Bacteriology and Mycology Flashcards by PATRICIA ONG

Specimen collection is collected during:

acute phase of illness; within 72 hours of illness

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Collected samples must be transported without delay within _________

30 mins to 2 hrs

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Level of prioritization: Critical/Invasive specimen (1)

CSF
Amniotic fluid
Blood
Pericardial fluid
Heart valves

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Level of prioritization: Unpreserved (2)

Feces
Sputum
Wound drainage

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Level of prioritization: Quantitation required (3)

Catheter tip
Urine
Tissues for quantitation

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Level of prioritization: Preserved (4)

Urine
Feces
Swab in holding media

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Level of prioritization: Batch processing (5)

Sputum
AFB culture

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Specimen for culture:

Blood
CSF
Sputum
Throat swab
Nasopharyngeal swab
Cotton swab
Endotracheal aspirate
Calcium alginate swab
Stool

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Specimen to rule out bacteremia, septicemia, and sepsis

Blood

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Most common cause of sepsis normal flora:

E.coli, S. aureus

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Blood pathogen that is never a normal flora

P. aeruginosa

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Cleansing procedure before collecting blood for blood culture

70-95% alcohol -iodine scrub- alcohol rinse

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Common contaminants of blood culture

Viridans
S.epidermidis
Cutibacterium acnes (P. acnes)

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Dilution of blood to media

1:10

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Result for blood culture:

7 days

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Detection days for Brucellosis:

3-4 weeks

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Detection days for leptospirosis

8 weeks

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Microscopic agglutination test for leptospirosis uses:

Live leptospira antigen to patient’s serum

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Macroscopic identification test for leptospirosis

Heat killed leptospira

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Rapid Test for Brucellosis

Serum Agglutination Test

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4 significant Brucella (scam)

B. Suis
B. Canis
B. Abortus
B. Melitensis

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of sets to be collected for blood culture

2-3 sets

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Hours of interval for blood cultures collected at 2 different sites

1 hour

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Amount to be collected for blood culture: Adult

> 20 mL

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Amount to be collected for blood culture: Pediatric

1-10 mL

Amount to be collected for blood culture: infants

0.5 mL to 1.0 mL

Anticoagulant for blood culture

0.025% SPS

SPS disadvantages

Inhibits other bacteria: Neisseria, G. vaginalis

Remedy for SPS inhibition

1% gelatin

Anticoagulant not used for bacterial culture

EDTA and Citrate

Procedure for blood culture that uses white top tube with EDTA

PCR procedure

Volume collected in blood culture in emergency cases

40 mL

Collect blood sample during:

peak of fever

Media for Blood culture (bacte)

TSB BHI Brucella broth Castañeda bottle (biphasic media- both liquid and solid)

specimen to rule out meningitis

CSF

CSF tube intended for micro

Tube 2

Storage for CSF

37 deg C

Specimen for CSF smear preparation and culture

sediments

Cause of neonatal meningitis

S. agalactiae

Cause of meningitis for children < 5 years old

H. influenzae serotype b

Cause of meningitis for adult meningitis >29 years old

S. pneumoniae

Cause of meningitis for 5-29 years old

N. meningitidies

Cause of meningitis for elderly/immunocompromised

L. monocytogenes

Added to CSF collection from a shunt

thioglycollate

Thioglycollate allows the growth of

Facultative anaerobes Strict aerobes anaerobes

Specimen of choice for urine

clean catch midstream

Unable to void urine specimen

catheterized

Specimen of choice for anaerobic culture of urine

Suprapubic urine

Specimen for molecular studies like PCR in urine

1st morning urine

Usual request for urine

culture and sensitivity

#1 cause of UTI

E. coli

UTI in females and elderly women with catheter

S. saphrophyticus

Other causes of UTI:

Klebsiella, E. faecalis (gram positive cocci)

Media used for susceptibility test for kirby bauer

MHA

Used for determining the dilution factor of urine sample

Calibrated loop

1 ul loop dilution factor

1000

10 ul loop dilution factor

100

Computation for colony count:

