2-2 Flame sterilization and tube transfer

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Flame sterilization is a very quick simple method of killing microorganisms on an inoculating loop or needle. The loop or needle is held inside a flame for a few seconds to bring it to redness and then cooled. Once cool, the loop or needle can be used for various culture manipulations. Make sure that the area that contacts the culture is flamed to redness. Also, be patient and let the loop cool down, this usually takes about 15-30 seconds. Learning this technique is essential to everything else you do in microbiology.

Transfer of culture from agar plates to tubes, or from tube to tube, is a common, simple procedure. It is important to perform these transfers in a consistent and rapid manner. The following protocols have been found effective.

To transfer a culture from an agar plate to a broth or agar slant:

  1. Place the Bunsen burner in front of you and assemble all necessary equipment with in arms reach. Position everything so that you will not burn yourself while trying to inoculate your tubes.
  2. Label the tube of broth or agar to be inoculated with identifying marks. The culture, the date, and your initials for example. Place it in a rack in front of you.
  3. Holding the inoculating loop handle, flame the entire wire to redness.
  4. When the wire cools (about 15-30 seconds) remove the lid of the plate with your other hand and obtain an inoculum by removing a small portion of the surface growth on the agar plate. In most cases you will be picking an isolated colony. Choose a well isolated one. Do not dig into the agar. Replace the lid of the plate immediately.
  5. Hold the tube to be inoculated with the free hand. Remove the cotton plug or cap of the tube with the little finger of the hand holding the needle holder. If a cotton-plugged tube is used, the mouth of the tube should be passed briefly through the flame to singe off dust and lint particles. (Dust or lint may fall into the tube and contaminate the medium.)
  6. Introduce the inoculum into the tube. When inoculating a tube of broth, rub the wire against the glass just above the fluid level and then tip the tube slightly to wash the inoculum into the broth. The wire should not be rattled against the sides of the tube to shake an inoculum into the broth; this is unnecessary and may create a dangerous aerosol. If the transfer is made to an agar slant, a single mid-line stroke over its surface is made with the wire or loop.
  7. Replace the cap or plug (the latter after reflaming the mouth of the tube).
  8. Flame the inoculating wire again to redness, slowly to avoid spattering. Put the loop holder down after the wire cools.

In tube to tube transfers by loop or straight wire, both the tube containing the inoculum source and the tube to be inoculated are usually in the hand at the same time. An easy procedure which prevents hand fatigue and the danger of dropping the tubes is illustrated in Figure 2-1.

Figure 2-1 Tube to tube transfer

The standard method for transferring microbes from one medium to another. Each of the steps is described in the text.

  1. The tubes are positioned in the hand as shown in Fig. 2-1 B-E. Plugs and caps can be loosened by twisting them.
  2. The needle holder is taken in the other hand and the wire flamed and allowed to cool (Fig. 2-1 A).
  3. The plugs or caps are removed with the last two fingers of the hand holding the inoculating wire leaving the thumb and index finger free to hold and to manipulate the loop holder with the second finger as a guide and support. Flame the tops of the tubes.
  4. Immerse the inoculating wire into the broth culture or scrape the wire across a portion of surface growth on an agar slant to obtain inoculum. Make the transfer from one tube to the other (Fig. 2-1 C).
  5. Flame the tubes (Fig. 2-1 D).
  6. Return the plugs or caps to the tubes (Fig. 2-1 E).
  7. Flame the inoculating wire to sterilize it (Fig. 2-1 F).

At first, this procedure will be awkward, but after some practice it becomes second nature. Figure 2-2 contains a movie that demonstrates the technique of flame sterilization and tube transfer.

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