India Ink Preparation- Purpose, Principle, Procedure and Result

 

Purpose:  Used for direct microscopic examination of capsules of many microorganism.

Principle: India ink or Nigrosin preparation is used for direct microscopic examination of capsules of many microorganisms.  The fine granules of the India ink or Nigrosin give an opaque background against which the clear capsules can be easily seen.  This technique is particularly useful in visualizing the capsule of Cryptococcus neoformans in cerebrospinal fluid, sputum and other secretions.

Required equipment and reagents:

Equipments:

1.    Bunsen burner

2.    Biological safety cabinet

3.    Light Microscope – 40 x magnifications

Reagents:

1.    Cover slip

2.    Microscopic slides

3.    Nigrosin stain

Procedure:

1.    Centrifuge CSF specimen lightly to concentrate any microorganisms in the specimen.

2.    Emulsify a small quantity of the sediment into India ink or nigrosin on a microscope slide and overlay with a coverslip.

3.    Do not make the contrast emulsion too thick, or the transmitted light may be completely blocked.

4.    Examine the mount directly under a microscope, using 10X for screening and 40X for confirmation of suspicious encapsulated microorganisms.

Result:

Positive: Capsulated organism or Positive for Cryptococcus sp.

Negative: No Capsulated organism or Negative for Cryptococcus sp.

 

Negative stain for Cryptococcus sp

Quality control procedures:

Gram positive cocci: Streptococcus pneumonia ATCC 6303

Gram negative bacilli: Escherichia coli ATCC 25922

References:

1. Practical Medical Microbiology by Mackie & McCartney 14^th Edition, Page No-796 – 798.
2. Bailey and Scott’s Diagnostic microbiology, 12^th edition by Betty et al., 2007, Page no: 822- 831.