3-6 Preparation of a Bacterial Smear for Staining

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Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure. Figure 3-4 demonstrates smear preparation.

The best smears are made from bacteria that have grown on a solid surface such as an agar slant or plate. A bit of growth from a culture is mixed with distilled or tap water to form a slightly turbid solution and this is spread on a clean grease free slide. When staining broth cultures, a drop of broth is transferred directly to a slide, using no extra water. The procedure for making a smear is as follows:

  1. If more than one culture is to be examined using the same stain, it is possible to prepare up to 6 smears on the same slide. Before preparing the slide, divide it into the appropriate number of sections and clearly label each section on the underside of the slide.

  2. If your culture has been grown on a agar slant or agar plate. Place a small drop of water on a clean, grease-free slide. Next, using a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water and rub the needle around until the material is evenly emulsified. Spread the drop over a portion of the slide to make a thin film. The suspension should be only slightly turbid.

  3. If you are using a broth culture, the broth culture must have clearly visible turbidity. Transfer a loopful of culture from the broth onto a clean grease free slide. Spread the drop over a portion of the slide to make a thin film.

  4. Allow the film to air-dry. To get a good stain, it is important to let the smear dry completely. Excess water left on the slide will boil during the fixing stage, causing most microbe present to rupture. Rushing this step will result in a poor final stain.

  5. Once dry, "fix" the smear to the slide by passing the bottom of the slide through the tip of the burner flame several times for a one second. After heat fixing, touch the heated portion of the slide to your hand. It should be comfortably warm, but not burning hot.

  6. Take care not to under-fix (the smear will wash off) or over-heat (the cells will be ruptured or distorted) the slide. The correct amount of heat fixing is learned by experience.

  7. Allow the smear to cool and apply the stain.

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