Serial dilution and colony counting

1. Serial dilution and colony counting • Serial dilution and colony counting • Also know as “viable cell counts” • Concentrated samples are diluted by serial dilution • The diluted samples can be either plated by spread plating or by pour plating

2. Serial dilution and colony counting • Serial dilution (cont.) • Diluted samples are spread onto media in petri dishes and incubated • Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual. • A simple calculation, with a single plate falling into the statistically valid range, is given below:

4. Serial dilution and colony counting Count the number of colonies on a plate that has between 25 and 250 colonies. Any plate which has more than 250 colonies is designated as "too numerous to count" (TNTC). Also any plate which has fewer than 25 colonies is designated as "too few to count" (TFTC). Notes:According to FDA Bacteriological Analytical Manual ( in rare situations) If there is no 25-250 colony plate, use plate having nearest to 25 or 250 colonies. If all plates from a sample show no colonies, record count as 0.

5. Bacterial Generation time

6. Growth in Batch Culture • “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction. • Growth is an increase in the number of cells, not an increase in size. • A “batch culture” is a closed system in broth medium in which no additional nutrient is added after inoculation of the broth. • Generation time:time it takes for a cell to divide and the population to double.

7. Growth in Batch Culture • Typically, a batch culture passes through four distinct stages: • Lag stage • Logarithmic (exponential) growth • Stationary stage • Death stage

8. Standard Bacterial Growth Curve in Batch Culture

9. Lag stage ( phase ) • During lag phase, bacteria adapt themselves to growth conditions. • It is the period where the individual bacteria are maturing and not yet able to divide. • During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs. • So in this phase the microorganisms are not dormant.

10. Logarithmic (exponential) growth • Is a period characterized by cell doubling. • This period in which the organisms are growing at the maximal rate possible. • Generation time can be easily obtained from the exponential phase of a growth curve. • Exponential growth cannot continue indefinitely, because the medium is soon depleted of nutrients and enriched with wastes.

11. Stationary stage (phase) • Stationary phase, the growth rate slows as a result of nutrient depletion and accumulation of toxic products. • This phase is reached as the bacteria begin to exhaust the resources that are available to them. • This phase is a constant value as the rate of bacterial growth is equal to the rate of bacterial death.

12. Death stage (phase) • The number of deaths eventually exceeds the number of new formed. • Cell death may result from nutrient limitation, Toxic waste accumulation,loss of cell's ability to detoxify toxins and O2 depletion.

13. Standard Bacterial Growth Curve in Batch Culture

14. Optical density is directly proportional to the number of cells. When the bacterial population doubles, the optical density of the bacterial suspension also doubles.