Introduction of India Ink Preparation
Table of Contents
Cryptococcal meningitis occurs in immunodeficient patients and when meningitis is clinically suspected, for example, patients with HIV/AIDS, or when yeast cells with lymphocytes are detected when performing Cerebrospinal fluid (CSF) cell count or examining 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 technique permits visualization of the usually transparent and unstainable capsules of various micro- microorganisms like Cryptococcus neoformans (most commonly), Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, etc.
Principle of India Ink
The capsules are non-ionic so the India ink used will not bind to it. Therefore, capsules appear as a clear halo around the yeast cells.
Composition of India Ink
- Black Pelican Drawing Ink No. 17
- Deionized Water
Requirements for India Ink Preparation
- India ink or nigrosin stain
- Clean and grease-free slide and coverslips
- Test specimen-CSF
- Droppers or inoculating loop
- Waste discarding container
- Bunsen burner
- Test tubes
- Compound Microscope
- Control stains (For positive control- Cryptococcus neoformans
and for negative control Candida albicans)
Procedure of India Ink Preparation
- Put on gloves.
- 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.
- Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
- If any suspicious objects encounter, examine with the high dry objective (40X).
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 figure. Very rarely capsules are absent.
Result Interpretation of India Ink Preparation
The encapsulated strain of Cryptococcus neoformans in India Ink preparation of CSF
- Encapsulated yeasts: Positive
- Non-encapsulated yeasts: Negative
- Positive control: Presence of encapsulated yeasts
- Negative control: Absence of encapsulated yeasts
- Test: Positive as shown above image.
Capsules of Cryptococcus neoformans like organisms seen
Importance of India Ink Preparation Assay
When encapsulated yeasts are detected in CSF a presumptive diagnosis of cryptococcal meningitis can be made.
Limitations of Ink Ink or Negative staining
- India ink or nigrosin preparation or negative staining 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.
Keynotes on India Ink Preparation
- 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.
- The evidence of capules may also be determined in Gram staining of bactertia but they can only be confirmed by negative staining.
Negative Staining Footages
Cryptococcus Capsules in Negative Staining or Nigrosin Preparation
Capsules of Cryptococcus neoformans in Nigrosin wet mount microscopy
Capsules and yeast cells of Cryptococcus neoformans in negative staining of CSF microscopy at a high magnification
Capsules and yeast cells of Cryptococcus neoformans in negative staining of culture microscopy at a high magnification of 1600X
Capsule of Klebsiella pneumoniae in negative staining or dry India ink preparation microscopy
Encapsulated Streptococcus pneumoniae in Gram staining of CSF showing Gram-positive diplococci bodies with clear zones
Capsulated strain of pneumococcus in Gram staining of sputum showing Gram-positive diplococci and bodies surrounded by clear zones
The capsulated strain of Haemophilus influenzae in Gram-staining of sputum showing pleomorphic Gram-negative coccobacilli to small and large rods and bodies surrounded by clear zones at a magnification of 2000X
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