Testing and Materials, Philadelphia, pp.
                      131-138


                      Levin, M. A., and Resnick. I. G. 1981. Bifidobacteriurn In: Dutka, B. J. (ed.)
                      Membrane filtration: ap&hcations, t
                                                         echnig-ues, and pMb_lem . Marcel Dekker, New York,
                      pp. 129-159..


                      Levin, M.A., Fischer, J. R., and Cabelli, V. J. 1975. Membrane filter technique for
 0                    enumeration of enterococci in marine waters. A-Rpl, Microbiol. 30-66-71.

                      Ley, A. N., Bowers, R. J., and Wolfe, W. 1988. Indoxyl-O-D-glucuronide, a novel
                      chromogenic reagent for the specific detection and enumeration of Escherichia coli in
                      environmental samples. Can. J. Microbiol. 34:690-693.


                      Li, W. K. W., and Dickie, P. M. 1985. Growth of bacteria in seawater filtered through
                      0.2 urn Nucleopore membranes: implications for dilution experiments. Mar, Ecol. BLo&
                      Se
                        ,r. 26:245-252.


                      Littel, K. J., and Hartman, P.A. 1983. Fluorogenic selective and differential medium for
                      isolation of fecal streptococci. A
                                                       4W-I. Environ. Microb . 45:622-627.


                      Loesch, W. J. 1969. Oxygen sensitivity of various anaerobic bacteria. AWL Microbiol.
                      18:723-727.


                      Logan, K. B., Rees, G. E., Seeley, N. D., and Primrose, S.D. 1980. Rapid
                      concentration of bacteriophages from large volumes of freshwater: evaluation of positively
                      charged, microporous filters. J. Virol, Methods. 1:87-97.






                                                           94


Lopez-Torres, A.J., Hazen, T.C., and Toranzos, G.A. 1987. Distribution and in situ
survival and activity of Klebsiella pneumoniae and Escherichia coli in a tropical rain forest
watershed. Curr. Microbiol. 15:213-218.

Ludwig, W., Seewaldt, E., Kilpper-Balz, A., Schleifer, K.H., Magrum, L., Woese, C.
R., Fox, G.E., and Stackebrandt, E. 1985. The phylogenetic position of Streptococcus
and Enterococcus. J. Gen. Microbiol. 131:543-551.

Lum, r. and Chang, G. 1990. Glucuronidase-negative Escherichia coli in the ECOR
reference collection. J. Food Protect. 53:972-974.

Mackey, B.M., and Derrick, C.M. 1986. Peroxide sensitivity of cold-shocked
Salmonella typhimurium and Escherichia coli and its relationship to minimal medium
recovery. J.Appl. Bacteriol. 60:501-511.

Macy, J.M. 1981. Nonpathegenic members of the genus Bacteroides. In: Starr, M.P.,
Stolp, H., Truper, H. G., Balows, A., and Schlegel H. G. (eds.) The Prokaryotes, Vol.
II. Springer-Verlag, Berlin, pp. 1450-1463.

Mara, D.D., and Oragui, J.I. 1981. Occurrence of Rhodococcus coprophilus and
associated actinomycetes in feces, sewage and freshwater. Appl. Environ. Mircorbiol.
42:1037-1042.

Mara, D.D., and Oragui, J. 1983. Sorbitol-fermenting bifidobacteria as specific
indicators of human faecal pollution. J.Appl.Bacteriol. 55:349-357.

Marshall, K.C. 1985. Mechanisms of bacterial adhesion at solid-water interfaces. In:
Savage, D.C., and Fletcher, M. (eds.) Bacterial adhesion. Mechanisms and physiological
significance. Plenum Press, New York, pp. 133-161.

Martinez, J., Garcia-Lara, J., and Vives-Rego, J. 1989. Estimation of Escherichia coli
mortality in seawater by the decrease in 3H-label and electron transport system activity.
Microb.Ecol. 17:219-225.

Matches, J.R. and Liston, J. 1974. Mesophilic clostridia in Puget Sound. Can.J.
Microbiol. 20:1-7.

						95


Matson, E.A., Hornor, S.G., and Buck, J.D. 1978.  Pollution indicators and other
microorganisms in river sediment.  J. Water Pollut. Cont. Fed. 50:13-19.

Matthew, W.S. and Smith, L.L. 1968.  Sterol metabolism-III.  Sterols of marine waters.
Lipids 3:239-246.

McCambridge, J., and McMeekin, T.A. 1979. Protozoan predation of Escherichia coli in
estuarine waters.  Water Res.  13:659-663.

McCambridge, J., and McMeekin, T.A. 1980.  Relative effects of bacterial and protozoan
predators on survival of Escherichia coli in estuarine water samples.  Appl. Environ.
Microbiol.  40:907-911.

McCambridge, J., and McMeekin, T.A. 1981.  Effect of solar radiation and predacious
microorganisms on survival of fecal coliforms and other bacteria.  Appl. Environ.
Microbiol. 41:1083-1087.

