[Federal Register Volume 64, Number 207 (Wednesday, October 27, 1999)]
[Notices]
[Pages 57893-57902]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 99-28016]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration
[Docket Nos. 99D-4488 and 99D-4489]


Guidance for Industry: Reducing Microbial Food Safety Hazards for 
Sprouted Seeds and Guidance for Industry: Sampling and Microbial 
Testing of Spent Irrigation Water During Sprout Production

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

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SUMMARY: The Food and Drug Administration (FDA) is publishing two 
related guidance documents entitled ``Guidance for Industry: Reducing 
Microbial Food Safety Hazards for Sprouted Seeds'' and ``Guidance for 
Industry: Sampling and Microbial Testing of Spent Irrigation Water 
During Sprout Production.'' These guidances are intended to provide 
recommendations to suppliers of seed for sprouting and sprout producers 
about how to reduce microbial food safety hazards common to the 
production of raw sprouts to ensure that sprouts are not a cause of 
foodborne illness and to ensure that they comply with the food safety 
provisions of the Federal Food, Drug, and Cosmetic Act (the act). The 
first guidance is based largely on recommendations from the National 
Advisory Committee for Microbiological Criteria for Food's report 
entitled ``Microbial Safety Evaluations and Recommendations on Sprouted 
Seeds'' (the 1999 NACMCF report) (Ref.1). The second guidance is 
intended to assist sprouters in implementing one of the principle 
recommendations (i.e., microbial testing) in the broader sprout 
guidance.

DATES: Written comments may be submitted at any time, however, comments 
should be submitted by December 13, 1999, to ensure adequate 
consideration in preparation of revised documents, if warranted.

ADDRESSES: Submit written requests for single copies of the guidance 
entitled ``Guidance for Industry: Reducing Microbial Food Safety 
Hazards for Sprouted Seeds'' and/or the guidance entitled `Guidance for 
Industry: Sampling and Microbial Testing of

[[Page 57894]]

Spent Irrigation Water During Sprout Production'' to the Office of 
Plant and Dairy Foods and Beverages (HFS-306), Center for Food Safety 
and Applied Nutrition, Food and Drug Administration, 200 C St. SW., 
Washington, DC 20204, 202-205-4200. Send one self-adhesive label to 
assist that office in processing your request. The guidances are 
attached to this notice as appendixes 1 and 2 and are also accessible 
via the FDA home page on the Internet: http://www.fda.gov. Submit 
written comments on the final guidance(s) to the Dockets Management 
Branch (HFA-305), 5630 Fishers Lane, rm. 1061, Rockville, MD 20852.

FOR FURTHER INFORMATION CONTACT: Michelle A. Smith, Center for Food 
Safety and Applied Nutrition (HFS-306), Food and Drug Administration, 
200 C St. SW., Washington, DC 20204, 202-205-2975, FAX: 202-205-4422, 
e-mail: [email protected].
SUPPLEMENTARY INFORMATION:

I. Background

    Since 1995, raw sprouts have been increasingly implicated in 
foodborne outbreaks. Between January 1995 and May 1999, there were 11 
reported outbreaks in the United States associated with sprouts from 
commercial growers, 9 of which were due to various Salmonella serotypes 
and 2 to Escherichia coli O157. The number of culture-confirmed cases 
in each of these outbreaks ranged from 8 to more than 500, and more 
than 1,300 cases have been reported overall (Ref. 1). Alfalfa and 
clover sprouts have been implicated most often, but, because all kinds 
of sprouts are produced under similar conditions, all raw (uncooked) 
sprouts may pose a risk. In all of the reported outbreaks, the likely 
source of the pathogen was contaminated seed. However, in a large 1996 
Salmonella Montevideo/Meleagridis outbreak, poor sanitation and 
unhygienic practices at the sprouting facility may also have 
contributed to the contamination of sprouts (Ref. 1).
    Sprouted seeds represent a food safety problem because the 
conditions under which sprouts are produced (time, temperature, water 
activity, pH, and nutrients) are ideal for the exponential growth of 
bacteria. If bacterial pathogens are present on or in the seed, 
sprouting conditions are likely to encourage their proliferation (Ref. 
2).
    FDA and other public health officials are working with industry to 
identify and implement production practices to ensure that seed and 
sprouted seed are produced under conditions that are safe. While these 
efforts have improved food safety awareness within the industry and 
have led to a significantly better understanding of the microbial 
ecology of sprout-associated foodborne illness, not all industry 
segments have been reached. Traceback investigations reveal that most 
of the firms associated with recent outbreaks were not using approved 
seed disinfection treatments, or were not using them consistently, and 
were not testing for microbial contamination during sprout production. 
Although currently approved treatments can significantly reduce 
pathogen levels in or on seeds, they have not been shown to completely 
eliminate pathogens (Ref. 1). Consequently, outbreaks continue to 
occur.
    On July 9, 1999, FDA issued a consumer advisory advising all 
consumers to be aware of the risks associated with eating any variety 
of raw sprouts, and advising persons at high risk of developing serious 
illness due to foodborne disease (children, the elderly, and persons 
with weakened immune systems) not to eat raw sprouts (Ref. 3). This 
advisory is updated from a previous advisory issued August 31, 1998 
(Ref. 4), and was prompted by information from clover and alfalfa 
sprout-associated salmonellosis outbreaks that occurred from January 
1999 through May 1999.

II. Guidance

    In 1997, FDA asked the National Advisory Committee for 
Microbiological Criteria for Food (NACMCF) to : (1) Review the current 
literature on sprout-associated outbreaks, (2) identify the organisms 
and production practices of greatest public health concern, (3) 
prioritize research needs, and (4) provide recommendations on 
intervention and prevention strategies. On May 28, 1999, NACMCF adopted 
a report entitled ``Microbial Safety Evaluations and Recommendations on 
Sprouted Seeds'' (Ref. 1).
    FDA is now issuing ``Guidance to Industry: Reducing Microbial Food 
Safety Hazards for Sprouted Seeds'' (the sprout guidance). This 
guidance incorporates some of the recommendations made by the 1999 
NACMCF report. The guidance identifies the most important steps, e.g., 
use of good agricultural practices (Ref. 5), seed disinfection 
treatment, and microbial testing, which the agency believes should be 
implemented immediately to reduce the risk of raw sprouts as a vehicle 
for foodborne illness and to ensure that sprouts comply with the Food 
Safety Provision of the act (21 U.S.C. 301-397).
    As noted in the sprout guidance, routine use of approved seed 
disinfection treatments (such as 20,000 parts per million of calcium 
hypochlorite in water) is likely to reduce the level of contamination 
if present in or on seeds and, in turn, reduce the risk of foodborne 
illness from the consumption of sprouted seed. However, current 
approved treatments do not guarantee total elimination of pathogens. If 
even a few pathogens survive a seed disinfection treatment, they can 
grow to high levels during sprouting and contaminate the entire batch. 
Therefore, although seed disinfection treatment is recommended, 
microbial testing of spent irrigation water from each batch or 
production lot of sprouts should also be conducted to prevent 
contaminated product from entering the food supply.
    In addition, FDA is issuing ``Guidance to Industry: Sampling and 
Microbial Testing of Spent Irrigation Water During Sprout Production'' 
(the microbial testing guidance). This guidance is designed to assist 
sprouters in designing a microbial testing program to ensure 
adulterated product does not enter commerce. Specifically, this 
guidance recommends testing spent irrigation water from each individual 
batch or production lot of sprouts for two pathogens, E. coli O157:H7 
and Salmonella. The microbial testing guidance also provides 
instructions for the sampling and testing of sprouts for those 
instances when it is not possible to test spent irrigation water. 
However, sprouts should not be tested in lieu of irrigation water when 
spent irrigation water is available.
    Sprouters should be aware that the microbial testing program 
described in this guidance involves a number of hazards. Microbial test 
procedures should only be run by qualified personnel, in a qualified 
independent laboratory that is separate from food production areas. 
Additional criteria for laboratories performing these tests are 
provided in the microbial testing guidance.
    As more effective treatments or other food safety controls are 
identified and implemented, the current recommendation to test spent 
irrigation water from each batch of sprouts produced may be changed, 
e.g., to periodic microbial testing as a tool for validating the 
effectiveness of food safety systems.
    These guidance documents do not provide detailed information on all 
individual steps that should be followed in the production of seeds and 
sprouts. References and resources for assistance are listed at the end 
of this notice and in the broader sprout guidance. Other materials, in 
the form of further

