Silver Impregnation Technique

SILVER IMPREGNATION TECHNIQUES

Diagnosis of Leptosirosis by silver impregnation staining for leptospires

  • Leptospires are very thin and they stain poorly with conventional basic aniline dyes.
  • Bright field microscopy is not useful in the diagnosis of leptospires.
  • Dark field microscopy is the procedure of choice for the demonstration of the organisms in serum and urine.
  • The staining technique to ensure impregnation of silver salts on the soma of the organism in order to increase its thickness, and render it easily visible by light microscopy.

Reagents

A. Fixative: Glacial acetic acid : 1mL

  • Formalin : 2mL (40% formaldehyde solution)
  • Distilled water : 100Ml

B. Mordant: Phenol : 1g

  • Tannic acid : 5g
  • Distilled water : 100mL

C. Silver stain

  • Prepare 0.6% silver nitrate solution and few drops of 10% ammonia solution until a precipitate appears but dissolved on shaking.
  • Again add silver nitrate solution until the precipitate reappears and remains stable.
  • Store the reagent stored in an amber coloured bottle at 4°C until use.

Staining procedure

  • Prepare the smear on a microscopic slide.
  • Place the slide in the fixative (Reagent-A) for 2 minutes.
  • Air dry the slide.
  • Dip the slide in Methanol or absolute alcohol.
  • Clean the slide with tissue paper and air dry the smear.
  • Dip the slide in the mordant (Reagent-B), which is preheated to 75°C for 1 minute.
  • Rinse the slide in distilled water and air dry.
  • Dip the slide in the silver stain (Reagent-C) preheated to 75°C for one minute.
  • Rinse the slide in the distilled water, air dry and examine under oil immersion of bright field microscope.

Interpretation of result

  • The leptospires stain brown and occur as long curved organisms with one or both ends characteristically hooked against a clear white or pale yellow background.

Leptospira_stained_with_silver_stain

Figure: Leptospira stained with silver stain

Questions
  • Why unstained leptospires could not be seen under light microscope?
  • Draw a neat diagram of a stained leptospires as seen under light microscope.
Last modified: Thursday, 5 May 2011, 9:27 AM