 The main reason for microbiological testing is that currently approved antimicrobial treatments cannot guarantee total elimination of all pathogens from seed. Even if only a few pathogens survive a seed disinfection treatment, they can grow to high levels during sprouting. Spent irrigation water from each production lot should be tested microbiologically to ensure that contaminated product is not distributed. Testing irrigation water rather than the sprouts themselves is recommended because it is fairly uniform and requires fewer sample preparation steps and fewer samples compared to sprouts. If the sprouts are grown in soil and spent irrigation water is not available, the sprouts themselves may be tested. The sprouts also may be tested in addition to the irrigation water. It is possible to obtain test results before shipping product without losing product shelf life because testing for pathogens can be done with irrigation water as early as 48 hours into what is generally a three to five day growing period. Testing may be done by the producer or contracted out, but it should be done by trained personnel in a qualified laboratory using validated methods. The microbial testing required involves a number of hazards including growing pathogenic bacteria and disposing of contaminated waste materials. While a variety of microbiological tests might be performed, the main pathogens of concern are Salmonella species and E. coli 0157H7 which have caused sprout associated foodborne illness outbreaks. Therefore, these will be the target microorganisms. A sampling plan should be decided upon before sample collection begins. The sampling plan will identify when the samples will be taken. The current recommendation is that samples are taken about 48 hours into the growing period. Next, it will identify the number and size of samples to be taken from each lot. A lot is defined as sprouts from a single lot of seed that were planted at the same time in a single growing unit, meaning a single drum or rack of trays. A lot number reflects the seed used and the production date and time. Careful record keeping will help minimize product loss in the event that a micro test is positive. Pooling or combining samples from different lots will make the interpretation of the results more difficult. If a positive sample is found, all lots represented by the pooled sample should be discarded or held for further testing. Further, pooling may dilute pathogens, if present, to a level where they may not be detected. The sampling plan should also state what test method will be used for each target microorganism. Sample collection should be done by personnel that have been trained to collect representative samples aseptically. A representative sample is one that accurately represents the whole lot. If sprouts are grown in drums or in trays with a common water outlet, a single one-liter sample should be collected as water leaves the growing unit. If sprouts are grown in trays and there is no common collection point for water from the trays, it will be necessary to collect water from individual trays. The amount taken from each tray should be approximately the same. Collecting the entire sample from one tray would not be representative of the whole lot. Samples should be collected at the beginning of the irrigation cycle, especially when irrigation uses a large volume of water or lasts a relatively long time. Aseptic sample collection means that the procedures are designed to avoid introducing microorganisms from the hands, sample containers, air, or anything except the sample itself. Aseptic procedures are critical to avoid contamination of the sample during sample collection, storage, and transportation. Equipment used to collect samples should be clean and sterile. Sample containers may include pre-sterilized plastic bags, tubes, cups, and flasks. Container should be properly labeled to identify the sample, lot, time, and date of sampling. Sample collectors should wear a clean lab coat, gloves, and hair covering in order to prevent contamination of the sample by the collector. Any sampling instruments, such as cups or tongs, should be protected from contamination before and during use, and used only once so that each sample is taken with a fresh sterile utensil. The sample container should be filled about three-quarters full to prevent overflow. Samples should be delivered to the laboratory promptly and not held for any length of time before analysis. Preferably, samples should be stored at temperatures no greater than 40 degrees Fahrenheit if analyses cannot be performed within the first two hours after sampling. A sample of spent irrigation water should be collected for each production lot or batch. At least one liter of spent irrigation water should be taken as the water leaves the drum or trays during the irrigation cycle. If sprouts are sampled, 32 sample units should be aseptically collected, each approximately 50 grams or about 2 ounces. Sample units should be collected throughout the entire production lot, meaning from top to bottom, side to side, and front to back of the tray or drums. Each 50 gram sample unit of sprouts should be placed directly into individual, clean, sterile, pre-labeled containers to facilitate subsampling at the testing lab. It is strongly recommended that all testing for pathogens be conducted in a qualified, independent testing laboratory that meets several key criteria. As mentioned earlier, such a laboratory should be physically separated from the sprouting facility to prevent cross-contamination of the sprouts from test materials. Conducting these tests requires the use of cultures of pathogenic bacteria which could pose a hazard both to the workers and the consumers if not done properly. The laboratory should be staffed by personnel with training and experience in