Spirochete Staining by Fontana’s Technique

Spirochete Staining by Fontana’s Technique

Introduction

  • Spirochete are spiral shaped Gram negative bacteria.
  • They are unique in morphology and locomotion.
  • The spirochete are spiral in shape so its size is measured by measuring its length and width.
  • Generally it is 5 to 500 µm in length and 0.1 to 3 µm in width.
  • Spirochete have endo-flagella that runs through periplasm.
  • The  endo-flagella may be 2 to 100 on number and these flagella are wound around the helical cells in between protoplast and outer membrane.
  • These spirochete are extremely delicate organism  so some special technique are used to stain them.

There are two technique to stain these spiral shape organism.

  1. Fontana’s staining technique.
  2. Becker’s staining technique.

Here we are going to study Fontana’s staining technique in detail.

Fontana’s staining technique

Aim

To stain spirochete by using Fontana’s staining technique.

Requirement

  1. Fontana’s fixative.
  2. Fontana’s mordant.
  3. Fontana’s stain.
  4. 95% Ethanol.
  5. Tartar sample.

Procedure

  1. Take a pin,dip in alcohol and flame sterilized the pin.
  2. This sterilize pin is used to to take a tarter sample present in inter-spaces of teeth.
  3. Now this tarter sample is mixed in the sterile distill water.
  4. A drop of these suspension is taken with the help of sterile wireloop on a clean grease free slide and smear is prepared.
  5. This smear is air dried but not heat fixed.
  6. This smear is fixed by using chemical fixative that is Fontana’s fixative.The slide is placed on colphin jar containing  Fontana’s fixative for 3 times at interval of 1 minute.
  7. Further the fixative is washed of and slide is treated with 95% alcohol for 3 minutes.
  8. After 3 minutes the excess alcohol is washed off and slide is hold with the help of forcep and passed through flame to remove excess alcohol.
  9. In the next step Fontana’s mordant is added on smear and heated till steam appears.
  10. After steam appear the slide is washed with water ,air dried.
  11. These air dried slide is now treated with Fontana’s stain and heated till steam rises.
  12. After steam rise the slide is washed with distill water,air dried and observed under oil immersion.

Flow Chart of smear preparation for Fontana’s staining procedure.

Flow chart of smear preparation of Fontana's staining technique
Flow chart of smear preparation of Fontana’s staining technique.

Flow chart for Fontana’s staining procedure

 

Flow chart for Fontana's staining procedure
Flow chart for Fontana’s staining procedure.

Mechanism

  1. Spirochete are very delicate organism they easily get destroy by heat fixation so here heat fixation step is avoided.
  2. Here smear is treated by Fontana’s fixative .This fixative contain chemicals like formalin and glacial acetic acid.
  3. After fixation the smear is treated to Fontana’s mordant .This mordant contains tannic acid which increases affinity of stain towards stain.
  4. Further the stain is treated with alcohol and  Fontana’s stain.
  5. Fontana’s stain contain ammonical silver nitrate and when these stain is heated it forms silver oxide is formed.
  6. This silver oxide precipitates on the organism and increases the diameter of cell and stains the cell.

Result

After completion of Fontana’s staining procedure the Spirochete are stain brownish black in colour.

One Comment

  1. Dr.S.R. Deshmukh March 25, 2014 Reply

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