#### U. S. Food and Drug AdministrationNational Advisory Committee on Microbiological Criteria for FoodAdopted May 28, 1999

Microbiological Safety Evaluations and Recommendations on Sprouted Seeds
TABLE OF CONTENTS
Executive SummaryIntroductionSprout-Associated Outbreaks Sprout outbreaks due to *Salmonella* Sprout outbreaks due to *E. coli* O157 Summary of perspectives gained from outbreak investigationsMicrobial Ecology of Foodborne Pathogens Associated with Sprouts  Seed production Sprout production PathogensCurrent Industry Practices Seed production Sprout production  Recent efforts to improve sprout safetyPrevention and Intervention Strategies for Pathogens on Seed and Sprouted SeedsFindings and Recommendations**Tables** Table 1.  Reported United States Sprout Outbreaks, 1995-1998 Table 2.  Reported International Sprout Outbreaks, 1988-1997 Table 3.  Distribution of the seed lot implicated in the 1998/99 *Salmonella* Mbandakaoutbreak and associated seed treatment strategy  **Figures** Figure 1.  Typical seed production process Figure 2.  Typical sprout production process**Appendices** Appendix 1 - Seed production Appendix 2 - Sprout production Appendix 3 - Rationale for the 5-log performance standard recommendationReferences

 EXECUTIVE SUMMARY
In 1997, the National Advisory Committee on Microbiological Criteria for Foods (NACMCF/theCommittee) was asked to review the current literature on sprout-associated outbreaks; identifythe organisms and production practices of greatest public health concern; prioritize researchneeds; and provide recommendations on intervention and prevention strategies.  In response tothis charge, the Fresh Produce Work Group (FPWG) documented the relevant epidemiology andmicrobial ecology of sprout-associated outbreaks and reviewed current industry practices andinitiatives related to the growing of seed and the production of sprouts.  

Sprouts have been identified as a special problem because of the potential for pathogen growthduring the sprouting process.  If pathogens are present on or in the seed, sprouting conditionsmay favor their proliferation.  There is no inherent step in the production of raw sprouts to reduceor eliminate pathogens.  Contaminated seed is the likely source for most reported sprout-associated outbreaks.  Research has been initiated on methods to reduce or eliminate pathogenicbacteria on seeds and sprouts and some treatments show promise.  However, to date, no singletreatment has been shown to completely eliminate pathogens under experimental conditionsused.

Finally, the Committee found that, at the time of the charge, there was a lack of fundamentalfood safety knowledge along the continuum from seed production through sprout consumption.More recently, many have become aware of the potential for this food to be a vehicle forfoodborne illness and the need for appropriate controls; however, such awareness is notuniversal.  Although seed appears to be the most likely source of contamination in sproutassociated outbreaks, practices and conditions at the sprouting facility may also impact on thesafety of the finished product.  In recent sprout-associated outbreak investigations, facilitiesassociated with outbreaks did not consistently apply seed disinfection treatments prior tosprouting.  Conversely, facilities that used seed from the same lot as an implicated facility, buthad not been associated with reported illnesses, appear to have been consistently using seeddisinfection treatments, such as 20,000 ppm calcium hypochlorite, to disinfect seed prior tosprouting.

The Committee has developed a number of specific recommendations, including:

1) The knowledge of all interested parties pertaining to the microbiological safety of sproutedseeds must be enhanced; government and industry should develop education programs for seedand sprout producers on basic principles for microbiological food safety, good agriculturalpractices, good manufacturing practices, and hazard analysis and critical control point (HACCP)systems,

2) Good agricultural practices should be systematically implemented to reduce the potential formicrobial contamination of seeds for sprout production,

3) Seed cleaning, storage, and handling practices that minimize the potential for microbialcontamination should be developed and implemented,

4) Seeds should be treated with one or more treatments that have been shown to reducepathogenic bacteria that may be present.  Intervention strategies that deliver less than a givenreduction (at this time, 5-log) in levels of *Salmonella* spp. and enterohemorrhagic *Escherichiacoli* O157 should be coupled with a microbiological testing program,

5) Establish good manufacturing practices and food safety systems, including regulatoryoversight, microbial testing, adoption of HACCP, and improved traceback, that systematicallylook for means to prevent seeds from serving as the vehicle for foodborne disease, and

6) Conduct research related to the microbiological safety of sprouted seeds, particularly in theareas of pathogen reduction or elimination, sources of contamination and its prevention, andpreventing or retarding pathogen growth during sprouting.
 INTRODUCTION
In 1995, the National Advisory Committee on Microbiological Criteria for Foods (NACMCF/theCommittee) was asked to evaluate the association between foodborne illness and fresh produce.The Committee was also asked to make recommendations that could be used to reduce the risk offoodborne illness outbreaks associated with these commodities.  NACMCF established a FreshProduce Work Group (FPWG) to address this issue and in 1998, published a report entitled"Microbiological Safety Evaluations and Recommendations on Fresh Produce" (NACMCF,1998). 

Within that document, sprouted seeds were identified as a special problem because of thepotential for pathogen growth during production.  Sprouts are produced by first soaking viableseed in water and then placing the seed in a warm, humid environment for an average of 3 to 7days to foster germination and sprout growth.  These conditions also provide nearly idealconditions for the exponential growth of bacteria.  If pathogens are present on or in the seed,sprouting conditions may allow for their proliferation. 

Since 1995, sprouts have been increasingly implicated in foodborne outbreaks.  In 1997, theCenters for Disease Control and Prevention submitted a request to the Food and DrugAdministration/Center for Food Safety and Applied Nutrition (FDA/CFSAN) suggesting thatthere be an expert consultation regarding the recent foodborne illness outbreaks associated withsprouts.  Subsequently, the FDA asked NACMCF to review the current literature on sprout-associated outbreaks; identify the organisms and production practices of greatest public healthconcern; prioritize research needs; and provide recommendations on intervention and preventionstrategies.  In response to this charge, the FPWG documented the relevant epidemiology andmicrobial ecology of sprout-associated outbreaks and reviewed current industry practices andinitiatives related to the growing of seed and the production of sprouts.  The FPWG toured seedmills and sprout manufacturing facilities in Northern California to better understand currentindustry practices.  Producers were very helpful in working with the FPWG to evaluate potentialmicrobial hazards associated with sprouts.  The Work Group also participated in a publicmeeting held September 28 and 29, 1998, to discuss the current state of science and technologyin relation to the control of pathogenic bacteria on sprouts. 

 This paper contains an overview of sprout-associated outbreaks, microbial ecology of bacterialpathogens associated with sprout outbreaks, current industry practices and recent efforts toimprove the safety of sprouts, prevention and intervention strategies, research needs andrecommendations for improving the microbial safety of sprouted seeds. 
 SPROUT-ASSOCIATED OUTBREAKS
Microbiological surveys have shown the presence of a variety of foodborne pathogens in sprouts.*Salmonella* spp.*, Listeria monocytogenes*, *Staphylococcus aureus, Bacillus cereus*and*Aeromonas hydrophila* have been isolated from sprouted seeds, including alfalfa, mung bean,cress, soybean, and mustard (Beuchat, 1996).  *Escherichia coli* O157, various serotypes of*Salmonella*, and*Bacillus cereus*have been the causative agents of documented outbreaks offoodborne illness associated with sprouts (Jackson, 1998).  This overview will focus on*Salmonella* and *Escherichia coli* O157 sprout-associated outbreaks in the United States from1995 through the spring of 1999. 

During this time frame, there were nine reported commercial sprout outbreaks in the U.S., sevenof which were due to different *Salmonella* serotypes, and two to *E. coli* O157.  The number ofculture-confirmed cases in each of these outbreaks ranged from eight to more than 500, and morethan 1,200 cases have been reported overall (Tables 1 and 2).  Some of the outbreaks involvedmultiple states and other countries, while others occurred in one or two states.  Alfalfa sprouts,the most common form of green sprouts available in the marketplace,  were implicated in alloutbreaks, although in two outbreaks, other types of sprouts were also either culture-positive, orwere mixed with the alfalfa sprouts.  In all of the reported outbreaks, the likely source ofcontamination was seed.  However, in the large 1996 *Salmonella* Montevideo/Meleagridisoutbreak unsafe sprouting practices may also have contributed to the contamination of sprouts.

The problem is not restricted to the U.S.; sprout-associated outbreaks have also been reportedfrom other countries, including the United Kingdom, Sweden, Finland, Japan, Denmark, andCanada.  Alfalfa sprouts were often implicated in these non-U.S. outbreaks, but mung bean andcress sprouts also were associated with outbreaks.  Radish sprouts were associated with a verylarge *E. coli* O157:H7 outbreak in Japan in 1996 and a smaller outbreak in 1997.  The frequencyof foodborne disease attributable to mung beans and soy bean sprouts is generally lower than thatassociated with "green" sprouts (e.g., alfalfa, clover, radish) since sprouts of beans are typicallycooked before consumption. 
 
Sprout-Associated Outbreaks Due to *Salmonella*

***Salmonella*****Stanley**

In 1995, a  *S*. Stanley outbreak raised concerns and provided important insights about sprout-associated salmonellosis (Mahon et al., 1997; Puohiniemi et al., 1997).  Between March and Juneof that year, there was a marked increase in the number of cases of  *S*. Stanley in Arizona,Michigan and Finland.  Ultimately, 242 culture-confirmed cases in 17 states and Finland wereattributed to this international outbreak.  Initially, 22 *S*. Stanley cases in Arizona were identifiedand a case-control study implicated consumption of alfalfa sprouts as being significantlyassociated with illness.  Alfalfa sprouts were also identified as the vehicle by independent case-control studies in Michigan and Finland (Mahon et al., 1997).

Laboratory analyses of *S*. Stanley patient isolates indicated that the U.S. and Finland cases wererelated.  The *S*. Stanley isolates from the U.S. and Finland had a unique antimicrobial resistancepattern (resistance to tetracycline, bactrim, and kanamycin, and sensitivity to ampicillin).Molecular subtyping by pulsed-field gel electrophoresis (PFGE) also indicated the same uniquepattern for the U.S. and Finnish isolates.  Analysis of alfalfa seeds and sprouts from the smallamount of the seed lot remaining did not yield *S*. Stanley.

Follow-up investigations of the outbreak indicated that contaminated alfalfa sprouts were grownby at least nine different sprout producers.  These producers obtained alfalfa seed from a singleU.S. seed supplier who purchased the seeds from a seed shipper in the Netherlands.  This sameDutch shipper also supplied the seed for the sprouts eaten by the patients in Finland.  The seedwas not grown in the Netherlands, but, due to mixing of seed lots, it was not possible to determinethe ultimate source of the seed.  Evidence of rodents and birds was observed within the seedconditioning facility. 

***Salmonella*****Newport**

In late 1995 and early 1996, an outbreak of 133 cases of *S*. Newport infection in Oregon andBritish Columbia was recognized.  Case-control studies implicated alfalfa sprouts.  *S*. Newportwas isolated from both alfalfa sprouts and seeds from which they were grown and molecularsubtyping by PFGE showed that a single strain caused the outbreaks.  A traceback showed that thecontaminated seed came from a single lot from a Dutch shipper.  Subsequent investigationindicated *S*. Newport outbreaks due to this contaminated seed lot occurred in six states andDenmark (Van Beneden et al., 1999).  Analysis of the implicated seed lot by the most probablenumber method (MPN) yielded 0.1-0.6 CFU of *S*. Newport/25 g of seed.  Sprouting of theimplicated seed lot also yielded *S*. Newport.  

***Salmonella*****Montevideo and *Salmonella* Meleagridis**

In May through July 1996, approximately 500 culture-confirmed cases of *Salmonella* serotypesMontevideo and Meleagridis occurred in Nevada and California (Farrar and Mohle-Boetani,1999).  One elderly patient died from sepsis.  A case-control study revealed that alfalfa sproutswere the only item associated with illness.  The same strain of *S*. Meleagridis was isolated frompatients as well as from sprouts obtained from retail stores and the sprouting facility.  Seedsamples, however, did not yield either serotype.  All of the implicated sprouts were produced atone facility and were sprouted from alfalfa seed grown locally in California.

Investigation of the sprout facility revealed unsanitary sprouting practices such as the presence offlies and rodent droppings, and use of the same plastic buckets to collect both finished sprouts andsprouts that had fallen on the floor.  Sub-optimal employee hygiene was also noted.  At the farmwhere the alfalfa seed was grown, several potential risk factors were observed including: 1) use ofchicken manure to fertilize the field, 2) use of canal water for watering crops, 3) transport ofalfalfa in unclean vehicles, and 4) the presence of livestock next to the alfalfa field. 

***Salmonella*****Infantis and *Salmonella* Anatum**

An outbreak of *Salmonella* serotypes Infantis and Anatum in Kansas and Missouri in 1997resulted in 109 culture-confirmed cases.  Alfalfa, rose, radish and snow pea sprouts yielded bothserotypes, and the alfalfa seeds yielded *S*. Anatum.  The implicated sprouts were produced at asingle facility.  The seed was locally grown and came from many surrounding farms (Pezzino etal., 1998; Slutsker, 1999). 

