Leifson staining method of flagella

Requirements

• Bacterial culture (Pseudomonas fluorescens)
• Pipette
• Formaline
• Centrifuge
• Inoculation needle
• Bunsen burner
• Microscope
• Glass slide
• Coveralls

Prepare the following reagents:

Solution A:

• Sodium chloride 1.5 g
• Distilled water 100 ml

Solution B:

• Tannic acid 3.0 g
• Distilled water 100 ml

Solution C:

• Pararosaniline acetate 0.9 g
• Pararosaniline hydrochloride 0.3 g
• Ethanol (95%) 100 ml

Solution A and B are mixed in equal volumes and then added its 2 part in 1 part of Solution C.

Procedure

1. Make a smear of bacterial culture on a clean glass slide.
2. Draw a rectangle with the help of glass marking pencil, around the film.
3. Flood dye Solution within the marked area of the wax line.
4. Leave the slide for 7 – 15 min. Till golden line develops on the dry surface.
5. Remove the stain by floating off the film with gentle flowing tap water.
6. Examine under oil immersion.

Result

The bacterial body and flagella stain red.

Reference

Dr. R. C. Dubey – practical microbiology