Overview
This lecture covers aseptic transfer techniques for safely moving bacteria between culture media without contamination, focusing on procedures, instruments, and key steps for inoculation.
Aseptic Transfer Principles
- Microorganisms are present on lab surfaces and in the air, so contamination is a constant risk.
- Aseptic transfer refers to handling bacteria without introducing contaminants.
- An inoculum is a small amount of bacteria transferred to a sterile medium (inoculation).
- Always start and finish by decontaminating the lab bench.
Inoculating Instruments
- Inoculating loop: a metal wire with a closed loop, used for liquid and surface transfers.
- Inoculating needle: a metal wire without a loop, used for solid media requiring stabbing.
- Instruments must be sterilized using a Bunsen burner until the wire is red hot.
- Allow instruments to cool (15–30 seconds) before use to avoid killing bacteria or creating aerosols.
Using the Bunsen Burner
- Attach the burner securely and keep the gas valve closed before lighting for safety.
- Control gas and air intake for an optimal blue flame with a bright inner cone.
- The hottest part is at the tip of the inner cone.
- Never leave an unlit burner open to gas.
Aseptic Transfer Procedures
- Label all tubes with inoculum identity, your name, and date.
- Flick tubes to suspend bacteria without spilling.
- Hold tubes at a slight angle and never set caps on the bench.
- Flame the mouth of tubes before and after inserting or removing an instrument.
- Avoid touching the instrument to any surfaces after sterilization.
Broth to Broth/Agar Slant Transfer
- Obtain a loopful of suspended broth culture.
- Flame tube mouths before and after transfer.
- For agar, gently zigzag the loop on the surface without digging into the agar.
Slant to Broth
- Touch only the edge of the loop to the slant’s growth streak.
- Swirl the loop gently in broth to release inoculum.
Agar Deep Inoculation
- Use a sterilized needle, not a loop.
- Gently pick up bacteria from slant; avoid stabbing slant.
- Stab the agar deep in the center about two-thirds down.
Petri Plate to Slant
- Circle and label desired colony.
- Open Petri plate minimally to prevent airborne contamination.
- Gently zigzag the loop on the slant surface.
Final Steps and Cleanup
- Flame instruments after each use.
- Discard used materials in appropriate containers.
- Decontaminate bench tops after procedures.
Key Terms & Definitions
- Aseptic transfer — Moving bacteria between media without contamination.
- Inoculum — Small sample of bacteria for transfer.
- Inoculation — Introducing bacteria into a sterile medium.
- Inoculating loop — Wire tool with a loop for transferring bacteria.
- Inoculating needle — Straight wire tool for stabbing solid media.
- Agar slant — Tube containing solidified agar at an angle.
- Agar deep — Tube with solid agar used for stabbing inoculations.
Action Items / Next Steps
- Practice flame sterilization and aseptic transfer techniques as shown.
- Prepare labeled media tubes before performing transfers.
- Review safety and cleanup protocols for future lab sessions.