Microbiology Practical: Culture Media Inoculation

Microbiology Practical (Inoculation of Culture Media)

• Aseptic Techniques are Crucial:
  • Prevent contamination of cultures and specimens
  • Prevent infection of lab workers and the environment
• Sterilize Instruments:
  • Bunsen burner flame sterilizes wire loops, straight wires, and metal forceps
  • Flame the necks of specimen bottles, culture bottles, and tubes before and after removing caps/plugs
  • When inoculating, don't allow caps or tubes to touch unsterile surfaces. Hold caps/plugs by hand to avoid contamination.
• Use Racks:
  • Use racks to hold tubes and bottles with specimens/media
• Slide Preparation:
  • Make slide preparations from specimens after inoculation
• Decontamination:
  • Decontaminate the workstation before and after work
• Safety Precautions:
  • Use a safety cabinet when handling hazardous pathogens
  • Wear protective clothing, wash hands after contact with infected material
  • Avoid eating, drinking, or smoking in lab
• Petri Dish Inoculation:
  • Dry the surface of the culture medium by removing the lid and placing it face-up on an indicator shelf
  • Invert the plate on its lid (at a 30-40 minute incubation at 35-37°C).
• Inoculation Technique:
  • Use a sterile loop or swab to apply inoculums to a small area of the plate
  • Flame sterilize the loop when spreading inoculums
  • Slopes: Streak inoculum in a zigzag pattern
  • Butt and Slope: Stab into the butt first, then streak the slope in a zigzag pattern
• Stab Media: Use a sterile straight wire to stab through the center, withdrawing it along the inoculation line, without creating additional stab lines
• Fluid Media: Use a loop, wire, or Pasteur pipette (dependent on medium type) to inoculate
• Labeling: Label inoculated media with the date, patient number, and other appropriate details (slope labeled on underside, stab culture above agar level). Note anaerobic (An O₂ or CO₂) incubations.
• Incubation: Incubate inoculated media promptly. Protect from light, insects, and dust. Control temperature, humidity, and gaseous atmosphere for optimal microorganism growth.
• Temperature: Incubate at the optimum temperature. Minimum temperature below which growth stops, and the maximum temperature above which growth stops and death occurs
• Humidity: Maintain adequate humidity. Dry conditions can reduce microbial growth.
• Gaseous Atmosphere:
  • Obligate aerobe: Requires free oxygen.
  • Microaerophile: Needs low levels of oxygen
  • Obligate anaerobe: Needs oxygen-free conditions.
  • Facultative anaerobe: Can grow with or without oxygen.
  • Carboxyphile: Requires carbon dioxide.
• Anaerobic Culture: Anaerobic Incubation helps to differentiate organisms and isolate facultative anaerobes from commensal microbes. Use techniques such as commercially produced sachets, copper-coated wool, or reducing agents in culture media.