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What kind of organisms are fungi?
eukaryotic
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If fungi is single-celled it is a
yeast
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If fungi is filamentous it is a
mold
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Candida A microscopic morphology
Ovoid
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Candida A gram stain reaction
Gram +
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Candida A on Sabouraud Dextrose Acid Agar
Moist, white, opaque
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Candida A in sabourauds dextrose acid broth
clear with sediment "+"
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Candida A chlmydocondia test
+ (terminal clomisdospore)
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Candida A species ID
Albican
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Candida B microscope morphology
Enlongated cigar shaped
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Candida B gram stain
Gram +
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Candida B on sabouraud dextrose acid agar
dry, off-white, translucent
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Candida B growth in sabouraud's dextrose acid broth
flaky, white surface growth
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Candida B chlamydoconidia test
"-" (cross sticks/pseudohyphae)
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Candida B species ID
Krusei
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What are the two media used to id dermatophytes?
- dermatophyte test medium (DTM)
- Sabouraud dextrose acid agar
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What are you looking for in the DTM? (Dermatophyte Test Medium)
Production of red color
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what are you looking for in sabouraud Dextrose Acid Agar?
observation of macroscopic appearnace of growth
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Microsporum audouinii appearance
white cottony colony
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Microsporum audouinii is the causative agent of ?
tinea capitis
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Trichophyton rubrum appearance?
white cottony colony, red on reverse side
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Trichophyton rubrumis the causative agent of?
tinea pedis
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Trichophyton tonsurans appearance?
colonies from a folded surface with yellowish color in the depressions. Surface is velevety to powdery with a variation in color.
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Trichophyton tonsurans is the causative agent of?
tinea capitis
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is cryptococcus neoformans yeast or mold?
always yeast
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What is needed to identify the capsule of a yeast?
india ink stain
-
Why is it hard to stain the capsules of the yeast?
very mucoid colony ( large amount of polysaccharide)
-
what test is used to id the genus of yeast?
Urease Test
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Cryptococcus sp. urease test result?
positive (pink color)
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Candida sp. urease test result?
Negative (No color change)
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Yeast cells other than _______ are urease negative
cryptococcus
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cryptococcus sp is a yeast or mold?
yeast
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candida sp is a yeast or mold?
yeast
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What is used to id species in yeast?
Nigerseed (birdseed) agar
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cryptococcus neoformans nigerseed agar result?
brown colored colonies
-
cryptococcus sp. (other than cryptococcus neoformans) nigerseed agar result?
White colonies
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What is dimorphic fungi at 25 C?
mold
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What is dimorphic fungi at 37 C?
Yeast
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What is sporothrix schenckii?
a mold colony that has characteristic mitocondria in daisy cluster
-
Mycobacteria
aerobic slow growing bacilli that possess a high lipid content in their cell wall
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What oganisms are "acid-fast bacilli"
Mycobacteria
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what is required for the decolorization of mycobacteria?
a spcial stain that utilizes acid alcohol
-
What are the four special chemicals used to stain mycobacteria?
- carbol fuschin solution (kinyoun) containing tergitol
- acid alcohol solution
- methylene blue, alkaline
- broth (Dubos) culture of mycobacterium grodonae
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list the 11 steps of mycobacteria staining
- prepare films of resuspended broth culture and fix slide by gently heating
- place slide on slide rack
- cover film with carbol fuchsin (kinyoun)
- allow to stand for 5 minutes
- wash off with water
- wash with acid alcohol until decolorized (grayish appearance)
- wash off with water
- counterstain with methylene blue, alkaline for (15-30 secs)
- wash off with water
- dry by blotting
- examine under oil immersion
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when staining mycobacteria what appears red?
acid-fast bacteria
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when staining mycobacteria what stains blue?
bacteria which are not acid-fast stain blue
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What part of the body is needed to stain for mycobacteria?
sputum
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rice extract agar is a suitable substrate for what?
chlamydospore production
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When candida albicans is grown on rice extract agar, what happens?
mycelium-bearing ball-like clusters of budding cells and the thick-walled round chlamydospores
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What is the general process of the germ tube testing?
- Take yeast cells and serum
- incubate it for (2-4 hours)
- wait for germ tube formations
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DTM is selective for the growth of what?
dermatophytes
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Why is the ph indicator (phenol red) added to DTM?
To monitor acidic/basic reactions
-
A yellow color in DTM means what?
reaction is acidic
-
A red color on the DTM means what?
Reaction is basic
-
What happens on the DTM to make its pH rise and turn it red?
dermatophytes utilize the keratin in the medium they produce alkaline end products.
-
what is the disease name of sporothrix schenckii?
