Maintenance of Microbial Cultures

• General Principles
  • Biological samples, including microbiological/microbial culture are delicate and require proper handling and storage to maintain viability and characteristics.
  • Standardise handling and storage procedures to minimise contamination or changes in growth behaviour.
  • Consistent treatment of stock cultures ensures reliable and reproducible microbiological test results.

• Culture Arrangement

Get cultures from national culture collections or qualified secondary suppliers. Use only micro-organisms approved for use in educational or laboratory settings.

• • Ensure documented equivalency to relevant reference strains.
  • Cultures may be acquired frozen, freeze-dried, on slants, or in ready-to-use formats.
  • Confirm both purity and identity of all cultures before use in quality control testing by vitek-2 or gene sequencing method.
  • Ready-to-use cultures must undergo incoming testing for both identity and purity.
  • For commonly used strains, confirm identity ideally to the genus and species level.

• Culture Preparation and Resuscitation
  • Follow the supplier’s instructions or validated in-house procedures for preparation and resuscitation.
  • Use the “seed-lot technique” for stock culture storage:
    • Resuscitate and grow the original sample in suitable medium.
    • Prepare aliquots from the first passage and suspend in cryoprotective medium.
    • Transfer to vials and freeze at –30°C or lower.
    • For long-term storage, keep at –70°C or in lyophilised form.

• Working Culture Practices
  • Use frozen stock cultures to inoculate working cultures on a monthly or weekly basis.
  • Do not refreeze unused portions of opened suspensions; discard them to prevent contamination and viability loss.
  • Track the number of transfers (passages) to avoid excessive subculturing.
  • Avoid more than 5 passages to prevent phenotypic changes or mutations.
  • A passage is defined as transferring organisms from a viable culture to fresh medium with subsequent growth.
  • Follow test-specific guidelines regarding maximum allowable passages.

 Maintaining Pure Cultures

• Prevent contamination during inoculation and handling—essential for both safety and scientific accuracy.
• Being able to recognise contamination is a key laboratory skill.

Maintaining Stock Cultures

• Maintain a stock to avoid frequent re-purchase.
• Cultures suitable for school or lab use are generally easy to maintain through regular sub-culturing.
• Maintain careful records and ensure transfer of cultures at least four times a year.
• Do not use streak plates as stock cultures.
• Use slope cultures in screw-cap bottles:
  • Prevents evaporation and drying.
  • Reduces risk of accidental opening.
  • Makes contamination detection easier.
• Prepare two types of stock cultures:
  • Working stock: Used routinely for sub-culturing.
  • Permanent stock: Opened only to prepare new stocks.
• Incubate at an appropriate temperature until adequate growth is observed.
• For strict aerobes, slightly loosen the cap during incubation; reseal tightly before storage.
• Store in a dedicated refrigerator, or at room temperature in cupboards/drawers (if suitable).
• Monitor regularly for contamination.

Checking Cultures for Contamination

• Inspect colony morphology on streak plates and cell uniformity in stained slides.
• Compare to known characteristics of the original culture.
• Regularly check the working stock and always check when preparing new stock.
• If contaminated:
  • Do not attempt to isolate colonies from the mixed culture.
  • Return to the uncontaminated working or permanent stock cultures.

Preventing Contamination of Cultures and the Environment

• Use non-absorbent cotton wool plugs for test tubes and pipettes.
  • These allow airflow but trap micro-organisms via electrostatic adsorption.
  • Plugs must stay dry to retain filtration properties.
• Ensure plugs are:
  • Properly shaped
  • Easy to handle
  • Durable enough to withstand repeated use
• Poorly made plugs compromise aseptic technique and increase contamination risk.

Aseptic Transfer of Cultures and Sterile Solutions

• Regular practice is essential to develop proficiency in aseptic techniques.
• Making a streak plate:
  • Reinforces multiple aseptic skills.
  • Reduces contamination risk by keeping Petri dish exposure time minimal.
• Use the correct tool:
  • Loop: Generally used for colony transfers.
  • Straight wire: Used for tiny colonies or isolating from natural samples.
  • Pipettes: For liquid transfers between tubes or bottles.