Overview
This lecture covers the procedure and key considerations for preparing and inoculating agar plates using the molten agar pour plate method.
Preparation of Molten Agar
- Molten agar should be kept at around 50°C in an incubator or water bath.
- Label the base of empty Petri dishes with all relevant sample information before starting.
- Remove molten agar from heat and allow it to cool until it is just comfortable to hold.
Inoculation Process
- Too hot agar can kill the inoculum; too cold agar may begin to set prematurely.
- Use aseptic technique close to a Bunsen burner for inoculation.
- Up to 1 ml (1000 microliters) of inoculum can be added, which is 10 times more than used in spread plating.
Mixing and Pouring
- Gently mix bacteria and molten agar to prevent bubble formation.
- Pour the mixed agar into a sterile Petri dish and allow it to set completely.
Incubation and Growth Observation
- Confirm agar is set before inverting and incubating plates.
- Incubate plates overnight as usual.
- Bacterial colonies will grow on the surface and inside the agar.
- Anaerobic and aerobic bacteria can both grow, but obligate aerobes may not grow if trapped within the agar.
Key Terms & Definitions
- Molten Agar — Agar that is liquefied by heating, used for pouring plates.
- Inoculum — The sample containing microorganisms added to the agar.
- Aseptic Technique — Procedures used to avoid contamination by unwanted microorganisms.
- Obligate Aerobes — Bacteria that require oxygen to grow.
Action Items / Next Steps
- Practice labeling technique and aseptic inoculation.
- Review procedures for temperature control and mixing to avoid killing organisms or air bubble formation.