Food Safety Practices Guidance for Sprout Manufacturers
Appendix A: Guidance on Sample Collection and Testing of the Spent Irrigation Water and Sprouts

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Note: The following is based on Health Canada's Policy Document entitled Guidance for Industry: Sample Collection and Testing for Sprouts and Spent Irrigation Water.

Microbial testing of spent irrigation water is considered to be one of the most practical and acceptable testing techniques currently available (refer to Health Canada's Policy Document entitled Policy on Managing Health Risk Associated with the Consumption of Sprouted Seeds and Beans). Health Canada recommends that sprout manufacturers regularly test spent irrigation water, because water that has flowed over and through the sprouts is a good indicator of the types of microorganisms in the sprouts themselves, including microbial pathogens of concern (salmonella spp., E. coli O157:H7). Sprouts should not be tested in place of spent irrigation water unless the production methods make it impossible to test the spent irrigation water. However, the recommendation to test spent irrigation water does not preclude additional testing of sprouts (either sprouts collected during production or finished product). Representative samples should be collected from each production lot and analysed for microbial pathogens of concern.

Sampling Equipment and Containers

Equipment and containers used to collect samples should be clean and sterile. They may be purchased pre-sterilized, or alternatively, they may be sterilized at 121°C (250°F) for 30 minutes in an autoclave, prior to use. Heat-resistant, dry materials may be sterilized in a dry-heat oven at 140°C (284°F) for 3 hours. Once sterilized, the sampling equipment and containers should be protected from contamination at all times before and during use. Handling personnel must ensure that the used sampling equipment, containers and the collected samples do not contaminate remaining sterile equipment and containers.

The type of sample containers to be used depends on whether spent irrigation water or sprout samples are being collected. Containers may include pre-sterilized plastic bags, bottles, tubes, cups and flasks. They should be dry, leak-proof, wide-mouthed, and of a size suitable for the samples. Containers should also seal properly to ensure the integrity of the sample. The containers should be properly labelled prior to collecting the sample.

When to Sample

Samples of spent irrigation water can be collected as early as 48 hours after the start of sprouting. If the seeds are pre-soaked (e.g. soaked in water for a short time and then transferred to growing units for sprouting), the pre-soak time must be included in the time allocation. Early results will allow the sprout manufacturer to take corrective actions sooner, thus minimizing the potential for one lot of sprouts to contaminate other lots.

Procedures for Sample Collection

Sample collection of spent irrigation water and sprouts should be done on site by trained personnel. Aseptic sampling procedures should be used to avoid contaminating the sample and the product being sampled.

Personnel should wear a clean lab coat, hair net and sterile gloves. Hands should be washed immediately prior to putting on sterile gloves. The sterile gloves should be put on in a manner that does not contaminate the outside of the glove. During sample collection, hands should be kept away from the mouth, nose, eyes and face. After sample collection, the gloves should be properly discarded.

The sterile sample container should be opened only sufficiently to allow for the sample to be collected. The sample should be placed directly in the container. Once the sample is collected, it should immediately be closed and sealed. If samples are collected in a container equipped with a lid, the lid should NOT be completely removed. The lid should not be held separately or placed on a counter.

The sample container should be filled no more than 3/4 full to prevent overflow. The air from the container should not be expelled when sealing, particularly for plastic bags.

Once collected, the samples should be delivered to the laboratory promptly. The sample should be kept at an appropriate temperature, preferably between 0°C and 4°C (32°C to 40°F). To avoid cross-contamination from melting ice, sealed coolant packs should be used.

Pooling samples from different sprout lots may reduce the number of lab analyses to be performed; however, if a presumptive positive is found, all sprouts lots represented by the pooled sample are considered suspect. The suspect sprout lots should either be discarded or each sprout lot should be analysed separately to determine which lot(s) is (are) contaminated.

Sample Size

The volumes given below for spent irrigation water and sprouts represent a sufficient sample size to test for the presence of the microbial pathogens of concern (i.e. Salmonella spp. and E. coli O157:H7).

A. Spent Irrigation Water

One (1) litre of water should be aseptically collected as the water leaves a drum or tray(s) during the irrigation cycle. Spent irrigation water samples should be collected directly into clean, sterile, pre-labelled containers.

