BISC209: Aseptic Transfer

Aseptic Transfer

Aseptic transfer technique is important to prevent contamination of the culture being maintained as well as yourself. Manipulation of the tubes, plates and transfer tools requires patience and practice and is vital to success in the microbiology laboratory.

Proper use of a Bunsen burner: YouTube demo [1]

Knowing how to adjust the Bunsen Burner flame for the most effective incineration is extremely useful. There are only two parts that you may need to adjust. Watch the YouTube video for a visual demonstration. Find the needle valve that controls the height of the flame on the bottom of the burner, check the integrity of the rubber tubing connecting the gas source to the gas inlet nozzle, and be sure to turn the gas on only when you are ready to light the burner and shut it off properly (at the gas source) whenever you are not using the flame.

Aseptic transfer
Broth to Broth example, YouTube demo [2]

1. Label the destination container for the culture.

2. Holding your loop like a pencil, insert the loop into the flame as illustrated in Figure 1. The orientation of the loop wire in the flame is important for proper incineration. Keep the wire in the flame until it is red-hot. Then move the adjacent nonwire part of the loop lightly through the flame. The wire will now be sterile, and the nonwire part will have any dust burned off that might have fallen into the media during the transfer procedure. Remember that the loop is now hot and sterile. Allow the loop to cool for a few seconds in the air.

Figure 1: Proper flaming of a loop. Note how the loop handle is held by only the thumb and first two fingers and the loop is inserted into the hottest part of the flame.

3. Pick up the stock broth tube of your organism with your other hand, while still holding the sterile loop. With the hand holding the loop, use your little finger against your palm to remove the cover from the culture tube as shown in Figure 2. Do not put the cover down on your bench. If the tube was closed with a cotton or plastic plug, lightly pass the lip of the tube through the Bunsen burner to burn off any adhering dust. If the tube was closed with a plastic cap the lip of the tube should be sterile and this flaming is probably unnecessary. Now, insert the loop into the broth, and then remove it, carrying a loopful of culture. Flame the top of the culture tube, replace the tube cover, and return the tube to a rack.

Figure 2: Transferring a culture. (a) Removal of a tube cap while manipulating a loop; (b) Obtaining inoculum from a broth tube while maintaining sterility of the cap (note cap in hand).

4. Pick up the labeled destination tube or plate. Remove its cover, (flame if appropriate), 5. Insert the loop containing the culture into the broth, swirl gently and remove.
6. Replace the cover and set the tube in the rack.
7. Resterilize the loop before putting it down to avoid contamination of the bench with the culture. Be careful. When the loop has liquid on it, you must insert the loop into the flame slowly to allow the liquid to evaporate rather than boil, which would splatter live bacterial cells all over the bench, your books and you.

Inoculation of a Slant
Agar slants are made from the liquid media best suited to maintain your organism and a small amount of seaweed extract (Agar) that solidifies the media. Slants are useful for growing and storing pure cultures of organisms because evaporation is minimized and they don't spill. After growing for 24-72 hours (depending on your organism)at the appropriate temperature the agar slants is stored in the cold room.

Once a pure culture is established on an agar slant, it is called a stock culture and is used to make the inoculations of all other media. Everytime a stock is used, the first inoculation is to prepare a replacement stock slant. This reduces the chance of contamination of your stock culture as well as maintaining a source of healthy cells over the long term.

Use the inoculating loop and aseptic transfer technique to make the slant in either one of these 2 patterns. The zig-zag pattern allows for abundant growth when planning to inoculate lots of media and the straight line slant is best for storing in the cold room.

The source culture for a slant can be an isolated colony on a plate, another slant or a broth. In all cases use the inoculating loop and aspetic transfer technique.

Fig. A-3: The tube on the left uses a zig-zag pattern to encourage lots of growth. The one on the right is useful for multiple inoculation.
Transfer from a pure colony to a slant
1. Use the growth media appropriate for your organism

2. Select an isolated “pure” colony from an isolation streak plate.

3. Following aseptic technique remove ½ or less of the colony using your sterilized, cooled loop by touching your loop to the surface growth.

4. Holding your loop in the top 3 fingers of your dominant hand, remove the cover of a slant tube. Remember: Hold the cover in the bottom two fingers and palm while you twist the tube with your other hand.

5. Insert the inoculated loop into the SLANT tube and starting at the lower surface of the agar slant, using a zig-zag or straight motion, drag the loop across and up the surface of the slant.

6. Incubate at the appropriate temperature for the appropriate time.

7. Refrigerate the stock or use it for further inoculations always making at least one replacement slant first.
Once a pure culture is established continue to use aspetic technique and standard transfer manipulations to maintain your stock cultures regularly and to inoculate other tubes or plates.