Gray staining method for flagella

Many bacteria are mobile due to the presence of one or more very fine thread-like, filaments structures called flag Ella’s, which are thin, proteinaceous structures, originating from the cytoplasm and project out from the cell wall.

Gray staining method is one of the most frequently used staining method for flagella. 

Requirements

Solution A:

  • Tannic acid (20%) 2 ml
  • Potassium alum (saturated aqueous) 5 ml
  • Mercurial chloride (saturated aqueous) 2 ml
  • Basic fuchsin (3%) in 95 % ethanol 0.4 ml

Add the fuchsin dye to other solutions and filter the solution.

Solution B:

  • Ziehl carbol fuchsin
  • Basic fuchsin 0.3 g
  • Ethanol 10 ml
  • Phenol 5.0 g
  • Distilled water 95 ml

Mix Solution A and B and store in a colored bottle.

Procedure

  1. Grow the flagellated bacteria (Pseudomonas, Rhizobium, etc.) On a suitable broth. If it is a solid media, add a small quantity of 1 % pertinent broth into which the organisms swim.
  2. Remove the medium by centrifugation to obtain the pellets.
  3. Resuspend the pellets in 10% formalize to produce a light, faint turbidity and for the prevention of flagella.
  4. Allow a loop full of  suspension to a slide so as to prepare a film.
  5. Flood the smear with solution A for 6 min and wash the slide with Distilled water.
  6. Place a piece of blotting paper over the film and flood the slide with solution B for 3 min.
  7. Remove the paper from the slide, wash and gently blot dry.

Result

Flagella will stain red and can be observed under oil immersion objective.

Reference:

Dr. R. C. Dubey – Practical Microbiology

Gaurav Singh

Editor in Chief Medical Microbiology & RDT Labs - RDT Labs Magazine

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