Basic Aseptic Technique and Inoculation

• Aseptic transfer

  Transfer of a culture from one culture vessel to another without introducing any contaminating microorganisms. A transfer may involve the transport of organisms from an isolated colony on a plate of solid medium to a broth tube or inoculating various media (solid or liquid) from a broth culture for various types of tests.
• Basic aseptic technique and inoculation

  1. Work area disinfection

  2. Loops and needles

  3. Culture tube flaming

  4. Liquid medium inoculation

  5. Final flaming

  6. Petri plate inoculation

  7. Final disinfection
• Aseptic technique

  • Transfer from tube media to tube media (broth, slant, buttslant, butt/deep agar)
  • Transfer from plate media to plate media
  • Transfer from tube media to plate media (and vice versa)
• Inoculation of bacteria

  • Pure culture technique
  • Subculture technique
  • Evaluation of slants
• Quadrant streak (method A)Step 1 and 2

  1. Streak one loopful of organisms over Area 1 near the edge of the plate. Apply the loop lightly. Don’t gouge in the medium

  2. Flame the loop, cool 5 seconds, and make 5 of 6 streaks from Area 1 through Area 2. Momentarily touching the loop to a sterile area of the medium before streaking insures a cool loop.
• Tube media inoculation

  • Broth - mix
  • Slant - streak
  • Deep agar - stab
  • Butt-slant - stab then streak
• Quadrant streak (method A)Step 3 to 5


  3. Flame the loop again, cool it, and make 6 or 7 streaks from Area 2 through Area 3

  4. Flame the loop again and make as many streaks as possible from Area 3 into Area 4, using up the remainder of the plate surface.

  5. Flame the loop before putting it aside
• Quadrant Streak (Method B) Step 1 to 3

  1.Streak one loopful of organisms back and forth over Area 1, starting at point designated by “s”. Apply loop lightly. Don’t gouge into the medium.

  2. Flame the loop, cool 5 seconds and touch the medium in sterile area momentarily to insure coolness,

  3. Rotate the dish 90 degrees while keeping the dish closed. Streak Area 2 with several back-and-forth strokes, hitting the original streak a few times.
• Quadrant Streak (Method B) Step 4 to 6

  4. Flame the loop again. Rotate the dish and streak Area 3 several times, hitting last area several times.

  5. Flame the loop, cool it, and rotate the dish 90 degrees again. Streak Area 4, containing Area 3 several times and drag out the culture as illustrated.

  6. Flame the loop before putting it aside.
• Radiant Streak

  1.Spread a loopful of organisms in a small area near the edge of the plate in Area 1. Don’t gouge medium.

  2. Flame the loop and allow it to cool for 5 seconds. Touching a sterile area of the medium will insure coolness.

  3. From the edge of Area 1 make 7 or 8 straight streaks to the opposite side of the plate.

  4. Flame the loop again, cool it sufficiently, and cross streak over the last streaks, starting near Area 1.

  5. Flame the loop again before putting it aside.
• Continuous Streak

  1.Starting at the edge of the plate (Area A) with a loopful of organisms, spread the organisms in a single continuous movement to the center of the plate. Use light pressure and avoid gouging the medium.

  2. Rotate the plate 180 degrees so that the uninoculated portion of the plate is away from you.

  3. Without flaming loop, and using the same face of the loop, continue streaking the other half of the plate by starting at Area B and working toward the center.

  4. Flame your loop before putting it aside
• Plate Media Inoculation

  1. Quadrant Streak (Method A)
  2. Quadrant Streak (Method B)
  3. Radiant Streak
  4. Continuous Streak