***Salmonella*****Senftenberg**

Two clusters of *Salmonella* Senftenberg infection occurred in Nevada and California in late 1997through July 1998.  The 60 culture-confirmed cases in both clusters were shown to be due to thesame strain of *S*. Senftenberg.  The implicated sprouts were an alfalfa/clover sprout mixture froma single local sprout producer.  The sprout producer associated with this outbreak inconsistentlyused a chlorine disinfection treatment on seed prior to sprouting (Farrar and Mohle-Boetani,1999).

Cultures of immature sprouts from a growing drum in the facility associated with the outbreakyielded the pathogen but analysis of  the clover and alfalfa seeds did not yield the pathogen.  Itwas not possible to determine definitively which type of sprout in the alfalfa/clover mix causedthe outbreak.  However, clover may be more likely, since clover seeds from one production lotwere used during the entire outbreak period, whereas the alfalfa seed source changed in March1998.  Further, 10,000 pounds of alfalfa seed from one of the implicated seed lots was alsodistributed to sprouting facilities throughout Washington without reports of *Salmonella*Senftenberg infections. 

***Salmonella*****Havana/Cubana**

An outbreak of 18 cases of *S*. Havana infection in Arizona and California in May of 1998 waslinked to consumption of alfalfa sprouts produced by one large California sprout producer and,possibly, one small producer.  The large sprouting facility claimed to soak seeds in 2,000 ppmchlorine for 30 minutes, followed by a 300 ppm chlorine soak for several hours prior to sprouting.However, documentation at the facility prompted questions about the consistency of theapplication of chlorine treatments.  The small sprouting facility utilized a pre-soak treatment,containing approximately 1,000 ppm chlorine, for seed disinfection (Farrar and Mohle-Boetani,1999).

Subsequently, a cluster of 22 cases of  *S*. Cubana occurred during May through August of 1998 inArizona, California, Maryland, New Mexico, and Utah that was linked to consumption of alfalfasprouts from the same large California sprout producer identified in the *S*. Havana outbreak.  Thesame seed lot was used by the implicated producer to produce the sprouts associated with bothoutbreaks.  Analysis of the implicated seed lot yielded *S*. Havana, *S*. Cubana, and *S*. Tennessee.The *S*. Havana and *S*. Cubana strains isolated from the implicated seed lot had PFGE patternswhich were indistinguishable from patient isolates.

***Salmonella*****Mbandaka**

In January through March 1999, a *Salmonella* Mbandaka outbreak occurred in Oregon,Washington, Idaho, and California that was associated with alfalfa sprouts.  A total of 75 caseswere reported (Oregon, 31; Washington, 19; Idaho, 5; California, 20) at the time that thismanuscript was prepared.  Cases occurring in Oregon, Washington, and Idaho were associatedwith sprouts produced from a single Washington sprouting facility that was not consistentlytreating seed with a disinfectant (Keene, 1999).  A single lot of seed, grown in SouthernCalifornia and obtained from a seed conditioning facility in California was used by theWashington sprouting facility.  *S.* Mbandaka was isolated from alfalfa seed from the implicatedseed lot, alfalfa sprouts from the Washington manufacturing facility, and from alfalfa sproutsfrom aseptically sprouted seeds.  Subsequent investigations of the distribution of the contaminatedseed lot provided interesting information concerning the effectiveness of seed treatments to reducepathogen levels.  The implicated seed lot was distributed to and sprouted by 4 other sproutproducers in California (3) and Florida (1) (Table 3).  Two of the California sprout producers useda calcium hypochlorite seed treatment (2,000 - 20,000 ppm) before germination of the seeds.  Thethird California sprouter was apparently not using a chlorine treatment for seeds.  The Floridasprouter presoaked the implicated seed lot in 20,000 ppm calcium hypochlorite, 3 times for 20minutes per soak prior to sprout germination.  Only the facilities that did not consistently pretreatseeds were linked to *S.* Mbandaka infections.
 
Sprout Outbreaks Due to *E. coli* O157

**Michigan/Virginia**

In 1997, an outbreak of 108 cases of *E. coli* O157:H7 in Michigan and Virginia wasepidemiologically associated with sprouts (CDC, 1997).  The isolates were indistinguishable onmolecular subtyping by PFGE, suggesting a common source.  Traceback revealed that allimplicated alfalfa sprouts were produced at a single sprouting facility in each state.  Sproutsgrown by the Michigan sprouter at the time of the outbreak came from two lots of seeds; one fromIdaho and the other from Australia.  The Virginia sprout manufacturer used the same lot of Idahoseeds as one of the lots used in Michigan.  Cultures from this seed lot did not yield *E. coli*O157:H7. 

Further investigations revealed that seed may have been contaminated at the farm where thealfalfa was grown.  On the alfalfa farm in Idaho where the seeds were harvested, several possiblesources of contamination from cow and deer manure were noted.  Some fields were irrigated withwater drained from neighboring fields where manure was applied and some alfalfa fields weredirectly adjacent to cattle feed lots.  Some alfalfa was grown next to a deer refuge, and deer wereobserved in these fields daily.

**California**

In June of 1998, another sprout outbreak occurred in Nevada and California involving anon-motile strain of *E. coli* O157.  Eight culture-confirmed cases were linked to consumption ofan alfalfa/clover sprout mixture from the same sprout producer implicated in the *Salmonella*Senftenberg outbreak.  This producer inconsistently used chlorine disinfection of seed prior tosprouting.  Laboratory analysis of seeds, sprouted seeds, and environmental samples did not yield*E. coli* O157:NM (Farrar and Mohle-Boetani, 1999).

**Japan**

In contrast to the size of the outbreaks in the U.S., a very large outbreak of over 6,000culture-confirmed cases of *E. coli* O157:H7 infections occurred in Japan in 1996.  Consumptionof radish sprouts was strongly linked to illness (Nat'l Inst. Inf. Dis., Ministry of Health andWelfare, Japan, 1997).  In 1997, a smaller outbreak occurred with 126 cases, in which an identicalstrain of *E. coli* O157:H7 was isolated from patients and from radish sprouts obtained from apatient's refrigerator (Gutierrez, 1997).
 
Summary of Perspectives Gained from Outbreak Investigations

Outbreak investigations identified several factors that affect the microbial safety of sproutedseeds.  To date, contaminated seed have been the likely source for most, if not all, outbreaks.Seed contamination could have occurred at the farm, seed processor, or sprouting facility.  Theprocesses used for the production of sprouted seed offer ample opportunity for crosscontamination from a few seeds or sprouts to the entire production lot.   Most seed used forsprouting is not grown for human consumption.  The seed is generally grown, milled, and storedunder conditions where contamination can readily occur.  Frequent failures to isolate pathogensfrom implicated seeds suggests that seed contamination may be intermittent, at very low levels, orunequally distributed within seed lots.  However, even low levels of human pathogens are aconcern.  Conditions during sprouting (time, temperature, water activity, pH, and nutrients) areideal for growth of pathogenic bacteria such as *Salmonella* and *E. coli*.   

In recent outbreaks, investigations have attempted to determine the extent to which certainpractices, such as seed disinfection treatments, are being used by sprout producers associated withan outbreak.  In general, facilities associated with recent outbreaks often did not apply seeddisinfection treatments, applied treatments inconsistently, or used disinfectants at relatively lowlevels.  Conversely, facilities that traceforward investigations have identified as having used seedfrom the same lot as an implicated facility, but that have not been associated with any reportedillnesses, appear to have been consistently using seed disinfection treatments, such as 20,000 ppmcalcium hypochlorite, to disinfect seed prior to sprouting.  While there may be other mitigatingfactors (such as product volume and amount of implicated seed used) these observations supportthe efficacy of seed disinfection treatments as a means to reduce the potential of sprout-associatedfoodborne illness outbreaks.
 
MICROBIAL ECOLOGY OF FOODBORNE PATHOGENS ASSOCIATED WITHSPROUTS
Prior to sprouting, seeds can harbor significant levels of microorganisms.  Various surveys haverevealed aerobic plate counts (APCs) of 3 - 6 x 104 CFU/g (Andrews et al., 1979), 9 x 105 CFU/g(Andrews et al., 1982), and 5 - 400 x 103 CFU/g (Prokopowich and Blank, 1991) on alfalfa seeds;1 - 20 x 104 CFU/g on mung beans (Andrews et al., 1982); 1 x 105 CFU/g on onion seeds(Prokopowich and Blank, 1991); and 3 x 107 CFU/g on rice seeds (Piernas and Guiraud, 1997a).

The microflora of the seeds increases during sprouting, and high levels of microorganismstypically associated with commercially sprouted seeds have been recognized for many years.Alfalfa sprouts and bean sprouts from retail outlets had APCs of 108 - 109 CFU/g (Patterson andWoodburn, 1980), and retail onion sprouts had > 109 CFU/g (Prokopowich and Blank, 1991).Mung bean sprouts obtained from restaurants had APCs of > 106 CFU/g (Sly and Ross, 1982).  Itis important to note that high microbial levels *per se* are not necessarily of public health concernin sprouts.  It is the presence and growth of pathogenic microbes that is the concern. 

While fresh produce, including sprouts, can serve as a potential source of many types offoodborne pathogens (e.g., bacterial, viruses, protozoa, fungi, and helminths) (NACMCF, 1998),bacterial pathogens are of particular concern with sprouts.  This reflects the fact that theenvironmental conditions and nutrients present during sprout production provide excellentconditions for the outgrowth of most pathogenic bacteria, if present.  Pathogens can grow toelevated levels since there are no inherent steps in the production of sprouts that either preventbacterial growth or eliminate them entirely.
 
Seed Production 
Pathogenic bacteria could be introduced to sprouted seed by a number of pathways, including viathe seeds, the water used during germination and sprouting, unsanitary production practices, ormishandling by the consumer (Patterson and Woodburn, 1980).  However, epidemiologicalinvestigations suggest that seed are the likely source in most, if not all, sprout-associated illnessoutbreaks (Puohiniemi et al., 1991; CDC, 1997a; Mahon et al., 1997).

Little information is available on how seeds become contaminated with bacterial pathogens.Since seeds are raw agricultural products, they could be contaminated by a variety of potentialsources of fecal contamination, including contaminated agricultural water, use of inadequatelytreated manure as a fertilizer, location of fields near animal rearing facilities, access by feralanimals, and inadequate agricultural worker hygiene  (NACMCF, 1998).

Once present on or in seed, pathogens are likely to survive for extended periods of time.  Studieshave shown that *Salmonella* can survive for months under dry conditions such as those used tostore alfalfa seeds (Mistcherlich and Marth, 1984).  The natural microflora on rice seed showedlong-term viability, decreasing by less than 0.5 log after 260 days of refrigerated storage andapproximately 2 logs after 277 days at ambient conditions (Piernas and Guiraud, 1997a).  *Erwiniaherbicola*, a common saprophyte of alfalfa seed, could be isolated for at least 3 years aftertreatment of seeds with 1% sodium hypochlorite (Moline and Kulik, 1997). 
 
Sprout Production 

Many of the sources of contamination described above (e.g., untreated or improperly treatedwater, animal waste or manure, poor sanitation of equipment and poor personal hygiene) couldalso be potential sources of contamination for seeds or sprouts at the sprouting facility.  Althoughseeds are suspected to be the most likely source of contamination, contamination of water usedduring sprouting could be a source of initial contamination or a vehicle for subsequent crosscontamination.  When only the roots of fully developed radish sprouts were immersed in watercontaining *E. coli*O157:H7, the pathogen was found throughout the edible portion (Hara-Kudo etal., 1997).  Likewise, *E. coli*O157:H7 was found both on the outer surfaces and the inner tissue ofradish sprouts grown from artificially inoculated seeds (Itoh et al., 1998).

The key aspect of sprouts that increases the risk of foodborne disease compared to other freshproduce, is the exponential growth of bacteria during sprouting.  Microorganisms on seeds cangrow quickly under the favorable conditions of the sprouting process (e.g., water activity,temperature, pH, time, and nutrients).  An overnight soak of seeds in tap water caused a 10-foldincrease in aerobic plate counts (Prokopowich and Blank, 1991).  Within the first two days ofsprouting, microbial populations on rice seeds increased approximately 2 logs (Piernas andGuiraud, 1997a) and approximately 3 logs on alfalfa seeds and mung beans (Andrews et al., 1982;Splittstoesser et al, 1983).  Sprouts germinated in sterile containers in the laboratory had APCs ashigh as 9.2 x 108 CFU/g, an indication that microbial growth during sprout production can occurunder sanitary conditions (Splittstoesser et al., 1983).  