Colonies x dilution factor = colony count/mL of urine

Colony count considered as UTI

>100,000

Urine without preservative: ___ priority

2nd

Urine with preservative: ______ priority

4th

Preferred preservative for urine culture

boric acid

Maintains accurate colony count preservative

Boric acid

Sputum is used to rule out:

Lower respiratory tract infections (pneumonia, bronchitis, TB)

Patient must be instructed to _______ with water prior to collection and cough deeply into container

rinse/gargle

Sputum volume requirement:

5 to 10 mL

Bartlett’s classification is used to differentiate

sputum and saliva

Bartlett's classification: not a respiratory sample no of SEC and PMN

no of SEC: >10 no of PMNs: <25

Cell that can be seen to confirm for true respiratory sample

Alveolar macrophage

Usual cause of pneumonia

K. pneumoniae P. Aeruginosa S. pneumoniae H. influenzae

Causes pneumonia to those with cystic fibrosis

P. aeruginosa

1 cause of ventilator associated/intubated pneumoniae

P. aeruginosa

Process that should be done to sputum for detecting M. tuberculosis

Decontamination and Digestion

Process of removing contaminants and normal flora

Decontamination

Process of liquefying sample and free any trapped organism to make it detectable

Digestion

Gold standard for decontamination and digestion

N-acetyl-L-cysteine (mucolytic agent to digest) + NaOH (acts as decontaminant)

Other agents for decontamination and digestion

Z-TSP 4% NaOH Cetylpyridium chloride Sodium chloride method 5% oxalic acid

BSL for contaminants

BSL 1

BSL for mucous membrane, ingestion, exposure

BSL 2

BSL for acquired aerosol transmission, inhalation

BSL 3

Specimen collection of SPUTUM according to Bailey's and positive result

1 sputum in 3 days (1 per day) positive: 2/3

DOH collection of SPUTUM; positive result

1st collection: morning 2nd collection: random Positive: 1/2

Specimen of choice for detecting streptococcal infection and C. diphteriae

Throat swab

Major throat pathogen

S. pyogenes

Pharyngitis is caused by bacteria

S. pyogenes

Major throat flora

Viridans strep (S. mitis, S. mutans, S. salivarius)

No.1 cause of SBE and causes dental caries

Viridans

Viridans produces ______ and ______ which enhances attachments to teeth

Glucans and Dextrans

Specimen of choice for detecting B. pertussis

Nasopharyngeal swab

Causes whooping cough

B. pertussis

N. meningitidies is a normal flora from

nasopharynx, oropharynx

N. meningitidis is pathogen seen

CSF

Capsulated form of N. meningitidis

pathogenic

No capsule form of N. meningitidis

normal flora

Toxic to neisseria

Cotton swab

To collect N. meningitidis use:

Dacron Calcium alginate

Component of cotton swab that is toxic to Neisseria

Fatty Acids

Specimen to collect when patient is on ventilator/intubated

Endotracheal aspirate

Not to be used for viruses as inhibits/prevent replication

Calcium alginate

Primarily used for aerobic bacteria

Swab

Anaerobic specimen should be rejected if it is collected using

SWAB

Specimen of choice for detecting GIT pathogens

Stool

Normally detected GIT pathogens

salmonella, shigella

Invasive bloody diarrhea

Shigella

4 serogroups of Shigella (d,f,b,s)

A: S. dysenteriae B: S. flexneri C: S. boydii D: S. Sonnei

Serogroup of shigella that is a late lactose fermenter

S. Sonnei

Human pathogen of Salmonella

S. enterica

Animal pathogen of Salmonella

S. bongori

If stool specimen is not possible, specimen can be collected in

rectal swab

Media for stool specimen

EMB MAC SSA

Semi-quantitation technique for stool:

isolation streak (4 quadrants)

Critical values in Microbiology: Positive

blood culture CSF gram stain or culture Cryptococcal antigen test or culture blood smear for malaria S. pyognes from a sterile site Acid-fast smears or Mycobacterium culture S. agalactiae or herpes simplex from genital site of a pregnant woman at term Detection of significant pathogen (i.e., B. pertussis, Brucella, Legionella)

mostly used in laboratory; readily available for decontamination and digestant of sputum

