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100 terms

MLS 370 MYCOLOGY LAB

STUDY
PLAY
Fungi characteristics
-Eukaryotic
- Mold, yeast, or dimorphic
-150 species cause disease
-Wide range of infections
Lab ID
-Macroscopic and Microscopic morphological characteristics
-Carb and extracellular enzyme production(physiological characteristics)
-Clinical picture and epidemiology
Use hood for this type of fungi
filamentous/mold
Shrink seals or fungi tape are used on plates with these fungi
fungi with aerial mycelium
perform this on all unknown molds before cell culture
wet prep
do not set up slide cultures on this type of fungi
dimorphic fungi
autoclave contaminated materials at
121oC for 1 hour at 15 psi
BSL 1
agents not known to cause disease in health adults
BSL 2
agents associated with human disease or hazard; percutaneous injury, ingestion, mucous membrane exposure
BSL 3
indigenous or exotic agents with potential for aerosol transmission; disease may have serious consequences
BSL 4
dangerous or exotic agents which pose high risk of life threatening disease, aerosol transmitted lab infections, or related agents with unknown risk of transmission
Many fungal infections are similar to which other infection and therefore is often ordered with fungal cultures
mycobacterial infection
rapid transport and processing is important because of contamination by...
bacteria and saprophytic fungi
deliver to lab within in ____ hours of collection
2
set up fungal culture within__hours of receipt into lab
2
sources for specimens include
respiratory, CSF, blood, cutaneous,urine, tissue, bone marrow, sterile body fluids, urogenital, and wounds
This percentage of healthy women have yeast normally
20%
Routine incubation temperature
30oC
2 sets of cultures for dimorphs are incubated at_____and _______ oC
25 and 37
routine fungal cultures must be kept __days before being called negative
30
yeast fungal cultures
keep 7 days and discard
Rapid grower
less than 5 days
Intermediate grower
6 to 10 days
Slow grower
greater than 11 days
Moniliaceous
clear, colorless, pastel color
Dematiaceous
darker staining on the reverse (also maybe on the obverse)
yeast-like
looks like Candida albicans
glabrous
smooth
velvety
suede fabric, short uniform length
cottony
long aerial mycelium/hyphae
granular
sugary; reproductive structures are heavily sporulating
septate hyphae
have septations
aseptate hyphae
rare septa; large, wide, ribbon-like
hyaline
moniliaceous; clear/pale reverse color and hyphal elements pastel color
dematiaceous
dark, olive, black, brown reverse colony color
special formations seen mostly in dermatophytes
1. Raquet hyphae
2. nodular organs
3. pectinate bodies (comb)
4. favic chandeliers (antlers)
5. spiral hyphae
6. chlamydoconidia
7. arthroconidia
Storage units, swollen cells found in pseudohyphae, refractile, appear empty, may be intercalary between blastoconidia of pseudohyphae or terminal (on end of pseudohyphae)
Chlamydospores
Asexual reproductive cells produced directly form true hyphae, appear to have contents in structure
Chlamydoconidia
Cyclohexamide
anti fungal agent
Chloramphenicol
antibiotic
BHI (Brain Heart Infusion agar)
nutritious, used to get fastidious dimorphs growing ; can add blood or antibiotics
Sabouraud Brain Heart Infusion agar (SABHI)
able to grow fastidious fungi
SABHI with blood
able to grow fastidious fungi, more nutrients
Sabouraud Dextrose agar (SDA)
supports most fungal growth; 4% glucose or 2% glucose in the modified version
BHI biphasic blood culture bottles
typically inoculate a blood culture bottle if you have blood
You want to give enough_____to grow, but not too much that the fungi don't _______.
