Laboratory Methods For Isolation And Identification Of Medically Important Yeast Flashcards
Eukaryotic, unicellular fungi
Reproduces by budding
Moist to waxy colonies
Round to oval, or less often elongate or irregular in shape
Yeast
Yeast cell wall
Chitin, glucans and mannans
Yeast cell membrane
Ergosterol
CULTURE MEDIA FOR ISOLATION
❏Primary isolation medium for yeasts
❏Acidic pH (5.0) inhibits growth of bacteria
❏High concentration of sugar
❏Sabouraud Dextrose Agar (SDA)
Acidic pH of SDA that inhibits the grow of bacteria
5.0
Chloramphenicol
Inhibits bacteria
inhibits saprobes
Cycloheximide
CULTURE MEDIA FOR ISOLATION
❏Selective medium for isolation of fungi
❏Mycosel Agar
(SDA with Chloramphenicol and Cycloheximide)
Test to determine if isolated colony is bacteria or yeast
-1 drop of sterile saline/water glass slide
Wet mount (from colony)
Germ tube Positive
No constriction
Germ tube Negative
Constriction
• Screening test used to differentiate Candida albicans and Candida dubliniensis from other yeast.
• Tests the ability of the yeast to produce tube-like projections from a conidia or spore in human serum or fetal bovine serum after 2-3 hours of incubation at 37°C.
No constriction
Germ tube Positive
GERM TUBE
• Used in the identification of different yeasts • Same as used in bacteriology
Urease Test
Intense pink color (Urease Test)
Positive
No color change (Urease Test)
Negative
• Corn meal with tween 80 agar test
• For distinguishing various species of Candida and other
yeasts
• Polysorbate (tween) 80 is added to cornmeal agar to reduce the surface tension to allow for the development of pseudohyphal, hyphal, blastoconidial growth, chlamydospores and arthroconidia.
Morphology Studies (Dalmau Plate Method)
Morphology Studies (Dalmau Plate Method)
Cornmeal Agar, Potato Dextrose Agar, Rice extract agar
Can be found on the terminal end of the pseudohyphae
Chlamydospore
Seen on cornmeal agar
Presence of blastoconidia, pseudohyphae, and chlamydospores
Organism that has Presence of pseudohyphae, blastocondia
C. albicans
C. dubliniensis
C. krusei
C. parapsilosis
C. keyfr (pseudotropicalis)
C. tropicalis
Organism that has presence of pseudohyphae, blastocondia, Chlamydoconidia/ Chlamydospores
C. albicans
C. dubliniensis
Organism that has presence of arthroconidia, yeast cells only
C. (Torulopsis) glabrata
Cryptococcus spp.
Rhodotorula spp.
Organism that has presence of pseudohyphae and yeast cells only
Saccharomyces spp.
Organism that has presence of pseudohyphae, arthroconidia
Trichosporon spp.
Determines the ability of a yeast to degrade a specific carbohydrate
Fermentation is demonstrated by production of gas Useful when other tests fail to identify the isolate
Carbohydrate Fermentation Test
• Determines the ability of a yeast isolate to use a particular carbohydrate substrate as its sole carbon in a medium
• For precise identification of frequently encountered yeasts
• Identification is obtained through Analytical Profile Index or using Identification Software.
Carbohydrate Assimilation Test
Carbohydrate Assimilation Test Identification is obtained through
Analytical Profile Index or using Identification Software.
Principle:
• Composed of 20 cupules for 19 assimilation tests.
• The cupules are inoculated with semi-solid minimal medium.
• Yeast will only grow if they are capable of utilizing each substrate as the sole carbon source.
• Reactions are read by comparing to growth control
API 20 C AUX
Carbohydate Assimilation Test
• API 20 C Interpretation if posotive
Will result to a turbid of the well
Carbohydate Assimilation Test
• API 20 C Interpretation if negative
No turbid of the well
Other ID Methods:
AUTOMATED IDENTIFICATION SYSTEM (Yeast)
Vitek 2 system
Microscan walkaway-96/40/ autoscan -4 systems
Phoenix system
Incubation of Api 20 C Aux
29°C ± 2°C for 48-72 hours
Positive color of Carbohydrate Fermentation Test
Yellow
Negative color of Carbohydrate Fermentation Test
Green
Used for isolation and differentiation of clinically significant Candida species
• Colonies of C. albicans,
C. tropicalis, and C. krusei produce different colors
Chromogenic Agars
Color produce of C. albicans/ Candida dubliniernsis in Chromogenic Agars
Green
Color of C. Tropicalis in chromogenic agars
Blue
Color of C. Krusei in chromogenic agars
Purple
Color of C. Glabrata in chromogenic agars
It varies (yellow, gold)
CGB Agar Used for the differentiation of :
Cryptococcus gattii versus Cryptococcus neoformans
CGB stands for
L-canavanine glycine bromthymol blue
Positive reaction of CGB agar
Yellow green to cobalt blue
Negative reaction of CGB agar
Yellow
Identification test for C. albicans and C. dubliniensis
Germ tube
Presumptive Identification Tests for Cryptococcus spp.,Rhodotorula spp., Trichosporon spp. (variable), C. krusei (variable), C. lipolytica
Rapid urease
Presumptive Identification Tests for Cryptococcus species
India ink
Other detection method
Serology
Why use serology?
Ag and Ab are easier to detect vs growing the organism
Ag and Ab are produced in large quantities and can be found in
Body fluids i.e. serum, CSF, urine
Ab test:
Immunidiffusion
Radioallergosorbent Test RAST
Ag test
Latex agglutination
Radioimmunoassay
Lateral flow
Latex agglutination system
Anti-cryptococcal antibody-coated latex particles
Lateral flow assay principle
Immunochromatographic dipstick assay
Advantages of lateral flow assay
- No specimen pre treatment
- 2 years shelf life, room temp storage, condensed packaging
- Internal control line eliminates external conteol requirements
- As easy as home pregnancy test
- Only product fDA-cleared for both C. Neoformans and C. Gattii
Lateral flow assay running time
10 mins
Limitations of latex agglutination system
Cross reaction with trichosporon beigelii
Latex agglutination system running time
45 mins
