2223Food Biology Lab4 Aerobic Plate Count

7/28/2019 2223Food Biology Lab4 Aerobic Plate Count

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Food Microbiology Lab Session 4 Page 1 of 4

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 underb)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 45 C.

3. Materials and special equipment

A) Plate count agar (PCA)B) Butterfield's phosphate-buffered (BPB) diluent.

KH2PO4

34 g

Distilled water 500 mlAdjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15

min at 121C. Store in refrigerator.

C) Sodium 2,3,5 triphenyltetrazolium chloride (0.1%) (optional)D) Stomacher or blenderE) Incubator capable of maintaining the growth temperature required for the specific typeof 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 CG)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.


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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 plateswith incubator temperature.

4.6. Counting Colonies


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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,

recordas 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 (


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Examples of the unusual calculationsDilution

Colonies 1:100 1:1000 EAPC/ml (g)Sample 1 18 2 1,900Sample 2 0 0 < 100 (< 1000)Sample 3 TNTC 640 640,000Sample 4 LA 640 640,000Sample 5 SPR 350 350,000Sample 6 TNTC 600(a) > 600,000

TNTC: Too numerous to count.

EAPC: Estimated aerobic plate count.

LA: Laboratory accident.SPR: Spreader.

(a): Based on 1/4 plate area.

2223Food Biology Lab4 Aerobic Plate Count

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