analytical microbiology techniques and handling of pathogens. The laboratory should periodically run positive controls to ensure that the tests used are capable of detecting pathogens when they are present. This can be done by adding a known quantity of pathogens to spent irrigation water or sprout samples and then testing these positive samples. The laboratory should have proper equipment and a demonstrated quality management system including appropriate record keeping and documentation. If testing is done by the sprout grower, then the facilities, personnel, and laboratory management system should also meet all of these criteria. Recommended sampling and testing procedures are described in more detail in the FDA guidance document Sampling and Microbial Testing of Spent Irrigation Water during Sprout Production, which is included in the written material supporting this video. The procedures were chosen to obtain results as simply and quickly as possible and provide evidence for the presence or absence of Salmonella and E. coli 0157H7. The test kits identified in the FDA guidance document are approved by the Association of Official Analytical Chemists International as screening tests or have been used in the past by FDA. If other methods are used, these alternative methods should also be validated or scientifically proven to give accurate results for this particular application. If chemical intervention treatments are applied to irrigation waters and note that currently none are sanctioned for use by regulatory agencies, then appropriate neutralization techniques such as use of DE broth should be included as part of the testing protocol. The rapid test kits described in the FDA guidance document all require use of an overnight enrichment step to promote growth of the target pathogen to detectable levels. These test kits cannot be used without this enrichment step to detect pathogens in irrigation water or sprouts. The testing procedures recommended by FDA were chosen to obtain results on the presence or absence of Salmonella and E. coli 0157H7 as quickly and simply as possible. They are screening tests. If the results are negative, these results are assumed to be correct. If the result is positive, however, it should be assumed that the product may be contaminated and further action is necessary. The grower should decide on an action plan for dealing with positive results before a positive is found. Two options exist. The grower should either accept the results of the screening test as true and discard the sprouts from the suspect lot or lots immediately, or ask the testing laboratory to run confirmatory tests and destroy the batch only if the confirmatory tests are also positive for the presence of the pathogen. The product should be held while confirmatory testing is completed. Product with a positive screening test should not be shipped until confirmatory testing has been completed. Rapid test kits are for screening only. Procedures from the FDA Bacteriological Analytical Manual, known as BAM, should be used to confirm positive results from the screening tests. When deciding whether to do confirmatory tests, remember that the extra time required will shorten the shelf life of the sprouts even if the confirmatory tests are negative. When a batch of sprouts is considered to be contaminated with a pathogen, the seed lot used to produce the sprouts and any other sprout production lots that were made from the same seed lot also may be contaminated and should be discarded. In addition, all equipment that has come in contact with the contaminated sprouts should be thoroughly cleaned and sanitized. The tests for pathogenic microorganisms as described previously may be conducted to screen incoming seeds. Other microbiological tests on seeds may be conducted as microbial indicators of quality. This activity is very often performed by the seed supplier or distributor and not necessarily by the sprout grower unless the grower wishes to include it as a further verification activity. Such testing may be written as a specification in the contract to purchase seeds. The effectiveness of testing seeds for pathogens is increased by growing sprouts from a representative portion of seeds from each lot and testing according to the methods described in the guidance document. However, it is difficult to find pathogens in seed and negative test results for a seed lot cannot guarantee that the seed is free of pathogens. Tests may also be done to monitor the hygiene and sanitation of the sprout production environment, particularly the effectiveness of pre-operational sanitation of food contact surfaces and non-food contact surfaces such as floors, walls and drains. Indicator organisms such as generic E. coli and total interobacteriaceae or possibly the coliform group may be sought for this purpose. Another type of test may be used to assess the general cleanliness of the facility and equipment surfaces using a technique known as ATP bioluminescence. Microbial testing may also be performed to monitor the hygiene and microbiological quality of in-process product, sprouts at different stages of growth or irrigation waters by testing for generic E. coli. Such monitoring activities are useful to identify trends in the quality of raw material and to assess the cleanliness, sanitation, and hygiene of the production environment. Results of such monitoring can be used to plan for improvements in effectiveness of quality and or sanitation controls. Additional information is included in the accompanying handouts. In this module, we have learned why it is important to test irrigation water for pathogenic bacteria, how to take samples of spent irrigation water and or sprouts for microbiological analysis, who should perform the test, how to interpret the results, and what action to take if a positive is found.