LEC3 - General Methods for Examination of Fungi Flashcards
fungi Incubation is at
22°C-30°C
- If suspected for dimorphic fungus, cultures should also be incubated
at what temperature
37 * C
fungi cultures are maintained for how many weeks
4-6 weeks
Record the ff upon culturing;
✓Number of days required to see fruiting structures
✓Mold or yeast
✓Media used
✓Temperature
✓Morphology of colonies
texture of colonies
velvety
cottony
powdery
hairy
what are the things to consider in Colony Morphology
- Texture
- Growth Measurements
- Center and Margin of Culture
- Sulcation
- Exudates
- Reverse Colony
- Any soluble pigments
- General medium
- Most commonly use
Sabouraud Dextrose Agar (SDA)
Sabouraud Dextrose Agar (SDA)
____% dextrose and an acidic pH
4%
pH of Sabouraud Dextrose Agar (SD
acidic
Sabouraud Dextrose Agar (SDA)
❖ Chloramphenicol
❖ Gentamicin
❖ Cycloheximide
Sabouraud Dextrose Agar (SDA)
modifications
- Emmon’s Modification
- Mycosel Agar
Sabouraud Dextrose Agar (SDA)
Modifications:
- Emmon’s Modification
describe the dextrose percentage and ph
2% DEXTROSE in neutral pH
Sabouraud Dextrose Agar (SDA)
Modifications:
Mycosel Agar
describe what is modified
SDA + Chloramphenicol and Cycloheximide
- Enrichment agar
Brain Heart Infusion (BHI) Agar
Brain Heart Infusion (BHI) Agar is used in
Recovery of Cryptococcus neoformans and dimorphic transitions of
Sporothrix schenckii and Paracoccidioides brasilliensis
Brain Heart Infusion (BHI) Agar are can be in what type of agar
tube or plates
Brain Heart Infusion (BHI) Agar
modifications
- BHI Broth + Penicillin
- BHI + Gentamicin + Chloramphenicol
Brain Heart Infusion (BHI) Agar
modifications :
BHI Broth + Penicillin
what is being isolated
growth of Zygomy
Brain Heart Infusion (BHI) Agar
modifications :
BHI + Gentamicin + Chloramphenicol
what is being isolated
Cryptococcus neoformans from
contaminated specimens
- Enriched selective medium
Inhibitory Mold Agar
Inhibitory Mold Agar
contains what substances
inorganic salts, chloramphenicol and gentamicin
- Primary recovery of pathogenic fungi
Inhibitory Mold Agar
- General purpose medium
Sabouraud Dextrose + BHI (SABHI)
- Addition of blood increases isolation of dimorphic fungi
Sabouraud Dextrose + BHI (SABHI)
Promotes conversion to the yeast stage
Sabouraud Dextrose + BHI (SABHI)
- Enhance growth of dermatophytes in cutaneous specimens and
inhibit other fungi and bacteria
Dermatophyte Test Medium
Dermatophyte Test Medium inhibitors
- Cycloheximide, Chloramphenicol and Gentamicin
Selective and differential for presumptive identification of genus
Candida from primary plates
CHROMagar Candida
isolation of Candida albicans specifically for the
growth of Chlamydospores
Cornmeal Tween 80 Agar
Cornmeal Tween 80 Agar
Addition of ____ to provide contrasting
background for observing morphologic features
of yeast
trypan blue
Christensen’s Urea Agar composition
Composed of 2% Urea, Phenol
Christensen’s Urea Agar is used in the isolation of
Cryptococcus, Trichosporon and Rhodotorula sp
agar used in * Differential identification of
Aspergillus spp
Czapek’s Agar
Czapek’s Agar components
Sodium nitrate, sucrose and yeast
extract
medium used for * Identification of Cryptococcus
neoformans
Niger Seed Agar/Birdseed Agar/Staib’s
Medium
Niger Seed Agar/Birdseed Agar/Staib’s
Medium composition
Potassium nitrate, peptone, meal
extract, sulfanilic acid, N,N-dimethyl1naphthylamine
Enhances pigmentation and sporulation of
dermatophytes
Potato Dextrose Agar
Potato Dextrose Agar composition
Potato extract, D-glucose
used in Differentiation of Microsporum canis (yellow pigment) to Microsporum auduinii (rice
grains turn brown)
Rice Medium
rice medium composition
White rice extract, polysorbate 80
medium used for Detection of Cryptococcus spp, differentiation of Trichophyton
mentagrophytes from Trichophyton rubrum
Urea Agar
component of urea agar
Peptone, dextrose, sodium chloride,
monopotassium phosphate, urea and
phenol red
positive result of urea agar
Pink
used for the Conversion of mold to yeast form of Blastomyces dermatitidis
Cottonseed Agar
Indirect Microscopic Examination through
Culture
things needed to observe
- Septate versus sparsely septate hyphae
- Hyaline or phaeoid hyphae
- Fruiting structures
- Type size, shape and arrangement of conidia
- Microscopic characteristics that should be observed:
- Septate/Aseptate
- Hyaline/Phaeiod
- Fruiting Structures
- Types, size, shape and arrangement of conidia
Methods for microscopic examination
- LPCB Tease Preparation (acto-phenol cotton blue)
- Scotch Tape/Adhesive/Cellophane Preparation
- Riddell/Microculture Technique
LPCB (Lactophenol Cotton Blue) Tease Mount
* Teasing needles are used to remove a portion of the mycelium from
the ____of the colony
middle third
Disadvantage of LPCB (Lactophenol Cotton Blue) Tease Mount
disruption of conidia during the teasing process
- gently touching a piece of clear tape, sticky side down, to the surface
of the colony and then removing it.
