Lab 4: Determination of Aerobic colony count in

Lab 4: Determination of Aerobic colony count in Foods ENUMERATION OF MICROORGANISMS IN FOOD Application This method is applicable to the enumeration of viable aerobic bacteria (psychrophilic, mesophilic and/or thermophilic bacteria) in foods.

The Aerobic Colony Count (ACC) estimates the number of viable aerobic bacteria per g or mL of product. A portion of the product is mixed with a specified agar medium and incubated under specific conditions of time and temperature. It is assumed that each viable aerobic bacterium will multiply under these conditions and give rise to a visible colony which

can be counted. Psychrophilic bacteria: an organism which grows optimally at or below 15oC, which has an upper limit for growth at ca. 20oC, and which has a lower limit of growth of 0oC or lower. Mesophilic bacteria: an organism whose optimum growth temperature lies within a range generally accepted as ca. 20 - 45oC.

Thermophilic bacteria: an organism whose optimum growth temperature is > 45oC. Materials and special equipment HCl pH meter NaOH Stomacher

Colony counting device Procedure Determine which type of aerobic bacteria are being enumerated. Analyze each sample unit individually.

The test shall be carried out in accordance with the following instructions: Handling of Sample Units 1. 2. During storage and transport, the following shall apply: with

the exception of shelf- stable products, keep the sample units refrigerated (0-5oC). Sample units of frozen products shall be kept frozen. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death. 3. Analyze sample units as soon as possible after Procedure Preparation of Media 1. Prepare plate count agar and dispense in appropriate quantities. Sterilize. 3. Clean surface of working area with a suitable disinfectant.

4. Clearly mark the duplicate Petri plates. Procedure Preparation of Dilutions 1. Prepare sterile 0.1% peptone water diluent. 2. To ensure a truly representative analytical unit, 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. 3. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in Table I. If a sample size other than 25 g or mL is used, maintain the 1:10 sample to dilution ratio, such as 11 (10) g or mL into 99 (90) mL. Procedure 4. In some instances it may be advantageous to prepare the initial dilution on a percent basis to

obtain a more accurate test material weight than is attained by the dilution ratio method; 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). Procedure 6. 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 NaOH or HCl. 7. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 8. Shake all dilutions immediately prior to making transfers to

ensure uniform distribution of the microorganisms present. Procedure Procedure Plating 1. Agitate each dilution bottle to re-suspend material that may have settled out during preparation. Procedure Incubation

Incubate plates in the inverted position for 48 h 4 h. Incubation temperature is dependent on the growth temperature requirements of the target organisms (for psychrophilic bacteria: 15 - 20oC, for mesophilic bacteria: 30 - 35oC, and for thermophilic bacteria: 55oC). Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature. 1. Counting Colonies Count colonies promptly after the incubation period.

2. If possible, select plates with 25-250 colonies (including pinpoint colonies). 3. If plates contain colonies which spread, select a representative portion of the plates free from spreaders, if possible, and count the colonies in this area.

The total count of the entire 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. Differentiation of Colonies from Interfering Particles 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.

2. Alternatively, after incubation flood plates with 2 mL of 0.1% 2,3,5, triphenyl-tetrazolium 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 colors

the colonies red and aids in distinguishing the food particles. Colonies cannot be picked for isolation after this method has been used. Recording Results Calculate the average count of the duplicate plates. End of lecture

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