Growth means more cells
In microbiology, growth means an increase in the number of cells. A bacterium dividing by binary fission doubles the population each generation time (also called doubling time), which depends on the species and conditions. After n generations, the number of cells is the starting number multiplied by 2 to the power n, provided growth stays exponential.
The growth curve
In a closed flask (a batch culture) four phases appear:
- Lag phase. Cells adjust to new medium and make enzymes; little division occurs.
- Exponential (log) phase. Cells divide at a constant, maximal rate, so the log of cell number rises in a straight line.
- Stationary phase. Nutrients run low or wastes build up; the number dividing roughly balances the number dying, so the count levels off.
- Death phase. Deaths exceed new cells.
Oxygen needs also vary: obligate aerobes need oxygen, obligate anaerobes are harmed by it, and facultative anaerobes grow either way.
Counting with dilutions
Cultures are often too dense to count, so they are diluted in steps. Moving 1 mL into 9 mL of sterile liquid gives a 1 in 10 dilution. A sample is spread on agar, and each colony is assumed to come from one colony-forming unit (CFU). The count is
CFU per mL = colonies counted divided by (volume plated in mL times the dilution factor).
Controlling microbes
Sterilisation destroys all life, including endospores. Disinfection reduces microbes on inanimate objects, and antisepsis does so on living tissue. An autoclave uses steam under pressure at 121 degrees Celsius, hotter than boiling, so it can kill endospores. The D-value is the time at a set temperature to kill 90% of the population, a one-log reduction.
Worked example
A broth starts with 4000 cells per mL, and the generation time is 40 minutes. How many cells after 4 hours?
- 4 hours is 240 minutes. 240 divided by 40 gives 6 generations.
- 4000 times 2 to the power 6 is 4000 times 64, giving 256,000 cells per mL.
Next, 0.2 mL of a 10 to the minus 4 dilution produces 62 colonies. Volume times dilution is 0.2 times 0.0001, which is 0.00002. Dividing 62 by 0.00002 gives 3.1 million CFU per mL in the original culture.
For D-values: if D is 3 minutes, then 6 minutes is two log reductions, so a population of 100,000 falls to 1000.