Culturing microorganisms and measuring growthEdexcel International A Level Biology: Revision notes

Aseptic technique prevents contamination of the culture by unwanted microorganisms and prevents the culture escaping into the environment. Key steps: sterilise media and equipment (autoclave); flame the inoculating loop until red hot before and after use; flame the neck of bottles and work near a Bunsen flame, which creates an upward current of air; open lids only briefly; use sterile pipettes; tape the lid of a plate (not sealing it completely) and incubate it inverted; and disinfect surfaces and wash hands.

Microorganisms are grown on agar (solid) or in broth (liquid) containing a carbon source, nitrogen source, minerals and growth factors, at a suitable temperature, pH and oxygen level. In schools cultures are incubated at or below 25 °C to reduce the chance of growing human pathogens, which grow best near 37 °C.

A viable count estimates the number of living cells able to divide, by counting the colonies that grow on agar. Because the concentration in a culture is usually too high to count directly, a serial dilution is made, in which a fixed volume of culture is transferred to a fixed volume of sterile diluent repeatedly (for example 1 cm³ into 9 cm³ gives a 10-fold dilution each step). A known volume of one or more dilutions is spread on agar and incubated. Plates with a countable number of colonies (about 30 to 300) are used.

Number of cells per cm³ = number of colonies ÷ volume plated (cm³) × dilution factor. Worked example: 42 colonies from 0.1 cm³ of a 10⁻⁴ dilution gives 42 ÷ 0.1 × 10⁴ = 4.2 × 10⁶ cells cm⁻³.

It assumes each colony grows from one viable cell, and it takes a day or more. A haemocytometer counts cells directly under a microscope in a grid of known volume, which is quick but counts both living and dead cells unless a stain is used.

Dry mass is a measure of total biomass: a known volume of culture is filtered or centrifuged, dried in an oven to constant mass and weighed. It needs a large sample, destroys the culture, and cannot distinguish living from dead cells.

Turbidity is the cloudiness of a liquid culture: as the cells multiply, less light passes through. A colorimeter measures absorbance (or percentage transmission) of a sample against a blank of sterile broth. It is quick and non-destructive, so the same culture can be followed over time. It counts living and dead cells, is not sensitive at low densities, and must be calibrated against cell counts to give numbers of cells.

When a few bacteria are placed in fresh broth, growth follows a characteristic pattern with four phases when the log of the number of viable cells is plotted against time.

• Lag phase: cells adjust to the medium and synthesise enzymes and other molecules, with little or no division.
• Exponential (log) phase: cells divide at a constant, maximum rate, doubling each generation time, because nutrients are not limiting.
• Stationary phase: the rate of division equals the rate of death, because nutrients are limiting and toxic waste has accumulated, so the number of viable cells is constant.
• Death phase: nutrients are exhausted and waste products build up, so the death rate exceeds the rate of division and the number of viable cells falls.

In the exponential phase the population doubles at a constant interval, so $N_t = N_0 \times 2^n$ where $n$ is the number of generations. Hence $n = \frac{\log_{10} N_t - \log_{10} N_0}{\log_{10} 2} = \frac{\log_{10} N_t - \log_{10} N_0}{0.301}$.

The growth rate constant is $k = \frac{n}{t}$, the number of generations per unit time, and the generation time is $\frac{1}{k}$. Worked example: a count rising from 5.0 × 10⁴ to 3.2 × 10⁶ cells cm⁻³ in 6 hours gives $n = \frac{6.505 - 4.699}{0.301} = 6$ generations, so $k = \frac{6}{6} = 1.0$ generation per hour and the generation time is 1 hour. The value of $k$ applies only in the exponential phase, when nutrients are not limiting.

A typical method uses yeast or bacteria in sterile broth in a shaking incubator at a fixed temperature. Use aseptic technique when setting up and sampling. Inoculate a known small volume of culture, take samples at regular intervals, and measure growth by turbidity with a colorimeter (zeroed with sterile broth), by a haemocytometer count, or by dilution plating for viable counts.

Control the volume and composition of the broth, temperature, aeration and pH, and replicate to give a mean. Plot the growth curve (log of count against time), identify the phases and calculate the growth rate constant in the exponential phase. Safety: treat all cultures as potentially harmful, disinfect equipment and dispose of cultures by autoclaving.