Drums:
One (1) litre of spent irrigation water may be collected from the drum.

Trays with Common Trough:
One (1) litre of spent irrigation water may be collected at the common trough.

Trays with no Common Trough:
If there is no common trough, spent irrigation water samples from individual trays should be collected and pooled. If the tray is large, spent irrigation water samples from different areas of the tray should be collected.

When ten (10) or fewer trays make up a production lot, approximately equal volumes of spent irrigation water should be collected from each of the ten (10) trays to make a total sample volume of one (1) litre.

For example:

  • Ten (10) trays: Collect 100 ml of spent irrigation water samples from each tray to make up a one (1) litre sample.
  • Eight (8) trays: Collect ~125 ml of spent irrigation water samples from each tray to make up a one (1) litre sample.

When there are ten (10) or more trays, collect ten (10) spent irrigation water samples throughout the entire production lot. For example, if there are 20 trays in a production lot, collect samples from every other tray in the rack, moving from top to bottom, side to side, and front to back.

B. Sprouts

Five (5) sample units of approximately 200 grams each should be aseptically collected from different locations in the drum or growing trays, to ensure the sample collected is representative of the lot. The sample units should be collected throughout the entire production lot (e.g. from top to bottom, side to side, and front to back of the drum or trays). Each 200 gram sample unit should be placed directly into individual clean, sterile, pre-labelled containers.

Microbial Testing Procedures

All microbial testing for pathogens should be conducted in an external, certified, independent laboratory, and meet the following criteria:

  • The laboratory should be physically separated from the food production facility to prevent cross-contamination.
  • Second, the laboratory should be staffed by personnel with training and experience in analytical microbiology techniques to ensure that tests are performed correctly and that all appropriate safety precautions, including appropriate waste disposal, are followed.
  • Third, the laboratory should have appropriate resources and be able to demonstrate that a quality management system is followed.

If the microbial analysis is done by the sprout manufacturer, the laboratory facilities, personnel, and quality management system should meet the above mentioned criteria, to ensure that testing is reliable and does not create food safety hazards.

The testing procedures described below can be used to obtain results as simply and quickly as possible on the presence or absence of the microbial pathogens of concern (i.e. Salmonella spp. and E. coli O157:H7). These methods are described in the Health Canada (HC) Compendium of Analytical Methods.

Please keep in mind that seasonal or regional differences in water quality, type of seed being sprouted, and variations in sampling and analytical conditions may all have an impact on the effectiveness of the screening tests.

Test Kits:

Escherichia coli O157:H7:

  1. MFLP-87 VIP EHEC. Biocontrol Systems, Inc., Bellview, WA.
  2. MFLP-94/95 Reveal E. coli O157:H7, Neogen Corp., Lansing, MI.
  3. MFLP-91 Tecra UVA method for E. coli O157:H7.
  4. Any other methods listed in the Compendium for E. coli O157:H7.

Salmonella spp. :

  1. MFHPB-24 Vidas SLM method, Biomerieux, Montreal.
  2. MFLP-96 Reveal kit for Salmonella.
  3. MFLP-97 Alert kit for Salmonella.
  4. MFLP-35 Tecra VIA for Salmonella.
  5. Any other methods listed in the Compendium for Salmonella spp.

General Laboratory Instructions

Follow instructions in each method.

Dividing Samples into Sample Units for Analysis

Spent Irrigation Water:
A total of one (1) litre of spent irrigation water should be collected. Two (2) 100 ml sample units should be analysed for the presence of E. coli O157:H7. Two (2) 375 ml sample units should be analysed for the presence of Salmonella spp. Any unused portion of spent irrigation water should be stored under refrigeration pending completion of the analysis.

Sprouts:
A total of five (5) sample units of 200 g of sprouts should be collected. For each sample unit, one 25 g sample unit should be analysed for the presence of E. coli O157:H7 and one 25 g sample unit should be analysed for the presence of Salmonella spp. Unused portions of the sprout sample units should be stored under refrigeration pending completion of the analysis.

When Pathogens of Microbial Concern Are Detected

When spent irrigation water or sprout samples are found to be positive for salmonella spp. or E. coli O157:H7, this is considered to be a health risk and in violation of Sections 4 and 7 of the Food and Drugs Act. The sprout manufacturer should notify the CFIA immediately.

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