4% NaOH

Agent added to sputum likely to contain P.aerugenosa

5% oxalic acid

Types of bacteria that is usually encountered in laboratory

Facultative anaerobes, aerobes, strict aerobes

Aerobes requirement:

21% O2 and 0.03% CO2

CAPNOPHILIC bacteria requires:

↑CO2

indicators of Gaspak Jar

Methylene blue and Resazurin

Color of Resazurin in presence of oxygen

Pink

Color of Methylene blue in presence of oxygen

Blue

Absence of oxygen color in gaspak jar

colorless

Component of Gaspak Jar that maintais anaerobiasis

Palladium pellets

Common failure of gaspak jar

inactivation of catalyst due to repeated use

Anaerobic culture % of components

5% CO2 10%H2 85% N2

For detecting capnophylic (Neisseria) and microaerophilic

Candle Jar

microaerophilic requirements

5-10% CO2 5% O2

Darting motility causes gastroenteritis wings of seagull appearance both microaerophilic and capnophilic

Campylobacter

Lowest concentration that inhibited bacterial growth of Antibiotic Susceptibility Testing

MIC (minimum inhibited)

Lowest concentration of antibiotic that can kill bacteria antibiotic susceptibility test

MBC (minimum bactericidal) /MLC (Minimum Lethal)

AST: can act against many bacteria

Broad Spectrum

AST: limited range of action

Narrow

Inhibitory effect of AST

Bacteriostatic

Killing effect of AST

Bactericidal

Media for Kirby Bauer Technique

Mueller Hinton Agar (MHA)

AST: Depth of agar

3-5 mm

AST: Size of antibiotic

6 mm

AST: streaking method

Overlap streaking

AST: Distance of disk from center

24 mm

AST: distance between 2 disk

15 mm

AST: incubation time and temperature

37 dec C for 16-18 hours

Plates should not be more than

5 stacks

Measure the zone of inhibition using

ruler or caliper

AST: sensitive measurement

> 16

AST: resistant measurement

< 16

Too thin, very dry agar surface, and too light inoculum effect in Kirby Bauer technique

False Sensitive/ Larger zone of inhibition

Too thick agar, too much moisture on agar surface, too heavy inoculum, and too long incubation time effect in Kirby Bauer technique

False Resistant/ Smaller zone

Swarming on Kirby Bauer should be

ignored

of antibiotic disk on a 150 mm plate

No more than 12 disk

Distance of disk on 100mm plate

5 mm

storage temp for antibiotic disk- working supply

2-8 deg C

Cell wall inhibitors: Beta-lactams

Penicilin Cephalosphorins Carbapenems

Cell wall inhibitors: Glycopeptide

Vancomycin

Protein synthesis inhibitors

Aminoglycosides Tetracycline MLS (Macrolide lincocymide streptogamin)

Protein Synthesis Inhibitors: Aminoglycosides

gentamycin

Protein Synthesis Inhibitors: MLS (Macrolide lincocymide streptogamin)

erythromycin clarithromycin

AST of Streptococci is fastidious; media is mixed with

Blood supplement

To detect inducible clindamycin resistance among strains of S. aureus

Double disk diffusion test (D-test)

Done if initial/after doing AST, erythromycin and clindamycin is discrepant

D-Test

D-test positive result

Blunting or Flattening (looks like D)

Gene that activates resistance to clindamycin

ERM gene

Quantitative - dilution method: Uses a strip with single antibiotic of different concentrations along its length

E-Test / MIC on a Stick

(+) result of E-test/ MIC on a stick

Ellipse of growth inhibition

Used for research rather than routine laboratory testing of antibiotic susceptibility

E-Test/ MIC on a stick

Serum bactericidal Test is also known as

Schlicter test

Measures activity of antibiotics in patient’s own serum against the pathogen, to detect if patient receiving effective treatment for infection

Schlicter Test (Serum Bactericidal Test)

Screening for carbapenemase producing bacteria Detect the ability of the organism to produce the enzyme carbapenemase

