Laboratory Investigations in Microbiology

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Chapter 2: Aseptic Transfer Techniques

As we have seen in the previous exercise, contamination is a serious concern in the microbiology laboratory. However, the risk of contamination of lab cultures or the environment can be greatly reduced by the use of proper aseptic techniques. Aseptic means "no infection" or the absence of microbial growth. Aseptic technique is not just one procedure, but rather an entire way of operating around microorganisms that prevents contamination and infection. The basic principles of this technique include:

The first of these principles can be achieved through proper hand-washing and disinfecting your lab bench before and after lab. Use hot water, soap and a brush for most effective hand washing. To disinfect your lab bench, pour/squirt some lab disinfectant on the lab bench and wipe across the entire work surface using a paper towel. 

A partially open Petri dish is an invitation for contamination!Minimizing exposure of culture media to the air and to contact with any foreign objects will also greatly reduce the chance of contamination. Keep test tubes capped and Petri dishes closed except for the few seconds it will take to transfer cultures into and out of them. When performing streak techniques on an agar plate, use one hand to hold the Petri dish lid between the agar plate and your face (kind of like a sneeze guard at a salad bar!). Avoid touching the agar surface unintentionally , especially with your fingers! Other things to avoid: tipping over liquid media (may cause backwashing of contaminants from the cap into the broth), sniffing media, and repeated/unnecessary opening and closing of tubes or plates. One other technique that reduces the risk of contamination in test tubes is passing the mouth of the test tube through the Bunsen burner flame after uncapping the tube and againFlame-sterilizing an inoculating loop just before recapping it.

Bacti-cineratorThirdly, all media, glassware, and tools MUST be sterile (devoid of any living organism). To achieve this, glassware (pipettes, Petri plates) is usually baked at 160 °C for 2 - 4 hours. Culture media and plastic ware are sterilized by autoclaving for 15 - 60 minutes. This temperature sterilizes liquids, melts agar so it can be poured into dishes, and kills microorganisms without melting plastic or boiling off water. Other tools for use in microbiology (especially inoculating loops & needles) are sterilized before and after each use with a Bunsen burner flame or a Bacticinerator. A bacticinerator heats metal wire with infrared in an enclosed chamber without the hazards of having an open flame in your work area. We will be using the Bacti-cinerator for most of our experiments.

Transfer of bacterial cultures from one medium/location to another should be done with caution and efficiency. The techniques discussed here can be adapted to Inoculating needletransfers from plates to broths, slants to plates, etc. At times, a different tool (e.g. inoculating needle) can be used, for instance when it is desirable to stab-inoculate a medium.Blow-out pipette (left) and To-deliver pipette (right)

Yet another transfer tool common to Microbiology is the pipette. When using glass pipettes, pipettes are heat-sterilized in metal canisters. Pipettes should always be tip-down in the can to avoid contamination of the pipette when removing the pipette from the can, and the tips of pipettes should never be touched to skin or lab benches. There are two types of pipettes commonly in The number at the top of a pipette tells you its sizeuse: the blow-out pipette and the to-deliver pipette. Blow-out pipettes require that every last drop is pushed out of the pipette to obtain the desired volume - the "10 ml" mark on a 10 ml pipette is the tip of the pipette itself!. To-deliver pipettes require you to leave the remainder of the liquid below the "10" mark in the pipette as waste - the "10 ml" mark can be seen near the tip of the pipette. [Pipettes come in 1 ml, 5 ml, and 10 ml sizes. Be sure to always check for the size of your pipette by reading the numbers at the top (the number before the word "in")]

Pipetting is accomplished using a pipette pump (image right). Blue-colored pumps accommodate 1 - 2 ml pipettes, and green-colored pumps accommodate 5 and 10 ml pipettes. Once securely pushed into the rubber socket, liquid is pulled into the pipette by turning the wheel, and is released again by turning the wheel in the other direction or pushing down on the top of the pipette.Holding a pipette pump

For smaller volumes (1.0 ml and less), you will usually use a micropipettor along with sterile plastic pipette tips. The two most common sizes of micropipettors are the P1000 (useful for pipetting 100 - 1000 ul) and the P200 (for 20 - 200 ul). The P1000 usually has a blue "P1000" symbol and uses blue-colored pipette tips. The P200 has a yellow "P200" symbols and uses yellow pipette tips. When using these pipettors, first turn the dial to the correct setting for the volume you need to pipette (keep in mind the numbers are in microliters). Next, attach the correct-size tip from the sterile container. Next, push the plunger down until you reach the stop (the plunger can go in further, but this is only for ejecting any left-over fluid from the pipette tip. Try getting a feel for when you reach the first and second 'stop' when you first pick up the pipettor). With the plunger held down, submerge the pipette tip in the liquid you wish to piette, then slowly allow the plunger to come out again. If you let go of the plunger too quickly, you may suck fluid into the barrel of the pipettor. Lastly, you can eject the liquid by pressing down on the plunger all the way to the second stop.

The objective of today's lab exercise will be to practice the aseptic transfer of bacteria from a test tube to other culture media, as well as the transfer of liquids using pipettes.

Note: These procedures are part of the Laboratory Skills Test

Materials & Methods

Materials per student: 
  1. Demonstration of aseptic technique - Bunsen burner method
    1. Disinfect your lab bench and set your test tubes into a test tube rack
    2. Sterilize your inoculating loop by holding the wire inside the Bacti-cinerator's ceramic core for at 8-10 seconds. Please avoid touching the sides of the chamber. 
    3. Allow your inoculating loop to cool off for ~ 20 seconds
    4. Pick up the bacterial broth (source) and remove the cap with your other (loop) hand
    5. Dip the inoculating loop into the source broth and withdraw the loop. Observe that there is a drop of liquid caught in the loop.
    6. Replace the cap onto the source tube and place the source tube into the rack
    7. Pick up the destination tube (TSB) and remove the cap with your other (loop-holding) hand
    8. Insert the inoculating loop with its drop into the broth. Shake the loop vigorously to stir in the transferred bacteria. Withdraw the loop.
    9. Re-cap the tube and place it into the rack
    10. Re-sterilize the inoculating loop (8-10 seconds)
  2. Aseptic transfer exercise
    1. Label your TSB tubes with your name (or initials) and the lab section you are in.
    2. Transfer 1 loopful of sterile TSB from one tube to the second tube.
    3. Turn in your TSB tubes "to be incubated" 
  3. Aseptic transfer of liquids: pipetting
    1. Compare the two types of glass pipettes available: to-deliver vs. blow-out
    2. Using a pipette pump, practice the accurate transfer of 8.0 ml of water into a plastic dish. 'zero' the balance before transferring; the weight of the water will tell you how much water you actually transferred (1 ml = 1.00 g). Record the amount you pipetted.
    3. Practice using a micropipettor by transferring 0.8 ml (use the P1000) and 0.08 ml (use the P200) of water into the plastic Petri dish. Again, the weight of the water you pipetted will tell you how accurate you were! Record the weights.
Next lab period
  1. Examine your TSB tubes (aseptic transfer) for cloudiness.
Data Sheet & Review Questions (printable)


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