If pathogenic bacteria are present, they can grow to high levels.  In artificial inoculations of 200 -500 spores/g,*B. cereus*increased to 103 - 107 CFU/g during sprouting of mung bean, alfalfa andwheat seeds (Harmon et al., 1987).  On naturally contaminated alfalfa, mung bean, and wheatseeds harboring <3 - >100 MPN *B. cereus* /g, pathogen levels increased 1,000- to 10,000-fold to<103 to >106 CFU/g during sprouting (Harmon et al., 1987).  Rice seeds artificially inoculatedwith *Listeria innocua*  (500 CFU/g)**and *B.  cereus*(3,100 CFU/g) supported growth of themicroorganisms to 108 and 107 CFU/g, respectively, after 48 hours of sprouting (Piernas andGuiraud, 1997a).  *S.*Stanley increased by 2.5 logs in 24 hours during the germination ofcontaminated alfalfa seed and by another 1 log during the first 18 hours of sprouting (Jaquette etal., 1996).  These levels decreased only slightly during 10 days of refrigerated storage of thesprouts.  Pooled *S.*Anatum and *S.*Montevideo**inoculated onto mung bean seeds increased by 4 -5 logs during sprouting, and pooled *Salmonella*Eimsbuettel and *Salmonella* Poona inoculated onalfalfa seeds increased by 3 - 4 logs during sprouting (Andrews et al., 1982). *E. coli*O157:H7inoculated onto radish seeds increased by 4 - 5 logs during 24 hours of sprouting (Hara-Kudo etal., 1997). 
 
Pathogens 

As indicated above, sprouted seeds represent a unique microbial food safety concern due to thepotential for certain pathogenic bacteria to grow rapidly during the germination and sprouting ofthe seeds.  For sprouts contaminated by pathogens that do not grow during sprouting (protozoa,viruses), the risks of adverse public health consequences are similar to those already noted forfresh produce (NACMCF, 1998).  In fact, the risks associated with those microorganisms mightbe reduced due to the extensive washing that sprouts receive during their production.  Thus,pathogenic bacteria that either cannot grow under the conditions encountered during sproutproduction (e.g., *Campylobacter jejuni*) or that are not likely to be competitive enough to reachthe levels needed to have an adverse public health impact (e.g., *Staphylococcus aureus*) are notconsidered to be an increased risk in sprouts compared to other fresh produce.

The following are pathogens that have either been implicated with sprouted seed-associatedoutbreaks or have been identified as being a potential source of increased risk due to their abilityto proliferate during sprouting. 

***Salmonella***

As noted in the preceding section, *Salmonella* has been responsible for several large sprout-associated outbreaks worldwide (O'Mahony et al., 1990; Oregon Health Division, 1995; Mahon etal., 1997; Puohiniemi et al., 1997; Farrar and Mohle-Boetani, 1999).  Reported outbreaks ofsalmonellosis associated with sprouted seeds suggest an initial low level contamination of theseeds, followed by growth during sprouting (Splittstoesser et al., 1983; Mahon et al., 1997).Salmonellae have been shown to grow during the sprouting process (Splittstoesser et al., 1983;Andrews et al., 1983).  

**Enterohemorrhagic*Escherichia coli***

Enterohemorrhagic *E. coli* have been responsible for several large sprout-associated (radish andalfalfa) outbreaks (CDC, 1997; Wantabe and Okasa, 1997).  Epidemiological and/ormicrobiological evidence suggest that seeds were the source of the pathogen (Itoh et al., 1998).  *E.coli* O157 can grow rapidly to large populations during sprout production.

***Listeria monocytogenes***

*L. monocytogenes* is associated with soil, plant and animal products, and food processingenvironments.  Because it is so ubiquitous, there are multiple opportunities for *L. monocytogenes*to contaminate either seeds or sprouts.  *L. monocytogenes* can grow at refrigeration temperatureson a variety of produce, including sprouts (Buchanan, 1999; Lovett, 1989).  This pathogen hasbeen isolated from commercially produced sprouted seeds but no cases of human listeriosis havebeen linked to those sprouts.  Whether the growth amplification kinetics seen with *Salmonella* and*E. coli* O157 during the sprouting process also occur with *L. monocytogenes* is unknown.  At thepresent time, it is unclear whether the presence and growth of *L. monocytogenes* in sprouts issignificantly different from the presence and growth of *L. monocytogenes* in other fresh produce.

***Bacillus cereus***

*B. cereus* is a ubiquitous spore-forming bacterium.  It is commonly found in soil and on plants(Kramer and Gilbert, 1989).  In 1973, an outbreak was associated with the consumption of sprouts(a mixture of soy, cress, and mustard seeds packaged in a seed sprouting kit) contaminated with *B.cereus*.  As with other pathogens associated with outbreaks from contaminated sprouts, the likelysource was seed.  *B. cereus* is capable of growth under seed sprouting conditions (Harmon et al.,1987).

***Yersinia enterocolitica***

*Yersinia enterocolitica* can be found in diverse foods of animal origin including pork, beef,poultry, and dairy products, and is commonly isolated from different environments such as lakes,rivers, wells, and soil (Kapperud, 1991).  An outbreak of yersiniosis was associated with eatingnon-commercially produced bean sprouts that were grown using pond water (Cover and Aber,1989).  As a psychrotroph, it must be assumed that *Y. enterocolitica* can grow on sprouted seedsduring refrigerated storage (Chao et al., 1988) just as it can with other products.  The bacterium islikely to be capable of growth during sprout production, but no specific data are available.

***Shigella***

*Shigella,* because of its low infectious dose (10-100 organisms), its dissemination in fecally-contaminated water, its ability to proliferate in vegetables, and the high degree of handling oftenassociated with the production and packaging of sprouts, must be assumed to be a potential causeof foodborne disease in sprouted seeds (Rafii et al., 1995).  However, no reports of this pathogenlinked to foodborne disease outbreaks associated with sprouted seeds were identified. 
 CURRENT INDUSTRY PRACTICES
Contamination of sprouted seed can occur at any point in the farm to fork continuum.  However,because of the special nature of sprouts, this discussion will focus on two areas of concern, i.e.,seed production in the field and seed conditioning facility, and sprouted seed production.  Asmentioned previously, seed appears to be the primary source of contamination in sprout-associated outbreaks.  Further, the conditions during the sprouting process are nearly ideal for theexponential growth of pathogenic bacteria if they are present on or in the seed.  After sprouting(e.g., during packaging, distribution, retail sale, and preparation for consumption) microbialhazards are similar to those previously identified for fresh produce (NACMCF, 1998). 

Between December 1997 and March 1998, the California Department of Health Services, Foodand Drug Branch (CDHS/FDB) and the United States Food and Drug Administration (FDA)conducted an inspection survey of 45 sprout producers in California.  The purpose of the surveywas to identify products produced and processes used, and to obtain baseline information oncurrent industry practices in California as they may impact on the microbiological safety ofsprouts. 

Information collected was compared against the Voluntary California Sprout Growers Guidelines(California Sprout Working Group, 1997) which was distributed to all known sprout producers in1997.  Information was also collected on demographics of the firm, and on subjects such as seedsource and handling, water source and quality, facility sanitation, and employee hygiene.  A reportof survey results and recommendations was released in May, 1998 (CDHS and FDA, 1998) and isfurther discussed in the sprouted seed production section below and in Appendix 2.

In July 1998, the FPWG, FDA, and CDHS toured seed production fields, seed distributors, andsprout manufacturers in California to further understand current industry practices and to observepractices used to reduce or prevent contamination of sprouts.   The basic processes included intypical seed and sprout production operations, and a discussion of recent efforts to improve thesafety of sprouted seeds follows.
 
I.  Seed Production

Plants for seed production are grown in typical agricultural environments and seed are generallytreated as a raw agricultural product.  Potential sources of contamination in the field includeagricultural water, improperly managed animal manure, contact with wild animals, and inadequateworker hygiene.  In addition, domestic animals may be allowed to graze on alfalfa fields.  Whilesuch contact is not likely to be a significant problem for the primary use of seed, i.e., seed forforage production, even low level, sporadic contamination of seed for food use may result insignificant public health concerns because the sprouting process amplifies pathogen levels.  

This overview focuses on alfalfa seed production, though many aspects of production (e.g., GoodAgricultural Practices (GAPs) in the field and Good Manufacturing Practices (GMPs) duringconditioning) would be equally applicable to other types of seed.  Seed production consistsbroadly of the steps described in Figure 1.  Some growers may modify some of these practicesdepending on many factors, such as the needs of the crop, resources of the operation, andrequirements, if any, imposed by the buyer or distributor.  Alfalfa seed production andconditioning are described in more detail in Appendix 1.

Harvesting procedures expose the seed to a substantial amount of dirt and debris and likely spreadlocalized contamination throughout the harvested seed.  The processes used to sort, clean, store,and package seeds at seed mills may reduce, but are unlikely to eliminate pathogenicmicroorganisms.  If performed incorrectly, these steps could serve as a source of contamination orcross contamination.  Further, the FPWG noted the possibility that damage to seeds, eitherinadvertently or purposefully to change the seeds' germination characteristics (e.g., scarification),could aggravate contamination by making removal of pathogenic microorganisms duringsubsequent steps more difficult.

For most crops, only a small proportion of harvested seed goes to sprout manufacturers.  Further,the decision whether to direct seed to agricultural uses or to sprouting is often not made until afterharvest.  Thus, the seed grower does not necessarily know whether seed will be sold for food useand, therefore, may have little incentive for following GAPs.  Finally, seed processing, shippingand selling practices often involve mixing multiple lots of seeds of different origins, complicatingtraceback and providing an opportunity for cross contamination. 
 
II.  Sprout Production

The CDHS/FDA survey found sprouts being produced in buildings, sheds, greenhouses, modifiedbuses, agricultural fields, or a combination of these.  The 45 firms covered by the inspectionsurvey produced 24 different types of sprouts, the most frequently observed products were mungbean, alfalfa, clover, and radish sprouts.  Only 25 firms reported producing over 5,000 pounds ofsprouts per day (CDHS/FDA, 1998).

Sprout production consisted broadly of the steps depicted in Figure 2.  Some firms may add to oromit some of these practices depending on a number of factors, including the type of seed beingsprouted and the size and resources of the firm.  Sprout production is described in more detail inAppendix 2.

Although seed appears to be the primary source of contamination in sprout-associated foodborneillness outbreaks, practices at the sprouting establishment may increase or decrease the extent ofthe microbial hazards.  Poor sanitation and inadequate hygiene at the sprout production facilitycan exacerbate the problem of sprout-associated foodborne illness.  The CDHS/FDA surveyindicated there were significant gaps in sprout manufacturers' understanding and knowledge offood safety, GMPs, and regulatory requirements.  In addition, most sprout producers were notregistered as food processing establishments as required by California regulations, and thus hadnot been previously inspected for compliance with GMPs. 

Examples of gaps in knowledge of GMPs observed during the inspection survey include, but arenot limited to:
- Raw materials and other ingredients should be inspected upon receipt to ensure they areclean and suitable for processing into food.  Bags of seed which have been contaminatedwith rodent urine will glow when viewed using a blacklight.  This is a useful tool to ensureincoming product is clean and to monitor contamination by rodents during seed storage.However, at the time of the survey, the majority of firms did not blacklight or visuallyinspect incoming seed.
Seed should be stored under conditions that will protect against contamination and preventdeterioration.

At the time of the survey, less than half of the firms applied disinfection treatments to seedbefore sprouting.

Approximately 22% of firms used non-municipal well water.  Half of these firms did nottest water for microbial quality.
Many firms did not have hot water for cleaning equipment or for hand washing.

At the time of the survey, many firms were observed to have equipment that was not easilycleaned and/or was not properly stored.  

Five firms had no cooling facilities for holding finished product.  Another five firms hadcoolers above 45°F.

Approximately half of the firms surveyed reported that employees had not receivedhygiene or sanitation training in the last year.  Almost as many firms reported that they didnot currently supervise employee hygiene or sanitation.  Inspectors noted that basicsanitary knowledge was lacking for many food workers who were observed handlingunsanitary objects and then handling sprouts. 

Restrooms at some facilities were inaccessible, inadequately stocked or maintained.  Fewsprout production facilities had hand dip stations.

None of the firms surveyed had sufficient records to facilitate a complete traceback fromfinished product to the field where the seed was grown.

In August 1998, FDA issued a national field assignment directing district offices 1) to inspect 100sprouting facilities, 2) to collect samples of alfalfa, radish, broccoli, and mung beans at differentstages of the sprouting process for microbial analysis, and 3) to conduct a survey to develop aprofile of the sanitary operations currently in use in the U.S.  Districts were also instructed toidentify firms operating under insanitary conditions, document the conditions, and takeappropriate regulatory actions.  Based on preliminary evaluation of data from this national survey,FDA expects that the current practices and needs of U.S. sprout producers are similar to thoseidentified by the CDHS/FDA survey of California producers (Smith, 1999).
 