Modified hodge test

First inoculated on MHA for Modified Hodge Test

E. coli

Carbapenem disk for modified hodge test

Imipinem, meropenem

Positive result of a modified hodge test

clover leaf like pattern of zone of inhibition

Fungi belong to plant kingdom without roots and stems and are referred to as

thallophytes

resembles keratin as to function but not composition

Chitin

Existing only as Yeast or Mold

monomorphism

Existing either as Yeast or Mold:

dimorphism

Temperature dependent fungi

dimorphism

Colonies are moist, creamy, opaque, and pasty

Yeast

Fungi that grows at 37 deg C

Yeast

Colonies are fluffy, cottony, wooly, or powdery

Mold

Grows at room temperature

Mold

Fundamental unit of fungi

hyphae

Hyphae function

absorption of nutrients in the environment

Mass of countless hyphae

Mycelium

mycelium that penetrates medium and absorbs nutrients

Vegetative (thalus)

Mycelium part extending above the surface of the colony; usually contains the fruiting buddies that produces spores

Aerial (reproductive)

structure for reproduction of fungi

spores

Type of hyphae: no cross walls

aseptate (coenocytic)

Aseptated hyphae are exhibited by

zygomycota

Hyphae divided into cells by crosswalls

Septate

Spiral (coiled) hyphae is seen in:

trichophyton mentagrophytes

Nodular bodies hyphae are seen in

Microsporum canis

Hyphae with club shaped areas

Racquet

Racquet hyphae are exhibited by:

Epidermophyton floccosum

Broken comb appearance of hyphae

Pectinate body

Pectinate body is seen in

M. audouinii

curved, freely branching hyphae

Favic chandeliers/antler

Favic chandeliers/antler is seen in:

T. schoenleini and T. violaceum

Sexual and asexual spores (perfect fungi)

Synanomorphs/Polymorphs

only sexual spores

teleomorphs

produced during asexual reproduction

anamorph

Made during sexual reproduction Produced through meiosis Produced as a result of nuclear fusion

Sexual spores

formed within a SAC like structure called ASCUS; usually in a fixed # of eight

Ascospores

formed within a club shaped structure called basidium

Basidiospores

Formed from the union of the two undifferentiated or identical hyphal cells (similar)

Zygospores

formed from the union of 2 differentiated (not identical)

Oospores

Produced during asexual reproduction Formed by/produced as a result of mitosis Without nuclear fussion

Asexual spores

asexual spores produced singly or in groups specialized vegetative hyphal strands called conidiophores

Conidia

type that is unicellular pyriform or elliptoid shape

Microconidia

larger, spindle shape, multicellular hyphae

Macroconidia

Formed through fragmentation of hyphal cells

Arthroconidia

Arthroconidia is exhibited by:

trichosporon geotichum coccidiodes

Produced through budding process

Blastoconidia

Blastoconidia is seen in

Candida albicans

formed from the enlargement of the hyphal cells

Chlamydoconidia

within the hyphae of chlamydoconidia

intercallary

Seen in the lateral part of the hyphae of chlamydoconidia

Sesile

Seen in the end part of the hyphae of chlamydoconidia

terminal

Formed within the sporangium

Sporangiospores

Sporangiospores is seen in

Zygomycetes

Zygomycota (conjugation fungi) genera:

Rhizopus Absidia Mucor

Ascomycota (sac fungi) genera/species:

Aspergillus Saccharomyces Histoplasma capsulatum

Basidiomycota (club fungi) genera/species:

C. neoformans

Deuteromycota (fungi imperfecti) genera/species:

Dermatophytes: I.e. Fusarium

Fungal infection affecting the lungs; collect respiratory secretions

Systemic mycosis

may be collected using fine needle and inoculated at bedside

corneal scrapings

may be collected by aspiration

pus

use of cotton swabs may give false ____ microscopic results for pus

positive

Most common specimen collected for fungal culture

Respiratory tract secretions

Recovery of fungal agents causing systemic mycoses

Respiratory tract secretions

Agent of fungal meningitis in immunocompromised people

C. neoformans

If the CSF quantity is not sufficient, the priority is for

Micro

Collected sample of CSF may be processed either through

Filtration or sedimentation (do centrifugation)