[[Page 57895]]

guidance, educational videos, etc. will be made available, as 
appropriate. For example, FDA and the California Department of Health 
Services, Food and Drug Branch, in cooperation with industry, are 
producing a comprehensive educational video outlining sprout specific 
practices, in a number of areas, including those practices recommended 
in this guidance. The agency expects the video to be available in early 
2000.
    The guidance documents (see appendixes 1 and 2) represent FDA's 
current thinking on prevention of microbial hazards in sprouted seeds. 
They do not create or confer any rights for or on any person and do not 
operate to bind FDA or the public. An alternative approach may be used 
if such approach satisfies the requirement of applicable statutes and 
regulations. Following the recommendations in this guidance will not 
shield any person or any food from appropriate enforcement under the 
act if adulterated food is distributed in interstate commerce. The 
guidances are being distributed in accordance with FDA's policy for 
Level 1 guidance documents as set out in the agency's Good Guidance 
Practices, published in the Federal Register of February 27, 1997 (62 
FR 8961).
    FDA believes this guidance and current education and outreach 
efforts will have a significant positive impact on those industry 
segments that still need tools to make a safer product. The agency will 
closely monitor the safety of sprouts and the adoption of enhanced 
prevention practices as set out in this guidance.
    Failure to adopt effective preventive controls can be considered 
insanitary conditions which may render food injurious to health. Food 
produced under such conditions is adulterated under the act (section 
402(a)(4) (21 U.S.C. 342(a)(4))). FDA will consider enforcement actions 
against seed and sprout producers who do not have effective preventive 
controls in place, in particular, effective microbial testing.
    On December 27, 1999, FDA plans to initiate a national field 
assignment, sending investigators to sprouting facilities to test water 
used to grow sprouts (i.e., spent irrigation water) and to assess the 
level of adoption of preventive controls.

III. References

    The following references are on display in the Dockets Management 
Branch (address above) and may be seen by interested persons between 9 
a.m. and 4 p.m., Monday through Friday.
    1. National Advisory Committee on Microbiological Criteria for 
Foods, 1999a. Microbiological Safety Evaluations and Recommendations 
on Sprouted Seeds, http://vm.cfsan.fda.gov/mow/
sprouts2.html.
    2. National Advisory Committee on Microbiological Criteria for 
Foods, 1999b. Microbiological Safety Evaluations and Recommendations 
on Fresh Produce. Food Control, 10:117-143.
    3. FDA, 1999, Press Release--Consumers Advised of Risks 
Associated With Raw Sprouts, P99-13, http://www.fda.gov/bbs/topics/
NEWS/NEW00684.html.
    4. FDA, 1998, Talk Paper--Interim Advisory on Alfalfa Sprouts, 
T98-47.
    5. FDA, 1998, Guidance for Industry--Guidance to Minimize 
Microbial Food Safety Hazards for Fresh Fruits and Vegetables, 
http://www.foodsafety.gov/dms/prodguid.html.

IV. Comments

    FDA is soliciting public comments, but is implementing this 
guidance document immediately because of continuing foodborne illness 
outbreaks associated with consumption of raw sprouts. Interested 
persons may, at any time, submit written comments on the guidance 
documents to the Dockets Management Branch (address above). Two copies 
of any comments are to be submitted except that individuals may submit 
one copy. Comments should be identified with the appropriate docket 
numbers found in brackets on each guidance document. A copy of the 
guidance documents and comments received may be seen in the office 
above between 9 a.m. and 4 p.m., Monday through Friday.

    Dated: October 21, 1999.
Margaret M. Dotzel,
Acting Associate Commissioner for Policy.
The text of the guidances follows:
GUIDANCE FOR INDUSTRY -
REDUCING MICROBIAL FOOD SAFETY HAZARDS
FOR SPROUTED SEEDS\1\
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    \1\ This guidance has been prepared by the Office of Plant and 
Dairy Foods and Beverages in the Center for Food Safety and Applied 
Nutrition at the Food and Drug Administration. This guidance 
represents the agency's current thinking on reducing microbial food 
safety hazards for sprouted seeds. It does not create or confer any 
rights for or on any person and does not operate to bind FDA or the 
public. An alternative approach may be used if such approach 
satisfies the requirements of the applicable statute and 
regulations. Following the recommendations in this guidance will not 
shield any person or any food from appropriate enforcement under the 
Federal Food, Drug, and Cosmetic Act if adulterated food is 
distributed in interstate commerce.
---------------------------------------------------------------------------

[Docket No. 99D-4488]
    All parties involved in the production of sprouts--seed producers, 
seed conditioners, and distributors, and sprout producers--should be 
aware that seeds and sprouted seeds have been recognized as an 
important cause of foodborne illness. The following recommendations 
identify the preventive controls that the Food and Drug Administration 
(FDA) believes should be taken immediately to reduce the risk of raw 
sprouts serving as a vehicle for foodborne illness and ensure sprouts 
are not adulterated under the food safety provisions of the Food, Drug, 
and Cosmetic Act (the act). Failure to adopt effective preventive 
controls can be considered insanitary conditions which may render food 
injurious to health. Food produced under such conditions is adulterated 
under the act (21 U.S.C. 342(a)(4)). FDA will consider enforcement 
actions against any party who does not have effective preventive 
controls in place, in particular, microbial testing.
     These recommendations are based on the recommendations of the 
National Advisory Committee on Microbiological Criteria for Foods 
(NACMCF, 1999) and elaborate on Compliance Policy Guide 7120.28 (CPG 
7120.28).
    Seed Production: Seeds for sprout production should be grown under 
good agricultural practices (GAPs) in order to minimize the likelihood 
that they will contain pathogenic bacteria. For more information on 
GAPs, see FDA's 1998 ``Guidance for Industry: Guide to Minimize 
Microbial Food Safety Hazards for Fresh Fruits and Vegetables''. Copies 
of this guidance are available on the internet (http://
www.foodsafety.gov/dms/prodguid.html) or by calling the number listed 
in the references and resources at the end of this guidance.
    Seed Conditioning, Storage, and Transportation: Seeds that may be 
used for sprouting should be conditioned, stored, and transported in a 
manner that minimizes the likelihood that the seeds will be 
contaminated with pathogens. For example, seed should be stored in 
closed or covered containers in a clean dry area dedicated to seed 
storage. Containers should be positioned off the floor and away from 
walls to reduce the possibility of contamination by rodents or other 
pests and to facilitate regular monitoring for pest problems.
    Sprout Production: Sprouters should implement appropriate practices 
to ensure that sprouts are not produced in violation of the act which 
prohibits the production of food under insanitary conditions which may 
render food injurious to health (21 U.S.C. 342(a)(4)). In addition to 
seed treatment and testing for pathogens (see below), sprouters should 
maintain facilities and equipment in a condition that will protect 
against contamination. Facilities with poor sanitation can 
significantly increase the risk of contaminating product. Sprouters 
should employ good

[[Page 57896]]

sanitation practices as a standard operating procedure to maintain 
control throughout all stages of sprout production. Inadequate water 
quality and poor health and hygienic practices can all increase the 
risk of food becoming contaminated with pathogens. Sprouters may wish 
to review 21 CFR Part 110 which sets forth good manufacturing practices 
(GMPs) in manufacturing, packaging, or holding human food that cover 
these aspects of food production.
    Seed Treatment: Seeds for sprouting should be treated with one or 
more treatments (such as 20,000 ppm calcium hypochlorite\2\) that have 
been approved for reduction of pathogens in seeds or sprouts. Some 
treatments can be applied at the sprouting facility while others will 
have to be applied earlier in the seed production process. However, at 
least one approved antimicrobial treatment should be applied 
immediately before sprouting\3\. Sprouters should carefully follow all 
label directions when mixing and using antimicrobial chemicals.
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    \2\ In 1998, the Environmental Protection Agency issued a 
``section 18'' for the temporary use of 20,000 ppm calcium 
hypochlorite to disinfect seed for sprouting. In the fall of 1999, 
the exemption was renewed for another year. However, in order to 
ensure continued availability of this treatment, registrants should 
be actively pursuing a full registration under section 3 in 2000.
    \3\ Antimicrobials are either pesticides chemicals or food 
additives, depending on where they are used. As such their use on 
seeds for sprouting must be approved by EPA or FDA. To find out what 
antimicrobials have been approved by EPA or FDA for use on seeds for 
sprouting, you can call 202-418-3098.
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    Testing for Pathogens: Because currently approved antimicrobials 
have not been shown to be capable of eliminating all pathogens from 
seed, sprout producers should conduct microbiological testing of spent 
irrigation water from each production lot to ensure that contaminated 
product is not distributed. Because testing for pathogens can be done 
with irrigation water as early as 48 hours into what is generally a 3 
to 10 day growing period, producers who plan accordingly can obtain 
test results before shipping product without losing product shelf-life. 
Testing, whether done by the producer or contracted out, should be done 
by trained personnel, in a qualified laboratory, using validated 
methods. Additional information on sample collection and microbial 
testing, including how to sample and test sprouts when testing spent 
irrigation water is not practicable (as may be the case with soil-grown 
sprouts), can be found in a companion guidance document referenced 
below.
    Traceback: Traceback cannot prevent a foodborne illness outbreak 
from occurring. However, being able to trace a food back to it's source 
quickly can limit the public health and economic impacts of an 
outbreak, if it occurs. Information gained in traceback investigations 
may also help prevent future outbreaks. Sprout producers, seed 
producers, conditioners and distributors should develop and implement 
systems to facilitate traceback and recalls in the event of a problem. 
All parties should test their systems in advance of a real problem.
References and resources:
    1. Food and Drug Administration. 1982. Compliance Policy Guide 
Sec. 555.750 Seeds for Sprouting Prior to Food Use, i.e., Dried Mung 
Beans, Alfalfa Seeds, etc. (CPG 7120.28 ) can be viewed and printed 
from the WWW at the following address http://www.fda.gov/ora/
compliance_ref/cpg/cpgfod/cpg555-750.html
    2. Food and Drug Administration. 1998. Guidance for Industry--
Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and 
Vegetables can be viewed and printed from the WWW at the following 
address http://www.foodsafety.gov/dms/prodguid.html or may be 
obtained by calling 202-401-9725.
    3. Food and Drug Administration, 1999. Press Release--Consumers 
Advised of Risks Associated with Raw Sprouts. P99-13. http://
www.fda.gov/bbs/topics/NEWS/NEW00684.html
    4. FDA, 1999. ``Guidance for Industry: Sampling and Microbial 
Testing of Spent Irrigation Water During Sprout Production'' can be 
viewed and printed from the WWW at http://vm.cfsan.fda.gov
    5. National Advisory Committee on Microbiological Criteria for 
Foods. 1999a. Microbiological Safety Evaluations and Recommendations 
on Sprouted Seeds. http://vm.cfsan.fda..gov/mow/sprouts2.html
    6. National Advisory Committee on Microbiological Criteria for 
Foods. 1999b. Microbiological Safety Evaluations and Recommendations 
on Fresh Produce. Food Control. 10:117-143.
    7. Copies of Federal regulations in the Code of Federal 
Regulations (CFR) may be purchased from the U.S. Government Printing 
Office or by telephone at (202) 512-1800. The CFR is also available 
at local branches of U.S. Government Printing Office Bookstores. 
Information on location of regional branches is available on the WWW 
at the following address: http://vm.cfsan.fda.gov/lrd/ob-reg.html
    8. Sections of the CFR, such as 21 CFR Part 110 Current Good 
Manufacturing Practices in Manufacturing, Packing, or Holding Human 
Food, can be viewed and printed from the WWW at the following 
address: http://www.access.gpo.gov/nara/cfr/index.html.
Appendix 2--Guidance for Industry: Sampling and Microbial Testing of 
Spent Irrigation Water During Sprout Production

GUIDANCE FOR INDUSTRY -
SAMPLING AND MICROBIAL TESTING OF SPENT IRRIGATION WATER
DURING SPROUT PRODUCTION\4\
---------------------------------------------------------------------------

    \4\ This guidance has been prepared by the Office of Plant and 
Dairy Foods and Beverages in the Center for Food Safety and Applied 
Nutrition at the Food and Drug Administration. This guidance 
represents the agency's current thinking on reducing microbial food 
safety hazards for sprouted seeds. It does not create or confer any 
rights for or on any person and does not operate to bind FDA or the 
public. An alternative approach may be used if such approach 
satisfies the requirements of the applicable statute and 
regulations. Following the recommendations in this guidance will not 
shield any person or any food from appropriate enforcement under the 
Federal Food, Drug, and Cosmetic Act if adulterated food is 
distributed in interstate commerce.
---------------------------------------------------------------------------

[Docket No. 99D-4489]

Introduction

    Raw sprouts have been associated with at least eleven foodborne 
illness outbreaks since 1995. FDA and other public health officials are 
working with industry to identify and implement production practices 
that will assure that seed and sprouted seed are produced under safe 
conditions. While these efforts have improved food safety awareness 
within the industry and have led to a significantly better 
understanding of the microbial ecology of sprout-associated foodborne 
illness, not all industry segments have been reached and outbreaks 
continue to occur. Consequently, FDA released a guidance document, 
entitled ``Guidance for Industry: Reducing Microbial Food Safety 
Hazards for Sprouted Seed'' (the ``sprout guidance''). The sprout 
guidance identifies a number of areas, from the farm to the sprout 
facility, where FDA believes immediate steps should be taken to reduce 
the risk of sprouts serving as a vehicle for foodborne illness and to 
ensure that sprouts are not adulterated under the Food, Drug, and 
Cosmetic Act (the act). Specific recommendations in the sprout guidance 
include: development and implementation of good agricultural practices 
and good manufacturing practices in the production and handling of 
seeds and sprouts, seed disinfection treatments, and microbial testing 
before product enters the food supply.
    The agency will closely monitor the safety of sprouts and the 
adoption of enhanced prevention practices as set out in the sprout 
guidance. FDA plans to send investigators to sprouting facilities to 
test water used to grow sprouts (i.e., spent irrigation water) and 
assess the adoption of preventive controls. Failure to adopt effective 
preventive controls can be considered insanitary conditions which may 
render food injurious to

[[Page 57897]]

health. Food produced under such conditions is adulterated under the 
act (21 U.S.C. 342(a)(4)). FDA will consider enforcement actions 
against any party who does not have effective preventive controls in 
place, in particular, effective microbial testing.
    This guidance document, ``Sampling and Microbial Testing of Spent 
Irrigation Water During Sprout Production,'' is intended to assist 
sprouters in implementing one of the principal recommendations in the 
broader sprout guidance, i.e., that producers test spent irrigation 
water for two pathogens (Salmonella and Escherichia coli O157:H7) 
before product enters commerce. Instructions are also provided for the 
sampling and testing of sprouts for those instances when it is not 
possible to test spent irrigation water. However, for the reasons 
discussed below, sprouts should not be tested in lieu of irrigation 
water.

Why Test

    Salmonella and Escherichia coli O157:H7 have been the major causes 
of sprout-associated illness outbreaks. Seeds are the likely source of 
contamination in most of these outbreaks. Routine use of approved seed 
disinfection treatments (such as 20,000 parts per million of calcium 
hypochlorite in water) is likely to reduce the level of contamination 
if pathogens are present in or on seeds and, in turn, reduce the risk 
of foodborne illness from the consumption of sprouted seed. However, 
current approved treatments cannot guarantee total elimination of 
pathogens. The same conditions that encourage germination and growth of 
seeds (e.g., temperature, available moisture, and nutrients), also 
encourage the growth of bacterial pathogens. Even if only a few 
pathogens survive a seed disinfection treatment, they can grow to high 
levels during sprouting and contaminate the entire batch. Therefore, 
seed disinfection treatments should be combined with microbial testing 
to ensure that pathogens are not present before sprouts enter the food 
supply.
    As additional food safety controls are identified and implemented, 
the current recommendation to test irrigation water from every batch of 
sprouts produced may be changed, e.g., to periodic microbial testing as 
a tool for validating the effectiveness of food safety systems.

Who Should Perform The Tests

Sample collection

    Sample collection should be done by personnel that have been 
trained to collect representative samples aseptically. Obviously, 
sample collection should be done on site, either by employees or by 
contract personnel. Aseptic sampling procedures are described below.

Testing

    FDA recommends that all testing for pathogens be conducted in an 
external, qualified, independent laboratory that should meet several 
key criteria. First, the lab should be physically separated from the 
food production facility to prevent cross-contamination from test 
materials. This is especially important where the materials used in the 
enrichment step required before testing and the positive controls 
(described below) can contain pathogens and if not properly handled may 
contaminate sprouts.
    Second, the laboratory should be staffed by personnel with training 
and experience in analytical microbiology techniques to ensure that 
tests are performed correctly and that all appropriate safety 
precautions, including appropriate waste disposal, are followed. Third, 
the laboratory should have appropriate resources and a demonstrable 
quality management system.
    If testing is done by the sprouter, then the laboratory facilities, 
personnel, and management system should also meet all these criteria to 
ensure that testing is reliable and does not create food safety 
hazards.

Why Sample Irrigation Water

    Procedures are provided for testing spent irrigation water and 
sprouts. Although each has advantages and disadvantages, FDA is 
recommending testing spent irrigation water.
    Spent irrigation water that has flowed over and through sprouts is 
a good indicator of the types of microorganisms in the sprouts 
themselves and the microflora in spent irrigation water is fairly 
uniform. Thus sampling procedures for spent irrigation wate are 
relatively simple. Furthermore, water can be used directly in the test 
procedures described here. The only potential disadvantage of testing 
spent irrigation water is that the level of microorganisms recovered in 
irrigation water is about 1 log less than the level in sprouts. If 
pathogens are present in sprouts at very low levels, it is possible 
that they might be missed in water but recovered in sprouts.
    Testing the sprouts themselves has several significant 
disadvantages. First, multiple sprout samples must be taken from 
different locations in the drum or trays to ensure that the sample 
collected is representative of the batch. Furthermore, additional 
preparation (e.g., selecting representative subsamples for analyses, 
blending or stomaching, and allowing sprout particles to settle out) is 
required when testing sprouts. Each additional step in any procedure 
(sampling or testing) introduces a possibility for error.
    Consequently, sprouts should not be tested in place of irrigation 
water unless production methods make it impossible to test spent 
irrigation water. For example, spent irrigation water may not be 
available when sprouts are grown in soil. [Note: The recommendation to 
test irrigation water does not preclude adding the testing of sprouts 
(either sprouts collected during production or finished product), to a 
food safety plan that includes testing irrigation water.] Sampling and 
testing steps specific to sprouts are given in italics and may be 
disregarded when testing spent irrigation water.

Sampling Plan

    Sprouters should have a sampling plan in place to ensure the 
consistent collection of samples in an appropriate manner. The 
following factors should be considered in determining when and how to 
sample.

When to Sample

     Pathogens are most likely to be present at detectable levels at or 
after 48 hours from the start of the sprouting process. Levels will not 
necessarily increase after 48 hours and may decline slightly. Thus, 
collecting samples for testing can be done as early as 48 hours after 
the start of sprouting. If seeds are presoaked (e.g., soaked in water 
for a short time and then transferred to growing units for sprouting), 
presoak time should be included in the 48 hour minimum.
    If you are using rapid test kits, samples may be collected as late 
as 48 hours prior to shipping and still provide an opportunity for the 
sprouter to obtain test results before product enters the food supply. 
However, early results will allow a sprouter to take corrective actions 
sooner, minimizing the potential for a contaminated batch of sprouts to 
contaminate other production batches. Earlier testing (i.e., 48 hours 
after the start of sprouting) will also minimize the time and resources 
spent on a batch of sprouts if a presumptive positive is found. If a 
firm's action plan includes running confirmatory tests on a presumptive 
positive before discarding product, testing earlier rather than later 
allows more time to run additional tests.

[[Page 57898]]

How to Sample

    Aseptic procedures are critical to avoid contaminating the sample 
during sample collection, storing the sample(s), and transporting the 
sample(s) to the lab. Aseptic sampling procedures, as described below, 
should be part of a firm's plan for sample collection.
     Equipment used to collect samples should be clean and sterile. 
Sampling tools and sample containers may be purchased pre-sterilized. 
Alternatively, tools and containers may be sterilized at 121  deg.C 
(250  deg.F) for 30 minutes in an autoclave prior to use. Heat-
resistant, dry materials may be sterilized in a dry-heat oven at 140 
deg.C (284  deg.F) for 3 hours.
    The type of sample containers used will depend on the type of 
samples collected but may include pre-sterilized plastic bags, tubes, 
cups, and flasks. Containers should be dry, leak-proof, wide mouthed, 
and of a size suitable for the samples. Sample containers should be 
properly labeled prior to starting sample collection.
    Sample collectors should wear a clean lab coat, sterile gloves, and 
a hair net to insure they do not contaminate the samples. Hands should 
be washed immediately before sampling, and prior to putting on sterile 
gloves. Sterile gloves should be put on in a manner that does not 
contaminate the outside of the glove. Gloves should be properly 
disposed of after use.
    Hands should be kept away from mouth, nose, eyes, and face while 
collecting samples.
    Sampling instruments should be protected from contamination at all 
times before and during use. Sampling instruments and samples moved 
between the sampling site and the sample container, should not be 
passed over the remaining pre-sterilized instruments.
    The sterile sample container should be opened only sufficiently to 
admit the sample, place the sample directly in the container, then 
immediately closed and sealed. If collecting samples in a container 
with a lid, the lid and container should be held in one hand while 
collecting the sample. The lid should NOT be completely removed. (The 
lid should not be held separately or placed on a counter).
    The sample container should be filled no more than 3/4 full to 
prevent overflow. The air from the container should not be expelled 
when sealing, particularly for plastic bags. Samples or sampling 
equipment should not be exposed to unfiltered air currents.
    Samples should be delivered to the laboratory promptly. Perishable 
material should be kept at an appropriate temperature, preferably at 0 
to 4.4  deg.C (32 to 40  deg.F). Sealed coolant packs should be used to 
avoid contamination from melting ice.

What to Sample and How Much to Collect

    FDA recommends that a sprouter test for pathogens by collecting a 
sample of spent irrigation water from each production lot or batch. For 
purposes of this guidance, a production lot or batch is defined as 
sprouts from a single lot of seed that were started at the same time in 
a single growing unit (i.e., a single drum or rack of trays). Pooling 
samples should be avoided as pooling from different production batches 
may decrease the sensitivity of the tests by diluting the level of 
pathogens in a contaminated sample with samples that are not 
contaminated. Pooling samples from different batches also complicates 
the interpretation of results. If a presumptive positive is found, the 
sprouter should discard all lots represented by the pooled sample or 
perform additional tests to determine which batch(s) are contaminated.
1. Sample Collection for Spent Irrigation Water
    The volumes given below for spent irrigation water (or sprouts) 
represent a sufficient sample size to test for both Salmonella and 
Escherichia coli O157:H7.
    If testing spent irrigation water, 1 liter of water (about 2 pints 
or one quart) should be aseptically collected as the water leaves a 
drum or trays during the irrigation cycle.
    If sprouts are grown in drums, a single 1 liter sample may be 
collected.
    If sprouts are grown in trays, and all trays in a production lot 
have a common trough for collecting spent irrigation water, a 1 liter 
sample may be collected at that point. If there is no common collection 
point for water from trays, it may be necessary to collect water 
samples from individual trays and pool these samples. In this case, a 
sampling plan should be devised to ensure collection of a sample that 
is representative of the production lot. When 10 or fewer trays make up 
a production lot, approximately equal volumes of water should be 
collected from each of the 10 trays to make a total sample volume of 1 
liter. For example, collect about 100 ml of water from each of 10 trays 
to make a 1 liter sample; about 125 ml from each of 8 trays; 167 ml 
from each of 6 trays, and so on. When more than 10 trays make up a 
production lot, ten samples should be aseptically collected, 
approximately 100 ml each from different trays. Again, samples should 
be collected throughout the entire production lot (e.g., if there are 
20 trays in a production lot, collect samples from every other tray in 
the rack moving from top to bottom, side to side, and front to back). 
Samples should be placed directly into a clean, sterile, prelabeled 
container.
2. Sample Collection for Sprouts
    If testing sprouts, thirty-two (32) sample units should be 
aseptically collected, approximately 50 grams each, from different 
locations in the drum or growing trays. The total sprout sample will be 
approximately 1,600 g (about 56.48 ounces or 3.53 pounds per production 
lot or batch). Sample units should be collected throughout the entire 
production lot (e.g., from top to bottom, side to side, and front to 
back of the drum or trays). Each 50 gram sample unit should be placed 
directly into individual clean, sterile, prelabeled containers. 
(Keeping the thirty-two sample units separate will make it easier for 
the lab to select representative analytical units for microbial 
analysis compared to pulling analytical units from a single 1,600 gram 
mass of sprouts.)

Microbial Testing

Testing Procedures

    The testing procedures described in this guidance are screening 
tests. They were chosen to obtain results as simply and quickly as 
possible (i.e., to provide answers in 48 hours or less) on the presence 
or absence of two major pathogenic bacteria, i.e., Salmonella and 
Escherichia coli O157:H7. Formal confirmation methods, which take 
longer than 48 hours, are described in the FDA Bacteriological 
Analytical Manual (published by AOAC International, Gaithersburg, MD).
    The kits identified in this guidance are AOAC approved screening 
tests and/or FDA has experience with their use. These are also the 
tests that FDA plans to use as screening tests to monitor spent 
irrigation water at sprouting facilities. If screening methods, other 
than those described here are used, they should first be validated 
either by formal collaborative studies or by comparative studies with 
standard methods using the specific commodity in question spent 
irrigation water or sprouts.
    Procedures for determining the presence or absence of Escherichia 
coli O157:H7 and Salmonella species using the test kits listed below 
are provided at the end of this guidance. These procedures should be 
performed separate from the food production

[[Page 57899]]

facility by a qualified laboratory, preferably an independent, 
certified lab.
    The rapid test procedures described in this guidance all involve an 
enrichment step to encourage the selective growth of pathogens, if they 
are present, in order to make their detection possible. These test kits 
will NOT detect pathogens in irrigation water or sprouts if the 
enrichment step is not performed.
    In addition, seasonal or regional differences in water quality, 
type of seed being sprouted, individual sprout production factors, and 
variations in sampling and analytical conditions may all impact on the 
effectiveness of the screening tests. Therefore, the lab should 
periodically run positive controls (i.e., sprout or water samples to 
which a known quantity of pathogens have been added) to ensure the 
tests used are capable of detecting pathogens when they are present in 
the samples being tested.

Test Kits

Escherichia coli O157:H7
    1. VIP EHEC, Biocontrol, Inc., Bellview, WA., (AOAC Official method 
 996.09)
    or
    2. Reveal E. coli O157:H7, Neogen Corp., Lansing, MI.

Salmonella

    1. Assurance Gold Salmonella EIA, (AOAC Official method  
999.08)
    or
    2. Visual Immunoprecipitate (VIP) Assay for Salmonella, (AOAC 
Official method # 999.09)
    (Both kits are manufactured by BioControl Systems, Inc., 12822 SE 
32nd Street, Bellevue, WA 98005).