McFeters, G.A., Bissonnette, G.K., Jezeski, J.J., Thomson, C.A., and Stuart, D.G.
1974. Comparative survival of indicator bacteria and enteric pathogens in well water.
Appl. Microbiol. 27:823-829.

Means, E.G. and Olson, B.H. 1981.  Coliform inhibition by bacteriocin-like substances
in drinking water distribution systems.  Appl. Environ. Microbiol. 42:506-512.

Meiman, J.R., and Kunkle, S.H. 1967.  Land treatment and water quality control. J.
Soil Wat. Cons. 22:67-70.

Metcalf, T.G., and Jiang, X. 1988.  Detection of hepatitis A virus in estuarine samples by
gene probe assay.  Microbiol. Sciences. 5:296-300.

Metcalf, T.G., Slanetz, L.W., and Bartley, C.H. 1973. Enteric pathogens in estuary
waters and shellfish.  In: Chichester, C.O. and Graham, H.D. (eds.) Microbial safety of
fishery products.  Academic Press, New York, p. 215-234.

									96





 0


                      Metcalf, T. G., Moulton, R, and Eckerson, D. 1980. Improved method and test strategy
                      for recovery of entericviruses from shellfish. A
                                                                    Wl. Environ. Mimbiol. 39:141-152.


                      Miescier, J. J., and CAbelli., V. J. 1982. Enterococci and other microbial indicators in
                      municipal wastewater effluents. J. Water Pollut. Control Fed. 54:1599-1606.


                      Miescier, J. J., Peeler, J. T., Clem, J. D., Read, K B., Jr., and Furfan, S. A. 1985.
                      Final report on a comparison of two methods for recovery Of Eschenchia coli Type I and
                      .fecal coliforms, from oysters, Food and Drug Administration. Division of Cooperative
                      Programs, Washington, D. C., 18 PP.


                      Milne, D. P., Curran, J. C., and Wilson, L 1986. Effects of sedimentation on removal
                      of faecal coliform. bacteria from effluents in estuarine water. Water Res 20:1493-1496.


                      Mitchell, K 1972. Ecological control of microbiological imbalances. In: Mitchell, R.
                      (ed.) Water WIlution microbigkU. New York: Wiley Interscience, pp. 273-288.


                      Mitchell, R. and Chamberlin, C. 1975. Factors influencing the survival of enteric
                      microorganisms in the sea: an overview'. In: Gameson, A. L H. (ed.) Discharge of
                      sewage- from sea oujfWL& Pergamon Press, Oxford, pp. 237-251.


                      Mitchell, R. and Jannasch, H. W. 1969. Processes controlling virus inactivation in
                      seawater. Environ. Sci. Tech, 3:941-943.


                      Mitchell, R. and Yankofsky, S. 1969. Implication of a marine ameba in the decline of
                      Eschg1ich coli in seawater. Environ. Sci. & Tech. 3:574-576.


                      Mitchell, K, Yankosky, S., and Jannasch, H. W. 1967. Lysis of Escherichia coli by
                      marine micro-organisms.          215:891-892.


                      Moberg, L J. 1985. F1  uorogenic assay for rapid detection of Escherichi coli in food.
                      AML Environ. Microbiol. 50:1383-1387.


                      Moebus, K. 1972. Bactericidal properties of natural and synthetic sea water as influenced
                      by addition of low amounts of organic matter,. Mar, Biol. 15:81-88.



                                                          97


			
Molitoris, E., McKinley, G., Krichevsky, M.I., and Fagerberg, D.J. 1985.
Comparison of conventional and miniaturized biochemical techniques for identification of
animal streptococcal isolates.  Microb, Ecol. 11:81-90.

Moore, W.E.C., and Holdeman, L.V. 1974. Human fecal flora: the normal flora of 20
Japanese-Hawaiians. Appl. Microbiol. 27:961-979.

Morotomi, M., Ohno, T., and Mutai, M. 1988.  Rapid and correct identification of
intestinal Bacteroides spp.  with chromosomal DNA probes by whole-cell dot blot
hybridization.  Appl. Environ. Microbiol. 54:1158-1162.

Mundt, J.O. 1963.  Occurrence of enterococci on plants in a wild environment. Appl.
Microbiol. 11:141-144.

Mundt, J.O. 1982.  The ecology of the streptococci.  Microb. Ecol. 8:355-369.

Mundt, J.O., Coogin, J.H.,Jr., and Johnson, L.F. 1962.  Growth of Streptococcus
faecalis var. liquefaciens on plants.  Appl. Microbiol. 10:552-555.

Munoa, F.J., and Pares, R. 1988.  Selective medium for isolation and enumeration of
Bifidobacterium spp. Appl. Environ. 54:1715-1718.

Munro, P.M., Gauthier, M.J., and Laumond, F.M. 1987.  Changes in Escherichia coli
cells starved in seawater or grown in seawater-wastewater mixtures.  Appl. Environ.
Microbiol. 53:1476-1481.

