Methods of Counting Bacteria

1. Methods of Counting Bacteria • Direct microscopic • Most probable number • Standard plate count • Coulter counter • Turbidity (optical density) – this is an indirect method

2. Standard Plate Count The basis of the standard plate count is that dilutions are made of the starting culture and each of these dilutions is plated onto solid agar (pour plate or spread plate). Each colony that grows up represents a single cell. Therefore the number of colonies can be used to back calculate the starting population density in cells/ml. Sometimes it is called cfu/ml (colony forming units per ml)

3. Standard Plate Count – Things you must know 1. How to calculate an individual dilution factor. It is the volume added divided by the final total volume. 2. How to move back and for between fractions and scientific notation 3. How to calculate the total dilution factor. It is the multiplication of each individual dilution factor. 4. You must include the plating dilution factor (if there is any) in your final total dilution factor.

4. Standard Plate Count Some Trial Dilution Problems

5. Optical Density Optical density (OD) is only an indirect measure of bacterial numbers. By itself, it can only tell us if one culture is more dense than another. It cannot give us absolute numbers. It is based on the fact that when a culture has a higher number of cells, it scatters more light. The problem with OD readings is that at high denisty the cells scatter almost all of the light and the cell density is no longer proportional to the optical density.

6. Optical Density However, if you do a standard plate count and an optical density reading at the same time, these data can be used to generate a line that will predict the absolute numbers for that particular bacterial strain. OD Cfu/ml