General Laboratory Instructions

Prepared Media Storage

Unless noted otherwise most media can be made in advance and stored at 
20-30  deg.C (68-86  deg.F) in the dark with a shelf life of at least 
one month. Media should be well wrapped or contained in order to reduce 
evaporation.
Equipment Sterilization
Safe and proper operation of sterilizing autoclaves requires specially 
trained personnel. The sterilization time is typically 121  deg.C (250 
deg.F) for 15 minutes.
Media and Equipment Decontamination
Used culture media and test kits should be decontaminated by 
autoclaving before disposal. Decontamination should be performed in an 
area that is totally separated from media preparation and 
sterilization. Trained personnel should be used to properly 
decontaminate used media.
Dividing Samples into Subsamples for Analyses
Spent Irrigation Water - A total of 1 L of spent irrigation should be 
collected for analysis. Two (2) 100 ml subsamples should be analyzed 
for the presence of E. coli O157:H7. Two (2) 375 ml subsamples should 
be analyzed for the presence of Salmonella. Any unused portion of the 
spent irrigation water should be stored under refrigeration pending 
completion of the analysis.
Sprouts - Thirty-two (32) 50 g analytical units of sprouts should be 
collected for analysis. Two (2) of the 50 g analytical units (25 g 
subsamples from each) should be analyzed for the presence of E. coli 
O157:H7 and thirty (30) of the 50 g sample units (25 g subsamples from 
each) should be analyzed for the presence of Salmonella. Unused 
portions of the sprout analytical units should be stored under 
refrigeration pending completion of the analysis.
Sample preparation (stomaching sprouts)
The procedures in this guidance use a blender to prepare sprouts for 
testing. As an alternative to blending, sprouts may be homogenized in a 
Stomacher (Model 400). To use a Stomacher, place 25 grams of sprouts in 
a sterile Stomacher bag, add 225 ml enrichment broth and process on 
medium speed for 2 minutes.

Interpretation of Results and Subsequent Actions

Interpreting Results

    Analyses should be performed in duplicate (two tests for each of 
the two pathogens). When results are negative for all tests, results 
are assumed to be correct. When results are positive for one or both 
tests for either pathogen, the results are considered presumptive and 
the grower should either:
1. Consider the presumptive positive result(s) to be true and take 
immediate corrective actions, as described below, OR
2. Ask the testing laboratory to run confirmatory tests and destroy the 
batch only if the confirmatory tests are also positive for the presence 
of a pathogen.
    In considering the second option, remember that confirmatory 
testing takes extra time and will lessen the marketable shelf life of 
the sprouts. (All product should be held until test results are 
available.) Rapid test kits are for screening ONLY. Confirmatory 
testing should be done using standard methods in the FDA 
Bacteriological Analytical Manual (Edition 8, Revision A-1998).

Corrective Actions

     Each facility should have a corrective action plan in place before 
a positive is found so that, if one does occur, corrective actions can 
be taken quickly. The following should be included in a corrective 
action plan.
    Seed is the likely source of contamination in sprout-associated 
foodborne illness outbreaks. Further, recent outbreak investigations 
have shown that a single contaminated seed lot can result in 
contamination of multiple production lots of sprouts. Therefore, when a 
batch of sprouts is considered to be contaminated with a pathogen, the 
batch of sprouts, the seed lot used to produce the sprouts, and any 
other sprout production lots that were made from the same seed lot and 
that are still under control of the sprouter, should be discarded.
    In addition, anything in the sprouting facility that has come into 
contact with the contaminated production lot or its water (e.g., drums, 
trays, bins, buckets, tool and other sprouting equipment, testing 
equipment, and other possible surfaces, such as floors, drains, walls, 
and tables), should be thoroughly cleaned and then sanitized to avoid 
contamination of subsequent batches of sprouts. Care must be taken in 
handling contaminated sprouts or water, equipment, and test materials 
to avoid accidental exposure to the pathogen(s).

A) Procedure for the Escherichia coli O157:H7 Rapid Analysis of 
Spent Irrigation Water or Sprouts.

I. Test Kits choose one. VIP EHEC\5\, Biocontrol, Inc., 
Bellview, WA., or  Reveal E. coli O157:H7\5\, Neogen Corp., 
Lansing, MI.
---------------------------------------------------------------------------

    \5\ The enrichment procedure described in this guidance for the 
tests for Escherichia coli O157:H7 have been modified by FDA to 
enhance the ability of the kits to detect Escherichia coli O157:H7 
in spent irrigation water and sprouts.
---------------------------------------------------------------------------

II. Equipment and Materials

 1. 1 Mechanical blender (capable of 10,000 to 12,000 rpm) or Stomacher 
Model 400 (with required stomacher bags)
 2. Sterile blender jars, with cover, resistant to autoclaving for 60 
min at 121  deg.C
 3. 1 Balance, with weights (2000 g capacity, sensitivity of 0.1 g)
 4. 1 L Erlenmeyer flask
 5. 2 Sterile graduated pipettes, 1.0 and 10.0 ml and pipette aids
 6. Sterile instruments for use in taking and handling of samples (such 
as knives, tongs, scissors, spoons, etc.)
 7. Sterile culture tubes, 16 x 150mm or 20 x 150mm
 8. Incubator/shaker platform, 35 + 1  deg.C
 9. pH meter or test strips
 10. Fisher or Bunsen burner
 11. Magnetic stirrer and stir bars

[[Page 57900]]

 12. Sterile syringes
 13. Sterile 0.2 m filters
 14. Distilled water

III. Ingredients

 1. Peptone
 2. NaCl
 3. Na2HPO4
 4. KH2PO4
 5. Casamino acid
 6. Yeast extract
 7. Lactose
 8. Acriflavin (antibiotic)
 9. Cefsulodin (antibiotic)
 10. Vancomycin (antibiotic)

Preparation of antibiotic stock solutions

Prepare a stock solution of each antibiotic (acriflavin, cefsulodin, 
and vancomycin) by dissolving 1000 mg of each antibiotic in a separate 
tube containing 10.0 ml of distilled water. Filter-sterilize the 
solution using a 0.2 m filter and syringe. The stock solution may be 
stored for several months in foil wrapped tubes at 4 C (39.2 C).
Prepare the modified Buffered Peptone Water as described below, 
autoclave, cool, add antibiotic supplements. Instructions for sprouts 
are given in italics.
Modified Buffered Peptone Water (mBPW)
Step 1. To make 1000 ml of mBPW, mix the following constituents into 
distilled water, stirring to dissolve. For spent irrigation water, 
prepare double strength (2X) mBPW, as follows: (If testing sprouts, use 
single strength enrichment broth base.)

 
------------------------------------------------------------------------
                 Modified Buffered Peptone Water (mBPW)
-------------------------------------------------------------------------
                                              Double
                                           strength (2X)      Single
                                           (For use with   strength (1X)
               Ingredient                      spent       (For use with
                                            irrigation       sprouts)
                                              water)
------------------------------------------------------------------------
Peptone                                        20.0 g          10.0 g
NaCl                                           10.0 g           5.0 g
Na2HPO4                                         7.2 g           3.6 g
KH2PO4                                          3.0 g           1.5 g
Casamino acid                                  10.0 g           5.0 g
Yeast extract                                  12.0 g           6.0 g
Lactose                                        20.0 g          10.0 g
Distilled water*                          1000 ml         1000 ml
------------------------------------------------------------------------
*pH 7.2  0.2 (Test pH of distilled water BEFORE adding the
  ingredients above. If necessary, pH may be adjusted with 1N HCl or 1N
  NaOH.

