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Flagella Stains

Flagella Stains
Extremely thin, hair appendages that protrude through the cell wall and originate from the granular basal body, embedded in the cell membrane of bacterial cell are called flagella. They are the organs of locomotion in bacterial cell. Another appendages are called as pili or fimbriae. They are shorter, thinner and straighter than flagella (Figure 5-8).

Bacterial flagella account for upto 2% of the dry weight of a cell. Anatomically, a bacterial flagellum consists of three parts a basal body, a hook and main filament
 FIGURE 5-8.
Electron micrograph of bacterial cell showing fimbriae (a) and flagella (b).
FIGURE 5-9.
Model demonstrating basal body, hook and filament part of flagellum from Escherichia coli. Dimensions are expressed in nanometers (Courtesy: M.L. De Pam­philis and J. Adler).

The length of flangellum is usually several times to that of the cell, but diameter is nearly in the range of 10-20 nm. Among bacteria only certain species of bacilli, vibrio and spirullum possess flagella. Cocci generally do not possess flagella, although there are exceptions, e.g., Nitrococcus mobilis. Flagellum is composed  f proteinic subunits called flagellin, having molecular weight in the range of 20,000 to 40,000 daltons.

The structure and function of flagella is very well discussed by Berg (20), DePamphilis and Adler (21) and Silverman and Simon (22). The arrangement and number
of flagella varies considerably (Figure 5-10) and is an impor­tant tool in bacterial taxonomy e.g. genus Pseudomonas is characterized by a polar flagellum. The antigenic property of flagella (H-antigen) is also useful in the identification of bacteria and in the diagnosis of certain diseases.

It has long been realized that bacterial flagella are actually below the visual limit in size. However, they can be seen under light microscope by employing special technique. Regardless of the method employed, in order to make flagella visible, their apparent thickness must be increased. For this purpose mordant is used which usually consists of tannic acid and some metallic salts. Deposition of mordant on flagella increases the apparent diameter of flagella which on staining becomes visible by light microscope.

The flagella stains are notoriously difficult to perform because of
(i) the tendency for stain to deposit on background material and indeed onto the surface of slide and
(ii) the ease witl;1 which bacteria shad these delicate appendages, if culture is not handled carefully.

For these reasons special care in cleaning of slide and preparing bacterial smear is essential.
(a) Cleaning of slide: Use new, heat resistant slide and immerse it in absolute alcohol for 10-15 minutes. Clean the slide with dichromic acid cleaning solution. Wash the slide with distilled water. Before use, pass the slide back and forth through a Bunsen flame until flame shows yellow colour. Cool the slide and use.
(b) Preparation of Smear: Good results can be obtained by preparing smear in following way. Take agar slant having, actively growing and young culture (18-20 hours old). Add carefully about 2-4 ml of sterile distilled water through the wall of tube keeping slant upwards. Rotate the tube slowly between palms. Incubate the tube at 30°C for 30-40 minutes. Check motility of the suspension by hanging drop preparation. If culture is motile then proceed further. Using sharp glass marker make four rectangular areas approximately of equal size. Transfer a small drop from the top of bacterial suspension by means of capillary pipette to the end of square on the reverse side of the marks. Tilt the slide to about 70° and allow the drop to run slowly to the other end and remove immediately the excess with the help of blotting paper. Allow the slide to air dry.

The important factors influencing the result of staining are
(i) skill of investigator,
(ii) the organism under study,
(iii) growth conditions like age, medium composition etc. and
(iv) staining method. The most important of these is the first. Therefore, whatever method is employed, practice improves markedly the result.

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