SOP: Enumeration of Coliforms in a Variety of Foods Using the TEMPO CC (Coliform Count) Method

GIFSQ — Global Institute for Food Safety & Quality

Health Canada, Compendium of Analytical Methods, Volume 3 — Laboratory Procedure MFLP-106, October 2018. (This method carries no “replaces” line.)

Principle

This method is applicable to the enumeration of coliforms to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. It has been validated for use in all foods, excluding strongly coloured foods.

The TEMPO CC test is intended for use exclusively with the TEMPO system for the enumeration of coliforms in 22 – 27 hours in all foods, excluding strongly coloured foods.

The test consists of a vial of culture medium and a card, which are specific to this method. The culture medium is inoculated with the sample to be tested. The inoculated medium is transferred by the TEMPO Filler instrument into the card containing 48 wells of three different volumes. The card contains 3 sets of 16 wells (small, medium and large wells) with a one log difference in volume for each set of wells. The card is designed to simulate the Most Probable Number (MPN) method. The card is then hermetically sealed in order to avoid any risk of contamination during subsequent handling.

The culture medium contains a fluorescent pH indicator which, when its pH is neutral, emits a signal detected by the TEMPO Reader instrument. The coliforms present in the card assimilate the nutrients in the culture medium during incubation, resulting in a decrease in pH and the extinction of the fluorescent signal. Depending on the number and type of the positive wells, the TEMPO system calculates the number of coliforms present in the original sample according to a calculation based on the MPN method.

Apparatus

• TEMPO system, which includes a TEMPO Filler, a TEMPO Reader and all necessary components for its intended use
• TEMPO CC (reference 80044) assay kit: contains the necessary reagents, including cards and culture medium in vials for the analysis of 48 test samples
• TEMPO Filter Bags (reference 80015)
• Pipette to dispense 0. 10mL or 1. 0mL and disposable tips
• Vortex type mixer
• Stomacher, blender or equivalent
• Incubator capable of maintaining 35°C
• TEMPO Control Kit (reference 80000)

It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. The following applies to steps of the method which apply to growth only. Where a temperature of ≤ 37°C is recommended in the text of the method, the temperature may be ± 1. 0°C, e. g., 35 ± 1. 0°C. However, where higher temperatures are recommended, it is imperative that the incubators or water baths be maintained within 0. 5°C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

Reagents

The method formulates no media or reagents itself; the following are used as named, prepared and sterilized according to the manufacturer’s instructions:

• 0.1% Peptone water
• To prepare: suspend 1.0 g of peptone in 1.0 L of distilled water; mix thoroughly and dispense; autoclave at 121 °C for 15 min. Final pH 7.0 ± 0.2.
• Sterile deionized or distilled water

Sample preparation

• In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
• Analyze sample units as soon as possible after their receipt in the laboratory.
• Have primary and secondary sterile diluent prepared, pre-warmed to room temperature before use.
• Clean the surface of the working area with a suitable disinfectant.
• To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.
• Prepare a 1 in 10 primary dilution of the food in a TEMPO Bag by adding aseptically 25 g or mL (the analytical unit) to 225 mL of 0.1% Peptone Water. Shake, stomach or blend according to the type of food. If a smaller sample size other than 25 g or mL is used, maintain the 1 in 10 dilution (e.g., 11 g in 99 mL or 10 g in 90 mL).

Note: The fluorescent signal may be affected if the primary dilution is strongly coloured (e.g., fruit purées and cocoa) or becomes coloured during incubation due to an oxidation reaction (ex. Raw mushrooms) or certain dehydrated products rendering the primary dilution turbid (ex: ready-to-use cake mixes). For the TEMPO CC test, a minimum dilution of 1 in 400 is recommended for these matrices.

• Blend/stomach for the minimum time required to produce a homogeneous suspension; to avoid overheating, blending time should not exceed 2 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface.

Note: The interval between the homogenization of the primary dilution and its transfer into the TEMPO card must not exceed 45 minutes.

Procedure

The test shall be carried out in accordance with the following instructions:

Set the dispenser(s) containing the secondary diluent to the appropriate volumes and prime the pump by eliminating the first two (2) volumes dispensed.

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Log on to the TEMPO preparation station.

