Sterilisation and Culture Techniques
Introduction
This domain distinguishes sterilisation from disinfection and covers physical and chemical methods, media preparation, aseptic transfer, and isolation of pure cultures. The exam tests method selection, parameters, and evidence of effectiveness.
Core rules
- Sterilisation destroys all microbial life, including bacterial spores; disinfection reduces pathogens but may not kill spores.
- Moist heat autoclaving is typically 121°C at 15 psi for 15 minutes for routine media, with longer times for large volumes or dense loads.
- Dry heat is used for glassware and metal at 160°C for 2 hours or 170°C for 1 hour.
- Heat-labile liquids are filtered through a 0.22 µm membrane; this removes bacteria but not viruses.
- Ethylene oxide or gamma radiation is used for heat-sensitive plastics and single-use items.
- A streak plate dilutes a sample across agar to obtain isolated colonies; aseptic transfer uses a sterile loop and flame or disposable sterile technique.
Worked example
A 1 L broth is autoclaved in a 2 L flask with a loose cap. The correct cycle is 121°C, 15 psi, 15 minutes or longer according to validated load size, using the liquid cycle. A chemical indicator strip shows exposure to heat, but a biological indicator containing Geobacillus stearothermophilus spores is needed to verify that the cycle actually killed spores.
Traps
Autoclave tape is not proof of sterilisation. Do not autoclave heat-labile antibiotics, vaccines, or volatile chemicals. Overfilled flasks and tight caps prevent steam penetration. A 0.22 µm filter does not remove viruses, and a sterile filter does not sterilise the receiving vessel.