India Ink Preparation: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses, Keynotes, and Negative Staining Footages
India Ink Preparation: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Uses and Keynotes
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 likeCryptococcus neoformans (most commonly), Klebsiella pneumoniae, Streptococcus pneumoniae,Haemophilus influenzae, etc.
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
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
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
Fig. Cryptococcus Capsules in Negative Staining or Nigrosin Preparation of CSF
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
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
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
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
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
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
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
Medical Mycology. Editors: Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia.
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.
Rippon’s JW: Medical Microbiology. The pathogenic fungi and the Pathogenic Actinomycetes. 3rd ed 1988 Publisher WB Saunder co, Philadelphia.
A Text-Book of Medical Mycology. Editor: Jagdish Chander. Publication Mehata, India.
Topley & Wilsons Medical Mycology. Editors: M.T. Parker & L.H. Collier, 8th ed 1990, Publisher Edward Arnold publication, London.
Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
Practical Laboratory Mycology. Editors: Koneman E.W. and G.D. Roberts, 3rd ed 1985, Publisher Williams and Wilkins, Baltimore.
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.
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
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Excellent blog you have here but I was wondering if you knew of any user discussion forums that cover the same topics discussed in this article? I'd really love to be a part of group where I can get feedback from other experienced individuals that share the same interest. If you have any suggestions, please let me know. Cheers!
I have seen lots of useful items on your site about computer systems. However, I've the thoughts and opinions that notebooks are still less than powerful enough to be a sensible choice if you generally do jobs that require loads of power, just like video enhancing. But for web surfing, microsoft word processing, and a lot other prevalent computer work they are all right, provided you may not mind small screen size. Many thanks sharing your ideas.
Pretty! This was a really wonderful post. Thank you for your provided information.
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Thank you for your articles. They are very helpful to me. May I ask you a question?
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