Introduction to Lab Ex. 19: Enumeration of Bacteria - Pour Plate Technique

Lab Ex. 19: Enumeration of Bacteria – Pour Plate Technique Bacterial growth may be determined based on increase in the number of cells. Reproduction and thus growth leads to increase in cell number and thus the population of the bacterial culture. Bacterial growth follows specific patterns as seen in the growth curve patterns for bacteria. The time it takes for a population of bacterial cells to double in number is called the generation time. Bacterial growth may be determined by using either Cell Mass methods or Cell Number methods.

Cell Mass methods: Turbidity measurements by Spectrophotometry Total Protein Total DNA Cell Number methods: Direct Microscopic Counts (hemocytometer) Pour Plate Most Probable Number Membrane Filter Enumeration methods may yield either total counts or viable counts. Total count is a count of cells including dead and live cells. Viable count is a count of only those cells that are alive in the sample.

Pour Plate method: This is the most commonly used method for enumeration of bacteria in a wide variety of samples including milk, food, meat, soil etc. Pour plate methods yield a count of only the living cells in the sample and thus are a viable count. There are two steps to the process: dilution of the sample so that various dilutions of the sample may be inoculated onto plates and a count of the colonies that grow made; the second step is the plating of the dilutions so that each cell in the diluted sample may then grow and form colonies that will in turn become visible to the naked eye and can be counted.

Dilution is important since the colonies will have to be counted and with a concentrated sample there may be too many colonies than can be accurately counted. Plating is important since a count of only the living cells is required in this procedure (only living cells will be able to multiply and form colonies) Samples of milk, meat and soil will be used in the exercise. Rules to keep in mind: - only living cells are counted. Why? - only plates with colony numbers between 30-300 are useable (<30 or >300 are not statistically valid) - use aseptic technique - dilutions to be done accurately

Procedure: - aseptically dilute sample with sterile water blanks - make 1:10 dilutions as instructed, based on sample - aseptically transfer 1ml volume of dilutions that are to be plated onto sterile Petri plates - pour molten agar onto the sample in Petri dish and mix thoroughly - incubate as instructed for growth of colonies - during next lab period, count colonies on plates - use the formula: cfu/ml = # of colonies x 1 dilution