Nematodes

Preparation of Bacterial Smear

 

Culturing of bacteria

To study the microorganisms and their characteristics, we need a pure culture. A pure culture contains only a single kind of microorganism. To obtain pure culture various techniques can be adopted.

• Pour plate method
• Streak plate method
• Spread plate method

Preparation of Bacterial Smear

Principle

 Various types of staining techniques are used for differential as well as simple visualization of bacteria. Bacterial smears must be prepared prior to the execution of staining. For smear formation following parameters should be taken care:

1. Preparation of glass slide: Clean glass slide is essential for smear preparation. Grease and oils should be removed. For this purpose soap washing and then rinsing is used. Additionally, acid alcohol (95% ethyl alcohol and 5% HCl) rinsing is also used to completely remove remaining dyes and oils.
2. Labeling of the slide: Proper labeling is also important for identification. The label should have at least an organism name and name or initials of the owner. For labeling, permanent glass markers should be used. Care should be taken that the label does not come into direct contact with the staining reagents.
3. Preparation of smear: It is important to avoid thick and dense smears. Normally thick and dense smear occurs when too many cultures are used. Such type of smear results in diminishing the amount of light to pass through and are difficult to visualize the morphology of a single cell. A good smear is one that when dried results in a very thin whitish layer or film.

• Broth cultures: re-suspend the broth culture by tapping the tube. Depending on the size of the loop one or two loop full cultures are normally sufficient. These should be evenly spread over the glass slide as maximum as can be done (normally the size of a coin), with the help of the loop. After smear formation, it should be air-dried.
• Solid medium: Direct transfer of solid culture to slide results in very much thick preparations. So, these cultures should be diluted by adding one or two loopful of water in the center of the slide, then transfer the bacterial cells with the help of a loop or needle. Avoid loopful culture transfer only try to shift minimum culture just by touching the bacterial colony. The suspension is formed by mixed in the circular motion of the loop or needle. This will help to avoid cell clumping. The finished smear should be semitransparent or translucent. Finally, smear should be allowed to air dry.

Heat fixation

Unless the smear is fixed on the glass surface, it will wash away during staining. So, to avoid washing the cells it should be heat fixed. Heat fixing is done by the rapid passing of the slide over the flame two to three times. Avoid long time exposure can burn the cells. During fixation, bacterial proteins are coagulated and fixed to the glass surface.

Materials and equipment

24h nutrient agar of staphylococcus and nutrient broth also, glass slides, Bunsen burner, inoculation loop and needle, glass marking pencil.