Aseptic Technique Practices and Procedures

Introduction to Aseptic Technique

• Aseptic technique is used to transfer a bacterial monoculture into a fresh medium without introducing contaminants.
• Key tools include:
  • Inoculation Loop
  • Inoculation Needle

Using a Bunsen Burner

• Lighting the Burner:

  • Close valve by covering vents.
  • Turn on gas slowly.
  • Ignite the burner.
  • Adjust flame height with gas amount.
  • Adjust flame temperature with oxygen control.
    • Yellow and orange = coolest
    • Blue = medium
    • Roaring blue with clear blue cone = hottest
  • Hottest part is the tip of the cone.

• Sterilizing Inoculation Tools

  • Pass through the hottest flame.
  • Do not set sterilized tools on any surface.

Transferring Bacteria

From Liquid Culture to Petri Dish

1. Flame the loop.
2. Remove container lid, flame the mouth.
3. Dip loop into liquid culture.
4. Flame the mouth again before replacing the lid.
5. Transfer to petri plate:
   • Use the lid as a shield.
   • Spread bacteria over plate surface.
   • Rotate plate a quarter turn between spreads.

From Liquid Culture to Liquid

1. Sterilize inoculation loop.
2. Remove liquid culture lid, flame the mouth.
3. Dip loop into culture, flame mouth again.
4. Flame tube of liquid media mouth.
5. Dip loop into tube.
6. Flame mouth before replacing lid.
7. Flame the loop for sterilization.

From Petri Plate to Stab Culture

1. Sterilize inoculation needle.
2. Cool the needle (test by tapping on clear auger area).
3. Use lid as shield, gently rub needle over single colony.
4. Transfer to culture tube:
   • Make three deep stabs in nutrient auger.
5. Replace lid, sterilize needle.

From Petri Plate to Liquid

1. Sterilize inoculation loop.
2. Cool the loop (test by tapping on clear auger area).
3. Use lid as shield, gently rub loop over single colony.
4. Remove liquid media lid, flame the mouth.
5. Dip loop into tube.
6. Flame mouth before replacing lid.
7. Sterilize the inoculation loop.