Nigrosin preparation showing capsule of Cryptococcus neoformans

Nigrosin preparation

Nigrosin preparation of CSF is showing capsules of Cryptococcus neoformans.

Yeast cells of C. neoformans  have surrounded  with capsules as shown above picture.

Principle of India Ink

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

Composition

Following  are the ingredients of nigrosin stain.

  • Nigrosin :  10.0 gm
  • Formalin:  0.5 ml
  • Distilled water:  100.0 ml

Test Requirements

  • Nigrosin stain/ India ink
  • Clean and grease free slides and cover slip
  • Test organism suspected specimens e.g. CSF in Crypotococcal meningitis, growth of bacteria on solid media
  • Inoculating loop
  • Bunsen burner
  • Microscope
  • Cedar wood oil ( optional for bacteria)

Test procedure

In CSF for Cryptococcus neoformans:

First centrifuge the CSF. Take a drop of sediment part  on a clean and grease free slide and add over it a drop of nigrosin. Mix it properly  and put cover slip over it .Observe under microscope , focusing at 10X objective and finally  40X objective.

In case of bacteria capsule

Take a  clean and grease free slide. Put a drop of nigrosin over it. Touch the colony with inoculating loop from solid medium  or take a loopful broth in case of liquid medium. Mix the nigrosin drop and make smear. Leave for air dry. Focus at 10 X objective and finally observe  at oi immersion lens i.e. 100 X objective.

Principle And Interpretation

Negative staining  permits visualization of the usually transparent and unstainable capsule of many organisms, most importantly Cryptococcus neoformans. Nigrosin consists of a suspension of fine particles of carbon. These form a dark background, against which capsules see clearly  as a result of displacement of the carbon particles.

Quality Control

Appearance blackish violet colored solution.

Clarity

Clear without any particles.

Results

It is clear halos surrounding the cells as shown above image and below video.

 Storage and Shelf Life

Store below 30°C in tightly closed container and away from bright light.

Use before expiry date on label.

Key Notes

  1. Nigrosin stain uses ion negative staining method permits visualization of the usually transparent and unstainable capsules of various micro- microorganisms  like Cryptococcus neoformans (most commonly), Klebsiella pneumonie, Streptococcus pneumoniae, etc.
  2. This stain uses in tests for viability because living cells exclude the dye, but it enters dead cells.
  3. Assessment of viability and motility parameters of sperms during semen alaysis: In vitro viability is generally assessed both before and after thawing by eosin–nigrosin stain.

Other Informations 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-

References

  1. Medical Mycology. Editors:  Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
  2. Rippon’s JW: Medical Microbiology. The pathogenic fungi and the pathogenic Actinomycetes. 3rd ed 1988 Publisher WB saunder co, Philadelphia.
  3. Clinical Microbiology Procedure Hand book Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. A Text Book of Medical Mycology. Editor: Jagdish Chandar.  Publication Mehata, India.
  5.  Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
  6. Topley & Wilsons Medical Mycology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
  7. Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  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. http://himedialabs.com/TD/S025.pdf
  10. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  11. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/nigrosin
  12. https://en.wikipedia.org/wiki/Nigrosin
  13. https://www.sigmaaldrich.com/catalog/product/sial/43925?
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