Flagella Staining Technique by Liefson’s Method

Flagella Staining Technique by Liefson’s Method

Introduction

  • Bacteria have two types of locomotory organs and that are Flagella and pili.
  • Here we are studying about flagella staining.
  • Flagella is a thin,hair like structure made up protein called as flagellin.
  • It size ranges from 20 µ to 200 µ in length.
  • Flagella is one of the most important locomotory organ .It is mainly made up of three parts- 1) Basal body 2) Filament 3) Hook.
  • Flagella is generally present in rod shape bacteria and very few cocci shape bacteria posses flagella.
  • As flagella are very thin and hair like they cannot be easily observed under microscope.
  • So a special technique is design to increase thickness of flagella as well as stain it.
  • Due to these technique we can observe structure of flagella easily under microscope.

Aim

To Stain Flagella of cell by using Leifson’s method

Requirement

  1. Flagellated cell culture slant.
  2. Leifson’s stain.
  3. 1 % Methylene blue.
  4. Distilled water.

Procedure

  • First of all take two hours old flagellated cell culture slant and add two to three drops of sterile distill water in the slant with the help of sterile pipette.
  • Note that the distill water is added slowly without disturbing the growth of cells.
  • After addition of distill water incubated the slant for 20 minutes.
  • Then take a drop of suspension from the slant and place the drop on a clean slide which is kept in slanting position.
  • The drop should flow slowly from one end of slide to other end to avoid folding of flagella on cell.
  • Allow smear to air dry here we don’t use heat fixation treatment .
  • After air drying the slide is flooded with Leifson’s stain till a thin film of shinny surface appear.
  • After this give a gentle stream of water wash treatment to a slide.
  • Now treat the slide with 1 % methylene blue treatment for 1 minute.
  • Give the slide water wash treatment ,air dry and observe under oil immersion lens.

Flow chart for preparation of smear for Flagella staining

Flow chart of smear preparation

 

Flow chart of Flagella staining procedure

flagella staining procedure

Mechanism

  • First of all in this procedure thickness of flagella is increase so it can be visible.
  • The Leifson’s stain is made up of tannic acid ,basic fuschin stain prepared in alcohol base.
  • When we treat Leifson’s stain with cell the tannic acid get attach to the flagella and alcohol get evaporated.
  • After evaporation of alcohol the thickness of flagella is increased due to deposition of tannic acid.
  • Where as Basic fuschin stain the Flagella.
  • After Leifson’s stain treatment cells are treated with Methylene blue stain.
  • This Methylene blue stains the cell.

Result

After observation under microscope we can observe that flagella appear red in colour and bacterial cell appear blue in colour.

 

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