Culturing Microorganisms

Spec Mapping — OCR H420 Module 6.2.1 — Cloning and biotechnology, content statements covering nutritional and environmental requirements for microbial growth, aseptic technique, the four phases of the bacterial growth curve (lag, log/exponential, stationary, death/decline), generation-time calculations, viable vs total counts, and batch vs continuous fermentation (refer to the official OCR H420 specification document for exact wording). This lesson follows directly from the microorganisms-in-biotechnology lesson and supplies the quantitative growth-kinetics framework underpinning every named fermentation in the previous lesson.

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 requires you to explain aseptic technique, the four phases of the bacterial growth curve, and the differences between batch and continuous fermentation.

The discipline of pure-culture microbiology was established in the late nineteenth century by Robert Koch at Berlin, who developed the techniques of solid-medium agar plating, streak inoculation and pure-colony isolation that remain the bedrock of school PAG 7 work today. Koch's postulates (1890) — the criteria for proving that a microorganism causes a specific disease — depended on the ability to grow that microorganism in pure culture, a capability he and his assistant Walther Hesse (whose wife Fanny Hesse suggested agar over gelatin) made routine. Paraphrased: Koch's contribution was procedural — he showed that microbiology could be a precise, replicable laboratory science rather than a speculative natural-history pursuit. The growth-curve mathematics in this lesson, including the four phases observed in batch culture, were systematised by Jacques Monod at the Institut Pasteur in the 1940s as a quantitative description of bacterial population dynamics.

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, providing both energy (via respiration) and the carbon skeletons for biosynthesis of proteins, nucleic acids and lipids. In industrial fermentation, cheap carbon sources include molasses (cane / beet sugar refining waste), corn-steep liquor and whey.
• Nitrogen source — usually ammonium salts, nitrates or amino acids, providing N for amino acids and nucleotides. Industrial sources include ammonium sulfate, soya flour, casein hydrolysate.
• Mineral salts — phosphate (for ATP, DNA, RNA, phospholipids), sulfate (for cysteine and methionine), magnesium (for enzyme cofactors, ribosome stability), potassium (cytoplasmic osmolyte), iron (cytochromes), trace metals (Mn, Zn, Cu, Mo).
• Vitamins and growth factors — for fastidious organisms (e.g. Lactobacillus) that cannot synthesise all their needs; B vitamins, biotin, p-aminobenzoic acid.
• Water — the solvent for all biochemistry; cells are typically ~70% water by mass.
• Oxygen — for aerobic organisms; strict anaerobes (Clostridium, methanogens) are killed by oxygen; facultative anaerobes (E. coli, Saccharomyces) grow either way; microaerophiles (Campylobacter, Helicobacter) need reduced O₂.
• Optimum temperature and pH — varies between species. Psychrophiles thrive at 0-20 °C; mesophiles at 20-45 °C (most human-relevant); thermophiles 45-80 °C; hyperthermophiles above 80 °C (Pyrolobus fumarii grows at 113 °C). pH optima range from acidophiles (pH 1-3, Acidithiobacillus) to alkaliphiles (pH 9-11, Bacillus alcalophilus).

Liquid media (broths) are used in large-scale fermentation; solid media (agar) are used for isolating and counting colonies. Agar's history is instructive: Robert Koch's wife and assistant Walther Hesse's wife Fanny Hesse suggested agar (derived from red seaweeds, Gelidium) in 1881 as a replacement for the gelatin Koch had been using; agar's much higher melting point (~85 °C) and bacterial-resistance made it the ideal solid-medium scaffold, and it remains the standard 145 years later.

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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. Autoclaving uses saturated steam under pressure to deliver moist heat sufficient to inactivate bacterial endospores (the most heat-resistant biological structures); 121 °C for 15 minutes is the established empirical minimum. Heat-sensitive materials (e.g. antibiotics, vitamins, serum) are filter-sterilised through 0.22 μm membrane filters that exclude all bacteria.
• Disinfect work surfaces with ethanol (70% v/v in water, the optimum concentration for protein-denaturation kinetics — pure ethanol is less effective because it precipitates surface proteins before penetrating cells) or with chlorhexidine, iodophors, or hypochlorite.
• Flame-sterilise the neck of bottles and inoculating loops before use. The loop must be heated to red heat along its entire length and allowed to cool briefly before inoculation, to avoid heat-shocking the sample.
• Work near a Bunsen flame — the upward convection updraught from the flame carries airborne contaminants away from the working zone, providing a low-tech sterile field.
• Lift lids only partially when inoculating Petri dishes — the "clam-shell" technique, opening only the side facing the worker and only as far as necessary to admit the loop.
• Seal agar plates with tape, but not all the way round, to allow some gas exchange and prevent the buildup of anaerobic conditions that favour pathogens. School practice in the UK is typically two short tape strips on opposite sides rather than a continuous seal.
• Wash hands and wear gloves to protect both worker and culture; remove gloves on leaving the work area.
• Incubate plates upside down (inverted, agar side up) to prevent condensation dripping onto the agar surface and smearing colonies.