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Reconstitute the culture medium (TEMPO vial) by dispensing the appropriate volume of the secondary diluent using the dispenser.

Note: For aromatic herbs, spices, teas and herbal teas, which may have inhibitory effect, a minimum dilution of 1 in 400 should be used.

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Prepare the secondary dilutions in the TEMPO vials:

• 1 in 40 dilution will enable enumeration between 10 and 4. 9 x 10⁴ CFU/g. Aseptically transfer 1 mL of the primary dilution into one TEMPO CC vial containing 3. 0 mL of sterile deionized or distilled water to create a 1 in 40 dilution.
• 1 in 400 dilution will enable enumeration between 100 and 4. 9 x 10⁵ CFU/g. Aseptically transfer 0. 1 mL of the primary dilution into one TEMPO CC vial containing 3. 9 mL of sterile deionized or distilled water to create a 1 in 400 dilution.
• If higher enumeration ranges are required, prepare serial dilutions of the original sample prepared at the sample-preparation step to achieve dilutions of 1 in 4 000, 1 in 40 000, 1 in 400 000, etc., according to the manufacturer’s instructions.

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Agitate each dilution to resuspend the material that may have settled during the preparation.

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Using a sterile pipette, transfer the appropriate volume (see the dilution options above) from the TEMPO Bag containing the sample to its corresponding vial with the reconstituted medium. Vortex or agitate manually for approximately 5 seconds.

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Following the instructions of the preparation station user interface, identify the sample to be tested.

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Remove one TEMPO CC card and scan the card information into the TEMPO system by following the prompts.

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Scan the TEMPO CC vial containing the sample, then insert the TEMPO CC fill tube into its corresponding TEMPO CC vial. Place the combination TEMPO CC card and vial into a filler rack.

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Repeat the card/vial scanning and assembly steps for each vial prepared.

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Fill the TEMPO cards using the TEMPO Filler. After the cycle, visually inspect that the vials are empty. Dispose of the used vials in an appropriate receptacle.

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Remove the filled TEMPO CC cards from the filler rack and insert them into the TEMPO incubation rack.

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Incubate the cards at 35 ± 1°C for 22-27 hours.

Note: The incubation time for the test is managed by the TEMPO Read software, which integrates a theoretical interval of 15 minutes between the reading of the card barcode and the start of incubation. If the real interval is greater than 15 minutes (without exceeding 2 hours), this extra time must be added to the remaining incubation time displayed by the TEMPO Read software. Reading must always be performed within the time limit authorized by the software.

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Remove the TEMPO incubation rack from the incubator.

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Log on to the TEMPO reading station.

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Introduce the TEMPO incubation rack containing the cards to be read into the TEMPO Reader. The reader scans the barcode of each card and interprets the results of fluorescence in the wells. It automatically associates the sample identifier with the type of test, the dilution and the enumeration results as colony forming units (CFU) per gram or milliliter of initial sample.

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At the end of the analysis, remove the cards from the rack and dispose of them into an appropriate receptacle.

Precautions

• The laboratory supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference “ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories”. (The source states this as a note with the materials rather than in a numbered Precautions section; it is carried here.)
• It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch).

References

• Health Canada. 2018. MFLP-106: Enumeration of Coliforms in a Variety of Foods Using the TEMPO CC (Coliform Count) Method. Volume 3. Compendium of Analytical Methods. Health Products and Food Branch.
• MFHPB-31: Determination of Coliforms in Foods Using Violet Red Bile Agar — Compendium of Analytical Methods, Volume 2, March 2001.
• American Public Health Association (APHA). (2004) 17th Edition. Standard Methods for the Examination for Dairy Products, APHA Washington DC.
• Blodgett R. BAM Appendix 2: Most Probable Number from Serial Dilutions. Bacteriological Analytical Manual (BAM). US Food and Drug Administration. (The reference’s website address is omitted per program rule.)
• International Standard EN ISO 6887-4 (2003) – Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. Part 4: Specific rules for the preparation of products other than milk and milk products, meat and meat products, and fish and fishery products.
• Woodward R.L. (1957) How probable is the most probable number? J. Am. Water Works Assoc. 49, 1060, 1068.