India Ink Preparation: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses, Keynotes, and Negative Staining Footages

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Introduction of India Ink Preparation

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.

India Ink Preparation: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses and Keynotes
Fig. India Ink Preparation Step

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

(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
  • Test specimen-CSF
  • Droppers or inoculating loop
  • Waste discarding container
  • Bunsen burner
  • Centrifuge
  • Test tubes
  • Compound Microscope
  • Control stains (For positive control- Cryptococcus neoformans
    and for negative control Candida albicans)

Procedure of India Ink Preparation

  1. Put on gloves.
  2. Centrifuge the CSF for 5 to  10 minutes.
  3.  Remove the supernatant fluid and mix the sediment.
  4.  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.
  5. Mix and cover with a coverslip.
  6. Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
  7.  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

The encapsulated strain of Cryptococcus neoformans in India Ink preparation of CSF
Fig. 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.

Report

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

Cryptococcus Capsules in Negative Staining or Nigrosin Preparation
Fig. Cryptococcus Capsules in Negative Staining or Nigrosin Preparation of CSF

Capsules of Cryptococcus neoformans in Nigrosin wet mount microscopy

Capsules of Cryptococcus neoformans in Nigrosin wet mount microscopy
Fig. Capsules of Cryptococcus neoformans in Nigrosin wet mount microscopy at a magnification of 400X

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 CSF microscopy at a high magnification
Fig. 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

Capsules and yeast cells of Cryptococcus neoformans in negative staining of culture microscopy at a high magnification of 1600X
Fig. 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

Capsule of Klebsiella pneumoniae in negative staining or dry India ink preparation microscopy
Fig. A capsule of Klebsiella pneumoniae in negative staining or dry India ink preparation microscopy at a magnification of 2000X

Encapsulated Streptococcus pneumoniae in Gram staining of CSF showing Gram-positive diplococci bodies with clear zones

Encapsulated streptococcus pneumoniae in Gram staining of CSF showing Gram positive diplococci bodies with clear zones
Fig. Encapsulated Streptococcus pneumoniae in Gram staining of CSF showing Gram-positive diplococci bodies with clear zones at a magnification of 1000X

Capsulated strain of pneumococcus in Gram staining of sputum showing Gram-positive diplococci and bodies surrounded by clear zones

Capsulated strain of pneumococcus in Gram staining of sputum showing Gram positive diplococci and bodies surrounded by clear zones
Fig. The capsulated strain of pneumococcus in Gram staining of sputum showing Gram-positive diplococci and bodies surrounded by clear zones at a magnification of 3000X

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

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
Fig. 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

Further Readings

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  3. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3rd ed 1988 Publisher WB Saunder co, Philadelphia.
  4. A Text-Book of Medical Mycology. Editor: Jagdish Chander.  Publication Mehata, India.
  5. Topley & Wilsons Medical Mycology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  6. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  7.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  8. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  9. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  10. http://himedialabs.com/TD/S025.pdf
  11. https://www.sciencedirect.com/topics/medicine-and-dentistry/indian-ink
  12. https://en.wikipedia.org/wiki/Cryptococcus_neoformans
  13. https://catalog.hardydiagnostics.com/cp_prod/content/hugo/indiaink.htm

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