Food Sample Analysis

Sample Selection

Instructions

• Selection of food items for collection and submission related to a foodborne outbreak should be determined based on all available environmental, epidemiological and clinical laboratory information related to the outbreak. Information about the clinical pathogen, if known or suspected, should be included to facilitate prioritization of food items both collected and tested.
• If unsafe food-handling practices are identified during the inspection of a food premise, selection of food items for a microbial risk assessment is based on the health inspector’s discretion. The submission of foods is not a requirement in these circumstances but may provide an additional tool available to support the assessment.

Considerations

• During a foodborne outbreak investigation, the results of an environmental investigation, such as a Hazard Analysis Critical Control Point (HACCP) review, and epidemiological analyses, such as the calculation of attack rate, will facilitate prioritization of food items that are most likely to be involved. If attack rates are unavailable, incubation period, clinical symptoms and epidemiologic information about related cases should be used.
• If risk stratification of food sources is not performed by the submitting public health inspector, and there is an abundance of samples submitted for testing, prioritization will be determined by PHO Laboratory Environmental Microbiology section. The communication of all available clinical, epidemiological and environmental information to PHO Laboratory is of great importance in guiding these decisions.
• Avoid submitting raw foods which require processing or cooking before consumption, unless they are linked to a specific outbreak or a confirmed etiological agent. If testing of raw foods is required, please contact the PHO Laboratory Customer Service Centre at 416-235-6556 or toll free 1-877- 604-4567.

Containers for Sampling

Instructions

• Sterile plastic bags with wire closure are the preferred sample container for all food types.
• Food samples found in the original container can be submitted in the original packaging providing that the container is air-tight and will not leak during transit.
• If the original container has been opened or if the packaging is fragile and may be damaged during transit, samples should be transferred from the original container to a sterile plastic sampling bag with wire closure.

Considerations

• If a commercial packaged product has been submitted, record the following product information on the Food Bacteriology Requisition: brand name, lot number, best before date, expiry date, use by date, Universal Product Code (UPC), name of manufacturer/importer/legal agent, unit size, non-intact (open) and intact (closed). Photographs may also be taken and maintained by the public health inspector to capture this information at the time of sample collection which may facilitate a food recall if required. Photographs should not be submitted to the laboratory.
• For foodborne outbreak investigations: when collecting an opened commercial packaged product, collection of a closed sample with the same lot may be useful to determine if the product was contaminated prior to consumer handling. PHO Laboratory will only analyze closed samples in conjunction with a foodborne outbreak investigation and only following consultation and approval by the laboratory. Please consult with the CFIA prior to submission as appropriate.
• Styrofoam and box-board packaging used for fast-food products are examples of containers that are not suitable for microbiological testing. They are not air tight and may leak, potentially resulting in cross contamination of sample(s). These containers should not be put inside a sterile sample bag since the outside surfaces of the containers are a potential source of contamination and the sample will be compromised.

Sample Preparation

Instructions

The following steps are recommended to organize and minimize food sample collection time:

• Investigate before sampling to determine a sampling plan.
• Ensure all materials required are available prior to collection.
• Label sample bags using a permanent marker.
• Complete requisition(s) before or after, never during, sample collection.
• After collection, double check to ensure the food sample unique identifier on the sample bag and requisition are legible and match.

Sample Collection

Instructions

• Use aseptic technique at all stages of sample collection. If sterile gloves or tongs are not available, an inverted sampling bag may be used as a glove to collect the sample.
• Collect samples that are representative of each item of food that is being investigated.
• Meals that contain multiple discreet items (e.g. rice and chicken) may be collected individually or combined as a single sample. If collected as a single sample, components should be collected in approximately equal amounts and will be tested as one sample.
• Foods that contain multiple items (e.g., sandwiches) may be collected as a single sample.
• If an individual component is of greater interest than other components of a meal or mixed food, sample the items individually so they can be tested as individual samples.
• Once collection is completed, close the mouth of the sterile plastic sampling bag with the wire closure, and roll down several times twisting the ends together tightly to prevent leaking during transport.
• Submit a minimum of 200 grams of each sample if possible, as multiple tests may be required. Samples that are less than 10 grams will not be tested.

Considerations

• Failure to follow these instructions will compromise the sample integrity and the laboratory results.
• Separation of meals or mixed foods must be performed at the sampling stage. The laboratory will not separate components of a sample once received in the laboratory.
• Leaking samples will not be accepted by the laboratory. Refer to the Food – Laboratory Acceptance Criteria section for a complete list of acceptance criteria.

Instructions

• Ship dry foods and other shelf stable foods in a closed container at ambient temperature.
• Ship frozen foods in an insulated container with sufficient ice packs to maintain the frozen state.
• Ship perishable foods in an insulated container with sufficient cold packs to maintain a temperature as close to 4°C as possible. If ice is used, contain the ice in a manner that does not allow water contact with the samples.
• Submit all food samples to the closest PHO laboratory as soon as possible after collection.

Considerations

• Foods that exhibit temperature abuse during shipping may be rejected and not processed. If the product appears to have undergone a change of state such as from a frozen state to a liquid state, the product will be rejected.

The accuracy of the test results may be affected by improper collection, handling and/or shipping. Food samples that do not meet the acceptance criteria may be rejected by the laboratory and a new sample would be required to be re-submitted with a newly completed requisition. The acceptance criteria are outlined below.

Satisfactory microbial limits for a food sample will be dependent on the food preparation, conditions (raw, ready-to-eat, and frozen, refrigerated, shelf stable) and commodity type (e.g., dairy, meat, etc.) in relation to a specific analysis. Refer to Health Canada’s Health Products and Food Branch (HPFB).

Standards and Guidelines for Microbiological Safety of Food - An Interpretive Summary for food commodity type details.