III.  Recent Efforts to Improve Sprout Safety

In August 1997, copies of the Voluntary California Sprout Growers Guidelines (the Guidelines)were mailed to 85 of the 109 firms initially identified as sprouting operations (California SproutWorking Group, 1997).  Of the 45 firms that were found to be sprouters and therefore covered bythe CDHS/FDA inspection survey, 11 stated that they did not receive the Guidelines and had noopportunity to make improvements.  Twenty four firms did not comment on whether they hadreceived the Guidelines or made improvements.  Eight firms reported making changes andimprovements; 7 as a result of the Guidelines and 1 based on sanitation guidelines received fromthe International Sprout Growers' Association (ISGA).  Changes made by these firms indicate thetypes of improvements that might be made by other firms that are effectively reached byeducation and/or inspection.  Changes included:
- 			Improving rodent control,  			Purchasing and using a blacklight,
		Replacing equipment to facilitate cleaning,
	Initiating sanitation programs,
			Creating a written standard operating procedures manual and/or HACCP program,
			Initiating a study to determine optimum procedures for seed disinfection and sanitation ofequipment and non-food contact surfaces,
		Purchasing and installing air curtains, and
	Initiating finished product testing for coliforms, 
E. coli
 O157:H7, and 
Salmonella
 sp.
CDHS/FDB developed and implemented a sprout specific training course to educate sproutedseed producers on the potential risk of their products, as well as federal, state, and localrequirements, GMPs and standard operating procedures (SOPs), appropriate sanitation anddisinfection procedures, and additional technical assistance resources available to assist sproutproducers.
In August 1998, CDHS and FDA's Center for Food Safety and Applied Nutrition (CFSAN)initiated activities that led to California obtaining clearance for the use of 20,000 ppm calciumhypochlorite for disinfecting seeds used for sprouting within California.  USDA's AgriculturalResearch Service (ARS) assisted in developing an emergency permit application, and theEnvironmental Protection Agency (EPA) quickly reviewed the application for the use of 20,000ppm calcium hypochlorite for disinfecting seeds for sprouting throughout the U.S.  Theexemption was granted by EPA in November 1998.  The exemption is only valid for one year.One of the stipulations on extending an emergency permit, if necessary, is that the registrants beactively pursuing a full registration.
The International Sprout Growers Association is instituting a voluntary quality assurance programin which members agree to follow ISGA sanitary guidelines based on GMPs.  Sprout producersthat pass a third party inspection may choose to label their products as ISGA-certified.  ISGAcertification requires that sprout producers apply a seed disinfection treatment of 20,000 ppmcalcium hypochlorite according to ISGA or EPA directions.  An alternative treatment may be usedif it reduces CFUs by  3.5 log as shown by data in a refereed scientific journal and complies withEPA and FDA requirements.  ISGA recommends that, at a minimum, third party inspectors lookfor general good sanitary conditions; current state and federal food processors registration; proofthat seed disinfection is being done; written SOPs and standard sanitary operating procedures(SSOPs); written pest control management procedures and evidence that procedures are beingfollowed (e.g., a pest control log); evidence of employee or manager GMP training within the lastyear; and verification of product cold chain and shipping at45°F.
In August 1998, a Sprouts Task Force initiated research at the National Center for Food Safetyand Technology (NCFST) in Illinois to study the effects of commercial sprouting conditions onpathogens and of potential seed treatment interventions (including chemical, heat, andirradiation).   Several treatments show potential for significant reduction, but not elimination, ofpathogens.  The task force is considering the feasibility of different treatment combinations andcriteria for microbial testing of seeds, sprouts, and irrigation water as an additional control insprout production.
Sprouts research projects have also been included in FDA's and USDA's research plans forproduce.  These projects include identifying and characterizing natural microflora, theincidence of pathogens on seeds and sprouts, and competitive interactions between naturalmicroflora and *Listeria monocytogenes* on sprouts.
 
