LAB 4:

Determination Aerobic Colony (Plate) Count

1. Application This method is applicable to the enumeration of viable aerobic bacteria (Psychrophilic, Mesophilic and Thermophilic bacteria) in foods. 2. Principle The Aerobic Colony Count (ACC or APC) 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 b) specific conditions of time and c) 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 <15 C (0-20 C). ^ Mesophilic bacteria: An organism whose optimum growth temperature between 20 45 C. ^ Thermophilic bacteria: An organism whose optimum growth temperature is > 45 C. 3. Materials and special equipment A) Plate count agar (PCA) B) Butterfield's phosphate-buffered (BPB) diluent. KH2PO4 Distilled water min at 121C. Store in refrigerator. C) Sodium 2,3,5 triphenyltetrazolium chloride (0.1%) (optional) D) Stomacher or blender E) Incubator capable of maintaining the growth temperature required for the specific type of aerobic bacteria being enumerated (i.e. for psychrophilic bacteria: 15-20 C, for mesophilic bacteria: 30-37 C, and for thermophilic bacteria: 55 C) F) Waterbath at 45 C G) Colony counting device (optional) 4. Procedure The test shall be carried out in accordance with the following instructions: 4.1. Handling of Sample Units 4.1.1. During storage and transport: a) Shelf stable products keep at Room temperature, b) refrigerated samples keep at (0-5 C) and c) frozen products keep frozen. 4.1.2. Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death. Food Microbiology Lab Session 4 Page 1 of 4 34 g 500 ml

Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15

4.1.3. Analyze sample units as soon as possible after receipt in the laboratory. 4.2. Preparation of Media 4.2.1. Prepare plate count agar then Sterilize. 4.2.2. Temper agar medium in a water bath to 45 C ensuring that the water level is 1 cm above the level of the medium in the bottles. 4.2.3. Clean surface of working area with a suitable disinfectant. 4.2.4. Clearly mark the Petri plates (Duplicate plates are recommended). 4.3. Preparation of Dilutions 4.3.1. Prepare sterile BPB diluent. 4.3.2. Ensure that the sample contents are homogeneous. If the sample is solid, obtain the analytical sample by taking a portion from several locations within the sample unit. 4.3.3. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL into 225 mL of the required diluent (Aq. 250 ml). If a sample size other than 25 g or mL is used, maintain the 1:10 sample to dilution ratio. 4.3.4. 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 an aliquot from below the liquid/foam interface. 4.3.5. If the pH is outside the range of 5.5-7.6, adjust the pH to 7.0 with sterile NaOH or HCl. 4.3.6. Prepare decimal dilutions as required, using a separate sterile pipette for making each transfer. 4.3.7. Shake all dilutions prior transfers to next dilution. 4.4. Plating 4.4.1. Resuspend each dilution bottle that may have settled out during preparation. 4.4.2. Pipette 1 mL of the dilutions to appropriately marked Petri plates. 4.4.4. Pour 12-18 mL of tempered agar into each plate, and mix by rotating. 4.4.5. Allow to solidify. Plates should be poured not more than 15 min after preparation of dilutions. 4.5. Incubation Incubate plates in the inverted position 15 20 C for psychrophilic bacteria; 30 35 C for mesophilic bacteria; or 55 C for thermophilic bacteria. Check for the results on daily basis up to 48 4 h for mesophilic bacteria. Psychrophilic and thermophilic bacteria may be incubated up to 5 days. Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature. 4.6. Counting Colonies Food Microbiology Lab Session 4 Page 2 of 4

4.6.1. Count colonies promptly after the incubation period. 4.6.2. If possible, select plates with 25-250 colonies (including pinpoint colonies) or plates that fall nearest to the 25-250 range. 4.6.3. If plates contain colonies which spread, select a representative portion of the plates free from spreaders. Then the total count of the entire plate can be estimated; e.g., 25 colonies counted on 1/4 of area of the plate; count for the whole plate: 25 x 4 = 100 colonies. Otherwise, record as Spreader (SPR). 4.6.4. When number of CFU per plate exceeds 250 or cannot be counted, record the counts as too numerous to count (TNTC). 4.6.5. When plate(s) from a sample are known to be contaminated or otherwise unsatisfactory, record the result(s) as laboratory accident (LA). 4.6.6. If the lowest dilution plated shows no colonies, the recorded value will be the lowest obtainable count preceded by a less than (<) sign. 4.7 Differentiation of Colonies from Interfering Particle 4.7.1. Food particles such as meat, milk powder, etc., often interfere with the count 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. 4.7.2. 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. Note: Colonies cannot be picked for isolation after this method has been used. 4.8. Reporting Results 4.8.1. Calculate the average count (arithmetic mean) if the duplicate plates used. 4.8.2. Calculate Aerobic Colony Count (ACC or APC), using the formula: N = A x D, where N is the Colony Forming Unit (CFU) per g (mL) of product, CFU/g (ml), A is the average count per plate, and D is the respective dilution factor. 4.8.3 When reporting results round-off the counts to two significant figures and record only the first two left hand digits; 11,300 CFU/g reported as 1.1 X 104 CFU/g 235,000 CFU/g reported as 2.4 X 105 CFU/g 4.8.4. Mark calculated APC with estimated aerobic plate counts (EAPC) to denote that it was estimated from counts outside 25-250 per plate range or a spreader plate. 4.8.5. Report test conditions. Food Microbiology Lab Session 4 Page 3 of 4

Examples of the unusual calculations Dilution Colonies Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 1:100 18 0 TNTC LA SPR TNTC 1:1000 2 0 640 640 350 600
(a)

EAPC/ml (g) 1,900 < 100 (< 1000) 640,000 640,000 350,000 > 600,000

TNTC: Too numerous to count. EAPC: Estimated aerobic plate count. LA: Laboratory accident. SPR: Spreader. (a) : Based on 1/4 plate area.

Food Microbiology Lab Session 4

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