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Government of Canada

Laboratory Procedure

Gouvernement du Canada
MFLP-44 April 1998

HEALTH PROTECTION BRANCH OTTAWA DETERMINATION OF AEROBIC AND ANAEROBIC SPOREFORMERS

John W. Austin Microbiology Research Division Bureau of Microbial Hazards Food Directorate Postal locator: 2204A2 Ottawa, Ontario K1A 0L2

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APPLICATION This method is applicable to the enumeration of viable sporeforming microorganisms in accordance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This revised methode replaces MFLP-44, dated May 1988. PRINCIPLE This Aerobic Sporeformers count (ASF) and Anaerobic Sporeformers count (AASF) estimates the numbers of viable sporeforming organisms per g or mL of product. A portion of the product is mixed with a specified agar medium and incubated under specified conditions of time and temperature. It is assumed that each viable microorganism will multiply under these specified conditions of incubation and give rise to a visible colony which can be counted. This revised method replaces MFLP-44 dated May 1988. DEFINITIONS OF TERMS See Appendix A of Volume 3. COLLECTION OF SAMPLES See Appendix B of Volume 3. MATERIALS AND SPECIAL EQUIPMENT 1) Refrigerator. 2) 3) 4) 5) 6) Trypticase soy agar. Water bath adjustable to 45oC and 75oC. Disinfectant. 0.1% peptone water. Blender.

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Published by: POLYSCIENCE PUBLICATIONS, P.O. Box 1606, Station St-Martin, Laval, Quebec, Canada H7V 3P9. TEL.: 1-800-840-5870. FAX: (450) 688-1930.

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7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17)

pH meter. 1N NaOH. 1N HCl. Thermometer reading to 75oC. Petri plates. Incubator adjustable to 75oC. Anaerobic jar. Colony counter. Tally register. 0.1% 2,3,5 triphenyltetrazolium chloride. "Standard methods for the examination of dairy products" A.P.H.A. 14th or latest Edition.

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PROCEDURE Analyze each sample unit individually. The test shall be carried out in accordance with the following instructions: 6.1 Handling of Sample Units 6.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated (0-5oC) or frozen depending on the nature of the product. 6.1.2 6.2 Analyze sample units as soon as possible after receipt in the laboratory.

Preparation of Media 6.2.1 Prepare trypticase soy agar and dispense in appropriate quantities. Sterilize. 6.2.2 Temper prepared melted agar in a water bath to 45oC ensuring that the water level is 1 cm above the level of the medium in the bottles. Clean surface of working area with a suitable disinfectant. Mark clearly the duplicate Petri plates identifying sample, sample unit, dilution and date of inoculation.

6.2.3 6.2.4

6.3

Preparation of Dilutions 6.3.1 Prepare sterile 0.1% peptone water diluent. 6.3.2 To ensure a truly representative analytical portion agitate liquid 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.

6.3.3

Prepare a 1:10 dilution of the food by aseptically adding 10 g or mL (the analytical unit) into 90 mL of the diluent. Blend or shake according to the type of food as indicated in Table I.

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MFLP-44 April 1998

6.3.3.1 If a homogeneous suspension is to be obtained by blending, the blending time should not exceed 2.5 min in order to prevent over-heating. With foods that tend to foam, use blender at low speed, and remove aliquot from below liquid/foam interface. 6.3.3.2 If a homogeneous suspension is to be obtained by shaking, shake the dilution bottles 25 times through a 30 cm arc in approximately 7 sec. 6.3.3.3 In some instances it may be desirable to prepare the initial dilution on a percent basis in order to obtain a more accurate weight of the material to be tested than is provided for by the customarily employed dilution ration basis, i.e., a 10% solution (suspension) is represented by 10 g (mL) per 100 g (mL) of solution (suspension), whereas a 1:10 dilution is based on 10 g (mL) of product (solute) plus 90 g (mL) of diluent (solvent). 6.3.4 Check the pH of the food suspension. If the pH is outside the range of 5.5-7.6, adjust the pH to 7.0 with sterile 1N NaOH or HCl. For each analytical unit, pipette 20-25 mL of 10-1 dilution (using 0.1% Peptone Water, see section 5.3) into a sterile tube. Also pipette 20-25 mL of a 10-1 dilution into an extra tube and place a thermometer into that tube (Control tube). Place the tubes into a water bath at 75oC. Shake the tubes intermittently until the thermometer of the control tube reads 75oC; then hold for 20 minutes. Prepare subsequent required dilutions from the heat-treated 10-1 dilution using Peptone Water (Suggested dilutions are 10-1, 10-2, and 10-3). Use a separate sterile pipette for making each transfer. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

6.3.5

6.3.6

6.3.7

6.3.8

6.4

Plating Note: Plates should not be poured more than 15 min. after preparation of dilutions. 6.4.1 Agitate each dilution bottle to resuspend material that may have settled out during preparation. Pipette 1 mL or 0.1 mL of the required dilutions to appropriately marked duplicate Petri plates. In the case of products that tend to adhere to the bottom of the plates, add the inoculum to 1.0 mL of sterile diluent previously placed in the Petri plate. For enumerating aerobic sporeformers, pour 12-15 mL of tempered agar into each plate, and mix by rotating and tilting. For anaerobic sporeformers use no more than 10 mL of the melted agar for pouring the plates. Allow to solidify.