PREVENTION AND INTERVENTION STRATEGIES FOR PATHOGENS ON SEEDSAND SPROUTED SEEDS
Prevention or intervention steps might be taken at one or more points during sprouted seedproduction.  Broadly, opportunities for prevention or intervention include strategies that 1)target seed (e.g., in the field, during conditioning, prior to distribution, or at the sproutingfacility prior to sprouting), 2) may be applied during sprouting (e.g., to processing water or toimmature sprouts), or 3) relate to the finished product.  Such strategies may include preventionof contamination, chemical and physical treatments to reduce or eliminate contamination,microbial testing, or combinations of these.
There is relatively little scientific literature available pertaining to techniques for the reductionor elimination of pathogenic bacteria on seeds and sprouts.  However, there has been a flurry ofresearch activity in the past two years which has been communicated directly to the FPWG.Since the seed used for sprouting appears to be a primary source of pathogenic bacteria causingsprout-associated illnesses, much of the research on prevention and intervention technologieshas focused on eliminating pathogens from the seed prior to sprouting.  
**Seed production**
Sources of seed contamination in the agricultural environment are similar to those described forfresh produce (NACMCF, 1998).  The likelihood of contamination in the field may be reducedby systematic implementation of GAPs.  
The FPWG found little scientific information available on the impact that seed conditioningand handling practices have on the microbiological safety of sprouted seeds.  However, asnoted earlier, opportunities for contamination or cross contamination exist.  Furthermore,damage to seed may make subsequent removal of pathogens more difficult.  Risk ofcontamination of seeds with foodborne pathogens at this stage might be reduced bydevelopment of systematic procedures to minimize contamination or cross contamination.
**Seed storage**
As noted earlier, once pathogens are present in seeds, they are likely to survive for extendedperiods of time under normal seed storage conditions.  Several researchers have investigatedpathogen survivability on seed stored under different conditions (Jaquette et al., 1996,Taormina and Beuchat, 1999).  In one study, *S.* Stanley was inoculated onto alfalfa seeds at 102-3 CFU/g.  *Salmonella* populations decreased 1 log after 8 - 9 weeks storage at 8°C.  Increasingstorage temperature to 21°C resulted in a 2 log reduction in *S.* Stanley after 9 weeks (Jaquette etal., 1996).  In another study, populations of *E. coli* O157:H7 inoculated onto alfalfa seeds(initial level of 103  log CFU/g) remained relatively unchanged after 38 weeks storage at 5°.However, within one week of storage at 25 and 37C, *E. coli* O157:H7 populations decreasedsignificantly.  When seeds were stored at 5, 25, or 37°C for 54, 13, or 8 weeks respectively,pathogens were not detectable by direct plating; however, culture enrichment revealed thepresence of pathogens in seeds after 38 weeks of storage at 25 and 37°C but not after 54 weeks(Taormina and Beuchat, 1999).  
**Seed testing**
Although epidemiological investigations have frequently identified seeds as the most likelysource of contamination of sprouts, laboratory analyses have often been unable to isolatepathogens from implicated seed.  This suggests that contamination may be sporadic and at lowlevels.  In the 1995-1996 *S.* Newport outbreak, analysis of the implicated seed lot by MPNyielded 0.1-0.6 CFU of *S*. Newport/25 g of seed (4 - 24 cells/1000 g seed) (Van Benden et al.,1999).  Analysis of the seed implicated in the 1998 *S.* Havana outbreak revealed *S.* Tennessee,*S.* Cubana, and *S.* Havana at levels of approximately 4 CFU/1,000 g (Farrar and Mohle-Boetani, 1999).  Nonetheless, sprout producers may find advantages to testing seeds forpathogens.  While a negative result does not guarantee the absence of pathogens, a positiveresult would allow a producer to avoid using seed lots that have been shown to containpathogens.
**Seed decontamination**
In general, sanitizing is more effective for reducing contamination on seeds than on sproutedseeds (Caetano-Anolles et al., 1990).  This may be due to a combination of lower levels of bothmicroorganisms and organic material present on seeds than on sprouts, and the internalizationof bacteria into sprout tissues during sprouting making them physically inaccessible tosanitizers (Hara-Kudo et al., 1997; Itoh et al., 1998).  
A successful seed decontamination treatment must inactivate microbial pathogens whilepreserving seed viability, germination, and vigor.  Seeds vary in sensitivity to antimicrobialagents and other treatments, which determines how well they germinate and grow aftertreatment.  In addition, a treatment that is effective for one type of seed may not be applicableto all types of seeds.  Seeds vary in surface features, which may influence how well anantimicrobial agent can access and inactivate pathogens on or in the seed.  Washing seed withwater alone decreases levels of *B. cereus, E. coli,*or *Salmonella*by 1 log and hence is only marginally effective in reducing pathogens (Harmon et al., 1987; Potter and Ehrenfeld, 1998).Several investigators indicated to the FPWG that alfalfa seeds are generally the most difficulttype of seed to sanitize effectively. 
A number of researchers have investigated the ability of chlorine compounds to inactivatepathogenic bacteria on seeds, particularly alfalfa seeds.  Pretreatment of the seeds prior togermination with 150 ppm of hypochlorite did not eliminate *S*. Newport (Aabo and Baggesen,1997).  Jaquette et al. (1996) examined the effects of chlorine (sodium hypochlorite treatmentsranging from 100 - 2040 ppm active chlorine) on alfalfa seeds inoculated with 102-3 CFU of *S.*Stanley/g.  Treating seed with 100 ppm active chlorine solution for 5 or 10 minutessignificantly reduced *S.* Stanley.  A further reduction, but not elimination, occurred aftertreatment with 290 ppm active chlorine.  Treatment with 1,010 ppm active chlorine did notresult in additional reduction compared to 290 ppm.  Treatment with 2,040 ppm active chlorinereduced *S.* Stanley to undetectable levels (<1 CFU/g).  A study of disinfection procedures forrice seeds showed that ethanol, hydrogen peroxide, or 1,000 ppm sodium hypochlorite reducedAPCs by 2-3 logs, but did not eliminate microflora (Piernas and Guiraud, 1997).
In another study, alfalfa seeds were inoculated with a mixture of 5 *Salmonella* serovars andsubjected to a variety of treatments at different concentrations using a variety of antimicrobialchemicals (i.e., calcium and sodium hypochlorite, hydrogen peroxide, and ethanol) (Beuchat,1997).  Significant reductions in *Salmonella* populations were observed with most increases inconcentration of the test chemical.  No adverse effects on percent germination were observedfor any treatment at any concentration.  Treatment solutions containing calcium hypochloriteand sodium hypochlorite at concentrations of 1,800 and 2,000 ppm active chlorine,respectively, 6% hydrogen peroxide, or 80% ethanol were effective in reducing populations by1000-fold after a 10 minute treatment (Beuchat, 1997).  Consistent with Jaquette et al. (1996),*Salmonella* were not detected by direct plating after treatment with 2,000 ppm active chlorine.However, in this study, where seed disinfection treatments resulted in < 1 CFU *Salmonella*/g,enrichment procedures were employed, and, in all cases, viable *Salmonella*were recovered.The author speculated that *Salmonella* trapped in cracks and crevices on the seed wereinaccessible to lethal concentrations of chemicals (Beuchat, 1997).
In a subsequent study, alfalfa seeds inoculated with*E. coli* O157:H7 at 1-2 X 106 were treatedfor 10 minutes with 2.0%, 2.5%, or 3.0% (w/v) calcium hypochlorite solutions containing13,800, 17,000, or 20,670 ppm active chlorine, respectively (Fett, 1998).  The lowestconcentration (2.0%) resulted in a 2 log reduction while higher concentrations (2.5 and 3.0%)reduced *E. coli* O157:H7 by approximately 4 logs.  No viable bacterial cells were detected onseed treated with 2.5 or 3.0% calcium hypochlorite when plating was done on selective *E.coli*/coliform Petrifilm count plates.  However, survivors were recovered by both direct platingonto Tryptic Soy Agar (TSA) as well as by enrichment.  Plating on both selective andnonselective medium indicated that some of the viable cells of *E. coli* present after treatmentwith calcium hypochlorite were injured.  The author suggested that additional reductions mayoccur when chemical treatment is used in combination with physical treatments such as lowdose irradiation or heat (Fett, 1998).
Another researcher treated seed with sodium- or calcium hypochlorite, ozonated water andhydrogen peroxide.  Of these treatments, 2% calcium hypochlorite showed the greatestreduction but did not completely eliminate the natural microflora.  Sodium hypochlorite atconcentrations greater than 1% inhibited seed germination.  Treatment of over a dozen types ofseeds in 2% calcium hypochlorite or 6% hydrogen peroxide resulted in variable germinationrates.  For example, 2% calcium hypochlorite allowed good germination of alfalfa seeds butdrastically reduced germination of onion seeds (Moline, 1999).  
Alfalfa seeds were artificially contaminated with *E. coli*O157:H7 and the effectiveness ofsodium hypochlorite, calcium hypochlorite, hydrogen peroxide, ethanol, chlorine dioxide,acidified sodium chlorite, and a consumer product (Vegi-CleanTM, Microcide, Inc., Detroit,Mich.) were tested for produce sanitation.  In no instance was *E. coli*O157:H7 eliminated (L.Beuchat, personal communication).  Calcium and sodium forms of hypochlorite at aconcentration of 2% appear to be equally effective for *E. coli*O157:H7 inactivation.  However,calcium hypochlorite may be preferable to sodium hypochlorite because it is not as volatile.  Inaddition, calcium hypochlorite solutions up to 20,000 ppm did not affect percent germination ofalfalfa seeds.  However, at high concentrations, alfalfa seed vigor (rate of germination) wasslowed (Beuchat, 1999).  Percent germination of alfalfa seed treated for 10 minutes in 3 %calcium hypochlorite solution was not significantly different from untreated seed after 2 days ofsprouting, however, the rate of germination was slower (Fett, 1998).  
The effectiveness of few other sanitizing agents for reducing the pathogen levels on seeds hasbeen reported in the literature.   A 1 - 2  log reduction in microbial counts on rice seed wasachieved with a treatment with 1% hydrogen peroxide  (Piernas and Guiraud, 1997b).Treatment with 70% ethanol was effective for reducing pathogen levels but significantlyinhibited germination.  Reducing the ethanol concentration to 10% improved germination butdecreased its microbiocidal effect (Piernas and Guiraud, 1997b).  Percent germination of alfalfaseed was also significantly decreased by treatment with 30 or 70% ethanol, precluding the useof ethanol as a sanitizer (Taormina and Beuchat, 1999).
Preliminary trials with commercially available sanitizers, such as GlycorineTM (a mixture oflauric acid and sodium lauryl sulfate), have been found to be effective in reducing  *E. coli*O157:H7 by > 2 logs per gram of alfalfa seeds.  Trials with these sanitizers have been at least aseffective as limited trials with hydrogen peroxide or other chemical seed treatments.  Researchis continuing to optimize exposure time and concentrations (Slade, 1999).  TsunamiTM (activeoxygen solution, Ecolab, Mendota Heights, Minn.) and VortexTM (active oxygen solution,Ecolab) at 80 ppm and Vegi-CleanTM at 20,000 ppm for 10 minutes reduced *E. coli* O157:H7 to< 0.5 CFU/g on alfalfa seeds.  However, *E. coli* O157:H7 was detected from treated seeds afterculture enrichment (Beuchat, 1999).
Ozone and ozonated water decreased microbial levels without adversely affecting the sproutingof black matpe and alfalfa seeds; and decreased the natural microflora on beans, peas, grain andspices by 1 - 3 logs (Naito and Shiga, 1989; Naito et al., 1988).  Hydronium ion treatment ofalfalfa seed has been initiated (Slade, 1999).
Potassium sorbate, calcium propionate, gallic acid, benzoic acid, salicylic acid,dihydroxybenzoic acids, hydroxybenzoic acids, riboflavin, phloxine b, eosin b, nisin, andcalcium-EDTA were also tested.  Of these treatment options, only calcium-EDTA and 4-hydroxybenzoic acid appear to warrant further study (Moline, 1999).  
**Surfactants**
It has been suggested that the barrier to disinfecting seeds is not in the lethality of the treatmentsolutions but in the ability of treatments to reach pathogens in the seeds.  Microbial cells inseed crevices may be protected from exposure to lethal concentrations of sanitizers and relatedsurface treatments (Caetano-Anolles, 1990; Beuchat, 1997).  The use of surfactants alonereduced *E. coli* O157:H7 populations by approximately 1 log (Beuchat, 1999).  Pre-treatmentsor co-treatment with surfactants have been proposed as a possible means of improving theeffectiveness of sanitizing agents.  Early research has not been promising.  The use ofsurfactants Tween 80 and benzalkonium chloride had little effect on improvingdecontamination of rice seed (Piernas and Guiraud, 1997b) or alfalfa seed (Beuchat, 1999)compared to disinfectant treatments alone.  However, research is continuing in this area..
**Heat**
Heat treatments have also been explored as a possible means for reducing pathogen levels onseeds.  *S.*Stanley levels were unchanged after a 10 minute soak in water at 21°C.  Soaking seedin 54°C water for 5 or 10 minutes significantly reduced but did not eliminate the pathogen.Soaking seed at 57 or 60° 5 minutes  reduced *S.*Stanley to < 1 CFU/g, without substantialloss of germination.  Higher temperatures and longer times (> 5 minutes) caused significantdeclines in germination.  Treatments up to 66°C for 10 minutes caused germination to declineto 6% (Jaquette et al., 1996).  The narrow temperature range between treatment efficacy andseed injury may make relying on heat alone to eliminate pathogens difficult on a commercialscale.
**Heat and disinfectants**
It has been suggested that pathogens that survive chemical disinfection treatments may beinjured (Fett, 1998).  Microbial populations (APCs) on rice seed decreased at least 5 log cycleswhen a 5-minute sodium hypochlorite soak was combined with heating at 60°C for 5 minutes(Piernas and Guiraud, 1997b).  Alfalfa seed were inoculated with *E. coli* O157:H7 and treatedwith various solutions at 55°C for 3 minutes.  Treatment with calcium hypochlorite (20,000ppm active chlorine) eliminated pathogens from three of three samples as determined by directplating.  When seeds were subjected to two 3-minute rinses at 55°C and at the sameconcentration of calcium hypochlorite, *E. coli* O157:H7 was detected in one of three samples.The pathogen was detected in all samples after culture enrichment (Taormina and Beuchat,1999).
In most patents associated with sprouting systems, the decontamination step, if listed, isrecommended primarily to inhibit spoilage rather than for pathogen reduction.  One U.S. patent,issued for sterilizing and cultivating seeds for sprouting, describes a procedure in which theseeds are exposed to heat for a short period, quickly cooled, and then soaked in chlorinesolution (Suzuki and Takizawa, 1997).  This treatment reduced but did not eliminate pathogens.Precise treatment conditions were not provided because of variability in the effects of heat onviability of different types of seeds, but 70°C for 20-30 seconds or 90°C for 10 seconds wasprovided as a guideline. 
**Radiation**
Gamma irradiation has been shown in preliminary studies to be an effective antimicrobialtreatment for both seeds and sprouts.   Using Cs137 and temperature control (5°C),  *E. coli*O157:H7 and *Salmonella*were inactivated while seed viability was maintained.  However, thedose required to inactivate these pathogens (up to 5 kGy) exceeds current allowable limits (1kGy) for treatment of produce.  Use of gamma irradiation in conjunction with chemicaltreatments (e.g., 20,000 ppm calcium hypochlorite) is being investigated (Thayer, 1999).   
Preliminary trials with electron beam irradiation of alfalfa seeds at 2.5 - 10 kGy at variousaccelerated voltages appear promising.  Researchers observed a linear reduction in the levels of*Salmonella* and natural microflora at increasing dose levels.  Pathogens were not detected inseeds treated at 10 kGy.  However, enrichment was not done prior to analysis.  Percentgermination was not reduced at the highest radiation level tested.  However, researchers notedphysiological changes (i.e., shortening, thickening, and curling) in the roots of sprouts grownfrom treated seeds.  Additional research on depth of penetration and combining electron beamirradiation at different levels with chemical treatments is planned (Slade, 1999).
**Other**
The use of a number of physical, non-thermal processing technologies, alone or in combinationwith antimicrobial chemicals, is virtually unexplored for seed decontamination.  Severalrelatively inexpensive technologies have not been investigated but may have potential use insprout production.  Ultraviolet light might be useful for surface disinfection of seeds, and itsactivity may be enhanced by antimicrobial chemicals such as hydrogen peroxide.  At certainultrasound frequencies, light is produced (soniluminescence), which might be useful inperoxide activation for microbial control.  Flashes of bright white light (PureBright®technology) have been effective for killing microorganisms on food surfaces.  The feasibility ofusing high pressure processing for reducing microbial levels on vegetables has been shown(Arroyo et al., 1997).  Pulsed electrical or magnetic field technologies are additional processesthat have not been tested and might be applicable to seeds.  Inactivation of microbial cellslodged in seed crevices might be enhanced by treatment of the seeds with gaseous antimicrobialcompounds (e.g., gas phase peroxide).   Incorporation of these compounds into packagingmaterials might also be useful for controlling microbial growth after sprouting.  In addition,vacuum infiltration of calcium or sodium hypochlorite may allow sanitation of surfaces ofseeds that have been resistant to current treatments.
Seed disinfection treatments may be done by the seed supplier or by the sprout producer.  Sometypes of treatments may be more easily done at one location than another.  In addition, becauseof the potential for survivors or for recontamination of treated seed, sprout producers may needto implement additional treatments just prior to sprouting. 
A reduction in pathogens of 1 - 2 logs has little practical significance because pathogenssurviving on seeds would be expected to grow during the spouting process.  Some disinfectionprocesses (e.g., 20,000 ppm calcium hypochlorite) have been identified that achieve higherreductions.  However, to date, no single treatment has been identified that can eliminatepathogens, if present, or one that can be applied to all types of seed.  It is possible thatcombining treatments may provide sufficient cumulative reductions to meet food safety goals.  
**Treatments/interventions during the sprouting process**
It may be possible to implement treatments during the sprouting process to prevent or inhibitthe growth of pathogens that survive seed treatments.  However, there is little publishedliterature in this area.  Use of rinse water, chlorinated at 100 ppm, during sprouting of mungbeans decreased counts of the natural microflora by < 1 log (Splittstoesser et al., 1983).Preliminary trials have begun for a number of other disinfectant treatments that might beapplied to sprouts or irrigation water during sprouting.  Treatments under investigation includethe use of sodium and calcium hypochlorite, hydrogen peroxide, TsunamiTM, sodium EDTA,and sodium chlorite (Fett,1999).  As with seed, successful treatments will need to be effectivein killing pathogens without being phytotoxic to growing sprouts.  
Biofilms tend to protect entrapped foodborne pathogens from the antimicrobial activity ofsanitizers and may also interfere with the activity of disinfectants applied to sprouted seedduring or after sprouting.  Studies on biofilm formation on sprouts have been initiated.Scanning electron microscopy has shown extensive formation of biofilms on commerciallyobtained sprouts and on 4-day old sprouts grown in the laboratory (Fett, 1999).
Competitive exclusion techniques, where non-pathogenic microorganisms are used to repressthe growth of pathogenic bacteria during sprouting have been suggested; however, there are fewreports in the literature.  When co-inoculated with *L. monocytogenes,*nisin-producinglactococci isolated from bean sprouts reduced the levels of the pathogen by 1 log (Cai et al.,1997).  Work has been initiated on using competitive exclusion during sprouting (Fett, 1999).Naturally occurring microbes from commercial alfalfa sprouts were isolated and tested for theirability to repress the growth of a mixture of *Salmonella*strains.  Preliminary trials have shown1 - 3 log reductions in *Salmonella*counts during a 7-day grow-out after challenge with thevarious microflora isolates.
While contamination of seeds may be at low levels and difficult to detect, microbialpopulations increase significantly during the sprouting process (Jaquette et al., 1996).Significant levels of microorganisms are recovered in the irrigation water during sprouting.Testing of sprout irrigation water for total counts and coliforms indicate that the water containsat least 90 % of the counts found in the sprouts themselves.  It is possible that testing spentirrigation water may be useful for monitoring microbial levels and detecting pathogens thatmay be present during sprouting.  Research is needed to determine an optimum testing protocol,including when and how to test, to maximize accuracy and achieve economic feasibility.  Theeffect of disinfectant treatments applied during sprouting on the level of pathogens andmicroflora in spent irrigation water is unknown.  If disinfectants kill pathogens in irrigationwater but allow pathogens to survive in sprouts, testing of irrigation water would bemeaningless (Tortorello and Fu, 1999).  
**Interventions for sprouted seed**
There have been few studies on reducing pathogen levels in finished product.   Washing maturesprouts with water decreased levels of *B. cereus, E. coli*or *Salmonella*by no more than 1 log **(Harmon et al., 1987; Potter and Ehrenfeld, 1998).  Soaking of fully developed mung beansprouts for 30 minutes in 0.5% sodium hypochlorite resulted in a 2  log decrease in counts(Splittstoesser et al., 1983).  Treating mung bean sprouts with 500 ppm sulfur dioxide reducedmicrobial counts by 1 - 2 log (Splittstoesser et al., 1983).								
As noted earlier, gamma irradiation has been shown in preliminary studies to be an effectiveantimicrobial treatment for both seeds and sprouts.  However, the dose required to inactivatethese pathogens (up to 5 kGy) exceeds current allowable limits (1 kGy) for treatment ofproduce (Thayer, 1999).
 
FINDINGS AND RECOMMENDATIONS
**Finding 1.  Food Safety Knowledge**
At the time of the charge from FDA, there was a lack of fundamental food safety knowledgealong the continuum from seed production through sprout consumption.  This included a generalperception that the sprouted seed industry was producing an agricultural commodity and not a"ready-to-eat" food.  Since then, most within the sprouted seed industry have become aware ofthe potential for this food to be a vehicle for foodborne disease and the need for appropriatecontrols.  This includes seed producers, processors and distributors, sprout producers, sproutretailers, and consumers.   However, such awareness is not universal. 

**Recommendation**

Industry and government should enhance the knowledge of all interested parties pertaining to themicrobiological safety of sprouted seeds by: 

a.	Ensuring that all stakeholders recognize that seeds and sprouted seeds could be a source offoodborne pathogens.

b.	Developing education programs for sprout growers and seed producers on basic principles offood safety microbiology, GMPs, GAPs and HACCP.

c.	Educating sprout retailers and consumers about the public health risk associated with theconsumption of sprouted seeds.

**Finding 2.  Seed Production**

Contaminated seed is the likely source for most, if not all, reported sprout-associated outbreaks.Seeds for sprouting are not currently produced in a manner different from that for seeds used forplanting.  There are multiple opportunities during seed production and harvest by whichcontamination with foodborne pathogenic microorganisms can occur.  Once present on seeds,these pathogens are likely to remain viable for extended periods.  The potential for seedcontamination is likely to be reduced by systematic implementation of good agriculturalpractices (FDA, 1998).  However, prevention of all contamination is unlikely.   

