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Nigrosin Preparation

Nigrosin preparation showing encapsulated Cryptococcus neoformans as shown above picture. Nigrosin stain (10% w/v ) is a negative stain. It uses for the observation of capsules. The capsule of organisms does not take stain and therefore clear halos surrounding the cells. It is clear as shown above picture. It uses for observation of various microorganisms capsules like Cryptococcus neoformans, Klebsiella pneumonie, Streptococcus pneumoniae, etc.

Composition

Following are the ingredients of nigrosin stain.

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

Test Requirements for Nigrosin preparation 

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

 Nigrosin preparation 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 a coverslip over it . Observe under a microscope, focusing at 10X objective and finally  40X objective.

In the case of Bacterial Capsule

Take a  clean and grease-free slide. Put a drop of nigrosin over it. Touch the colony with an inoculating loop from a solid medium or take a loopful broth in case of a liquid medium. Mix the nigrosin drop and make a 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 of Nigrosin Preparation 

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 a tightly closed container and away from bright light.

Use before the expiry date on the label.

Keynotes

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 pneumoniae, 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 analysis: In vitro viability is generally assessed both before and after thawing by eosin–nigrosin stain.

Bibliography

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. 3^rd ed 1988 Publisher WB Saunder co, Philadelphia.
3. 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.
4. A Textbook of Medical Mycology. Editor: Jagdish Chander.  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. Textbook 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?