VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

DIAGNOSIS

Specimens

• Scrapings from lesions, centrifuged milk samples, biopsy or tissue samples in 10% formalin for histopathology.

• Based on morphology
  • C.albicans grows as oval, budding yeast cell on agar cultures & in animal tissues.
  • Pseudohyphae are also produced in animal tissue by elongation of yeast cells that fail to separate.
  • In Gram stained smears C.albicans appear as purple-blue yeast cell.
  • It can also be demonstrated in specimens by 10% KOH (or) by lacto phenol cotton blue.
  • The tissue sections stained by PAS-haematoxylin (or) methaneamine silver stains, the C.albicans appear as thin walled oval, budding yeast cells and/or in the form of pseudohyphae.
• Based on isolation and identification
  • C.albicans grows well on blood agar or SDA without inhibitors (Candida spp may be inhibited by cycloheximide).
  • The plates are streaked with a small inoculum as for bacteria. The cultures are incubated at 37ºC, aerobically, for upto 5 days.
  • Colonies of C.albicans are white to cream, shiny, high convex and have a pleasant beery smell.
  • Smears from the colonies stained with Gram's or lactophenol cotton blue or methylene blue stain reveal thin walled budding yeast cell and pseudohyphae.
  • BiGGy agar (Bismuth-sulphite-glucose- glycine- yeast agar) can also be used for the isolation and identification of C.albicans.
  • Most bacterial contaminants are inhibited by the Bismuth sulphite. C.albicans and C.tropicalis strongly reduce the Bismuth sulphite to Bismuth sulphide.
  • C.albicans gives smooth, circular, brownish colonies and no color diffusion into the surrounding medium.
  • The colonies of C.tropicalis are similar but there is diffuse blackening of the medium after 72 hrs.
• Germ tube or serum tube test
  • A small inoculum from an isolated colony is suspended in 0.5 ml of sheep, bovine, rabbit or human serum and incubated at 37⁰C for 2-3 hrs.
  • A drop of the preparation is examined under phase contrast or high objective of the light microscope.
  • Small, thin walled tubes will be seen projecting from some of the yeast cells. This is charcteristic of C.albicans.
• Demonstration of Chlamydospore (Dalmu's technique)
  • Subsurface inoculation is made on corn meal- tween 80 or chlamydospore agar and the plates are incubated at 30ºC for 2-4 days.
  • A thin coverslip is placed on the surface of the agar and examined under high power microscope to demonstrate thick walled chlamydospores borne on the tips of pseudohyphae.
  • Clusters of smaller blastospores may also be present.
• Based on biochemical test

Ability to utilize	C.albicans	C. tropicalis	C. pseudotropicalis	C. parapsilosis
Glucose	Acid (A) and Gas (G)	A & G	A & G	A & G
Sucrose	A	A & G	A & G	-
Maltose	A & G	A & G	-	-
Lactose	-	-	A & G	-
Chlamydospore on corn meal agar	+	-	-	-

• Animal inoculation
  • Rabbits and mice are susceptible to intra venous and intra peritoneal inoculation respectively. Abscesses develop in the kidney.