Munro, P.M., Gauthier, M.J., Breittmayer, V.A., and Bongiovanni, J. 1989. Influence
of osmoregulation processes on starvation survival of Escherichia coli in seawater.  Appl.
Environ. Microbiol. 55:2017-2024.

Murtaugh, J.J. and Bunch, R.L. 1967. Sterols as a measure of fecal pollution, J. Water 
Pollut. Cont. Fed. 39:404-409.

										98







                       Nikaido, H. and Vaara, M. 1987. Outer membrane. In: F. Neidhardt, C.(ed.)
                       Escherichia coli and Salmonella W-himurium Cellular and Molecular biology, Vol. 1
                       American Society for Microbiology, Washington, D. C., pp. 7-22.


                       Nishimura, M. 1982. 50-isomers of stanols and stanones as potential markers of
                       sedimentary organic quality and depositional paleoenvironments. Geochim. Cpsmochim.
                       Acta 46:423-432.


                       Nishimura, M. and Koyama, T. 1977. The occurrence of stanols in various living
                       organisms and the behavior of sterols in contemporary sediments. Geochim, Cosmochim,
                       Acta 41:379-385.


                       O'Keefe, B. and Green, J. 1989. Coliphages, as indicators of fecal pollution at three
                       recreational beaches on the Firth of Forth. Wat. Res. 23:1027-1030.


                       Olivieri, V. P. 1980. Microorganisms from nonpoint sources in the urban environment.
                       In; Overcash, M. R. and Davidson, J. M. (eds.) Environmental imp= of flgppgLinA
                       Vg1lution. Ann Arbor Science, Ann Arbor, pp. 125-158.


                       Olson, B. H. 1978. Enhanced accuracy of coliform testing in seawater by a modification
                       of the most-probable-number method. A
                                                               MI. Environ. Microbiol. 36:438444.


                       OMalley, M. L., Lear, D. W., Adams, W. N., Gaines, J., Sawyer, T. K., and Lewis, E
                       J. 1982. Microbial contamination of continental shelf sediments by wastewater. J. Water
                       Pollut, Control. 54:1311-1317.


                       Onderdonk, A. B., Johnston, J., Mayhew, J. W., and Gorbach, S. L. 1976. Effect of
                       dissolved oxygen and Eh on Bacteroides fragilis during continuous culture. App_l.
                       Environ. Microbiol. 31:168-172.


                       Oppenheimer, C. H. and Kelly, A. 1- 1952. Escherichia coli in the intestine of a wild sea
                       lion. Sci     115: 527-528.


                       Oragui, J. I., and Mara, D. D. 198 1. A pelective medium for the enumeration of
                       St=jQggc
                                 &us bovis by membrane filtration. J. A@Ml. Bacteriol. 51:85-93.



                                                            99


Oragui, J.I., and Mara, D.D. 1983.  Investigation of the survival characteristics of
Rhodococcus coprophilus and certain fecal indicator bacteria.  App. Environ. Microbiol.
46:356-360.

Oragui, J.I., and Mara, D.Dl 1984. A note on a modified membrane-Bovis agar for the
enumeration of Streptococcus bovis by membrane filtration.  J. Appl. Bacteriol. 56:179-
181.

Orlob, G.T. 1956.  Viability of sewage bacteria in seawater.  Sewage Ind. Wastes.
28:1147-1167.

Osawa, R. and Mitsuaoka, T. 1990.  Selective medium for enumeration of tannin-protein
complex-degrading Streptococcus spp. in feces of koalas.  Appl. Environ. Microbiol.
56:3609-3611.

Osawa, S., Furuse, K., and watanabe, I. 1981a.  Distribution of ribonucleic acid
coliphages in animals.  Appl. Environ. Micriobiol.  41:164-168.

Osawa, S., Furuse, K., Choi, M.S., Ando, A., Sakuari, T., and Watanabe, I.  1981b.
Distribution of ribonucleic acid coliphages in Korea.  Appl. Environ. Microbiol. 41:909-
911.

Pagel, J.E., and Hardy, G.M. 1980.  Comparison of selective media for the enumeration
and identification of fecal streptococci from natural sources.  Can. J. Microbiol. 26:1320-
1327.

Pagel, J.E., Qureshi, A.A., Michael Young, D., and Vlassoff,L. 1982.  Comparison of
four membrane filter methods for fecal coliform enumeration.  Appl. Environ. Microbiol.
43:787-793.

Palmer, L.M., Baya, A.M, Grimes, D.J., and Colwell, R.R. 1984.  Molecular genetic
and phenotypic alteration of Escherichia coli in natural water microcosms containing toxic
chemicals.  FEMS Microbiol. Lett. 21:169-173.

Perez-Rosas, N. and Hazen, T.C. 1988. In situ survival of Vibrio cholerae and
Escherichia coli in tropical coral reefs.  Appl. Environ. Microbiol. 54:1-9.

									100







                     Pettibone, G. W.., and Cooney, J. J. 1986*Effect of organotins on fecal pollution
                     indicator organisms. A 1. Environ. Microbiol. 52:562-566.


