Microbiology Practical: Culture Media Inoculation

Study Notes

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.