(3) Collection, Handling, Processing of Clinical Mycology Specimens Flashcards
Enumerate the Techniques to obtain culture material for slide preparation
- Tease Mount Method
- Cellophane Tape Method
- Slide Culture/ Microculture/ Riddel’s Method
Techniques to obtain culture material for slide preparation
- One of the most common technique done in the laboratory.
- A dissecting needle, commonly a bent wire, is used to pull apart a fungal colony which is placed on a slide.
- Usually done when placing an LPCB stain on a culture sample.
Tease Mount Method
Go over the procedure
Techniques to obtain culture material for slide preparation
Cellophane tape is used to transfer aerial hyphae from the colony to a microscope slide for examination
Cellophane Tape Method
Go over the procedure
Techniques to obtain culture material for slide preparation
Allows for in-situ analysis of fungi with as little disruption on the structures as possible
Slide Culture/ Microculture/ Riddel’s Method
go over the procedure
Enumrate the Biochemical Tests
- Urease Hydrolysis Test
- Germ Tube Production
- Carbohydrate Assimilation Test
- Red Colonial Pigmentation
- Woods Lamp Examination
Biochemincal Tests
Tests an organism’s ability to produce exoenzyme, called urease, which hydrolyzes urea to ammonia and carbon dioxide.
Urease Hydrolysis Test
Biochemincal Tests
what is the positive reaction for Urease Hydrolysis Test
PINK
Urease (+) organisms generate enough ammonia to turn the phenol red indicator in the media into PINK.
Biochemincal Tests
what are the Urease positive fungies
Cryptococcus neoformans and
Trichophyton mentagrophytes
Biochemincal Tests
hyphae-like extensions of young yeast cells showing parallel non-septate sides
Germ Tube Production
Biochemincal Tests
what are teh fungi positive for Germ tube
Candida albicans and Candida dubliniensis
go over the procedure
Biochemincal Tests
- Determine the ability of a yeast to use a carbohydrate as its sole carbon source.
- Uses a yeast nitrogen base broth
Carbohydrate Assimilation Test
Biochemincal Tests
A (+) result for Carbohydrate ASsimilation test indicated by the growth of yeast means
the fungi can use carbohydrate as its carbon source
Biochemincal Tests
- Use the potato dextrose
agar to enhance the pigment production in some dermatophytes
red pigment is then observed in the sub-surface of the agar surrounding the
fungi
Red Colonial Pigmentation
Biochemical Tests
What fungi are red pigment producers
Trichophyton rubrum
and Rhodotorula species
Biochemical Test
Diagnostic test in which the skin or hair is examined while exposed to the black light emitted by a wood lamp
Woods Lamp Examination
Biochemical Test
Infected skin or hair (+) for wood lamps examination, dermatophytes will fluoresce what color?
bright green to yellow-green
The test works for both zoonotic and human infections.
Important in cases of some fungal infections where the fungal polysaccharide or proteins are shed out in body fluids
Serology-Antigen Detection Test
Serology- Antigen used
Polysaccharide capsular antigen of Cryptococcus neoformans
Glucuronoxylomannan
Serology- Antigen used
Mannan Antigens
For Candida species
Serology- Antigen used
Galactomannan Antigens
For Aspergillus species
Match the antigen
- Galactomannan
- Glucuronoxylomannan
- Mannan
a. Polysaccharide capsular antigen
of Cryptococcus neoformans
b. Aspergillus species
c. Candida species
- b
- a
- c
BACcla
Provides information that aids the physician in selecting the appropriate anti-fungal medicine totreat a specific fungal infection.
Antifungal Susceptibility Testing (AST)
AST results for fungi are easily influenced by methodological factors such as?
- Isolate growth phase and inoculum size
- Incubation time and temperature
- Type of media used
- Method of reading
Enumerate the Type of AST
- Brothe Dilution
- Disk Diffusion Method
Types of AST
- Different concentrations of one anti-microbial agent (AMA) against one fungal isolate.
Broth Dilution
Types of AST
Determines the minimum inhibitory concentration (MIC)—the lowest concentration of AMA that can inhibit the growth of fungi
Broth Dilution
Type of AST
- AMA are impregnated onto paper disks.
- Several AMA with standardized concentrations against one isolate
Disk Diffusion Method
These standards were formed due to the wide factors that can affect the results of antifungal susceptibility test
CLINICAL LABORATORY STANDARDS INSTITUTE (CLSI)
STANDARDS FOR AST
AST
what document pertains to reference method for broth dilution antifungal susceptibility testing of YEASTS
Document M27-A4
AST
Reference method for broth dilution antifungal susceptibility testing of FILAMENTOUS FUNGI
Document M38-A3
AST
DOCUMENT M38-A3 is of Microdilution method for molds that causes invasive and cutaneous infections including what spp?
Aspergillus species, Fusarium species, and Zygomycetes
AST
Method for antifungal DISK DIFFUSION susceptibility
testing of yeasts
Document M44-A2
DOCUMENT M44-A2 are intended only for testing this spp and not for other genera
Candida species
identify what AST document
Final Inoculum: 0.5-2.5x 103 CFU/mL
Test Medium:Roswell Park Memorial Institute (RPMI) 1640 with 0.2% glucose
Growth Temperature: 35 ‘C
Incubation Time: 24 to 48 hours
Endpoint: 50% inhibition
Reading of Endpoint: Visually
DOCUMENT M27-A4
identify what AST document
Final Inoculum: 0.4-5 x 104 CFU/mL
Test Medium: Roswell Park Memorial Institute (RPMI) 1640 with 0.2% glucose
Growth Temperature:** 35 ‘C**
Incubation Time: 48 hours
Endpoint: 100% inhibition
Reading of Endpoint: Visually
DOCUMENT M38-A3
Identify the Antifungal agents
- Polyene Macrolide Antifungals
- Azole Antifungal Drugs
- Polyene Macrolide Antifungals
Antifungal agents
This group of antifungal agents BINDS TO THE ERGOSTEROL of the fungal cell membrane causing the membrane to weaken and results to fungal cell death
Polyene Macrolide Antifungals
Antifungal agents
what are the Polyene Macrolide Antifungals
- Amphotericin B
- Nystatin
- Griseofulvin
- 5-Fluorocytosine or Flucytosine
Antifungal agents
This group of antifungal agents INHIBITS THE FUNGAL CYTOCHROME P450 ENZYMES
o These enzymes are needed for the synthesis of ergosterol in the cell membrane of fungi
Azole Antifungal Drugs
Antifungal agents
what are the Azole Antifungal Drugs
- Clotrimazole and Miconazole
- Fluconazole
- Ketoconazole
- Itraconazole
- Voriconazole
- Posaconazole
- Anidulafungin
- Micafungin
Antifungal agents
This group of antifungal agents INHIBITS BETA-(1,3)-D-GLUCAN SYNTHASE which is an important enzyme for the synthesis of the components of fungal cell wall.
Polyene Macrolide Antifungals
Antifungal agents
What are the polyene macrolide antifungals used
- Caspofungin
- Selenium Sulfide
- Potassium Iodide
Antifungal agents
INHIBITS SQUALENE EPOXIDASE which is needed for the synthesis of ergosterol in the fungal cell membrane
Terbinafine or Lamisil
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