Overview
This lecture covers proper aseptic techniques for transferring bacterial cultures to different media without introducing contaminants, including correct tool use, flame sterilization, and step-by-step transfer procedures.
Tools and Flame Use
- The inoculation loop and inoculation needle are main tools for bacterial transfer.
- Mastery of the Bunsen burner is essential for aseptic technique.
- Light the Bunsen burner by closing the base valve, turning on gas, and adjusting flame height.
- Adjust flame temperature via collar; blue flame is hotter, yellow/orange is coolest.
- The tip of the blue cone in the flame is the hottest part.
- Sterilize tools by passing them through the hottest part of the flame.
- Do not set sterilized tools on any surface before use.
Liquid Culture to Petri Dish Transfer
- Flame sterilize the loop.
- Remove the liquid culture lid and flame the mouth of the container.
- Dip loop into liquid culture, re-flame container mouth, and replace lid.
- Lift Petri plate lid as a shield and spread bacteria on agar surface.
- Rotate plate a quarter turn between spreads for even distribution.
Liquid Culture to Liquid Media Transfer
- Sterilize the inoculation loop in flame.
- Remove and flame the mouth of the liquid culture container.
- Dip loop into culture, re-flame, and close container.
- Open tube of liquid media, flame its mouth, and transfer bacteria with loop.
- Flame mouth of tube before replacing lid and sterilize loop again.
Petri Plate to Stab Culture Transfer
- Sterilize inoculation needle in flame and allow it to cool.
- Test coolness by touching clear area on agar.
- With lid as shield, gently rub needle over a single colony.
- Insert needle into nutrient agar tube, performing three deep stabs.
- Replace lid and re-sterilize needle after use.
Petri Plate to Liquid Media Transfer
- Sterilize the inoculation loop and allow it to cool.
- Test for coolness by tapping loop on agar away from colonies.
- Use lid as shield, rub loop over a single colony.
- Open liquid media tube, flame mouth, and transfer bacteria.
- Flame mouth before closing and sterilize loop after use.
Key Terms & Definitions
- Aseptic technique — set of practices to prevent contamination by unwanted microorganisms.
- Inoculation loop/needle — tools used to transfer bacterial cultures.
- Bunsen burner — device that produces a controllable flame for sterilization.
- Stab culture — inoculation method involving stabbing bacteria into solid media.
Action Items / Next Steps
- Practice all described aseptic techniques under supervision.
- Review steps for each transfer method before performing in lab.