Culturing Microorganisms

Growing microorganisms in the laboratory and in industry requires understanding of their nutritional needs, growth kinetics and the control of contamination. OCR A-Level Biology A specification 6.2.1 (f)–(h) requires you to explain aseptic technique, the four phases of the bacterial growth curve, and the differences between batch and continuous fermentation.

Key Definitions:

• Culture — a population of microorganisms grown in or on a nutrient medium.
• Aseptic technique — practices that prevent contamination by unwanted microorganisms.
• Batch culture — a closed system in which all nutrients are added at the start and the product is collected at the end.
• Continuous culture — an open system in which nutrients are added and products removed continually.
• Lag phase / log phase / stationary phase / death phase — the four stages of population growth in a closed culture.

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Nutrient Requirements

Microorganisms need:

• Carbon source — usually glucose or another sugar (energy and building blocks).
• Nitrogen source — usually ammonium salts, nitrates or amino acids (for proteins and nucleic acids).
• Mineral salts — phosphate, sulfate, magnesium, potassium (for enzymes and cell structures).
• Vitamins and growth factors — for fastidious organisms that cannot synthesise all their needs.
• Water — the solvent for all biochemistry.
• Oxygen — for aerobic organisms (but anaerobes are inhibited by oxygen).
• Optimum temperature and pH — varies between species.

Liquid media (broths) are used in large-scale fermentation; solid media (agar) are used for isolating and counting colonies.

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Aseptic Technique

Asepsis means the absence of unwanted microorganisms. It is essential because:

• Contaminants can out-compete or overgrow the desired culture.
• They may produce toxins, interfere with products, or ruin the batch.
• In industry, a contaminated fermenter can cost millions to decontaminate and restart.
• In medical settings, contamination can cause infection.

Standard Aseptic Practices

• Sterilise media and equipment in an autoclave at 121 °C (15 psi) for 15 minutes. Heat-sensitive materials are filter-sterilised.
• Disinfect work surfaces with ethanol or chlorhexidine.
• Flame-sterilise the neck of bottles and inoculating loops before use.
• Work near a Bunsen flame — the updraught carries airborne contaminants away.
• Lift lids only partially when inoculating Petri dishes.
• Seal agar plates with tape (but not all the way round, to allow gas exchange and prevent anaerobes).
• Wash hands and wear gloves to protect both worker and culture.
• Incubate plates upside down (inverted) to prevent condensation dripping onto the agar.

School and College Safety Rules

In schools, cultures must be incubated at 25 °C, not 37 °C, to discourage growth of human pathogens. Only specific non-pathogenic species (e.g. Micrococcus luteus, Bacillus subtilis) are used. Plates are sealed and autoclaved before disposal.

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Measuring Microbial Growth

Three main methods:

1. Direct counting — using a haemocytometer to count cells under a microscope. Gives total count (alive + dead).
2. Viable count — serial dilution and plating on agar; only living cells form colonies (CFUs — colony-forming units).
3. Turbidity (optical density) — measuring cloudiness with a colorimeter. Quick and easy but includes dead cells.

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The Bacterial Growth Curve

In a closed (batch) culture, bacterial populations follow a characteristic growth curve with four phases:

flowchart LR
    A[Lag phase] --> B[Log phase]
    B --> C[Stationary phase]
    C --> D[Death phase]

Plotted as log(number of cells) against time, the curve has a distinctive S-shape followed by a decline.

1. Lag Phase

Cells are adjusting to their new environment. They take up water, synthesise enzymes, membrane components and nucleic acids. Little or no cell division occurs. Length depends on previous culture conditions and the organism.

2. Log (Exponential) Phase

Cells divide at their maximum rate. Numbers double every generation (every 20–30 minutes for E. coli at 37 °C). On a log plot, this phase appears as a straight line. Nutrients are abundant and waste products are still low.

Generation time (g) is calculated as:

$g = \frac{t}{\log_2(N_t / N_0)}$g=log2​(Nt​/N0​)t​

where t is the elapsed time, N₀ is the starting number and Nₜ is the number after time t.

3. Stationary Phase

The rate of division equals the rate of death, so population size stays constant. This happens because: