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# Optimising and analysing growth

Once the right environment is set up (often this means 37 deg. Celsius, shaking the flasks to introduce oxygen bubbles into the solution and optimise growth, leaving plates incubating overnight, etc.), growth can finally be monitored. There are many ways of doing this, such as cell counts, dilution plating, mass and optical methods that detect turbidity.

Cell counts involve pipetting a small volume from a liquid culture under a slide with a grid and looking at it under a microscope. The smaller sections of the grid can be used to count the number of cells, then multiply it by the number of sections and by the factor corresponding to the volume taken from the main solution, to obtain the total number of cells present.

For example, if we count 15 cells in one of 25 grid sections from a 10 microlitre sample of a total volume of 10 mililitres (there are 10,000 microlitres in 10 mililitres, so our sample is 10,000 / 10 = 1,000 times less than the total volume), we would compute:

15 cells x 25 sections x 1,000 = 375,000 cells in our total volume

Another method of measuring your cultured microorganism is dilution plating. This is done when the initial amount is unknown, as well as to find out various things about the organism. Sequential agar plates of increasingly diluted microorganism samples are set up, and the emerging colonies are counted (usually grown overnight). Colonies form from just a single or few cells, so are good for tracking growth and estimating numbers. On some plates nothing might grow, while others might have far too many colonies to easily count.

The mass of course reflects the grow of microorganisms too, and can be measured from liquid cultures usually after centrifugation to separate the cells from the liquid, disposing off of the liquid an then weighing the solid mass of cells that have grown.

Finally, and the most used measurement technique, is measuring the absorbance or optical density (OD) of a liquid sample. As cells grow, the solution becomes increasingly turbid (opaque, cloudy) so how much light can pass through is a measure of how many cells have grown. For example, 1 ml samples from large flasks (of around 1 or 2 litres) of E. coli culture are taken every half an hour. They are pipetted into special clear cuvettes that are placed into a spectrophotometer. This passes a beam of light through the sample and detects the light passing through on the other side at a wavelength of 600 nm, specific to E. coli.

A value of 0.1 shows the beginning of bacterial growth, while by 0.8 they are growing exponentially. The trajectory of their growth curve is highly reproducible, and indicates specific growth stages in a culture.

The lag phase represents the beginning of their growth. Once they get adjusted to their new environment and start thriving, they are ready to divide. This takes place actively during the log phase when their growth is exponential (because 2 cells become 4, and 4 become 8, and 8 become 16). Once their expansion into the media has reached its maximum potential, and they begin to run out of space and nutrients, they reach the stationary phase where division halts.

If the medium is left the same, with excretory products ever increasing and nutrients running out, they begin to die. This is the death phase.

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