[Federal Register: October 27, 1999 (Volume 64, Number 207)]
[Notices]
[Page 57893-57902]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr27oc99-98]
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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: msmith1@bangate.fda.gov.
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/<difference>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/<difference>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.
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[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\
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\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
<greek-i> 996.09)
or
2. Reveal E. coli O157:H7, Neogen Corp., Lansing, MI.
Salmonella
1. Assurance Gold Salmonella EIA, (AOAC Official method <greek-i>
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.<bullet> VIP EHEC\5\, Biocontrol, Inc.,
Bellview, WA., or <bullet> 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 <plus-minus> 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
<bullet> Assurance Gold Salmonella EIA, or
<bullet> 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
October 25, 1999 Press Release: FDA Issues Guidance to Enhance Safety of Sprouts