Chap 8

• Culture media


  Media used for growing microorganisms in the laboratory
• Preparation of culture media

  1. Mixing with water & boiling

  2. Sterilisation (autoclaving)

  3. Fine-tuning (supplements and pH)

  4. Dispensing

  
• Tap water will affect selectivity and pH, so distilled water must be used
• Most media require sterilisation, so only bacteria from patient specimens will grow and not contaminants from water or powdered media
• Some media cannot be autoclaved (e.g., SS agar, Cary Blair agar)
• Liquid media are distributed to individual tubes or bottles before sterilization
• Agar media are sterilised in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave
• Items are positioned with enough space for the steam to pass through during autoclaving
• Autoclave tape


  Changes from white to black to indicate that the autoclave's interior temperature is high enough for sterilisation
• Membrane filtration


  Used to sterilise components that cannot withstand steam sterilisation (e.g. serum, certain carbohydrates and antibiotics, other heat-labile substances)
• Membrane filtration with pores ranging in size from 0.2 to 0.45 µm in diameter effectively removes most bacterial and fungal contaminants, but does not remove viruses
• If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, growth promoters, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates
• Culture media should be cooled down in a water bath (45-50°C) or hot plate stirrer before dispensing to minimize condensation
• Dispensing at too high temperature leads to excessive evaporation
• If media stay too long in a water bath, it may cause precipitation (reheat, but do not overheat)
• Cold water should not be used to cool down agar media, as it may lead to flakes or cloud formation
• Culture media storage guidelines

  • Stored away from light, heat sources, and at 10-30°C (certain sensitive media at 2-8°C)
  • Sealed, unopened containers to avoid moisture absorption
  • Not stored at or below 0°C to avoid freezing damage
• Culture media plates should be wrapped in sealed, labeled plastic bags (max 10 per bag), stored upside down at 2-8°C in the dark
• Refrigerators are used to store food and beverages, while freezers are used to store frozen food
• Using culture media


  1. Bring to room temperature before use

  2. Dry plates for 20-30 minutes at 35-37°C if condensation is present

  3. Visual sterility check before use is mandatory
• Most common errors & possible causes


  • (not provided)