Smear Preparation

Bacteria are, for the most part, transparent so stains are used to impart contrast and visibility in microscopic observation. Much of the success in staining microbes will come from preparing a good bacterial smear. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure.

A smear can be prepared from a solid or broth medium. During smear preparation, three things should be taken care of.

First, adhere the cells to the slide so that they are not washed off in subsequent procedures.

Second, do not overheat the smear so that the cells shrink and result in distortion and artifacts.

Third, prepare a thin smear, because the thickness will determine whether or not individual cells, their arrangement, or details regarding gram reaction or internal structure can be visualized.

Below are some guidelines for preparing a good bacterial smear. 

Procedure

1. Prepare lab bench by removing extraneous items and cleaning surface with table disinfectant.

2. Wipe slide clean with paper towel

3. Draw a circle on the bottom of the slide to indicate area of smear.

4. Transfer culture to top of slide:

From a solid media

• Ø  Place one drop of distilled water on the center of the circle using a loop
• Ø  Transfer a small amount of solid inoculum into the drop of water and disperse thoroughly.
• Ø  Spread both into a thin area about the size of a coin
• Ø  Allow smear to air-dry.

From a liquid media

• Ø  Place one to two loopful of liquid suspension/broth on the center of the circle.
• Ø  Spread the suspension into a thin area about the size of a coin
• Ø  Allow smear to air-dry.

 

                                                    a                                                            b

c                                                            d

5. When the smear is completely dry, heat-fix by quickly passing the smear over the flame two to three times.