                     Pierce, R. H. and Brown, R. C. 1984. Coprostanol distribution from sewage discharge
                     into Samsota Bay, Florida. Bull. Environ, Contam. Toxicol, 32:75-79.


                     Pisano, M. A., Sommer, M. J., and Lopez, M. M. 1986. Application of pretreatments for
                     the isolation of bioactive actinomycetes from marine sediments. AMI, Microbiol.
                     Biotechnol. 25:285-288.
                     Pocldington, FL, Leonard, J. D., and Crew.e, N.1 F. 1987. Le coprostanol comme
                     indicateur de la contamination fecale dans, reau de mer et es sediments marins. Oceano.
                     Acta 10. 83-89.


                     Poppell, C. F. 1979. Enumeration and occurrence of RNA coliphages, in wastewater.
                     Masters thesis, Johns Hopldns University, Baltimore, Maryland.


                     Post, F. J., Allen, A. D., and Reid, T. C. 1967. Simple medium for the selective
                     isolation of           and related organisms, and their occurrence in sewage. Avnl.
                     Microbiol. 15:213-218.


                     Postgate, J. R. 1967. Viability measurements and the survival of microbes under
                     minimum stress. In: Rose, A. H. and Willdnson, J. R. (eds.) Advances in microbial
                     physiology. Academic Press, London, pp. 1-23.


                     Power, U. F., and Collins, J. K. 1990. Tissue distribution of a coliphage and Escheric
                     coli in mussels after contamination and depuration. App-1. Environ. Mcrobiol, 56. 803-
                     807.


                     Powelson, D. K., Simpson, J. R., and Gerba, C. P. 1990. Virus transport and survival
                     in saturated and unsaturated flow through soil columns. J. Environ, Qual. 19:396-401.


                     Prauser, H. 1984. Phage host ranges in the classification and identification of gram-
                     positive branched and related bacteria. In: Ortiz-Ortiz, L., Bojalil, L. F., and Yakoleff, V.



                                                        101


			(eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes.  Academic
			Press, New York, pp. 617-633.

			Presswood, W.G., and Strong, D.K. 1978.  Modification of M-FC medium by
			eliminating rosolic acid.  Appl.  Environ. Microbiol. 36:90-94.

			Pugsley, A.P. and Evison, L.M. 1975.  A fluorescent antibody technique for the
			enumeration of feacal streptocci in water.  J. Appl. Bacteriol. 38:63-65.

			Purdy, R.N., Dancer, B.N., Day, M.J., and Stickler, D.J. 1984. A novel technique
			for the enumeration of bacteriophage from water.  Microbiol. Lett. 21:89-92.

			Purdy, R.N., Dancer, B.N., Day, M.J., and Stickler, D.J, 1985. A note on a
			membrane filtration method for the concentration and enumeration of bacteriophages from
			water. J. Appl. Bacteriol. 58:231-233.

			Rao, V.C., Seidel, K.M., Goyal, S.M., Metcalf, T.G., and Melnick, J.L. 1984.
			Isolation of enteroviruses from water, suspended solids, and sediments from Galveston
			Bay: survival of poliovirus and rotavirus adsorbed to sediments.  Appl. Environ.
			Microbiol. 48:404-409.

			Ray, B. 1989. Injured index and pathogenic bacteria: Occurrence and detection in foods,
			water and feeds.  CRC Press, Inc., Boca Raton.

			Reneau, R.B., Hagedorn, C., and Degen, M.J. 1989.  Fate and transport of bioligical
			and inorganic contaminants from on-site disposal of domestic wastewater.  J.Environ.
			Qual.  18:135-144.

			Rhodes, M.W., and Kator, H. 1988.  Survival of Escherichia coli and Salmonella spp. in
			estuarine environments.  Appl. Environ. Microbiol. 54:2902-2907.

			Rhodes, M.W., and Kator, H. 1990.  Effects of sunlight and autochthonous microbiota
			on Escherichia coli survival in an estuarine environment.  Curr. Microbiol. 21:65-73.




								102
	

0






                       Rhodes, M. W., Anderson, I. C., and Kator, H. 1. 1983. In situ development of
                       sublethal stress4m'            coli effects on enumeration. -A-4MI. En0viron. Microbiol,
                       45:1870-1876.


                       Richards,. P. 1985. Outbreaks of shellfish-associated entezic virus illness in the United'
                       States: reuisite for development of viral guidelines. J. Food          48:815-823.


                       Roberts, M. C., Moncla4 B., and Kenny, G. E. 1987. Chromosomal DNA probes for the
                       identification of Bacteroi    species. J. Gen. Microbiol. 133:1423-1430.


                       Rodrigues, U. M. and Kroll, R. G. 1988. Rapid selective enumeration of bacteria in
                       foods using a micr2ocolony epifluorescence microscopy techniue. 1. AWL Ba8Ltg6d0d
                       64:65-78.