nutrients, sporulate
Inhibitory Mold Agar (IMA)
selective, primary isolation medial; doesn't grow dermatophytes, has chloramphenicol but not cyclohexamide
Dermatophyte Test Medium (DTM)
screen only, not definitive, pH indicator (phenol red); stimulates conidia formation and pigment formation
Potato Flake Agar
makes fungi sporulate
Mycosel or Mycobiotic agar
selective for saprophytes; contains cyclohexaminde and chloamphenicol, primary dermatophytes
Yeast extract phosphate agar
inhibits saprophytes but grows slow growing fungi
Mycosel
SDA with chloramphenicol and cycloheximide
Inhibitory mold agar
yeasts will grow on this but bacteria are inhibited
A good specimen for using BHI with blood is___
sputum
Ascospore agars
1. V8 Juice agar
2. Malt Extract agar
Corn Meal Tween 80 agar
used to ID Candida albicans; stimulates clamydospore production
Cotton Seed conversion agar
used for Blastomyces dermatidis; converts dimorphs from mold to yeast form
Czapek's agar
Aspergillus recovery and identification
Niger/Bird Seed agar
contains caffeic acid and used for ID Cryptococcus neoformans-turns brown
This enzyme causes C. neoformans' positive reaction on NSA
phenoloxidase
Nitrate reduction agar
helps ID nitrate reducing organismsm, esp. yeasts
Potato Dextrose Agar (PDA)
stimulates pigmentation and conidia formation
Rice Medium
Microsporum audouinii doesn't grow on this
Trichophyton agars 1-7
Different chemicals in all 7 agars; ID species; hard to read
Urea agar
salmon=neg
magenta pink=positive
Yeast fermentation broth
anaerobic, sugars fermented
Yeast Nitrogen base agar
contains oxygen, simulation?
CHROMagar
selective and differential for Candida species
Which organism is green on CHROMagar
Candida albicans
Direct exam benefits
allows a preliminary report to physician, may enable fungal therapy, may lead to special media inoculation, may prevent overtreatment
Saline wet mount used for to see...
budding yeasts
hyphae/pseudohyphae
conidia
fungus-like bacteria
granules
spherules in Coccidioides immitus
Lactophenol Cotton Blue (LPCB)
Phenol-kills organisms
Lactic acid- preserves fungal structures
Cotton blue-stains chitin in fungal structures to make them visible
Can lactophenol cotton blue be made permanent?
yes
KOH prep
-used for hair, skin, nails, sputum, vaginal, and bronchial washings when a lot of cellular material is present
-dissolves keratin
What is the purpose of the KOH prep dissolving keratin?
make fungi more visible
Is the KOH prep permanent?
no. but you can add glycerol to save for 2-3 days
What color do fungi and yeasts stain in the Gram stain?
crystal violet/blue (Gram positive)
Budding yeasts indicate ____
infection
Gram Stain
-Yeasts and pseudohyphae are the most common elements observed
In the Gram Stain, Cryptococcus neoformans may be overlooded in which 2 specimens?
CSF or respiratory specimens
Modified Kinyoun Acid Fast stain
-ID aerobic, branching GPRs or fungus like bacteria, especially Norcardia species
-uses weaker acid to decolorize: 1%H2SO4
Norcardia appears what color under Modified Kinyoun acid fast stain?
red. everything else counterstains blue
Kinyoun Acid Fast Stain
used for staining ascospores-look magenta/red
3% HCl Decolorizer
India Ink prep
Use blue/black ink, india ink, negrosin
used for observation of capsules, esp. Cryptococcus neoformans and other encapsulated yeasts appear positive
Calcofluor white stain
dye absorbed by chitin in the fungal cell wall
may be used in conjuction with 10% KOH
Observe this color fluorescence for Calcofluor white stain
bluish white or apple green (depending on wavelength)
Calcofluor white stain is not good for these specimens
vaginal specimens or Coccidioides spherules
Gomori methenamine silver (GMS)
histology; stains fungi and actinomyces; stain black with green background
Periodic acid Schiff (PAS)
fungal elements stain magenta against a pink or green background
H&E
background pink
fungal elements stain purple
Wright Stain
Hematology; look for Histoplasma capsolatum in blood or bone marrow; stain purple
Fontana-Masson Stain
rare; dematiaceous if positive (melanin-like pigment stains positive)
What stain is better than India ink?
Mayers mucicarmine-stains capsle of Cryptococcus red, the background is yellow
Tease mount advantages
can be performed on a mature colony from a primary isolation plate; can be turned into a permanent mount
Tease mount disadvantage
rough teasing may disrupt conidia and make structural morphology difficult to see
Cellophane tape mount advantage
preserves the integrity of conidia and sturctures more than a tease mount
Cellophane tape mount disadvantage
tape acts as a coverslip, so you can't seal it for permanent mounting
Slide culture method (and advantages)
best method for observation of fungal structures; can be preserved for permanent mounting; two mounts can be preserved by permanent mounting
Slide culture advantage
fungal growth onto the slide and coverslip maintains the morphological position of structures to make ID easier
Slide culture disadvantages
Technical to set up; must wait for culture to grow after initial time of fungus isolation; Zygomycetes grow past the edge of the coverslip before formation of reproductive structures