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Microbial Cultivation: Operational Guide and Precautions

2025-06-26

Experimental Principles

Most bacteria are heterotrophic, requiring nutrient-rich media and controlled conditions (temperature, humidity, gas) for growth. By mimicking their natural habitat, we enable rapid ex vivo proliferation.

Growth Requirements

Nutrient-rich Media: Carbohydrates, proteins, vitamins, and minerals.
Optimal Temperature: Typically 20–40°C, varies by species.
pH Balance: Most thrive at pH 6.5–7.5.
Gas Environment: Aerobes need oxygen; anaerobes require oxygen-free conditions.

Medium Preparation

Key Steps:
1. Accurately measure components per recipe.
2. Record details: medium type, reagent grade, pH, volume, sterilization parameters, and preparation date.
Example Recipe Note:
"LB Medium (500mL): 10g tryptone, 5g yeast extract, 10g NaCl, pH 7.2, autoclaved at 121°C for 30 min on 2025-06-26 by Operator A."

Sterilization Techniques

Method	Principle	Applications	Temperature/Time
Chemical	Protein denaturation	Glassware, surfaces (ethanol, Lysol)	Not for media
Radiation	Nucleic acid photochemical damage	Air and surface sterilization (UV)	15–30 min exposure
Dry Heat	Oxidation and protein denaturation	Glass/metal tools (140–180°C)	1–2 hours
Moist Heat	Steam-induced bond disruption	Media and equipment (121°C)	30 min at 15 psi
Filtration	Physical barrier (0.01–0.45μm pores)	Heat-sensitive solutions	Ambient temperature

Inoculation Methods

Streaking: Linear spreading on solid media (inoculation loop).
Three-point Inoculation: Triangular placement for fungal morphology study.
Stab Inoculation: Anaerobe preservation via deep agar 穿刺.
Pour Plate: Mixing culture with 45°C molten agar.
Spread Plate: Uniform plating using a glass spreader.
Liquid Transfer: Pipetting between liquid/solid media.
Injection Inoculation: For live organisms (e.g., vaccine administration).
Live Host Inoculation: Virus culture in embryos or animal tissues.

Strain Preservation

Goal: Minimize metabolic activity to prevent mutation or death.
Key Conditions: Low temperature, dryness, anoxia, nutrient limitation.

Preservation Methods

Agar Slant Refrigeration:
- Inoculate slants, incubate, wrap in foil, store at 4°C.
- Renew every 2–3 months; semi-solid slants last 6–12 months.
Liquid Paraffin Preservation:
- Sterilize paraffin (121°C/30 min), dry at 110°C.
- Overlay cultures with 1cm paraffin, store upright at 4°C.

Critical Precautions

Sterility Maintenance:
- Flame tools before/after use; work within 15cm of Bunsen burner.
Environmental Control:
- Maintain lab humidity <60%; avoid drafts during inoculation.
Safety Measures:
- Use biosafety cabinets for pathogenic cultures; autoclave waste before disposal.