                       Roper, M. M., and Marshall,, K. C. 1974. Modification of the interaction between
                                   8m0h6Land bacteriophage in saline sediment hfic-rgh, Ecol. 1: 1- 13.


                       Roper, M. M., and Marshall, K. C. 1977. Lysis of Escherichia coli by a marine
                       myxobacter.           -2Ecol, 3:167-171.


                       Roper, M. M. and Marshall, K. C. 1978. Effects of a clay mineral on microbial predation
                       and para sitism of Escherichi p6& Microbiol. Ecol, 4:279-289.


                       Roper, M. M., and Marshall, K. C. 1978. Biological control agents of sewage bacteria in
                       marine habitats. Aust. J. Mar. Freshwater       .29:335-343.


0
                       Roper, M. M., and Marshall, K. C. 1979. Effects of salinity on sedimentation and of
                       particulates on survival of bacteria in estuarine habitats. Geomicrobi6ol. J. 1:103-116.

0                      Roszak, D. B. and Colwell, R. R. 1987. Survival strategies of bacteria in the natural
                       environment. Micr36obi12ol. Rev. 51:365-379.


                       Roth, W. G., Leckie, M. P., and Dietzler, D. N. 1988. Restoration of colony-T2forming
0                      activity in osmotically stressed Esch       32mi by betaine. Appl. Environ. Mi4c.robiol.
                       54:3142-3146.



0                                                           103
 

0






                       Rowbotham, T. J. and Cross, T. 1977a. Rhodococcus coprophilus sp. nov.: an aerobic
                       nocardiofonn actinomycete belonging to the "rhodochrous" complex. J. Gen. Microbiol.
                       100:123-138.


                       Rowbotham, T. J. and Cross, T. 1977b. Ecology of Rhodococcus coprophilus and
                       associated actinomycetes in fresh water and agricultural habitats. J. Gen, Microbiol,
 0
                       100:231-240.


                       Rubentschik, 2L, Roisin, M. B., and Bie1jansky, F. M. 1936. Adsorption of bacteria in
                       salt lakes. 1. Bacteriol. 32:11-3 1.


                       Sadler, R. 1889. Urease as a possible tracer for sewage effluent plumes. Wat. 0Sci. Tech,
                       21: 93-97.


                       Sadzikowski, M. R-, Sperry, J. F., and Wilkins, T. D. 1977. Cholesterol-reducing
                       bacterium from human feces. A 1. Envir2on. Microbiol, 34:355-362.


                       Santo Domingo, J. W., Fuentes, F. A., and Hazen, T. C. 1989. Survival and activity of
0
                       S4g4V_t8g4w2m faecali and                  coli in petroleum-contaminated tropical marine
                       waters. Environ, Pollut. 56:263-281.


                       Sartory, D. P. 1986. Membrane filtration enumeration of faecal clostridia and Ctostridium
0                      4pgr6f6f6ingg4ns in water. Water Res, 20:125  5-1260.

                       Savage, D. C. 1977. Microbial ecology of the gastrointestinal tract. In: Starr, M. P. (ed.)
0                      Ann.* Rev. Micro4biol. Annual Reviews Inc., Palo Alto, pp. 107-133.

                       Sayler, G. S. and Layton, A. C. 1990. Environmental application of nucleic acid
                       hybridization. In: Ornston, L. N. (ed.) Ann. Rev, Microbiol. Annual Reviews, Palo Alto,
0                      pp. 625-648.

                       Salyers, A. A. 1984. Bacteroides, of the human lower intestinal tract. Ann. Rev.
                       Microbiol, 38:293-313.


                       Sawyer, T. K. 1980. Marine amoebae from clean and stressed bottom sediments of the
                       Atlantic Ocean and Gulf of Mexico. J. Protozool. 27:13-32.



                                                           104
 

0









                        Saw
                           yer, T. K., Lewis, E J., Galass, M., Lear, D. W., O"Malley, M. L., Adams, W. N.,
                        Gaines, J. 1982. Pathogenic am oeba in ocean sediments near wastewater sludge disposal
                        sites. J. Water 8P0b1hy8t Control E0, 54:1318-1323.


                        Sawyer, T.  K.,Visesvara, G. S., and Harke, B. A. 1977. Pathogenic amebas from
                        braclash and ocean sediments, with a description of             a ha
                                                                           Acanthamoeb      0f0t2hetti -n. sp.
                        S      .196:1324-1325.


                        Sawyer, T. K., Nerad, T. A., Daggett, P.M., and Bodammer, S. M. 1987. Potentially
                        pathogenic protozoa in sediments from oceanic sewage-disposal sites. In: Capuzzo, J. M.,
                        and Kester, D. P, (eds.) Oceanic Processes in Marine Pollution, Vol, 1, Biolo
                        Processes and Wastes in the Ocean.. Robert. R Krieger Publishing Co., Malaar, Florida.,
                        pp. 183-194.


                        Scardovi, V. 1981. Chap   er 149. The genus Bifidobacterium. In: Staff, M. P., Stolp, H.,
                        Truper, H. G., Balows, A., and Schlegel, H. G. (eds.) The 2RL2-ktygks. Vol. 4U.
                        Springer-Verlag, New York, pp. 1951-1961.


                        Scarpi8no, P. V. 1975. Human enteric viruses and bacteriophages as indicators of sewage
                        pollution. In: Gameson, A. 8L H. (ed.) Dischme of sewage from sea outfall Pergamon
                        Press, Oxford, pp. 49-61.
 0

                        Schleifer, K. H., and Kilpper-Balz, R. 1984. Transfer of S80832844 facalis and
                        S4Mtococcu faec        to the genus Enterococcus nom. rev. as Enterococcus faecali comb.
                        n8ov. and Enter4ococcus faecium com. nov. Int. 1. Syst. Bacteriol. 34:31-34.


                        Seeley, N. D. and Primrose, S. B. 1980. The effect of temperature on the ecology of
                        auatic bacteriophages. J. Gen.      .46:87-95.


                        Seeley, N. D. and Primrose, S. B. 1982. The isolation of bacteriophages from the
                        environment. J. A44MI. Bact2eriol. 53:1-17.

0                       Servais, P., Billen, G., and Vives-Reg484, J. 1985. Rate of bacterial mortality in auatic
                        environments. 72A48W-81. Environ. Microbiol. 49:1448-1454.



0                                                          105
 

0






                        Sharpe, A. N. 1981. Hydrophobic grid-membrane filters: the (almost) perfect system. In:
                        Dutka, B. J. (ed.) Membrane filtration; avuhstations. techniues. and problems, Marcel
                        Dekker, New York, pp. 513-535.


                        Sharpe, A. N., Peterkin, 8P. I., and Malik, N. 1979. Improved detection of coliforms and
                                    coh in foods by a membrane filter method. -Appl. Environ. Mficm- -biol. 38:431-
 0                      435.


                        Shaw, B. G., Harding, C. D., Hudson, W. H., and Farr, 6L 1987. Rapid estimation of
                        microbial numbers on meat and poultry by the direct Pifluorescent filter techniue L
                        Food Protect, 50:652-657.


                        Shear, C. L and Gottlieb, M. S. 1980. Shell2f2ishborne disease control in the United
                        States: a commentary. Medical H38Mg2f2tse 6:315-327.


                        Shiaris, M. P., Rex,, A. C., Pettibone, G. W., Keay, K., McManus, P., Rex, M.A.,
                        Ebersolej., and Gallagher, E. 1987. Distribution of indicator bacteria and Vibrio
                        8p6gaha2@e4m8dl 2@cus in sewage-polluted intertidal sediments. A8ML Environment, Microbiol
                        53:156-1761.


                        Shig        G. and Price, J. 4F. 1988. Correlation of coprostanol to organic contaminants
                        in coastal and estuarine sediments of the US. Wat, Res. Bull. 24:989-998.


                        Sieburth, J. M., and Pratt, D. M. 1962. Anticoliform. activity of sea water associated with
                        the termination of Skeletonema           m blooms. Trans, N. Y. Acad. Sci. 24:498-501.


                        Simard, R. 8R 1971 Yeasts as an indicator of pollution. Mar. Poll. Bull. 2: 123-125.


                        Singh, A., Pyle, B. H., and Mc6Feters, G. A. 1989 Rapid enumeration of viable bacteria
                        by image analysis. J. Microbiol, method 10: 91-101.


                        Singh, S. N. and Ge0rba, C. P. 1983. Concentration of coliphage from water and sewage
                        with charge-modified filter aid. 72A44W-01, Envir08on. Micr16o48b6jo-0'l 425:232-237.


                        Sinton, L. W. and Ching, S. B. 1987. An evaluation of two bacteriophages as sewage
                        tracers. Water. Air, and Soil Pollut. 35: 347-356.


0                                                            106
 








                       Sjogren, R. E., and Gibson, M. J. 1981. Bacterial survival in a dilute environment
                       Appl. Environ. Microbiol, 41:1331-1336.


                       Skiba, U. and Wainwright, M. 1982. Assay of urease activity in marine sands-its use as
                       an indicator of sewage contamination of beaches. Enzyme Microb. Tech, 4:310-312.


                       Slanetz, L W., and Bartley, C. H. 1957. Numbers of enterococci In water, sewage and
                       feces determined by the membrane filter technique with an improved medium. J. Bacteriol.
                       74:591-595.


                       Slanetz, L W., and Bartley, C. H. 1965. Survival of fecal streptococci in sea water.
                       Health Lab, Sci. 2:142-148.


                       Smith, E. M., Gerba, C. P., and J. L. Melnick. 1978. Role of sediment in the persistence
                       of enteroviruses, in the estuarine environment. Appl, Environ. Microbiol. 35:685-689.


                       Smith, L DS. 1975. Common mesophilic anaerobes, including Clostridium botulinum
                       and Clostridium tetani. in 21 soil specimens. Appl. Microbiol. 29:590-594.


                       Snowdon, J. A. and Cliver, D. 0. 1989. Coliphages as indicators of human enteric
                       viruses in groundwater. Crit. Rev. Environ. Control 19:231-249.


                       Sobsey, M. D. 1987. Methods for recovering viruses from shellfish, seawater, and
                       sediments. In: Berg, G. (ed.) Methods for recovering viruses from the environment
                       CRC Press, Inc., Boca Raton, Florida, PP. 77-108.


                       Sobsey, M. D., Schwab, K. J., and Handzel, T. R. 1990. A simple membrane filter
                       method to concentrate and enumerate male-specific RNA coliphages, Jour.AWWA 82:52-
                       59.


                       Sodergren, A. 1989. Biological effects of bleached pulp mill effluents. National Swedish
                       Environmental Protection Board, Report 3558. Sohia, Sweden.




                                                          107









                      Somerville, C C., Knight, I. T., Straube, W. L, and Colwell, R. R. 1989. Simple,
                      rapid method for direct isolation, of nucleic acids from aquatic environments. Appl
                      Environ, Microbiol. 55:548-554.


                      Steffan, R. J., and Atlas, R. M. 1988. DNA amplification to enhance detection of
                      genetically engineered bacteria in environmental samples. Appl Environ, Microbiol,
                      54:2185-2191.


                      Strange, R.E.  1976. Microbial response to mild stress. Meadowfield Press Ltd.,
                      Durham, England.


                      Stuart, D. G., McFeters, G. A., and Schillinger, J.E. 1977. Membrane filter. technique
                      for the quantification of stressed fecal coliforms in the aquatic, environment. Appl.
                      Environ. Microbiol 34:42-46.


                      Switzer, R. F., and Evans, J. B. 1974. Evaluation of selective media for enumeration of
                      group D streptococci in bovine feces. Appl Microbiol. 28:1086-1087.


                      Tabor, P. S., and Neihof, R. A. 1982. Improved microautor  adiographic: method to
                      determine individual microorganisms active in substrate uptake in natural waters. Appl
                      Environ, Microbiol. 44: 945-953.


                      Tanaka, R. and Mutai, M. 1980. Improved medium for selective isolation and
                      enumeration of Bifidobacterium, A-MI. Environ. Microbiol. 40: 866-869.


                      Tartera, C. and Jofre, J. 1987. Bactmiophages active against Bacteroides fragilis in
                      sewage-polluted waters. AWL Environ. Microbiol 53:1632-1637.


                      Tartera, C., Bosch, A., and Jofre, J. 1988. The inactivation of bacteriophages infecting
                      Bacteroides fragilis by chlorine treatment and UV-radiation. FEMS Microbiol. Let
                      56:313-316.


                      Tartera, C., Lucena, F., and Jofre, J. 1989. Human origin of Bacteroides fragilis
                      bacteriophages present in the environment J. Appl. Microbiol. 55:2696-2701




                                                         108









                       Teshima, S., and Kanazawa, A. 1978. Conversion of cholesterol to coprostanol and
                       cholestanol in the estuary sediment Mem, Fac. Fish. Kagoshima Univ. 27:41-47.


                       Toranzo, A. E. Barja, J. L. and Hetrick, F. M. 1982. Antiviral activity of antibiotic-
                       producing marine bacteria. Can. J. Microbiol. 28:231-238.


                       Tornabene, T. G. 1974. Sterols, aliphatic hydrocarbons, and fatty acids of a
                       nonphotosynthetic diamtom, Nitzschia alba 9:279-284.


                       Trollope, D. R. 1984. Use of molluscs to monitor bacteria in water. In: Grainger, J. M.
                       and Lynch, J. M. (eds.) Microbiological Methods for Environmental Biotechnology
                       Academic Press, London, pp. 393-408.


                       USEPA. 1985. Test methods for Escherichia coli and Enterococci in water by the
                       membrane filter procedure. United States Environmental Protection Agency,
                       Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, EPA-600/4-
                       85/076.


                       USEPA. 1986. Ambient Water Quality Criteria for Bacteria-1986. US Environmental
                       Protection Agency, Washington, DC, EPA-440/5-84-002.


                       Vasconcelos, G. J., Jakubowski, W., and Ericksen, T. H. 1969. Bacteriological changes
                       in shellfish maintained in an estuarine environment Proceedings National Shellfisheries
                       Association 59:67-83.


                       Vasconcelos, G. J., and Swartz, R. G. 1976. Survival of bacteria in seawater using a
                       diffusion chamber apparatus in situ. Appl. Environ. Microbiol. 31:913-920.


                       Vaughn, J. M. and Metcalf, T. G. 1975. Coliphages as indicators of enteric viruses in
                       shellfish and shellfish raising estuarine waters. Water Re 9:613-616.


                       Vaughn, J. M., Landry, E. F. Thomas, M. Z. Vicale, T. J., and Penello, W. F. 1979.
                       Survey of human enterovirus occurrence in fresh and marine surface waters on Long
                       Island. Appl. Environ, Microbiol 38:290-296.