School and College Safety Rules

In UK schools, CLEAPSS guidance requires cultures to be incubated at 25 °C, not 37 °C, to discourage growth of human pathogens that might be present as contaminants on apparatus or in air-handling. Only specific non-pathogenic Containment Level 1 species (e.g. Micrococcus luteus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus niger) are used in routine school practical work. Plates are sealed with tape, never opened after incubation, and autoclaved at 121 °C for 15 minutes before disposal in clinical waste streams.

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Measuring Microbial Growth

Three main methods:

1. Direct (total) counting — using a haemocytometer to count cells under a microscope. Gives total count (alive + dead). Modern variants include flow cytometry with viability stains (propidium iodide for dead cells, SYTO9 for all).
2. Viable count — serial dilution and plating on agar; only living cells form colonies (CFUs — colony-forming units). The standard "Miles and Misra" technique plates 20 μL drops at multiple dilutions and counts the dilution giving 5-30 colonies per drop. Disadvantage: cannot count unculturable / VBNC (viable but non-culturable) cells, which are a major fraction of natural samples.
3. Turbidity (optical density) — measuring cloudiness with a colorimeter at 600 nm (OD₆₀₀). Quick and easy but includes dead cells, and the relationship between OD and cell density is non-linear at high biomass because of light scattering.

Method	Counts	Speed	Sensitivity	Drawback
Haemocytometer	Total (live + dead)	Minutes	$\sim 10^4$∼104 – $10^7$107 cells/mL	Tedious, doesn't distinguish living
Viable count (CFU)	Living, culturable only	24-48 h	$\sim 10$∼10 – $10^9$109 cells/mL	Slow, misses VBNC cells
OD₆₀₀	Total mass / scatter	Seconds	$\sim 10^7$∼107 – $10^9$109 cells/mL	Includes dead; non-linear at high density
Flow cytometry + stain	Live and dead, distinct	Minutes	$\sim 10^2$∼102 – $10^7$107 cells/mL	Requires specialist instrument

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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.

The same formula can be rearranged to give population size after n generations:

$N_t = N_0 \cdot 2^n \quad \text{where} \quad n = \frac{t}{g}$Nt​=N0​⋅2nwheren=gt​

For E. coli growing optimally at 37 °C in rich medium, g ≈ 20 minutes, so in 6 hours of unrestricted growth a single founding cell would in principle give rise to $2^{18} = 262{,}144$218=262,144 daughters — an exponential that the closed-batch environment quickly halts as nutrients deplete.

Worked generation-time calculation

A Lactobacillus culture grows from $5 \times 10^4$5×104 cells/mL to $4 \times 10^7$4×107 cells/mL in 4 hours of log-phase growth. Calculate the generation time.

$N_t / N_0 = \frac{4 \times 10^7}{5 \times 10^4} = 800$Nt​/N0​=5×1044×107​=800 $\log_2(800) = \frac{\log_{10}(800)}{\log_{10}(2)} = \frac{2.903}{0.301} \approx 9.64$log2​(800)=log10​(2)log10​(800)​=0.3012.903​≈9.64 $g = \frac{240 \text{ min}}{9.64} \approx 24.9 \text{ min per generation}$g=9.64240 min​≈24.9 min per generation

SVG — bacterial growth curve

3. Stationary Phase

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

• Nutrients become limiting.
• Waste products (e.g. lactic acid) accumulate to toxic levels.
• pH changes inhibit enzymes.
• Oxygen may run out.

Many secondary metabolites (including penicillin) are produced during the stationary phase.

4. Death (Decline) Phase

Death rate exceeds division rate. Cells die because of nutrient exhaustion and toxic waste build-up. The culture will eventually be empty of living cells unless rescued.

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Batch vs Continuous Fermentation

Batch Fermentation

A closed system. Nutrients are added at the start, the culture grows through its four phases, and the product is harvested at the end.

Advantages:

• Simple setup and operation.
• Good for making secondary metabolites like penicillin (produced in stationary phase).
• Easier to maintain sterility between batches.
• If contamination occurs, only one batch is lost.
• Flexible — different products can be made in the same vessel.

Disadvantages: