Preparation of Specimens for Microscopic Examination

The following points highlight the two techniques applied for the preparation of specimen for microscopic examination. The techniques are: 1. Hanging Drop Technique 2. Fixed and Stained Smears.

Technique # 1. Hanging Drop Technique:

Hanging drop or wet preparations permit examination of organisms in a normal living condition. A wet mount is made by placing a drop of fluid containing the organisms onto a glass slide and covering the drop with a cover slip.

To reduce the rate of evaporation and exclude the effect of air currents, the drop may be ringed with petroleum jelly or a similar material to provide a seal between the slide and cover glass. A special slide with a circular concave depression is often used for examination of wet preparations (Fig. 4.1.).

A suspension of microbial specimens is placed on a cover slip, and then inverted over the concave depression of the slide to produce a “hanging drop” of the specimen.

This technique is often issued to study the following:

(a) To study the morphology of spiral bacte­ria in particular along with other forms.

(b) To study the mobility of bacteria.

(c) To study cytological changes occurring during cell division and to determine the rate at which the division occurs.

(d) To study some cell inclusions viz. vacuoles and lipid materials.

Requirements:

1. Grooved Slide

2. Petroleum Jelly

3. Freshly prepared bacterial suspension

4. Cover-glass, forceps, pipettes

5. Microscope with bright field, dark field and phase contrast facilities.

Procedure:

A suspension of bacteria is placed on a cover- slip, and then inverted over the concave depression of the slide to produce a “hanging drop” of the speci­men. Then the slide is examined by microscope with dark field and phase contrast facilities.

Observation:

The morphology, mobility, cell inclusions of bacteria etc., can be visualized.

Technique # 2. Fixed and Stained Smears:

Fixed and stained preparations are most frequently used for the observation of morphological characteristics of bacteria.

The advantages of this procedure are that:

1. The cells are made more clearly visible after they are coloured, and

2. Differences between cells of different species and within the same species can be demonstrated by use of appropriate staining solutions.

The essential steps in the preparation of fixed stained smear are:

(i) Preparation of the film or smear

(ii) Fixation (heat fixation), and

(iii) Ap­plication of one or more stain solution.