                                                           109
 








                     Vaughn, J. M. and Landry, E F. 1983. Viruses in soils and groundwaters. In: Berg, G.
                     (eds.) Viruses in soils and groundwaters. CRC PRess, Boca Raton, pp. 163-210.


                     Venkatesan M. I., Ruth, E, and Kaplan, I. R. 1986. Coprostanols in Antarctica marine
                     sediments: a biomarker for marine mammals and not human pollution. Mat Pollut. Bull.
                     17:554-557.
 
                     Verstraete, W. and Voets, J. P. 1976. Comparative study of  E coli survival in two
                     aquatic ecosystems. Water Res   10:129-136.


                     Vlassoff, L 1977. Klebsiella       In: Hoadley, A. and Dutka, B J. (eds.) Bacterial
                     indicators/health hazards associated with water     Special Technical Publication 635,
                     American Society for Testing and Materials, Philadelpia, pp. 275-288.


                     Volterra, L, Bonadonna L, and Aulicino, F. A. 1985. Comparison of methods to detect
                     fecal streptococci in marine waters. Water. Air, Soil Pollut     26:201-210.


                     Wade, T. L, Oertel, G. F., and Brown, R. C. 1983. Particulate hydrocarbon and
                   coprostanol concentrations in shelf waters adjacent to Chesapeake Bay. Can, J, Fish,
                     Aquat. Sci, 40:34-40.


                     Walden, W. C. and Hentges, D. J. 1975. Differential effects of oxygen and oxidation-
                     reduction potential on the multiplication of three species of anaerobic intestinal bacteria.
                     AWL Microbiol. 30:781-785.


                     Walker, R. W., Wun,C. K., and Litsky, W. 1982. Coprostanol as an indicator of fecal
                     pollution. CRC Crit. Revs. Envir, Control 10:91-112.


                     Watkins, W. D., Rippey, S. R., Clavet, C. R., Kelly-Reitz, D. J., and Burkhardt, W. I.
                     1988. Novel compound for identifying Escherich coli. Appl Environ. Microbiol.
                     54:1874-1875.


                     Weiss, C. M. 1951. Adsorption of E coli on river and estuarine silts. Sewage Ind.
                             23:227-237.





                                                      110










                         Wheater, D.W.F., Mara, D.D., and Oragui,J. 1979. Indicator systems to distinguish
                         sewage from stormwater run-off and human from animal faecal material. In:James,A.,
                         and Evison,L.(eds.)Biological indicators of water quality. John Wiley and Sons,
                         Chichester,p.21-1-21-25.


                         Wheater,D.W.F.,Mara,D.,Opara,A.,and Singleton,P. 1980. Anaerobic bacteria as
                         indicators of faecal pollution. Proc. Royal Soc. Edinburgh 78B,s161-169.


                         Wilkins, T. D. and Hackman, A. S. 1974. Two patterns of neutral steroid conversion in
                         the feces of normal North Americans. Cancer Res. 34: 2250-2254.

                         Wright, R. T. and Coffin, R. B. 1984. Measuring microzooplankton grazing on
                         planktonic marine bacteria by its impact on bacterial production. Microbial Ecol. 10:137-
                         149


                         Wun, C.K., Rho, J., Walker, R.W., and Litsky, W. 1979. A simplified method for the
                         silmultaneous extraction of phytoplanktonic chlorophyll and fecal sterol from water. Wat.,
                         Air, and Soil Pollut. 11:173-178.


                         Xu, H. S., Roberts, N., Singleton, F.L., Attwell, R.W., Grimes, D.J., and Colwell, R.
                         R. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio choloerae in
                         the estuarine and marine environment. Microb. Ecol. 8:313-323.


                         Yde, M., Wulf, E. De, De Maeyer-Cleempoel, S., and Quaghebeur, D. 1982.
                         Coprostanol and bacterial indicators of faecal pollution in the Scheldt Estuary. Bull.
                         Environ. Contam. Toxicol 28:129-134.


                         Yoovidhya, T. and Fleet, G. H. 1981. An evaluation of the A-1 most probable number
                         and the Anderson and Baird-Parker plate count methods for enumerating Escherichia coli in
                         the Sydney rock oyster, Crassostrea commercialis. J. Appl. Bacteriol. 50:519-528.


                         Yoshpe-Purer, Y. 1989. Evaluation of media for monitoring fecal streptococci in
                         seawater. Appl. Environ. Microbiol. 55:2041-2045.

                      Zimmerman, R., Iturriaga, R., and Becker-Birck, J. 1978. Simultaneous determination of
                      the total number of aquatic bacteria and the number thereof involved in respiration. Appl.
                      Environ. Microbiol. 36:926-935.





















































                                                          112




























                                                                                                   DATE DUE






















                                                                                 GAYLORD No. 2333                                  PRINTED IN U.S.A.






















                                                                                                  36668           14107 733'