Microbial techniques and culturingEdexcel A-Level Biology B: Revision notes

Aseptic technique stops unwanted microorganisms contaminating a culture and stops the culture escaping. Equipment and media are sterilised (an autoclave at 121 °C for 15 minutes). The bench is wiped with disinfectant, hands are washed, and work is done near a Bunsen flame, whose updraught carries airborne microorganisms away. An inoculating loop is flamed until red hot, then cooled before use, and flamed again after. The bottle neck is flamed, lids are opened only briefly, and Petri dishes are taped with two or four short pieces (not sealed all round), so oxygen can enter and anaerobic pathogens are discouraged. Dishes are incubated upside down at no more than 25 °C in schools, to reduce the chance of growing human pathogens.

A broth is a liquid nutrient medium, used to grow large numbers of cells. Agar is a polysaccharide gel with nutrients added, used to grow separate colonies on a surface. A selective medium contains something that allows only some microorganisms to grow, for example a high salt concentration or an antibiotic. Media must supply a carbon source, a nitrogen source, minerals and sometimes growth factors, at the right pH.

• Cell counts: a haemocytometer counts total cells (living and dead) under a microscope.
• Dilution plating: serial dilutions are spread on agar, and each colony is assumed to come from one viable cell. Cells per cm³ = colonies ÷ volume plated × dilution factor. Example: 46 colonies from 0.1 cm³ of a 10⁻⁵ dilution gives 460 × 10⁵ = 4.6 × 10⁷ cells per cm³. It counts viable cells only, but clumps give underestimates.
• Mass: dry mass of cells after filtering and drying; it is destructive and cannot distinguish live from dead cells.
• Turbidity (optical): a colorimeter measures how much light a culture scatters; more cells make it more turbid. It is quick and does not destroy the culture, but it counts dead cells as well.

Lag phase: little increase while cells adapt and make enzymes. Log phase: nutrients plentiful, cells divide by binary fission and numbers double each generation. Stationary phase: the rate of division equals the rate of death, because nutrients run low and wastes build up. Death phase: death exceeds division.

In the log phase N = N₀ × 2ⁿ, where n is the number of generations, so n = (log₁₀ N − log₁₀ N₀) ÷ 0.301. The exponential growth rate constant is k = n ÷ t (generations per unit time), and the generation time is 1 ÷ k.

Example: N₀ = 2.0 × 10⁴, N = 3.2 × 10⁷ after 5 h. N ÷ N₀ = 1600, log₁₀ = 3.204, n = 10.6, k = 2.1 h⁻¹, generation time = 28 minutes.

Core Practical 12 (liquid culture): inoculate sterile nutrient broth aseptically, incubate at constant temperature, and remove samples at intervals to measure turbidity with a colorimeter (or make viable counts). Plot growth against time.

Core Practical 13 (streak plating): streak a mixed culture across agar, flame the loop between sections so the bacteria are progressively diluted, tape the dish, invert and incubate. Isolated colonies can be subcultured.

Safety and ethics: use non-pathogenic strains such as E. coli K12, carry out a risk assessment, never open incubated dishes, disinfect the bench, and autoclave or disinfect all cultures before disposal. Wash hands after the work.