Sediments of CSF are used for

smear preparation and culture

Supernatant of CSF is used for

serologic testing

CSF uses filter with a pore diameter of

.45 micra

BACTEC for blood is used to rule out

disseminated infection

blood lysis centrifugation system is used for the recovery of

dimorphic fungi

Vaginal specimens and urine must be transported within ____ following collection

24 hours

Vaginal secretion should be screened for

yeast

These are collected for recovery of dermatophytes

Hair, Skin, Nails

Tissue, Bone Marrow, and sterile body fluids must be minced or use high speed blender to get

cytoplasmic content

Tissue samples must be inoculated on

agar

Temporary mount for microscopic examination of fungi (%,L,I,C)

10-20% KOH Lactophenol Cotton Blue India ink/ Nigrosin Calcofluor white stain

dissolve the non-fungal material, digest the debris, make fungal structure more visible

10-20% KOH

Concentration of KOH for skin and hair

10%

Concentration of KOH for nails

20%

Purpose of heating KOH smears

increase the rate of clearing

Most widely used staining method for fungi (both preservative and stain)

Lactophenol Cotton Blue

acts as preservative in lactophenol cotton blue

lactic acid

acts as killing agent in lactophenol cotton blue

phenol

acts as stain in lactophenol cotton blue

cotton blue

Negative stain (only background is stained); to demonstrate the capsule of C. neoformans

India ink/ Nigrosin

India ink in histopath is used as

intravital stain

Fluorochrome dye; best for direct microscopy; will produce fluorescence; demonstrate chitin in cell wall

Calcofluor white stain

Permanent mounts:

Gram Stain (Hucker modification) Periodic acid Schiff Gomori Methenamine silver Acid Fast (Kinyoun's ) Giemsa or Wright's Acridine Orange Fontana Masson H&E Mayer's Mucicarmine Gridley's stain

Components of Hucker modification

Primary dye: Crystal Violet Mordant: Iodine Decolorizer: Alcohol/Acetone Counterstain: safranin Modified: ammonium oxalate to Crystal violet

to demonstrate details of fungal elements; a histologic stain;

Periodic acid Schiff

PAS will impart color

pink-red color bright magenta

PAS background stains

Green

best permanent mount

Gomori Methenamine Silver

Gomori Methenamine silver imparts

brown to black (melanin)

Outlines of the fungi in gomori methenamine silver

black

Internal of the fungi in gomori methenamine silver

pink black

background stain of the fungi in gomori methenamine silver

light green

GMS-stained sections are essential for

Tissue pathology

stain for detection of Nocardia (branching bacteria) and B. dermatitidis

Acid Fast (Kinyoun's )

routinely in hematology to stain blood cells

Wright

stain blood parasite

Giemsa

demonstrates yeast of histoplasma capsulatum

Giemsa/Wright stain

Stain for detection of M. furfur (Tinea versicolor)

Acridine Orange

Acridine orange result color

green fluorescent for fungal elements orange for epithelial cells

stain that detects hyphal pigmentation

Fontana Masson and H and E

non-pigmented or lightly pigmented hyphae

Hyaline

darkly pigmented hyphae

Dematiaceous

demonstrate C. neoformans; wood’s lamp to detect fluorescence

Mayer's Mucicarmine

Gridley's stain color for hyphae and yeasts

dark blue-pink

Gridley's stain color for tissues

deep blue

Gridley's stain color for background

yellow

Mycology plates and tubes should be incubated in ambient air at what temperature

25-30 deg C (room temperature)

some fungi grow slowly, cultures should not be discarded for

4-6 weeks

Culture is held for

30 days

prevents saprophytic fungi in fungi culture

cycloheximide

General purpose media

Sabaouraud's Dextrose Agar

pH of Sabaouraud's Dextrose Agar

5 to 6

Screening media for dermatophytes phenol red

DTM/ Dermatophyte Test Medium

Recovery of dermatophytes Sabaorauds with chloramphenicol and cyxloheximide

Mycosel/ Mycobiotic agar

inhibit bacterial growth in mycosel

chloramphenicol

prevents saprophytic fungi in mycosel

cyxloheximide

Used for stimulating chlamydospore formation of C. albicans

Cornmeal agar

Isolating aspergillus spp.