Step 2. Sterilize mBPW by autoclaving at 121  deg.C (250  deg.F) for 15 
minutes. Remove from autoclave and allow to cool until cool to the 
touch.
Step 3. Once the medium is cooled and immediately prior to the addition 
of a subsample, add the following filter-sterilized antibiotics to 1000 
ml of medium. For spent irrigation water, add the quantity of 
antibiotics listed in the column labeled double strength to the double 
strength (2X) mBPW. (If testing sprouts, add the quantity of 
antibiotics listed in the column labeled single strength to the single 
strength mBPW.)

 
----------------------------------------------------------------------------------------------------------------
                                         Antibiotic supplements for mBPW
-----------------------------------------------------------------------------------------------------------------
                         Double
                      strength (2X)
  Antibiotic Stock    (For use with
      Solution            spent                      Single strength (1X) (For use with sprouts)
                       irrigation
                         water)
----------------------------------------------------------------------------------------------------------------
Acriflavin (A)             0.2 ml          0.1 ml
Cefsulodin (C)             0.2 ml          0.1 ml
Vancomycin (V)             0.16 ml         0.08 ml
----------------------------------------------------------------------------------------------------------------

IV. Testing - Perform analysis in duplicate: For microbial testing, 
duplicate sub-samples (analytical units) need to be removed from 
the sample and placed in enrichment broth. Enrichment broth 
containing sub-samples are allowed to incubate for a period of 
time, and a small quantity of the enrichment broth/sample is 
applied to the test kit device. Specific directions follow:

Step 4.

Water: Two (2) 100 ml subsamples of spent irrigation will be analyzed. 
From the 1000 ml sample of spent sprout irrigation water, aseptically 
transfer 100 ml of sample into a sterile 1L flask containing 100 ml of 
2X mBPW+ACV. Repeat with second subsample.
Sprouts: Two (2) 50 g analytical units of sprouts will be analyzed. 
From two of the thirty-two 50 g analytical units collected, aseptically 
remove and weigh out a 25g subsample of sprouts. Transfer each of the 
25 gram subsamples of sprouts into separate sterile blender jars or 
sterile stomacher bags. Add 225 ml of single strength enrichment broth 
with added antibiotic supplements (1X mBPW+ACV) and blend at 10,000 to 
12,000 rpm until homogenized (at least 60 seconds) or stomach for 2 
minutes on medium setting in a Stomacher Model 400. Transfer sprout 
homogenate to a 1L Erlenmeyer flask.

[[Page 57901]]

Step 5. Incubate the enrichment broth/sample mixtures overnight at 
37  deg.C (98.6  deg.F) with shaking at 140 RPM.

Step 6. Test each enrichment broth sample for the presence of E. 
coli O157:H7, using either the VIP EHEC device or the Reveal E. 
coli O157:H7 device. Use 0.1 ml from the inoculated and incubated 
mBPW +ACV to inoculate VIP or 0.12 ml for the Reveal. Follow the 
manufacturers instructions for the inoculation of test kits.

Step 7. Observe test results within 10 minutes to avoid possible 
fading of bands which could lead to false negative results. A band 
in both the test and control chambers is a positive test for 
contamination. A band in only the control chamber is a negative 
test. If a band does not appear in the control chamber, the test 
was not done correctly and must be repeated.

B) Procedure for the Salmonella Rapid Analysis of Spent Irrigation 
Water (or Sprouts)

I. Test kits choose one

     Assurance Gold Salmonella EIA, or
     Visual Immunoprecipitate (VIP) Assay for Salmonella
Both are manufactured by BioControl Systems, Inc., (12822 SE 32nd 
Street, Bellevue, WA 98005). For purposes of pre-enrichment and 
selective enrichment, these test kits provide different instructions 
for each of three types of foods: (a) processed foods, (b) dried powder 
processed foods, and (c) raw foods. For the analysis of sprouts and 
spent irrigation water, use the pre-enrichment/selective enrichment 
procedures described for (c) raw foods.

II. Equipment and materials1. Blender and sterile blender jars OR 
Stomacher Model 400 with appropriate stomacher bags.

 2. Sterile, 16 oz (500 ml) wide-mouth, screw-cap jars, sterile 500 ml 
Erlenmeyer flasks, sterile 250 ml beakers, sterile glass or paper 
funnels of appropriate size, and, optionally, containers of appropriate 
capacity to accommodate composited samples
 3. Balance, with weights; (2000 g capacity, sensitivity of 0.1 g)
 4. Balance, with weights; (120 g capacity, sensitivity of 5 mg)
 5. Incubator, 35  deg.C (95  deg.F)
 6. Refrigerated incubator or laboratory refrigerator, 4 + 1  deg.C (39 
+ 1  deg.F)
 7. Water bath, 42 + 0.2  deg.C (107.6 + 0.2  deg.F)
 8. Sterile spoons or other appropriate instruments for transferring 
food samples
 9. Sterile culture dishes, size 15 x 100 mm, glass or plastic
 10. Sterile pipettes, 1 ml, with 0.01 ml graduations; 5 ml with 0.1 ml 
graduations and 10 ml with 0.1 ml graduations and pipette aids
 11. Inoculating needle and inoculating loop (about 3 mm id or 10 l), 
nichrome, platinum-iridium, chromel wire, or sterile plastic
 12. Sterile test or culture tubes, sizes 16 x 150 mm and 20 x 150 mm
 13. Test or culture tube racks
 14. Vortex mixer
 15. Sterile shears, large scissors, scalpel, and forceps
 16. Fisher or Bunsen burner
 17. pH test paper (pH range 6-8) with maximum graduations of 0.4 pH 
units per color change
 18. Sterile syringe
 19. Sterile 0.2 m filters