**Recommendation**

Seeds for sprout production should be grown under conditions that minimize exposure tofoodborne pathogenic bacteria.

a.	Growers should consider the presence of animal production facilities and related factors(e.g., slope of land, runoff controls) that could lead to the crop being inadvertentlyexposed to untreated manure and take steps to avoid contamination.

b.	The use of manure or domestic animal grazing on fields destined for seed productionshould be managed in accordance with good agricultural practices (FDA, 1998).

c.	Sources of agricultural water should be evaluated and managed in accordance with goodagricultural practices (FDA, 1998).

**Finding 3.  Seed Handling**

In addition to contamination during their production, seeds for sprouted seed production can becontaminated during post-harvest operations such as transport, conditioning, grading, storage,and packaging.  The risk that contamination with foodborne pathogenic microorganisms occursduring  this phase can be reduced by developing procedures and barriers that prevent introductionof the pathogen.

**Recommendation**

The potential for seed to become contaminated after harvest can be reduced by developing andimplementing seed cleaning, storage and handling practices that minimize potential sources ofcontamination that could lead to the introduction of pathogenic microorganisms.

**Finding 4.  Seed Treatment**

A number of treatments have been shown to reduce levels of pathogenic bacteria present onseeds, but none have totally eliminated pathogenic microorganisms.  Their routine use is likely toreduce the level of contamination if present and, in turn, decrease the risk for foodborne diseasewith sprouted seeds.  Some of these technologies can be applied at the sprouting facility whileothers can be applied at earlier steps in the seed production continuum. 

**Recommendation**

Seeds should be treated with one or preferably more than one treatment that has been shown toreduce the level of pathogenic bacteria on seeds.

a.	Just prior to sprouting, seeds should be subjected to one or more treatments that caneffectively reduce or eliminate pathogenic bacteria (e.g. 20,000 ppm calciumhypochlorite).

b.	The Food and Drug Administration and/or other appropriate regulatory agencies shouldexpedite the review of irradiation and other promising new technologies for the treatmentof seeds to reduce or eliminate pathogens prior to or at the sprout production facility.

c.	Based on currently available data on quantitation of pathogens in seeds and achievablereductions, seeds should undergo a combination of treatment  strategies that will achievea 5-log reduction in the levels of *Salmonella* spp. and enterohemorrhagic *Escherichia coli*O157*.*(See Appendix 3.)  This recommendation should be reevaluated as additional databecome available.  Intervention strategies that deliver less than a 5 log reduction shouldbe coupled with microbiological testing of sprouts or spent irrigation water.

**Finding 5.  Sprout Production**
While seeds have been identified as the primary source of foodborne pathogens on sproutedseeds, the procedures and practices used by sprout producers have a substantial impact on thelikelihood that pathogenic bacteria will survive and proliferate in sprouts.  This was aggravatedby what appears to have been an incomplete understanding by a segment of the sprouted seedindustry of the requirements for manufacturing and marketing of ready-to-eat foods.  There is aclearly identified need to establish good manufacturing practices and food safety systems thatsystematically look for means for preventing sprouted seeds from serving as the vehicle forfoodborne disease.

**Recommendation**

The microbiological safety of sprout production could be enhanced by:

a.	Classifying sprout production facilities as food processing operations.

b.	Ensuring that the sprout industry, in consultation with the Food and Drug Administrationand other appropriate regulatory agencies, establish and implement good manufacturingpractices for sprout production.

c.	Requiring the use of validated microbiological assays to test sprouts or their irrigationwater prior to harvest for pathogenic bacteria (see recommendation 4c).

d.	The adoption of HACCP programs by sprout producers.

e.	Development, implementation, and testing of systems that can be used by sproutproducers and seed mill operators to conduct traceback and recalls.

f.	The Food and Drug Administration expediting the review of irradiation and otherpromising new technologies that can be used to treat sprouts to reduce or eliminatepathogens.

**Finding 6.  Research**

There were substantial data gaps in the scientific information related to the microbiologicalsafety of sprouted seeds, particularly in relation to intervention technologies that can be used toprevent contamination or growth of pathogenic bacteria during seed germination and sprouting.

**Recommendation**

It is recommended that the government and industry fund research related to the microbiologicalsafety of sprouted seeds, particularly in the areas of:

a.	The development of additional technologies that can reduce or eliminate pathogenicbacteria on seeds or finished sprouts.  The research should focus on technologies that areappropriate for the small business character of the sprout industry.

b.	Identification of the sources of contamination of seeds and means for prevention.

c.	The development of means for preventing or retarding the growth of pathogenic bacteria(e.g., competitive exclusion) during seed germination and sprout outgrowth.

d.	Evaluation of seed characteristics and handling practices to determine their contributionto seed contamination, ease of decontamination, or internalization of pathogenic bacteria.

e.	Design of sprouting equipment that is easily cleaned and sanitized.

f.	Evaluate the additional risk reduction that could be achieved through the use of variouslabeling strategies. 

g.	Development of better methods for evaluating sprouts or spent irrigation water for thepresence of pathogenic microorganisms.

 TABLE 1. REPORTED UNITED STATES SPROUT OUTBREAKS, 1995-1998
**Year****Pathogen****No. of Cases****Location of Outbreak****Type of Sprout****Likely Source of Contamination****Reference**1995*S.* Stanley24217 states and FinlandAlfalfaSeedMahon et al., 1997
1995-96*S.* Newport>1337 states and CanadaAlfalfaSeedVan Beneden et al., 1999
1996*S.* Montevideo/ Meleagridis>500CaliforniaAlfalfaSprouter/SeedFarrar and Mohle-Boetani, 1999
1997*S.* Infantis/ Anatum90Kansas, MissouriAlfalfaSeedSlutsker, 1999
1997*E. coli* O157:H7108Michigan, VirginiaAlfalfaSeedCDC, 1997
1997/98*S.* Senftenberg60California, NevadaClover/AlfalfaSprouter/SeedFarrar and Mohle-Boetani, 1999
1998*E. coli* O157:NM8CaliforniaClover/AlfalfaSeedFarrar and Mohle-Boetani, 1999
1998*S.* Havana/Cubana18CaliforniaAlfalfaSeedFarrar and Mohle-Boetani, 1999
1999*S.* Mbandaka75Oregon, WashingtonAlfalfaSeedKeene, 1999

 TABLE 2. REPORTED INTERNATIONAL SPROUT OUTBREAKS, 1988-1997
**Year****Pathogen****No. of Cases****Location of Outbreak****Type of Sprout****Likely Source of Contamination****Reference**1988*S.* Saint-Paul143United KingdomMung BeanSeedO'Mahony et al., 1990
1989*S.* Gold-Coast31United KingdomCressUnkownJoce et al., 1990
1994*S.* Bovismorbificans492Sweden, FinlandAlfalfaSeedPonka et al., 1995Puohiniemi et al., 1997
1995*S.* Stanley114FinlandAlfalfaSeedKontiainen et al., 1996Mahon et al., 1997
1995*S.* Newport????Denmark, CanadaAlfalfaSeedOregon Health Division, 1995Aabo and Baggesen, 1997
1996*E. coli* O157:H7>6,000JapanRadishUnknownNat'l Inst. Infect. Dis. and Infect. Dis. Ctrl Div., Ministry of Health and Welfare of Japan, 1997
1997*S.* Meleagridis78CanadaAlfalfaSeedBuck et al., 1998
1997*E. coli* O157:H7126JapanRadishUnkownGutierrez, 1997
TABLE 3. Distribution of the seed lot implicated in the 1998/99 *Salmonella* Mbandaka outbreak, associated seed treatment strategy, and association with foodborne illness outbreak*.
**Sprouter****Pounds of implicated seed lot received****Pounds of implicated seed lot sprouted****Pounds of implicated seed lot embargoed****Seed treatment strategy****Facility implicated in outbreak**Sprouter #1 - WA2,0001,800200no consistent treatmentYES
Sprouter #2 - CA2,00002,000N/AN/A
Sprouter #3 - CA2,0001001,9002,000-20,000 ppm calcium hypochloriteNO
Sprouter #4 - CA1,0006753252,000-20,000 ppm calcium hypochloriteNO
Sprouter #5 - CA4,0003,850150no consistent treatmentYES
Sprouter #6 - FL2,0002,000020,000 ppm bleach, 3X for 20 min. eachNO
*A total of 40,000 pounds of the implicated seed lot was obtained from farmer A and processed at seed mill B.  A total of 15,000 pounds of the original 40,000 pounds of the implicated seed lot remained under the possession of farmer A and was embargoed by the state of California.  A total of 12,000 pounds of the original 40,000 pounds of the implicated seed lot was under the ownership of  seed distributor C and physically located at seed mill B and was embargoed by the state of California.  A total of 13,000 pounds of the implicated seed lot was distributed by seed distributor C to six sprouters as described above.  

 
 

 APPENDIX 1 -- SEED PRODUCTION
Specific agricultural practices may vary with the type of crop.  Some crops are grown exclusivelyfor seed while other crops may be grown for both foliage and seed.  For example, beans,including mung beans, are annuals with a single harvest per crop.  Other crops, such as alfalfa,are biennials or perennials and may be subject to repeated harvests of foliage (hay or silage) orseed or both. 

**Alfalfa Seed Production**
Approximately 80 million pounds of alfalfa seed are produced each year in the United States(Mueller, 1999).  California, Idaho, Oregon, Washington, and Nevada provide 85% of thisproduction.  Non-dormant varieties are planted for forage in areas where winters are mild.  Over90% of the seed produced in California are non-dormant seed (i.e., seed from varieties thatcontinue to grow, albeit slowly, during the winter months) and approximately 9% are seed ofsemi-dormant varieties (i.e., varieties that stop growth for a shorter time (e.g., 2 months vs. 5),compared to dormant varieties).  Seed of the more dormant varieties (i.e., varieties that do notgrow from about November through March) is typically produced in the Pacific Northwest.  

Current seed certification programs are a marketing and quality assurance tool for the foragemarket.  Use of certified seed is a guarantee to customers that the seed is of the stated variety andwill perform to expectations in terms of yield and forage quality.  Many of the quality attributesof certified seed have significantly less relevance for sprout producers compared to forageproducers.  Thus, uncertified, or common seed, is seed that most often is used for sprouting.However, there may be aspects of the current seed certification system, such as the ability totrace seed back to its source, that might be considered for incorporation into programs to improvethe safety of seed for sprouting.  

Seed for certification must pass a series of inspections and be grown under specified conditions.Each state has a seed certifying agency.  In California, seed is certified by the California CropImprovement Association (CCIA).  Growers wishing to certify seed submit an application toCCIA with information regarding previous crop history, seed lot planted, etc.  CCIA agentsinspect the fields for weed control, genetic purity, isolation from other fields, and other factors.CCIA agents certify that the seed is of the stated variety, purity, and quality.  Certified seedretains its identity through sale to the end user.  Certified seed can be traced back to the field inwhich it was grown.  More than 70% of California's total seed production is certified. 