6.4.2

6.4.3

6.4.4

6.5

Incubation 6.5.1 For aerobic sporeformer enumeration, incubate plates in the inverted position at 35oC 0.5o for 48 2 hr. Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature. 6.5.2 For enumerating the anaerobic sporeformers, incubate the poured plates in an anaerobic atmosphere such as that produced by the BBL Gaspack system.

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6.6

Counting Colonies 6.6.1 Count colonies promptly after the incubation period. 6.6.2 If possible, select plates with 20-200 colonies (including pinpoint colonies). If counts do not fall within this range select plates that fall nearest to the 20-200 range. Count colonies using a colony counter and a tally register. If plates contain spreaders, select a representative portion of the plates free from spreaders and count colonies in this area. Total count of the whole plate is estimated by multiplying the count for the representative area counted by the reciprocal of the fraction of the plate counted, e.g., 30 colonies counted on 1/4 of area of the plate; count for the whole plate: 30 x 4 = 120 colonies.

6.6.3 6.6.4

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Differentiation of Colonies from Interfering Particles 6.7.1 Food particles such as meat, milk powder, etc., often interfere with the enumeration of the plates. This can be eliminated by making one extra plate of each dilution containing interfering particles and holding it under refrigeration as a control for comparison during counting. 6.7.2 Alternatively, after incubation flood plates with 2 mL of 0.1% 2,3,5, triphenyltetrazolium chloride. Gently rock plates from side to side to cover the entire area with solution. Pour off excessive solution and allow the plates to remain at room temperature for 3 hrs. in an inverted position. The bacteria reduce the indicator to a formazan which colours the colonies red and aids in distinguishing the food particles. Colonies cannot be picked for isolation after this method has been used. Recording Results 6.8.1 Calculate the average count (arithmetic mean) of the duplicate plates, following the examples in Table 5-1: "Standard Methods for the Examination of Dairy Products", A.P.H.A., 14th Edition (E.H. Marth, Editor 1978). 6.8.2 When reporting results (Table II) round-off the counts to two significant figures and record only the first two left hand digits (E.g., record 2,850 as 2,900). If the lowest dilution plated shows no colonies, the recorded value will be the lowest average obtainable with given volume plated onto a given set of replicate plates preceeded by a "less than" (<) sign, e.g., for one millilitre and a set of duplicate plates (1 mL/plate) the value is < 0.5. The lowest possible average with one colony on one of the two duplicate plates is: 1 + 0 = 0.5. 2 This value is for a 10o dilution (Dilution Factor = 1). For other dilutions, the numerical value of 0.5 must be multiplied by the reciprocal of the dilution, i.e., the Dilution Factor, e.g. 1 = 10. 10-1

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6.8.3

6.8.4

To compute the number of sporeformers, use the formula: N = A x D, where N is the number of colonies per g (mL) of product, A is the average count per plate, and D is the respective dilution factor.

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PREPARATION OF MEDIUM

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7.1

When steam sterilization is used, it is essential that sufficient time be allowed for the load to reach the required temperature before the actual sterilizing period commences. This varies considerably with the nature and size of the load. Hence proper exposure times should be followed to ensure sterilization of solutions in flasks and heat stable culture media (Refer to sterilizer manual). Trypticase Soy Agar Tryptone Soy peptone Sodium chloride Agar Final pH 15 g 5 g 5 g 15 g 7.3

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Add ingredients to one litre of distilled water. Thoroughly mix. Carefully heat to dissolve medium. Distribute in test tubes or flasks, and sterilize at 121oC for 15 minutes. Mix well before pouring. Medium is commercially available. 7.3 Peptone Water (0.1%) Diluent Weigh 1.0 g of meat or casein peptone in 1000 mL of distilled water. Dispense in dilution bottle to obtain 99 mL after sterilization at 121oC for 15 min. Peptone is commercially available.

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TABLE I Type of Food Liquids Milk, water, etc. Viscous liquids Solids: Water soluble solids Powder, meats Processed cheese Preparation pipette directly to petri dish and/or weigh into diluent weigh into diluent Treatment shake shake

weigh into diluent weigh into diluent weigh into previously warmed (45oC) 2% sodium citrate (Na3C6H5O7.2H2O) weigh into diluent

shake blend blend

Spices

shake

TABLE II Examples for Recording Results Examples of the average number of colonies Counts between 30-300, e.g., 244 Counts higher than 300, e.g., 440 Counts lower than 30, e.g., 25 No count 0 Dilution 1:1000 Report as no. of bacteria per g(mL) 240,000

Highest dilution

440,000 E

Lowest dilution

25,000 E +500

Lowest dilution 1:1000