Czapek agar

Culture medium for C. Neoformans (develops brown pigment)

Niger seed agar/ Bird seed agar or Staib’s medium

Used for demonstrating pigment production of T. rubrum - capable of producing red pigment

Potato dextrose agar

unable to produce red pigment in potato dextrose agar

T. mentagrophytes

Identification of M/ audouinii differentiating it with M. canis

Rice medium

Not capable of growing in rice medium

M. Audouinii

Biochemical test media - differentiates T. mentagrophytes (+) from T. rubrum (-)

Urea agar

Urea agar is also used to isolate

C. neoformans

General media for yeast species

Brain Heart Infusion

Medium for Nocardia

Casein medium

Isolation of M. furfur

Malt extract agar

Recovery of saprobic(saprophytic) and pathogenic fungi

Potato Flake agar

Not a fungal media

Hay infusion agar

All mold cultures must be processed in BSL

BSL 2

Special test that differentiate T. mentagrophytes (+) and T. rubrum (-)

Hair perforation or baiting test Rapid urease test

Hair perforation or baiting test positive result:

V shape or conical hair perforation

Positive result of Rapid Urease Test:

development of pink to purple color

All candida are urease negative except

C. krusei

Positive control for Urease Test

C. neoformans

Negative control for Rapid Urease Test

C. albicans

For rapid identification of C. neoformans

L-DOPA ferric citrate test

Presumptive test for C. albicans and C. dubliniensis

Germ Tube Test

Positive result for L-DOPA ferric citrate test

development of black color (phenol oxidase)

Positive result for Germ Tube Test

fingerlike extensions/ hyphae like extension of young yeast cell- sperm like

a microdiffusion test for serologic confirmation of systemic fungi (+) result presence of precipitin

Exoantigen test

Test to differentiate C. dubliniensis and C. albicans

Temperature studies

Able to grow at 42 deg C

C. albicans

Identify particular carbohydrate that the fungi can use as sole carbon source

Carbohydrate assimilation test

Positive for: Glucose Maltose Sucrose Lactose Cellubiose Galactose Trehalose Xylose Negative for: Melibiose

C. albicans

Positive for: Glucose Maltose Sucrose Cellubiose Galactose Trehalose Xylose Negative for: Melibiose Lactose

C. tropicalis

Positive for: Glucose Maltose Sucrose Cellubiose Galactose Xylose Negative for: Melibiose Lactose Trehalose

C. keyfer

Positive for: Glucose Maltose Sucrose Galactose Xylose Trehalose Negative for: Cellubiose Lactose Melibiose

C. parapsillosis

Positive for: Glucose Maltose Sucrose Galactose Xylose Trehalose Cellubiose Melibiose Negative for: Lactose

C. guillermondi

Positive for: Glucose Xylose Negative for: Lactose Maltose Sucrose Galactose Trehalose Cellubiose Melibiose

C. krusei

Positive for: Glucose Xylose Trehalose Negative for: Lactose Maltose Sucrose Galactose Cellubiose Melibiose

C. glabrata

Fungi that may be detected through serological test

Agents of systemic mycosis: Blastomyces, Histoplasma Opportunistic fungis: Cryptococcus, Aspergillus

Particularly useful in diagnosing cryptococcal meningitis from CSF specimens

Antigen detection

Antigen detection test is performed by

latex agglutination or immunodiffusion tests

Antigen detection is helpful in detecting _____ and _____ in systemic infections

aspergillus and candida

Delayed hypersensitivity reactions to fungal antigens can be demonstrated by

Skin Test

Skin test may be performed in the ff:

histoplasmosis candidasis sporotrichosis coccidiodomycosis blastomycosis paracoccidiodomycosis dematophytosis

Diagnosis of mycoses in a shorter period as well as detect those fungi that are difficult or dangerous to cultivate in vitro

Molecular Techniques