Cellophane Tape/Scotch Tape Preparation
The tape is placed onto a drop of LPCB on a slide and examined
Cellophane Tape/Scotch Tape Preparation
Advantage of Cellophane Tape/Scotch Tape Preparation
retention of conidial arrangement
Disadvantage of Cellophane Tape/Scotch Tape Preparation
potential contamination of the colony, should be read within 30 minutes and then discarded
Useful for demonstrating the natural morphology of fungal structure
Slide Culture Technique/Riddell Technique
Direct Microscopic Examination perks
- Directly from clinical specimen
- Rapid report to the physician
- Provides clue to the genus of the organism
- Might provide evidence of infection despite negative cultures
- Most common routine test
10-20% Potassium Hydroxide (KOH) Test
10-20% Potassium Hydroxide (KOH) Test sample sources
Nail scrapings, hair, skin scales, thin slices of tissue
- Presumptive test for fungal infections
10-20% Potassium Hydroxide (KOH) Test
Upon collection of sample, 10-20% KOH is added to the slide, wait for
__, then look for yeast or hyphae.
15-30 minutes
-rapid breakdown of cellular debris
without the need for heating
DMSO (Dimethyl sulfoxide)
Uses a fluorescence dye
KOH with Calcofluor White Stain
KOH with Calcofluor White Stain
In need of a ____ microscope to
visualize formation of yeast and hypha
fluorescence
Superior to KOH test
KOH with Calcofluor White Stain
india Ink/Nigrosin Stain
what sample is needed
csf sample
India Ink/Nigrosin Stain
* CSF sample is used to examine presence of ___
Cryptococcus neoformans
In india ink/nigrosin ink, what is the appearance of yeast
Yeast surrounded by a halo (capsule) against a black background.
periodic acid schiff color of fungal element
magenta in pink or green background
gomori methenamine silver color of fungal element
black in green background
giemsa color of fungal element
purple blue yeast with clear halo or capsule in pink-purple background
india ink color of fungal element
yeast with clear halo in black background
KOH color of fungal element
REFRACTILE
KOH - cakcofluor white
fluorescent
masson-fontana color of fungal element
brown in pink-purple background
Sterile 5- to 10-mm hair fragments are floated on sterile water
supplemented with a few drops of sterile 10% yeast extract
Hair Perforation T
Differentiate T. mentagrophytes from T. rubrum -
5-day ____test at
room temperature
Urease Test
urease test uses what agar
Christensen urea agar
Single most useful nutritional test for dermatophytes
Thiamine Requirement
Only ____ are identified to be
germ tube positive
Candida albicans and Candida dubliniensis
7 different ____ number 1 through 7 are
used to determine the nutritional requirements of the
Trichophyton spp
Trichophyton agars
can
be used as an alternative in Germ Tube Production
Brain Heart Infusion Broth, Trypticase Soy Broth, Nutrient Broth
Germ Tube Production
- Incubated at 37°C and must be read not beyond_
3 hrs
Assimilation tests identify which carbohydrates a yeast can use
aerobically as a sole carbon source
Carbohydrate Assimilation
Serological Test
- Use of blood samples
- Complement Fixation
- Agglutination
- Complement Fixation
- Histoplasma capsulatum
- Coccidioides immitis
- Blastomyces dermati
Agglutination
Cryptococcus neoformans
* Other dimorphic fungi
Molecular Detection
- PCR
- Use the DNA of fungi as a confirmatory in identification
Positive for Urease
- Cryptococcus and Rhodotorula
- Most strains of Trichosporon spp
Negative for Urease test
- Candida spp
Temperature Studies of Cryptococcus spp
*
Weak growth at 35°C, no growth at 42°C, optimal growth at 25°C
Temperature Studies of * Candida sp
- Most can grow up to 45°C
- Candida dubliniensis cannot grow at 45°
Potassium Nitrate Assimilation
*___ (positive control)
Cryptococcus albidus
Potassium Nitrate Assimilation
__ (negative control)
Candida albicans
accurate method to determine the ability of yeast to use nitrate as
the sole source of nitrogen
Potassium Nitrate Assimilation
Potassium Nitrate Assimilation
Positive test result turns the medium __, if negative it turns ;__
blue; yellow