**Agricultural Production Practices**

Properly timed cultural practices are the keys to any successful agricultural production system.Among the most important production factors that have an impact on seed yield and quality arepollination, irrigation, insects, and weed infestations.  For alfalfa seed production in California,this includes:
- Winter irrigation to provide 40-50% of the annual water requirement  Fertilization with required nutrients as determined by soil or tissue tests
Application of herbicides to control problematic weeds
Spring clipping to initiate the seed crop
Application of insecticides to eliminate harmful pests
Pollination using honey bees, leafcutter bees, or alkali bees
Crop irrigations timed to promote seed set and avoid stress that may reduce yield
Hand weeding as necessary to control noxious weeds
Desiccation of the seed crop prior to harvest
Harvest and destruction of crop residue including volunteer alfalfa plants resulting from theprevious seed crop.
**Irrigation**
An essential component of alfalfa seed production is timely irrigation scheduling.  Highest seedyields are obtained when irrigation practices prevent severe plant stress and promote slow,continuous growth through the entire production period without excessive stimulation ofvegetative growth.  Deciding when to terminate irrigation for the season is also critical.  Enoughwater is required to mature the seed, but soil moisture must be depleted prior to desiccation or theplant will not dry down adequately to prepare for harvest. 
**Insect Control**
The most important pests in alfalfa seed fields are lygus bugs that feed on the buds, blooms, anddeveloping seeds of alfalfa throughout the season though other pests may also cause significantyield reductions in some years.  Cultural and biological control options are limited in availabilityand effectiveness for controlling the most significant insect pests.  Currently, growers must relyon chemical control of pests in order to maintain economic yields.
Insecticides must be carefully selected and applied to kill the target insects without harming beesrequired for pollination.  There are few chemicals registered for alfalfa seed that effectivelycontrol insect pests.  Furthermore, in order to use them, growers must comply with labelrestrictions to insure none of the treated seed or crop residue enters the food chain.  This meansthat seed may not be used for sprouting if the grower relies on currently registered chemicals tocontrol insect pests.  Chemical companies typically cannot afford to establish tolerances for theirmaterials to allow them to be registered on alfalfa seed as a food crop. 
**Weed Control**
Control of weeds in alfalfa seed production is important to prevent competition and encouragethe growth of the alfalfa.  In addition, seed for certification must meet stringent requirements forpurity. Weed control in alfalfa seed fields is continuous from stand establishment to the finalcleaning process.  Mechanical cultivation and use of herbicides registered for alfalfa effectivelycontrol most winter and summer annual and perennial weeds.  Herbicides can be applied byground or by air.  Rainfall or irrigation is often required to activate them.  In some areas, sheepgraze seedling fields as a means of weed control.
Since chemical control is not always 100% effective, large weeding crews are often employed toremove remaining weeds from seed fields late in the season.  Modern seed cleaning equipment isavailable to separate and remove weed seeds from alfalfa seed, although a significant quantity ofalfalfa seed can be lost in the process.  Therefore, control of weeds in the field is a more efficientand less expensive practice.
**Pollination**
Alfalfa must be cross-pollinated to maintain high forage and seed yields.  Pollinator activitysignificantly affects seed yield.  Honeybees, leafcutter bees, alkali bees, or a combination areused to pollinate alfalfa for seed production in the United States.
Honeybees are used almost exclusively to pollinate alfalfa for seed production throughoutCalifornia.  They are relatively inexpensive and readily available, but they are not the mostenthusiastic pollinators of alfalfa.  Because of their inefficiency, it typically requires a longpollination season to set seed with honeybees.
**Desiccation and Harvest**(1)
Harvesting alfalfa seed involves either 1) cutting and windrow curing followed by threshing witha pickup combine, or 2) chemically desiccating followed by direct combining the standing crop.Desiccants currently registered restrict using the seed from treated plants for sprouting.Therefore, this discussion will focus on harvesting seed from plants that have been air-dried.
Alfalfa seed should be swathed during periods of high humidity or dew after 2/3 or 3/4 of thepods have changed from green to a dark brown color.  The swather cuts the alfalfa just above thecrown, and lays it in long strips, or windrows, on the stubble in the field.  Physiologically matureseed will ripen in the windrow.  Windrows are ready for threshing when the moisture content ofthe foliage is from 12-18%.   Harvesting and combining equipment may occasionally "clip" theground, taking in dirt and any contamination that is present.
Mechanical damage caused by excessive cylinder speed and lack of proper clearance betweencylinder bars and concave has been related directly to seed damage and subsequent poor seedgermination.  Damaged seed may be more difficult to disinfect as pathogens in cracks andcrevices may be more difficult to reach with liquid treatments.  Mechanical damage can beidentified and monitored with a hand lens or microscope.  Damage can be reduced andpercentage germination maintained above 90% by the following methods:
maintaining proper cylinder speed;
maintaining proper cylinder-concave clearance and clearance between augers and theirhousings;
installing rubber flights in all elevators;
disengaging the cylinder drive when no plant material is going into the header, i.e. whenturning at the end of a field; and
maintaining the proper load in the cylinder.
Seed is transported from the field to the conditioning facility in steel bins loaded on a flatbedtruck or in bottom unloading bulk trucks.  Storage bins may be uncovered or covered with metal,paper, or plastic to prevent birds or rodents from entering the bin.  Bins are often stacked, and insome cases only the top bin is covered.  Bins hold 2,700 pounds of field run seed or 3,200-3,300pounds of clean seed.
**Seed Conditioning**
Conditioning removes soil, weed seeds, and other debris from the alfalfa seed.  This isaccomplished by equipment that utilizes differences in physical characteristics of alfalfa seed andthe non-seed fraction, such as particle size, shape, density, and surface texture.  Separatingmachines include 1) air-screen cleaner, 2) specific gravity separator, 3) velvet roll seed separator,4) magnetic separator, 5) indent disc and cylinder separators, and 6) spiral separators.
The air-screen cleaner makes separations on the basis of particle size, shape, and density.  Thefield-run seed is fed into an air stream that removes lightweight seeds and impurities.  Theheavier seed and impurities are directed to and flow down or fall through a series of inclinedreciprocating screens with different diameter circular and/or rectangular slotted holes.  The topscreen removes impurities larger than the seed; the last screen separates the seed from fineparticles of soil, broken seed, and other impurities that fall through.  Cleaned seed is then passedthrough an air stream that removes any remaining light material.
The specific gravity separator separates on the basis of specific gravity and particle size.  Theseparating unit of the gravity table is a quadrangle-shaped, perforated, tilted, and wire mesh orcloth-covered surface.  Air is forced up through this surface as the deck mechanism oscillates onboth horizontal and vertical planes.  Seed is introduced at the lower corner of the deck and movesup the inclined plane to the opposite sides.  The path of the seed and foreign material isdependent upon the specific gravity of each particle and the output is divided into a series ofdistinct fractions.
The velvet roll seed separator is used for more precise cleaning and separation based uponsurface texture and shapes.  A series of paired velvet covered rolls are mounted with a fewdegrees tilt from the horizontal with spacing between the rolls so that at the upper end the velvetmakes substantial contact and at the lower end the rolls are completely separate.  This separatorremoves dirt, sand, rough-coated weed seeds, and other foreign materials that have rough edgesthat cling to the velvet.  In most plants, the velvet roll seed separator is used as a finishingmachine; however, in a modern installation it is an in-line operation following the gravityseparator.
The magnetic separator also makes separations based upon surface texture.  Seeds are fed into ascrew conveyor with a regulated amount of water and finely ground iron powder.  The ironpowder clings to rough seed coats, cracks in the seeds, chaff, soil particles, and seeds whosesurface becomes sticky when moistened.  The mixture is passed over a high-intensity,magnetically charged, rotating drum.  Sound seed does not attach to the drum, whereas thoseparticles to which the iron powder adheres are attracted to the drum and later removed.  Themagnetic separator is used extensively to remove rough-coated dodder seed.
The indent disc and indent cylinder separators are used to remove seeds with the same width orthickness on the basis of length differences.  The short seeds are lifted into pockets orindentations in a rotating disc or cylinder.  The sizes of the indentations are shallower than alfalfaseed; thus alfalfa seed is not picked up.
The spiral separator is a nonmoving spiral, and its function is based on differences in movementcharacteristics of particles.  Spherical seeds or particles roll down the spiral, whereas thecharacteristically flat, irregular shaped alfalfa seeds tend to slide.  Round particles travel fasterand discharge at different distances from the center of the spiral.
**Scarification**
Some legume seed has a hard seed coat that is more impermeable to water compared to other("non-hard") seed.  Consequently, this hard seed does not readily absorb the moisture needed forgermination.  Mechanical scarification, or scratching the seed coat, improves percent anduniformity of germination of hard seed.  
Seed is generally scarified only if it has a high hard seed percentage.  The proportion of hard seedin a seed lot varies with the type of seed (dormant > semi-dormant > non-dormant seed) andwhere the seed is grown.  Seed grown in California from non-dormant varieties is generallybetween 2 and 12 percent hard seed.  When the same non-dormant varieties are grown outsideareas of their adaptation (e.g., in the Pacific Northwest), the percentage hard seed increasesgreatly (to 20 - 40%).
**Equipment**
All equipment should be thoroughly dry cleaned between seed lots.  When seed is intended forsprouting, some conditioners run additional raw seed between lots to clean out lines andminimize risk of contamination from agricultural seed treated with chemicals that are notapproved for food use. 
**Seed Quality**
All seed lots are tested for purity, germination rate, and noxious weed content prior to marketing.Seed is stored in metal bins in the warehouse until it is bagged. 

1. Information for this section was taken from Rincker, C.M. et al., 1988.  Seed ProductionPractices.  In: Alfalfa and Alfalfa Improvement.  Hanson, A.A., D.K. Barnes, and R.R. Hill, eds.Agronomy Series 29.  American Society of Agronomy, Crop Science Society of America, SoilScience Society of America.  Madison, WI. 

 APPENDIX 2 - SPROUTED SEED PRODUCTION
The following discussion is based on results of the CDHS/FDA survey and inspections and onobservations by the FPWG during site visits.
**Seed Receipt
Seed were usually received by the firm in bags or sacks.  The most common size noted was 50pounds (74% of firms), and the most common type bag was cloth.  Seed bags usually bore a taglisting the following information: supplier name, supplier address, lot number, seed type name,and country of origin.  
Approximately 83% of the manufacturers surveyed stocked seed sacks that were traceable to thesource.  Ninety-seven % of suppliers were identifiable from labeling and 71% of seed sacksshowed imprinted lot codes.  Fifty-one % of the bags originated from the United States.  Therewere two firms that did not have supplier identification.  One of these firm received seed fromChina.  The other bought its seed as needed from a health food store.
Seven producers said they regularly perform microbiological testing.  Amount of seed testedvaried from under one ounce to three pounds.  One manufacturer followed testing procedures inthe FDA Bacteriological Analytical Manual.  The majority of manufacturers (51 %) replied thatthey conducted no testing; though their suppliers provided them purity test analysis for suchitems as % Germination, % Dormant, and % Hard Seed.  These purity tests do not include assaysfor the presence of pathogenic organisms. 
The majority of firms surveyed (41/45) reported that they did not blacklight seed sacks orvisually inspect incoming seeds. 
**Seed Storage
It is important that seed are stored off the floor and away from walls to reduce rodentcontamination and facilitate inspection for signs of rodent contamination.  Open bags should bestored in a receptacle with a tight fitting lid or otherwise protected from contamination.  Eightfirms were cited for seed storage concerns.  Citations included failure to exclude pests, seedstorage in a garage among personal items, and standing water and debris in the seed storage area.
Types of pest control used were closely divided (51% in-house and 47% contractor).  Firms thatreported using contract pest control still showed evidence of rodent, insect, and bird activity.One of five firms showed evidence of domestic pets, three of nine firms showed evidence ofrodents, four of nine firms showed evidence of insects, and five of ten firms showed evidence ofbird activity.
**Pre-germination Soak
Soaking causes seeds to swell and softens hulls so a sprout will grow out of the seed.  This stepmay incorporate chlorination/disinfection.  Eighteen of the 45 firms surveyed reported that theyapplied a disinfection treatment during soaking and/or rinsing.
Seeds were soaked in tubs, bins, and drums in either chlorinated or non-chlorinated water.  Seedswere soaked for up to 12 hours in non-chlorinated water.  When chlorine was used, soak timewas generally shorter.  However, there was no set relationship between chlorine concentrationand length of soak time.  Chlorine concentrations ranged from 25 to 5,000 ppm and soak timeranged from 5 to 120 minutes.  Several firms reported using chlorine but did not report knowingchlorine concentration.
**Rinse
Rinsing removes residues from soaking.  Chlorinated or non-chlorinated water may be used. Buckets and hand held screens were typically used for rinsing.  Firms used differentcombinations of soak and rinse water type.  Reported practices included 1) chlorinated watersoak and non-chlorinated water rinse, 2) non-chlorinated water soak and chlorinated waterrinse, 3) chlorinated water for both soak and rinse, and 4) non-chlorinated water for both steps.
**Germination and Growth
Methods used for germination and growth, and subsequent harvest and washing, varied dependingon many factors, including the size of the operation and the type of sprout grown.  Few of thefirms surveyed utilized nutrients or other additives in their growing procedures. 
Surveyed firms reported the following germination processes by sprout type:
Rotating DrumAlfalfa, Broccoli, Clover, and RadishGrowing RoomMung Bean, Onion, Adzuki, Lentils, Garbanzo, Peas*, and Wheat berry Soil Tray	Wheat Grass, Sunflower, Peas*, Buckwheat, Daikon (White radish),Broccoli and Sungreens*Peas were grown both hydroponically and in soil.
Germination and growing times varied with type of sprout, time of year, and germinationprocess used.  Minimum and maximum ranges reported by product were:
Alfalfa3-7 daysOnion6-10 daysAdzuki2-9 daysPeanuts5-6 daysBroccoli4-7 daysRadish3-14 daysClover3-7 daysSoybean4-6 daysGarbanzo2-9 daysSunflower6-14 daysLentils2-9 daysWheat6-7 daysMung beans3-8 daysWheat grass3-14 days
**5a. Hydroponic Processes**

Hydroponic processes fell into two general categories; rotating drums, generally in a lightedroom, and stationary containers (e.g., bins, beds, or buckets) in environmentally controlledrooms or free standing growth chambers which were often kept dark.

Rotating Drums

Rotating drums were usually hard plastic cylinders measuring approximately 5 feet indiameter by 5 feet deep.  Drums were positioned sideways and off the ground with a chainattached to the back closed end for rotation to prevent clumping of sprouts.  The inside of thedrum was typically partitioned into quarters with the same plastic.

Typically, alfalfa, broccoli, clover, and radish seed were sprouted in rotating drums.  Eachdrum can hold eight to 100 pounds of seeds.  One hundred pounds of seed can yieldapproximately 500 pounds of finished product.  Once the seed is placed in the drum, the lid isclosed and the drum begins to rotate.  Water is sprayed intermittently on the seed.  Forinstance, seed may receive a water spray every 10 to 15 minutes for 10 seconds.  Temperatureof the water may be either ambient or heated to approximately 75°F.  The purpose of thewater is to 1) keep the sprouts from overheating, 2) remove by-product residues of growth,such as ethylene gas, and 3) irrigate the product. 

