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Aseptic Transfers in the Microbiology Wetlab

In the wetlab, aseptic transfers are a routine technique that must be mastered by microbiology students.  There is a stepwise process followed to aseptically obtain the inoculum and transfer it to a sterile medium.  The steps are repeated in reverse order as the process is completed. 

A typical transfer would look like this for tube-to-tube inoculation.

  1. Start the burner and flame the inoculating loop.
  2. Remove the tube caps and flame the mouths of the tubes.
  3. Pick up the inoculum with the sterile loop from the tube of inoculum.
  4. Transfer the inoculum to the sterile medium.
  5. Flame the mouths of the tubes and replace the caps.
  6. Flame the inoculating loop and turn off the burner.

In short, it is:

  1. Burner and loop
  2. Flame tubes
  3. Transfer
  4. Flame tubes
  5. Loop and burner.

Watch this video to see how all actions described above are performed during a tube-to-tube transfer. Note that the tubes remain closed as much as possible and only remain open during the actual transfer to reduce the possibility of airborne contamination entering either the inoculum or the sterile broth being inoculated.

 

A typical transfer would look like this for tube-to-plate inoculation.

  1. Start the burner and flame the inoculating loop.
  2. Remove the tube cap from the inoculum and flame the mouth.
  3. Pick up the inoculum with the sterile loop from the tube of inoculum.
  4. Flame the mouth of the tube and replace the cap.
  5. Remove the lid from the plate and streak the agar with the inoculum.
  6. Replace the lid on the plate.
  7. Flame the inoculating loop and turn off the burner.

In short, it is:

  1. Burner and loop
  2. Flame tube
  3. Pick up inoculum
  4. Flame tube
  5. Remove lid, deposit inoculum

  6. Streak plate, replace lid

  7. Loop and burner.

Watch this video to see how all actions described above are performed during a tube-to-plate transfer.  Note that containers remain closed as much as possible and are only opened when the transfer or streak are being performed to reduce the possibility of airborne contamination.  

 

A couple of things to note about the tube-plate transfer in the video.  First, you will note that the sterile loop goes into the inoculum and then onto the plate ONCE, and then after that the loop is sterilized and used on the plate alone several more times.  The tube-plate inoculation shown is called a "streak plate", a special technique used to dilute bacteria on the agar surface to physically separate them so they can grow into separated, isolated colonies that are pure cultures of a single species of bacterium.  Whatever bacteria are picked up on the loop are distributed onto the surface of the plate as the loop glides by. The initial inoculum of bacteria is spread over approximately one quarter of the plate.  Then the loop is sterilized and passes over the first streak lines a few times to pick up some cells that will be moved by the loop to the next region of the plate and then spread out on the streak likes in section two.  The loop is sterilized again and a small number of cells are picked up from region two and moved to region three by the loop just as before.  And this is repeated to take a few cells into region four where they are spread so far apart that they can form isolated colonies.  

So this is an example of a mechanical dilution, picking up a small fraction of cells from one region and moving them to the next to spread them out, and then repeating this to take an even smaller number to the next region and so on.  The result is dilution from thousands of cells being deposited per centimeter of streak line to very few, thus giving each an opportunity to grow into an isolated, pure colony of the unknown microbe.

Since a species of bacterium is genetically capable of only replicating copies of itself, all cells in an isolated colony are the genetic offspring of the cell that was deposited there during the streaking of the plate.  This is a pure culture.

When you are done, take a quiz on the process for completing aseptic transfers in the wetlab.