"Rose Gardner's disease"
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how do you concentrate sputum for culture, film and antibiotic observations (mycobacterium)
- Get a sputum specimen of at least 10ml
- add 10ml of digestant = (NaOH/NALC)
- vortex 30-60 seconds (liquified)
- let stand at R.T for 15-20 minutes
- add 30ml phosphate buffer (pH = 6.8)
- centrifuge at 3,000 rpm for 30 minutes
- discard supernatant
- take the sputum sediment ( for antibiotic sediment,for culture = lowenstein Jensen & Middlebrook 7H-10/7H-11, for film= acid fast stain )
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viruses are obligate ________ _________
intracellular parasites
-
what do viruses require for replication?
Host cells
-
can be viruses be grown or cultured in artficial media?
no, only bacteria and fungi
-
what are the three nonimmunological methods to IDing viruses?
- electron microscopy
- virus growth
- detection of viral genome
-
what virus is readily diagnosed by electron microscopy of stool samples?
rotaviruses
-
what is the causative agent of infantile gastroenteritis ?
rotavirus
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Whtat can viruses be grown in?
- tissue culture
- embroyonated chicken eggs
- experimental animals
-
What media is the prime choice for growing and isolation of the influenza viruses?
embryonated chicken eggs
-
where are most viruses grown?
Tissue cultures
-
What is the ID of a virus based on?
the CPE followed by id of virus specific antigens by immunoflorescence of other immunological technique
-
CPE
degenerative changes in cells, especially in tissue culture, and may be associated with the multiplication of certain viruses.
-
what is used to detect viral genome during viremia?
(PCR & RT-PCR) - Polymerase chain reaction and reverse transcriptase
-
(PCR & RT-PCR) can be used to id what
DNA or RNA
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Immunologic methods are used to what?
ID viruses and quantify the immune response(antibodies) in response to the etiologic (causative) agent during the acute and convalescent stages of the illness.
-
what are the immunological methods that can be used to id viruses?
- immunofluorescence
- enzyme immunoassay (EIA)
- enzyme-linked immunosorbebt Assay (ELISA)
- western blot analysis
- hemagglutination- inhibition test
- neutralization of viral infectivity
-
what does NALC stand for?
N-acetyl-L-cystenine
-
In the concentrated sputum process what is NALC?
digestant (mucolytic)
-
In the concentrated sputum process what is NAOH?
decontaminate
-
what inhibits the growth of microorganism other than mycobacteria on the loenstein jensen culture media?
additive malachite green
-
what is the medium of choice to test mycobacterium antibiotic susceptibility?
middlebrook 7h-10/7h-11
-
mycobacterium staining process called?
Ziehl-Neelsen (ZN) method
-
total magnification for electron microscope?
x165,000
-
two types of immunoflourences?
indirect and direct
-
direct IF?
when a fluorescence molecule is covalently linked to the antibody molecule
-
indirect IF?
in addition to first antiobdy in the direct version, a fluroescent second antibody specific for the primary antibody,
-
EIA (enzyme immunoassay)
An enzyme like horseradish peroxidase or alkaline phosphatase is conjugated to the antibody after the antibody-antigen reaction, enzyme substrate is added which converts the substrate into a chromophore at the site of the antigen-antibody complex.
-
what does ELISA stand for?
Enzyme-linked-immunosorbent assay
-
ELISA positive reaction steps?
- 1. antibody present in serum
- 2. binding of secondary conjugated antibody
- 3. enzyme reacts with substrate
-
ELISA Negative reaction steps?
- 1. antibody absent from serum
- 2. conjugated antibody does not bind
- 3. no conversion of substrate
-
ELISA is used for screening for what?
HIV antibodies
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The darker the color for ELISA means ...?
the more Antibodies there is
-
in ELISA color means
antibodies (usually yellow)
-
what kind of test is the western blot analysis?
confirmatory
-
in the western blot anaylisis, viral proteins are seperated by what?
polyacrylamide electrophoresis
-
in the western blot anaylsis, the protein bands are transferred to where?
nitrocellulose paper
-
viral proteins in the western blot analysis are .?
antigens
-
what is added to the nitrocellulose paper to react with the protein bands?
enzyme-conjugated anti-immunoglobulin reagent
-
What type of test is the oraquick rapid HIV?
screening
-
What samples can be used for the oraquick rapid HIV test?
blood, plasma or oral fluids
-
what is the confirmatory test that should be used with the oraquick rapid hiv test?
western blot analysis
-
The virus hemagglutination (HA) titer indicates that how many HA units be used for the hemagglutination-Inhibition (HI)
4 HA units
-
How do you calculate the dilution at which the tests are obtained?
divide the HA titer by 4
-
how do you calculate the fold increase in the acute and covalescent titers?
divide the covalescent titer by the acute titer.
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What is the main point/process of the neutralization of viral infectivity
neutralization of viral CPE by antibodies.
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