India Ink Stain – A Comprehensive Guide
Introduction
The India ink test is a rapid, cost-effective diagnostic tool used to detect Cryptococcus species, particularly Cryptococcus neoformans and Cryptococcus gattii, in clinical specimens. These fungi are known to cause cryptococcal meningitis, a severe infection primarily affecting immunocompromised individuals, such as those with HIV/AIDS.
Table of Contents
Principle
Staining of the Cerebro spinal fluid (CSF) sediment with India Ink to detect the polysaccharide capsule surrounding the yeast Cryptococci.
India ink (a colloidal carbon suspension) does not penetrate the polysaccharide capsule of Cryptococcus.
This creates a halo or clear zone around the yeast cells when viewed under a microscope.
The contrast between the dark background and the unstained capsule makes the organism easily identifiable.
Primary Sample
The test is primarily performed on:
- Cerebrospinal fluid (CSF) – For suspected cryptococcal meningitis.
- Blood, pleural fluid or tissue samples – In disseminated cryptococcosis.
It is especially useful in resource-limited settings where advanced tests like cryptococcal antigen testing (CrAg) or culture may not be readily available.
Required Container or additive types
Collect in a sterile, leak proof container.
Reagents and Consumables
Fresh clinical specimen (CSF is most common)
India ink (sterile)
Microscope slides and coverslips
Light microscope (40x–100x magnification)
- Autoclave
- Bio safety cabinet.
Procedure
- Centrifuge a specimen at 1500 rpm for 10–20 minutes in the sterile screw- capped test tube.
- Decant the supernatant in a separate test tube.
- Make wet mount of the deposit and add 1 drop of India Ink and cover with a cover slip.
- Observe under the microscope for the encapsulated, spherical yeast cells and record the results.
Interpretation of results
Positive Test: Round or oval yeast cells (5–10 µm) with a clear halo (capsule) against a dark background.
Negative Test: No encapsulated yeast cells visible.
Frequently Asked Questions
Is India ink test confirmatory for cryptococcosis?
No. It is a screening test. Confirmation requires:
Cryptococcal antigen test
Culture
Molecular methods
What are the limitations of the India ink test?
Low sensitivity, especially in early infection
False negatives in low fungal load
Operator-dependent interpretation
Does India ink stain the organism?
No. It stains the background, not the organism or capsule.
