India Ink Preparation when Cryptococcal meningitis is suspected

Introduction

Cryptococcal meningitis occurs in immunodeficient patients and when meningitis is clinically suspected, for example, patients with HIV disease, or when yeast cells with lymphocyte are detected when performing C.S.F. cell count or examine Gram smear, examine India ink preparation for encapsulated yeasts. India ink is used as a negative stain in negative staining that uses ion negative staining method permits visualization of the usually transparent and unstainable capsules of various micro- microorganisms like Cryptococcus neoformans (most commonly), Klebsiella pneumoniae, Streptococcus pneumoniae, etc.

Principle of India Ink

The capsule is non-ionic so that the India ink used will not bind to it. This is the reason, capsule appears as a clear halo around the yeast cells.

Composition of India Ink

( Hardy Diagnostics)

Black Pelican Drawing Ink No. 17
Deionized Water
Thimerosal

Requirements for India Ink Preparation

India ink or nigrosin stain
Clean and grease-free slide and coverslips
C.S.F. specimen
Droppers or inoculating loop
Waste discarding container or Bunsen burner
Centrifuge
Test tubes
Microscope
Control stains ( For positive control Cryptococcus neoformans
ATCC® 32045 while for negative control Candida albicans ATCC® 10231)

Procedure of India Ink Preparation

Centrifuge the CSF for 5 to 10 minutes.
Remove the supernatant fluid and mix the sediment.
Transfer an equal amount of sediment and India ink i.e. a drop of the sediment to a slide and add a drop of India ink.
Mix and cover with a coverslip.
Now, examine the preparation under a microscope using the 40 X objective.

Observation of India Ink Preparation

Look for oval or round cells, some showing budding, irregular in size, measuring 2-10 µm in diameter, and surrounded by a large unstained capsule as shown in the above figure. Very rarely capsules are absent.

Result Interpretation

Encapsulated yeasts are seen: Positive
Non-encapsulated yeast cells: Negative
Positive control: Presence of encapsulated yeasts seen
Negative control: Absence of encapsulated yeasts seen
Test: Positive as shown above image.

Report

Capsules of Cryptococcus neoformans like organisms seen

Importance of this test

When encapsulated yeasts are detected in C.S.F. a presumptive diagnosis of cryptococcal meningitis can be made.

Key Notes

Pelikan black drawing ink is suitable for this test.
When India ink is not available, use the nigrosin (20% w/v) solution.
Do not make the preparation too thick otherwise, the cells and capsules will not be seen.

Limitations of Ink Ink or Negative staining

India ink preparation is only for presumptive identifications of organisms and therefore it needs other tests like biochemical, immunological, molecular, or mass spectrometry testing that must be performed on colonies from pure culture for complete identification.
Fat droplets, white blood cells, and tissue cells are sometimes confused with organisms like Cryptococcus neoformans cells. Leukocytes and tissue cells may be dissolved by adding a drop of 10% KOH.
Some strains of Cryptococcus neoformans , as well as other cryptococci, may not produce discernible capsules in vitro.

Other Information related to Cryptococcus

Cryptococcus capsule in Giemsa stained smear as shown below-

Cryptococcus neoformans growth on Sabouraud Dextrose Agar (SDA) Bird Seed Agar (BSA)- Niger seed is replaced by Nescafe Coffee and Urea Hydrolization Test-Positive as shown in Video-

Cryptococcus neoformans in LPCB tease mount as shown below-

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