Air may be blown into the drum to keep the product ventilated.   Some manufacturers havedrums automatically set for rotation speed, water temperature, and air ventilation.

After sprouts are removed from the rotating drum, they are washed with cool water to removeexcess hulls.  Sprouts may be washed in a bucket of water and sieved through a hand screen,or they may be placed in a bubbling tub of water to loosen and float off hulls.  Sprouts areraked out and spin dried using a centrifuge for one minute, or, in some instances, a clothesdryer for two minutes.  Sprouts may also be placed in a cold room or otherwise treated toremove heat generated during the sprouting process.  When cool and dry, fully germinatedsprouts are ready for packaging.

Some manufacturers germinate sprouts in drums, allow up to 60% of growth to occur, thenrinse the sprouts, and transfer them to trays, cups, or final packages (such as clamshell typepackages) for further growth.  These containers are placed on a tiered growing table or in agrowing rack for an average of three to five days.  With this process, sprouts will grow in amore vertical, uniform manner and turn greener.   Some processors opt to not expose thesesprouts to light until the last day of growth.

Growing Rooms and Chambers

Mung beans and soybeans were generally grown in bins or beds in separate rooms.  Roomswere kept dark, humid, and with temperatures between 70 to 79°F.  Some bins were onwheels so that when the product finished growing, it could be wheeled out of the room toanother area for rinsing and packaging. 

Seeds were grown in bins or beds for approximately four to seven days.  Stationary beds wereusually separated with plastic dividers to separate sprouts at different stages of growth.  Anintermittent spray was applied either manually with a hose or by an automated wateringsystem.  The average water spray lasted less than a minute every four hours.  Non-chlorinatedmunicipal or well water was generally used.  

Mung and onion seed were germinated and grown in large (32-gallon) barrels.  Barrels hadair ventilation through a pipe, typically had holes at the bottom to drain excess water, andwere on pallets.  Water (80 to 85°F) was delivered to each line for one minute every fourhours.  Germination and growth averaged 5 to 6 days.  Eight pounds of mung beans yielded75 pounds of sprouts; 12 pounds of onion seed yielded 25 pounds of sprouts.

Adzuki, lentils, garbanzos, peas, and wheat berry seeds were generally sprouted in 5-gallonbuckets.  Bucket germination and growth times averaged 2 days.  Each 5-gallon bucket held12 pounds of beans or seeds.  Racks were used to hold the buckets of germinating beans orseeds.  Temperature of the rooms containing buckets and barrels ranged from 68 to 75°F,with an average room size of 16' by 16'.

Seed may also be sprouted in a free standing growth chamber.  A Sprouter AutomaticSprinkler System for germinating mung beans was seen at 1 firm.  It was an 8' by 2' by 4'device to hold the seed in a dark, controlled environment in a lighted room.  Growthchambers may be used for low poundage production, e.g., about 200 pounds of sprouts perproduction cycle.

**5b. Soil**

Ten of the firms surveyed were growing sprouts in soil.  These firms were also producinghydroponically grown sprouts.  Typically, soil-grown and hydroponic products were grownin separate rooms.

Sprouts were planted and grown in plastic trays containing a composted soil that was mixedto the producer's preferences.  In one case, the soil mix included composted horse manure,bone meal, and oyster shell flour. 

Seed used for soil trays was soaked and rinsed as described above for hydroponic operations.After rinsing, wet seed was held for 12 to 24 hours in buckets to allow for initial germination.Meanwhile, plastic trays (approximately 2' by 3') were filled with soil.  When the seeds wereready, they were spread on top of the soil and leveled out.  Growing usually took place in agreen house.  Water was sprayed on trays two times daily, usually by an automated pipesystem overhead.  Sprouts can take 5 to 10 days for optimal growth in wintertime, or 3 to 10days in summertime.

Most soil-grown sprouts were harvested at the facility, washed, packaged, and delivered tothe customer.  Typically, soil-grown products were brought into the cutting and packagingroom in soil trays.  They were often there at the same time as the hydroponic items.  At onefirm the following was observed.  Dirt trays were placed on one of three tables and thesprouts were cut from the stems and placed in large totes.  Dirt trays were piled beside thetable until cutting was completed.  Soil-grown sprouts were then mixed with other types ofsprouts and called "sprout salad" (sunflower, onion, radish, and clover) and "mixed beansnack" (whole peas, garbanzos, adzuki, and lentils.) 

Alternatively, some processors delivered the product as is, in the tray, directly to the retailer.At the retailer the tray was inserted in a plastic bag and placed in a cooler.  Typical retailersof this product were juice blending shops.  These shops cut sprouts, primarily wheat grass,directly from the tray and drop them into a blender with other fruits, vegetables, and variousingredients.  Apparently, some sprout producers and retailers view product that is allowed tocontinue growing in soil and is not harvested until just before use as more healthy andorganic compared to product that is harvested and packaged by the producer.  

When the retailer is finished cutting/snipping sprouts from the tray, the tray of soil withremaining product (e.g., roots) is returned to the sprout producer.  The producer may pick upthese trays while delivering new trays of freshly grown, uncut product.  Soil and remainingspout parts were then deposited into a compost mound.  The soil may be eventually reusedfor subsequent sprout production.

**Packaging
Firms reported various packaging processes, including 1) harvesting and washing sprouts atthe facility and placing sprouts into either bulk or individual retail containers, 2) 60% growthin a drum followed by further growth in individual trays, cups, or clam shell type packaging,then closing and distributing as is, and 3) soil tray growth without harvest.  Packaging wasusually done manually, in a separate packaging room.
Types of packaging used to transport product during distribution included 1) plastic bags(with and without holes for respiration) with heat-sealed or wired-twist closures, resealableziploc or tamper evident plastic bags; 2) heat sealed cellophane bags with partially-sealedfilm covers to allow respiration; 3) rigid plastic cup or box-type containers with completelysealed film cover; 4) clam-shell type packages; 5) uncovered and plastic-covered soil trays;and 5) used produce boxes. 
Packaging usually occurred at the growing site.  However, occasionally, product wastransported in bulk to another location to be packaged.  Some firms selling at farmers'markets transported the product in bulk and packaged the product into retail containers as itwas sold.
**Cooling and Storage
Finished product may be sent directly to the retailer with no refrigeration, or may be placedinto a cooler to await distribution.  Of firms surveyed, time in the cooler ranged from onehour to five days.  One firm, that was cited with many other objectionable conditions, keptproduct in their cooler for up to two weeks.  
Temperatures in most coolers ranged from 34°F to 45°F.  Five coolers were over 45°F.  Somecooler thermometers were not operating or had inaccurate readings.  Five firms had norefrigeration at the sprouting facility.
**Distribution
The 45 firms surveyed used a total of 116 trucks, vans, and cars for product delivery.  Ofthese 116 vehicles, 51 percent were refrigerated.  Some vehicles had refrigeration andthermometer gauges.  The general condition of 64 percent of the delivery vehicles was ratedby inspectors as "adequate". 
Reported delivery times ranged from 20 minutes up to 9 hours.  Ninety-seven percent of firmssurveyed distributed to firms within California while 11 percent distributed outside California.  Onefirm reported up to 100 miles for delivery distance.  Maximum delivery times depended not only ondelivery distance, but also on the number of delivery stops made.
Delivery distance, time, and refrigeration status of vehicles must take into account dock time andconditions.  Dock time is the time between removal from refrigeration at the firm and the timeproduct is placed in the delivery vehicle.  Thirty-seven of the 45 firms surveyed answered thequestion on dock time.  Fifty percent of these firms stated they had no dock time; 32 percent statedthey had less than 30 minutes; and 21 percent stated they had dock time from one hour to 10 hours.Often, temperature was not controlled on loading docks.  Where temperature was controlled, the setpoint was usually above 45°F.
**General Facility, Sanitation, and Employee Hygiene
**Water Quality**
Twenty two percent of firms used non-municipal well water.  Half of these firms tested theirwater at least for coliforms.  Seventy three percent used municipal water, and 4 % used bothwell and city water. 
**Sanitation**
Facilities were evaluated by state or Federal investigators and subjectively assigned a score of 1(poor) to 10 (good).  Nineteen of 45 firms scored no more than 5.  Many firms (23/45) firms didnot have hot water for cleaning.  Of those reporting to have hot water, only 10 had hot waterbetween 110 and 160°F.
At 9 firms, equipment and/or utensils were broken or cracked potentially allowingmicroorganisms or mold to grow in crevices.  Seventeen firms did not store equipment in amanner that would prevent contamination.   Six firms had equipment that were made of wood,pitted plastic, or were not easily accessible for cleaning.  Equipment surfaces had buildup ofbrown residue.  At several firms, totes were noted in direct contact with the floor in foot trafficareas after washing and sanitizing.  Some firms also reused produce boxes to distribute product.Used produce boxes may not have been adequately cleaned or protected during storage. 
**Restrooms**
Restrooms were evaluated by state or Federal investigators and subjectively assigned a score of1 (poor) to 10 (good).  Fifteen of the 45 firms scored no more than 5.
Food residue, oil, and bacteria on hands can best be removed with hot water and soap.However, 15 firms had no hot water.  There was no soap at 9 firms; no hand washing signs at23; and no paper towels at 16.  One firm had no hot water, soap, or paper towels.  One firm hadonly hot water.  
Restrooms need to be clean and easily accessible.  They need to have hot water, soap, andsanitary hand drying.  At a number of firms, restroom facilities were far away from the growingand processing facilities, in owners' homes, or were port-a-pots across fields.  One had a toiletthat opened into the processing area and had no door or vent to the outside of the building.  Atthis firm, used toilet paper was held in an open 5-gallon container.  A second firm also had usedtoilet paper stored in an open bucket in the bathroom.  One firm had a portable toilet, no handwashing station, and eight employees.
Hand dip stations are important to sanitize clean hands and gloves.  However, 30 firms did nothave hand dip stations.
**Training**
Twenty-three firms reported that employees had received hygiene or sanitation training in thepast year.  Length and type of training ranged from one day for new employees at 5 firms tocontinuous on the job training at 7 firms.  Twenty firms reported no current hygiene/sanitationsupervision.  There was a close correlation between the "no" answer on the question forwhether training was given and the "no" answer to the question for whether there was currentsupervision. 
Lack of awareness about contamination was a major issue on the FDA 483s (InspectionalObservations) and CDHS/FDB R.O.s (Report of Observations).  Many individuals wereobserved handling unsanitized items and then returning to packaging sprouts without washingand sanitizing their hands and/or gloves.  Food containers were placed on the floor to fill andmove product from one operation to the next.   
**Airborne Hazards**
The sprout processing facility was not fully enclosed at 11 firms.  Some firms operated withopen unscreened doors or windows and traffic pollutants, dirt, and dust could enter into thegrowing and packaging areas.  At one firm, a wall humidifier was improperly maintained in away that could trap and propagate molds, bacteria, and spores which could then blow across thegrowing trays.
**Testing of Finished Product**
A testing program is most effective when based on a written plan that specifies types of sproutsto be tested, sample size, frequency of testing, tests to be performed, and limits.  The planshould include actions to be taken when limits are exceeded. 
Ten firms reported that they had tested their finished product in the past, primarily for generic*E. coli* and *Salmonella*.  Five of these firms stated they tested with a regular frequency of onceeach week or once each month.  Though some firms grew many types of sprouts, alfalfa sproutswere the predominant sprout tested. 
**Record Keeping/Traceback Mechanisms**
As mentioned previously, seed suppliers were identifiable from tags on seed bags at most firms(43/45).  Further, 36 firms had seed receiving documents available for the past six months, and 32firms could identify lot numbers from tags on seed sacks.  Only 5 firms used a production lotnumber or color coding system on finished product.  
Thorough record keeping allows traceback from finished product on the market optimally back tothe seed grower.  None of the firms surveyed kept adequate records for a complete traceback.This means that in the event of a foodborne illness outbreak, none of the firms surveyed have thecapability to isolate when, where, and how the problem may have occurred.

 
APPENDIX 3 -- RATIONALE FOR 5-LOG REDUCTION PERFORMANCE STANDARDRECOMMENDATION
Limited quantitative data is available on the level of foodborne pathogens present in seeds usedfor sprouting.   Quantitative analyses performed on seeds associated with illness attributed tosprout consumption found pathogen levels ranging from <1 -  6/100 g of seed (Inami and Bryant,1999).  Therefore the worse case scenario for seed contamination was assumed to be 1pathogen/10g of seed.  It was also assumed that fifty kg of seeds is the amount of starting materialfor each batch of sprouts.  This yields an initial level of 5,000 pathogens per batch of sprouts.Thus, a 1- log treatment reduction will yield 500 pathogens/batch; a 2-log treatment 50pathogens/batch; a 3- log treatment 5 pathogens/batch; a 4-log treatment 0.5 pathogens/batch (onebatch out of every two will contain a pathogen); and a 5-log treatment 0.05 pathogens/batch (onebatch out of every twenty will contain a pathogen).  It is realized that the actual extent of riskreduction achieved will likely be greater than this because the extent of initial contamination andthe amount of seed used per batch of sprouts will typically be less than the values assumed in thecurrent worst case calculation.

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