III. Media and reagents

For preparation of media and reagents, refer to sections 967.25 to 
967.28 in Official Methods of Analysis (published by AOAC 
International, Gaithersburg, MD USA). Designations within parentheses 
refer to Appendix 3, Media and Reagents, of the Bacteriological 
Analytical Manual (BAM), Edition 8, Revision A ( also published by AOAC 
International).
1. Buffered peptone water (commercially available-Oxoid, BBL, or Difco)
2. Buffered peptone water + novobiocin
3. Tetrathionate (TT) broth (M145)
4. Rappaport-Vassiliadis (RV) medium (M132)
5. Trypticase soy broth (commercially available)
6. Trypticase soy broth + novobiocin
7. Trypticase soy broth + 2, 4 dinitrophenol + novobiocin
8. 1 N Sodium hydroxide solution (NaOH) (R73)
9. 1 N Hydrochloric acid (HCl) (R36)
10. Novobiocin solution, 0.1%
11. Sterile distilled water
Buffered peptone water (Number 1.), Buffered peptone water with 
novobiocin (Number 2), Trypticase soy broth with novobiocin (Number 6) 
and Trypticase soy broth with 2,4 dinitrophenol and novobiocin (Number 
7), are not included in the BAM. Their preparation is described below.
1. Buffered Peptone Water (BPW)
Dissolve 20 grams of commercially available buffered peptone water 
medium in 1 liter distilled water. Mix thoroughly. Dispense 225 ml 
portions into 500 ml Erlenmeyer flasks. After autoclaving for 15 min at 
121  deg.C, and just before use, aseptically adjust volume to 225 ml 
with sterile distilled water. Adjust pH, if necessary, to 7.2 + 0.2 
with sterile 1 N NaOH or 1 N HCl.
2. Buffered Peptone Water + novobiocin (BPW + n)
Immediately prior to the addition of a 25 g subsample, add 4 ml of 0.1% 
novobiocin solution to each 225 ml volume of buffered peptone water.
6. Trypticase soy broth + novobiocin (TSB+n)
Suspend 30 g of commercial available trypticase soy broth medium in 1 L 
of distilled water. Mix thoroughly. Warm gently on a temperature 
controlled hot plate until the medium is dissolved. Dispense in 10 ml 
aliquots in 20 x 150 mm tubes and autoclave 15 min. at 121  deg.C.
Just prior to sample addition, add 0.1 ml of 0.1% novobiocin solution 
to each tube containing 10 ml of Trypticase soy broth.
7. Trypticase soy broth + 2, 4 dinitrophenol + novobiocin (TSB+DNP+n)
Suspend 30 g of commercial available trypticase soy broth medium and 
0.1 g of 2, 4 dinitrophenol in 1 L of distilled water. Mix thoroughly. 
Warm gently on a temperature controlled hot plate until the medium is 
dissolved. Dispense in 10 ml aliquots in 20 x 150 mm tubes and 
autoclave 15 min. at 121  deg.C (250  deg.F).
Just prior to sample addition, add 0.1 ml of 0.1% novobiocin solution 
to each tube containing 10 ml of Trypticase soy broth + 2, 4 
dinitriphenol.
9. Novobiocin solution, 0.1%
    Novobiocin, sodium salt 0.1 g
    Distilled water 100 ml
Dissolve novobiocin in distilled water. Do not autoclave. Sterilize by 
filtering through a 0.2 m filter. Store solution at 4  deg.C (39.2 
deg.F), protected from light (e.g. wrap container in aluminum foil). 
Solution can be stored for one week.

IV.Testing

A. Irrigation water-From the 1 L spent irrigation water sample, two (2) 
375 ml subsamples will be analyzed for the presence of Salmonella.
1. Aseptically transfer a 375 ml subsample directly to a 6 L Erlenmeyer 
flask containing 3,375 ml BPW + n. Swirl to mix thoroughly. Repeat 
procedure with second 375 ml subsample of spent irrigation water.
2. Allow flasks to stand for 60 min at room temperature. Mix well and 
determine pH with test paper. Adjust pH, if necessary, to 6.8 + 0.2 
with sterile 1 N NaOH or 1 N HCL.
3. Incubate flasks without shaking for 18-26 hours at 35-37  deg.C (95-
98.6  deg.F). Each flask is considered to contain pre-enrichment broth.

[[Page 57902]]

4a. If using the Assurance Gold Salmonella Enzyme Immunoassay, transfer 
0.1 ml pre-enrichment broth to 10 ml RV medium and transfer another 1.0 
ml of pre-enrichment broth to 10 ml TT broth. Incubate in a water bath 
5-8 hours at 42  deg.C (107.6  deg.F). Incubation of the RV medium and 
TT broth in the water bath is termed the selective enrichment process. 
Following selective enrichment, transfer and combine 1.0 ml TT broth 
and 0.5 ml RV medium into a single tube containing 10 ml of prewarmed 
[42  deg.C (107.6  deg.F)] TSB + n broth. Incubate in a water bath 16-
20 hours at 42  deg.C (107.6  deg.F). Continue as described in this 
kit's instructions for (c) raw foods.
4b. If using the VIP Assay for Salmonella, transfer 0.1 ml pre-
enrichment broth to 10 ml RV medium and transfer another 1.0 ml of pre-
enrichment broth to 10 ml TT broth. Incubate in a water bath 18-24 
hours at 42  deg.C. Incubation of the RV medium and TT broth in the 
water bath is termed the selective enrichment process. Following 
selective enrichment, transfer and combine 0.5 ml of TT broth and 0.5 
ml RV medium into a single tube containing 10 ml prewarmed [42  deg.C 
(107.6  deg.F)] TSB+DNP+n broth. Incubate in a water bath 5-8 hours at 
42  deg.C (107.6  deg.F). Continue as described in this kit's 
instructions for (c) raw foods.
B. Sprouts - Thirty 50 g analytical units of sprouts were collected for 
Salmonella analysis. Aseptically weigh out a 25 g subsample from each 
analytical unit and transfer each subsample to a sterile blending jar 
(or stomacher bag). Add 225 ml buffered peptone water plus novobiocin 
(BPW + n). Blend the 25 g sprout subsample with 225 ml BPW + n for 2 
min. Repeat procedure for remaining twenty-nine analytical units.
1. The thirty 25 g sprout subsamples may be analyzed by either of the 
following two options:
Option A:
    Each 25 g/225 ml blended sprout homogenate is poured into a 500 ml 
Erlenmeyer flask, or equivalent container, and analyzed individually.
Option B:
    Fifteen of the thirty 25 g/225 ml blended sprout homogenates are 
poured into a 6 L Erlenmeyer flask, and analyzed collectively. Repeat 
with the remaining 15 blended sprout homogenates . Thus, each sample 
consists of two 375-g composites .
2. Allow flasks to stand for 60 min at room temperature. Mix well and 
determine pH with test paper. Adjust pH, if necessary, to 6.8 + 0.2 
with sterile 1 N NaOH or 1 N HCL.
3. Incubate flasks without shaking for 18-26 hours at 35-37  deg.C (95-
98.6  deg.F). Each flask is considered to contain pre-enrichment broth.
4a. If using the Assurance Gold Salmonella Enzyme Immunoassay, transfer 
0.1 ml pre-enrichment broth to 10 ml RV medium and transfer another 1.0 
ml of pre-enrichment broth to 10 ml TT broth. Incubate in a water bath 
5-8 hours at 42  deg.C (107.6  deg.F). Incubation of the RV medium and 
TT broth in the water bath is termed the selective enrichment process. 
Following selective enrichment, transfer and combine 1.0 ml TT broth 
and 0.5 ml RV medium into a single tube containing 10 ml of prewarmed 
[42  deg.C (107.6  deg.F)] TSB + n broth. Incubate in a water bath 16-
20 hours at 42  deg.C (107.6  deg.F). Continue as described in this 
kit's instructions for (c) raw foods.
4b. If using the VIP Assay for Salmonella, transfer 0.1 ml pre-
enrichment broth to 10 ml RV medium and transfer another 1.0 ml of pre-
enrichment broth to 10 ml TT broth. Incubate in a water bath 18 24 
hours at 42  deg.C. Incubation of the RV medium and TT broth in the 
water bath is termed the selective enrichment process. Following 
selective enrichment, transfer and combine 0.5 ml of TT broth and 0.5 
ml RV medium into a single tube containing 10 ml prewarmed [42  deg.C 
(107.6  deg.F)] TSB+DNP+n broth. Incubate in a water bath 5-8 hours at 
42  deg.C (107.6  deg.F). Continue as described in this kit's 
instructions for (c) raw foods.
[FR Doc. 99-28016 Filed 10-25-99; 8:45 am]
BILLING CODE 4160-01-F