Enumeration Procedure

I. PURPOSE

1.0 This procedure contains general instructions to count and identify microorganisms on agar plates. A consistent method enables Test Section to have more accurate records of colony counts.

II. COUNTING AND DIFFERENTIATING MICROBIAL COLONIES

1.0 Flip agar plate over, bottom side up, and put a felt tip pen dot on the back of each colony. This keeps you from counting the same colony more the once, or recounting the same colonies after an interruption

1.1 Note: Plates have a capacity of 30-300 (cfu/plate). If the number of colonies exceed 300, then the plate may be labeled as “too numerous to count”.

2.0 Colony Color

2.1 If two different colors or shades of color coexist in the same large “colony” it must be considered two.

3.0 Surface Texture

3.1 Some bacteria produce smooth, creamy colonies, while others may develop colonies with regular concentric ridges, and other bacteria produce colonies with a very irregular rough texture. This is an important clue as to whether an apparent spreader consists of one or several colonies

4.0 Shape

4.1 Spreading colonies can vary enormously in their shape. However, if they consist of two or more colonies which have coalesced, the locations of the centers of each original colony can often be determined, allowing them to be counted as separate colonies.

III. FILTER MEMBRANE

1.0 A membrane filter of the appropriate pore size is placed in a filter holder, and the sample is filtered. In this process microorganisms in the test sample are retained on the filter surface by the screening action of the membrane filter. Growth inhibitors can be removed by flushing the membrane with sterile NaCl. Afterwards, the membrane filter is placed on a culture medium and incubated.

2.0 An estimate can be made of the concentration of cells from the filter membrane plate using the following formula:

2.1

IV. DILUTIONS

1.0 Take 6 dilution tubes, each containing 9 ml of sterile saline and label the tubes 1-6. Dilute 1 ml of a microbial sample.

1.1 Withdraw 1 ml of the sample and dispense into the first dilution tube.

1.2 Using the same procedure, withdraw 1 ml from the first dilution tube and dispense into the second dilution tube. Continue doing this from tube to tube until the dilution is complete.

2.0 Transfer 0.1 ml from each of the last three dilution tubes (4-6) onto the surface of the corresponding agar plates.

3.0 Incubate the agar plates at 30-35°C for 48 hours and choose the plate that appears to have between 30 and 300 colonies.