Laboratory Diagnostic in Medical Mycology

Dr Mohamed S. Ellabib 7April 2006

INTRODUCTION
Mycology is a well-recognized, valuable component of the daily responsibilities
of the clinical microbiology laboratory. The list of the opportunistic fungal
pathogens is increasing at an impressive rate that is related to the expanding size
of the immune-compromised patient population. As a result, laboratory
technologists must be able to recognize an increasing large group of potential
fungal pathogen

Detection and recovery of fungi from clinical specimens

Dermatophytes and Agents of Superficial Mycosis

Specimen and direct microscopic examination:
Skin, nail scraping and hair shaft is placed in one or two drops of 10-20% KOH
or NaOH aqueous solution on a clean glass slide. A cover slip is placed on top
and the preparation is heated gently. Nails may require a strong alkali solution
(25ý%KOH) and long clearing time
N. B. combination of KOH plus Dimethyl sulfaoxide (DMSO) may be used for
nail specimens

OBSERVATIONS
Hair

Endothrix hair invasion Ectothrix hair invasion

Skin

KOH microscopic examination of skin or nail

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Nails

Isolation media:
1. SDA plus antibiotics and cycloheximide (SDACGC
2. SDACG (SDA Plus Antibiotics

Identification of etiologic agents

Colony characteristics:
Color of the surface and on the reverse of colony texture of the surface
(powdery, granules, woolly, cottony), and rate of growth.

Observation :
Trichophyton violaceum
Color Pink
Texture Waxy

Microscopic canis
Color White with yellow reverse color
Texture Woolly

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Trichophyton rubrum
Color White with brown to red reverse color
Texture downy to fluffy

Microscopic morphology:
Arrangement of the conidia (macroconidia or macroconidia) and other structure
may determine by teased mounts or slide culture preparations mounted in
lactophenol cotton blue. Some times special media may be used such as Corn
Meal Agar or Potato Dextrose Agar may be used to stimulate sporluation.

T. violaceum

M. canis

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Physiological tests
a. In vitro hair perforation test:
A number of dermatophytes have the ability to penetrate hair in vitro. This test
becomes helpful in distinguishing atypical isolates of dermatophytes, especially
T. mentagrophytes from T. rubrum. The hyphae of T. mentagrophytes penetrate
the hair perpendicularly, which results in conical, wedge like, or holes called
perforations. The presence or absence of perforation is the end point for this test.
The test is not read as negative until 28 days.

b. Rice grain test:

The test is useful in distinguishing atypical isolate of M. canis from M.
audouinii. M. canis rapidly grow on the rice grain, typically producing many
conidia and bright yellow pigment. The test also is useful in distinguishing T.
rubrum from T. mentagrophytes.

c. Urea hydrolysis:
Using Christensen’s Urea Agar or Broth the test is useful for differentiating T.
rubrum (urease negative) from T. mentagrophytes (urease positive), at the end of
seven days after incubation period at 25C. A color change from pale blue to
violet purple indicates an alkaline reaction.

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Pityriasis versicolor (Tinea versicolor):
a. Direct microscopic examination :
Skin scrapings are mounted in 10% KOH plus India ink or in lactophenol Cotton
blue.
Observation :

b. Isolation media:
SDACG plus 2% Olive oil or Tween 80

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Identification of yeast of medical importance with reference to
Candida spp using the following scheme

Germ tube test

Negative positive

Corn meal agar mostly C. albicans

+Chlamydospores
Hyphae absent hyphae present

Urease test arthroconidia (+) blastoconidia (-)

Trichosporon species or geotrichum spp Candida spp

Biochemical tests
(+) (-)

Small cells or large cell

C. glabrate S. cervisiae

Mucoid with Capsule or red with capsule

Cryptococcus spp Rhodoturula spps

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Identification of C. albicans and other Candida spps:
a. Germ tube test:
A small portion of an isolate colony of the yeast is suspended in a test tube
containing 0.5 ml of rabbit or human serum or plasma. The test tube is
inoculated at 30C for 2-3hr and examined microscopically for the presence of
germ tubes.

Observations:
C. albicans produce germ tube

Other Candida species produce pseudohyphae and true hyphae

C. glabrate produces yeast form only

b. Chlamydospore production:
For the differentiation of C. albicans from other Candida spps using Corn Meal
Agar plus 0.02 % Tween 80
Observation:

C. albicans produce chlamydospore after 24hr incubation at room temperature,

while other Candida spps produce only pseudohyphae and true hyphae.
N. B. C. glabrate produces yeast form only

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Capsulated Cryptococcus neoformans