Clinical Microbiology Procedure Handbook Vol.3 rd ed 1988 Publisher WB Saunder co, Philadelphia. The pathogenic fungi and the Pathogenic Actinomycetes. Editors: Emmons and Binford, 2nd ed 1970, Publisher Lea and Febiger, Philadelphia. Other Information related to CryptococcusĬryptococcus capsule in Giemsa stained smear as shown below-Ĭryptococcus 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-Ĭryptococcus neoformans in LPCB tease mount as shown below. Some strains of Cryptococcus neoformans , as well as other cryptococci, may not produce discernible capsules in vitro.Leukocytes and tissue cells may be dissolved by adding a drop of 10% KOH. Fat droplets, white blood cells, and tissue cells are sometimes confused with organisms like Cryptococcus neoformans cells.India ink preparation 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.Limitations of Ink Ink or Negative staining Do not make the preparation too thick otherwise, the cells and capsules will not be seen.When India ink is not available, use the nigrosin (20% w/v) solution.Pelikan black drawing ink is suitable for this test.a presumptive diagnosis of cryptococcal meningitis can be made. When encapsulated yeasts are detected in C.S.F. Negative control: Absence of encapsulated yeasts seenĬapsules of Cryptococcus neoformans like organisms seen Importance of this test.Positive control: Presence of encapsulated yeasts seen.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 above figure. Now, examine the preparation under a microscope using the 40 X objective.a drop of the sediment to a slide and add a drop of India ink. Transfer an equal amount of sediment and India ink i.e.Remove the supernatant fluid and mix the sediment.Centrifuge the CSF for 5 to 10 minutes.Control stains ( For positive control Cryptococcus neoformansĪTCC® 32045 while for negative control Candida albicans ATCC® 10231).Waste discarding container or Bunsen burner.Clean and grease-free slide and coverslips.This is the reason, capsule appears as a clear halo around the yeast cells. The capsule is non-ionic so that the India ink used will not bind to it. India ink is used as a negative stain in negative staining that 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. cell count or examine Gram smear, examine India ink preparation for encapsulated yeasts. Cryptococcal meningitis occurs in immunodeficient patients and when meningitis is clinically suspected, for example, patients with HIV disease, or when yeast cells with lymphocyte are detected when performing C.S.F.
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