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C. INDIA INK PREPARATION FOR CRYPTOCOCCUS
USE
The India Ink is provided in a reagent dispenser for use in the identification of Cryptococcus neoformans.
PRINCIPLE
The capsular material of C. neoformans does not absorb the carbon particles of the ink, resulting in a negative stain.
SPECIMEN
The specimen is usually CSF, pleural, synovial, peritoneal, or pericardial fluid sent in a sterile container.
PROCEDURE
Centrifuge CSF, pleural, synovial, peritoneal, pericardial fluid for 10 minutes at about 3000 r.p.m.
Use the ampule crusher for breaking the SpotTest ampule and for squeezing the ampule to dispense the reagent. Holding the ink dispenser upright and with the tip pointing away from you, squeeze gently to crush the ampule inside the dispenser.
Invert the dispenser and squeeze gently to dispense a drop of the ink on a glass slide.
Add 1-2 drops of specimen sediment to the slide, mix, and coverslip.
Using reduced light, examine the preparation for oval to spherical, single-budding, thick-walled yeast-like cells (5-10 microns in diameter) surrounded by a wide refractile, gelatinous capsule.
RESULTS
C . neoformans appears as a round, oval, or elongated, single-budding, thick-walled, yeast-like organism, 5-15 um in diameter, surrounded by a wide refractile capsule. If these yeast-like cells are seen, report as positive.
QUALITY CONTROL
Check the performance of the ink by using positive and negative controls.
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Positive control: |
C . neoformans |
ATCC 76484 |
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Negative control: |
Candida albicans |
ATCC 14053 |
REAGENT
The dispensers contain Pelikan Drawing Ink #17 Black.
STORAGE
Store the dispensers at 15-30 degrees C until the expiration date on the label.
LIMITATIONS
A positive India Ink test is a presumptive identification of C. neoformans. Additional biochemicals (Yeast Biochemical Card) are necessary for a definitive identification.
Leukocytes, fat droplets, and tissue cells are sometimes confused with C. neoformans' cells.
PRECAUTIONS
For in Vitro diagnostic use.
Discard all contaminated materials into a biohazard container.
REFERENCES
Bailey, W.R. and E.G. Scott. 1974. Diagnostic Microbiology. The C.V. Mosby co., St. Louis, Mo.
Difco/Detroit, Michigan. SpotTest India Ink. TI NO. 1116. May, 1985.
Lennette, L.H., A. Balows, W.J. Hausler Jr., and H.J. Shadomy, eds. 1985. Manual of Clinical Microbiology. 4th ed. American Society for Microbiology, Washington, D.C. |