The tables below describe the reporting limit and unsatisfactory level for ready-to- eat foods for indicator bacteria and foodborne pathogens according to Health Canada, and physicochemical testing available at PHO Laboratory. If a board of health would like further interpretation on food results, contact PHO Laboratory Customer Service Centre at 416-235-6556 or toll free 1-877-604-4567, and provide the sample identification number.

INDICATOR BACTERIA

^a CFU (colony forming units)
^b MPN (most probable number) is used for this test method
^c Microbial guidelines as per Health Canada
^d Microbial guidelines as per Alberta Health Services 

Unsatisfactory levels of indicator bacteria indicate improper food preparation, handling, storage or holding conditions, temperature abuse, and/or sanitation. Refer to specific test details below for more information.

Aerobic Colony Count (ACC): It is reasonable to expect properly prepared and handled ready-to-eat processed or cooked foods, where no additional food handling was required after food preparation, to have an ACC of < 10⁴ CFU per gram. An ACC of > 10⁵ CFU per gram is usually evidence of post processing temperature abuse, inadequate cooking or improper storage conditions. ACC cannot be applied to raw, uncooked, unprocessed foods (e.g., fruits or vegetables), cultured or fermented products (e.g., cheese, yogurt or salami, etc.) since these foods are known and expected to have naturally occurring microbial flora. There are three categories of ACC listed in the table above, which are based on food type and the processing/handling the food has undergone.

Category 1 – cooked foods that do not require handling or processing; i.e., soups, bread, quiche, cooked meat, fish & seafood, vegetables, etc.

Category 2 – cooked foods that require further handling prior to or during the preparation of the final product; i.e., hot dogs, sandwiches, burgers, etc.

Category 3 – foods that have a high ACC due to the normal microbial flora associated; i.e., pitas, potato or pasta salad, salad rolls, fresh fruits, raw vegetables, cheese, yogurt, deli meats, etc.

Total Coliform: Presence of coliforms in a processed, cooked, ready-to-eat food is indicative of inadequate processing or post processing contamination resulting from poor handling of the product. A satisfactory limit cannot be applied to uncooked or raw foods such as salads and salad ingredients, raw fruits and vegetables, raw meats as well as some fermented foods, etc., since coliforms are regularly found in and/or on these foods.

Escherichia coli: The presence of Escherichia coli in food is indicative of direct or indirect contamination of the food product with fecal material, thus, enteric pathogens may also be present. The presence may be indicative of inadequate processing, post processing contamination and poor sanitation.

Total gram negative: Total gram negative (TGN) count has the same significance as presence of coliforms. Elevated TGN count should not be detected in cooked or processed ready-to-eat foods. Absence of gram negative organisms in a food sample is another indicator of food safety.

Yeast and mould: The presence of yeast and moulds can cause various degrees of deterioration and decomposition of food, resulting in abnormal odours, flavours and colours. Both yeast and mould can also be added intentionally to a product for a desirable flavour such as mould for blue and brie cheeses and yeast for beer, ciders and wines.

FOODBORNE PATHOGENS

^a CFU (colony forming units)
^b Microbial guidelines as per Health Canada

Foodborne pathogens should not be detected from a ready-to-eat food and would indicate improper food preparation, poor handling, inadequate sanitation practices or possible contamination from a positive food source or an ill food handler. Appendix C: Major Foodborne Diseases: General Features provide additional information regarding the organisms incubation period, clinical symptoms, mode of transmission and associated foods.

PHYSIOCHEMICAL

pH: The pH is a function of the hydrogen ion concentration in the food and is a measure of food acidity and alkalinity, which varies with food type. pH is measured on a scale of 1 to 14, with 7 being neutral, lower numbers indicating greater acidity and higher numbers indicating greater alkalinity. pH is just one factor that influences bacterial growth, and can interact with the following to inhibit growth of pathogens and other organisms: water activity (a_w), salt, redox potential, preservatives and temperature. As the pH decreases, a lower temperature is needed to inactivate microbes.5 Foods with an equilibrium pH ≤ 3.7 will not support the growth of bacterial foodborne pathogens, regardless of the aw. However, if the pH of the food (jarred/canned products) is ≤ 4.6, microorganisms are inhibited providing the aw is ≤ 0.85. According to Public Health Ontario’s Home Canning Literature Review, June 2014, a hermetically sealed acidic canned or jarred food with an equilibrium pH ≤ 4.6 that has gone through sufficient heat treatment to eliminate vegetative microorganisms (bacteria, yeasts and molds), will not support the germination and growth of botulinum spores and production of the toxin.

Phosphatase: The enzyme alkaline phosphatase (ALP) is associated with the cream or fat globules of raw milks. It is inactivated below the detection limit of conventional methods following complete pasteurization. The detection of residual alkaline phosphatase indicates a reduction in pasteurization temperature of at least 1.5°C, a 5 minute reduction in holding time, or the presence of ≥ 0.3% raw milk. Since ALP is more heat stable than most pathogens (which may be present in milk), it is used as an indicator of pasteurization; however, a negative ALP test does not guarantee that the product is pathogen free.

Water activity (a_w): The availability of water for microbial, enzymatic or chemical activity determines the shelf life of foods. This water availability is measured as water activity (a_w). Water activity is the ratio of water vapor pressure of the food substrate to the vapor pressure of pure water at the same temperature. Water activity is measured on a scale of 0 to 1; pure water has an aw of 1.00 and the aw of a completely dehydrated food is 0.00. For most foods to be considered safe to store at room temperature, they should have an a_w ≤ 0.85. Bacterial foodborne pathogens are inhibited at this aw; however, for those microorganisms that can cause spoilage (e.g., yeasts and moulds), the a_w should be  < 0.60 to prevent food spoilage by inhibiting growth of these organisms.