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Aseptic Technique and the Transfer of Microorganisms
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 Transfer of Inoculum from a Plate Culture to a Sterile Broth Tube:

 

Materials Required:

 

  1. Nutrient agar plate (Petri dish) with bacterial colonies (inoculum).
  2. Tube of sterile Nutrient Broth.
  3. Inoculating loop.
  4. Bunsen Burner.
  5. Test tube Rack.
  6. Disinfectant.
  7. Marking pen.
     

Procedure:

 

  1. Prepare your work space (Laminar Air Flow Cabinet) or lab bench by wiping down the area with disinfectant.
  2. With a marking pen, label a tube of sterile nutrient broth with your initials, the organism's name and the date of inoculation. Place in the test tube rack.
  3. Sterilize your wire inoculating loop by passing it at an angle through the flame of a Bunsen burner until the entire length of the wire becomes glowing red/orange from the heat.
  4. Important: Never lay the loop down once it is sterilized or it may become re-contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum.
  5. Partially lift the lid of the plate culture and open it just enough to insert the inoculation loop. Do not completely open the lid and expose the surface to the air.
  6. Touch the loop to an area of the agar with no growth in order to cool down the loop.
  7. Choose an isolated colony and scrape off a very small amount of culture with the loop.  Be careful not to  gouge into the agar with the loop as you pick up the organisms Close the lid immediately once you have picked up the organisms and turn the plate upside down on the work surface.
  8. Pick up the tube of sterile nutrient broth with your free hand, carefully remove the cap (cotton plug) with your little finger of the hand holding the loop, and flame the mouth of the tube.
  9.  Insert the loop into the sterile broth and inoculate it by gently moving the loop back and forth in order to disperse the cells.
  10.  Remove the loop from the tube and flame the mouth of the tube again. Replace the cap on the tube and place it on the rack.
  11. Sterilize the loop by flaming it. Now you may lay it down on the lab bench or return it to its container.
  12.  Incubate the culture you just inoculated at 37°C for 24-48 hours.

 

Transfer of Inoculum from a Broth Tube to a Sterile Broth

 

Materials Required:

 

  1. Broth culture of microorganism (inoculum).
  2. Tube of sterile Nutrient Broth.
  3. Inoculating loop.
  4.  Bunsen Burner.
  5. Test tube rack.
  6. Disinfectant.
  7. Marking pen.

 

Procedure:



Much of the technique of transferring a broth culture to a tube of sterile broth is similar to transferring inoculum from a plate to a broth.

Please note that both procedures require the same guidelines for handling of the loop.

 

  1. Prepare your work space (Laminar Air Flow Cabinet) or lab bench by wiping down the area with disinfectant.
  2. With a marking pen, label a tube of sterile nutrient broth with your initials, the organism's name and the date of inoculation. Place in the test tube rack.
  3. Sterilize your wire inoculating loop by passing it at an angle through the flame of a Bunsen burner until the entire length of the wire becomes glowing red/orange from the heat.
  4. Important: Never lay the loop down once it is sterilized or it may become re-contaminated. Allow the loop to cool a few seconds to avoid killing the inoculum.
  5. Using your other hand, pick up the tube containing the culture (inoculum) and gently shake it to disperse the culture.
  6. Grasp the cap (cotton plug) of the inoculum tube with the little finger of your hand that holds the inoculating loop and remove it from the tube. Flame the mouth of the tube.
  7. Keeping the culture tube at an angle, insert the loop into the broth and remove a loopful of inoculum.
  8. Flame the mouth of the tube again and recap the tube. Place the culture tube back on the test tube rack.
  9. Pick up a tube of sterile nutrient broth with your free hand, carefully remove the cap (cotton plug) with your little finger of the hand holding the loop, and flame the mouth of the tube.
  10. Insert the loop into the sterile broth and inoculate it by gently moving the loop back and forth in order to disperse the cells.
  11. Remove the loop from the tube and flame the mouth of the tube again. Replace the cap on the tube and place it on the rack.
  12. Sterilize the loop by flaming it. Now you may lay it down on the lab bench or return it to its container.
  13. Incubate the culture you just inoculated at 37°C for 24-48 hours.

 

Differences Encountered in a Real laboratory:


In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:

 

  1. While inoculating agar media (plates and agar slants), make sure not to gouge or tear  the surface of the agar with the loop. Always label all tubes and plates with:
                      1. The name of the organism
                      2. The type of media 
                      3. Your initials 
                      4. The date
  2. Make sure to place agar plates in the incubator upside down.  This prevents water condensation from dripping onto the agar surface and spreading over the colonies. This will also prevent contamination and aid in the formation of isolated colonies.  
  3. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.
  4. While flaming the loop (or needle), be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame. Be careful; the metal will get extremely hot.
  5. Once you have flamed your loop (or needle), do not lay it down, blow on it, touch it with your fingers, or touch it to any surface other than your inoculum or the sterile media.  If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed with your inoculation.
  6. Allow your loop or needle to cool before you try to pick up your organism.  If you pick up organism with a hot tool, your cells will be killed. To cool your loop or needle quickly, place it on a section of agar that is uninoculated or is at least different from the area from which you will remove cells.
  7. Ensure that you are transferring the correct organism into your labeled tube by double-checking the name of the organism on the stock culture from which you are collecting your inoculum.
  8. When removing the caps from tubes, always keep the caps in your hand.  Never set them on the table, as they could pick up contaminants. 
  9. Always handle open tubes at an angle; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.  Also, keep the lid over a plate when removing inoculum, as this will help prevent environmental contamination.
  10. Always flame the lip of a culture tube when you open it and before you replace the cap.
  11. As soon as you are done inoculating, flame your loop or needle. Never place a contaminated tool on your workbench.
  12. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.
  13. Always disinfect your work area when you are finished.








     

 

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