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 or Broth to Plate Transfer
YouTube demo [2]

1. Label the destination container for the culture (uninoculated sterile broth in a tube or solid medium in a plate).

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 should be at ~ a 30 degree angle 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. Allow the loop to cool for a few seconds in the air before touching it to your culture or medium.

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 donor broth culture tube 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 or plug from the culture tube as shown in Figure 2. Do not put the cover or plug down on your bench. If the tube is glass, lightly pass the lip of the tube through the Bunsen burner to burn off any adhering dust and to create a temperature differential that temporarilty prevents dust from falling into your tube. Now, insert the loop into the broth without touching the sides of the tube, and then remove it, carrying a loopful of culture. Pass the top of the culture tube through the flame, replace the tube cover or plug, 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 sterile destination tube or plate. Remove its cover, (if it's a glass tube, pass the lip of the tube through the flame),
5. Insert the loop containing the culture into the destination tube of sterile broth, swirl gently and remove. If the destination is a plate of solid medium, follow the directions for streaking for isolation or for a spread plate found in the protocols section of the wiki.
6. Replace the cover and set the newly inoculated broth tube in the rack.
7. Resterilize the loop before putting it down by inserting the loop into the flame very slowly. Doing this slowly allows any liquid remaining on the loop to evaporate rather than boil and avoid splattering live bacterial cells all over the bench 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 and is useful when you are inoculating numerous samples. The one on the right encourages less growth and is useful for carrying stock cultures.
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 lift the cover of the plate and remove ½ or less of one 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, use your other hand to pick up a slant tube and the little finger and palm technique of your dominant hand to 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, gently drag the loop across and up the surface of the slant. try to avoid digging into the agar, but if this happens just lighten your touch and continue with the inoculation.

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.
This tools for transfer may change or be modified in many ways for specific types of media, tests, or procedures. Follow the specific instructions for these procedures always keeping in mind the principles of aseptic transfer.