Laboratory Diagnosis, Specimen Collection & Serologic Test 2 Flashcards
Important specimen if the infection is a disseminated type
BLOOD
temp and storage of blood
30 deg C up to 21 days
in Lysis centrifugation technique, for pathogenic fungal elements, it is best incubated for as long as
____ days
10-15
- Contains both solid and liquid media
- If bottle is inverted, specimen will be inoculated on the solid media
Biphasic broth-agar system
in Lysis centrifugation technique, As fast as ___ days of incubation, fungal elements can be already be detected on the plated media
4
this clinical specimen use culture media with cycloheximide
RESPIRATORY SECRETIONS
respiratory secretions examples
Sputum
Bronchoalveolar lavage (BAL)
Bronchial washings
Tracheal aspirate
- Optimal for isolation of H. capsulatum & filamentous fungi
- Centrifugation of concentrates is best before culture
Lysis centrifugation technique
Collection is not done by medical technologists
Lumbar tap
csf must be kept at what temp, and why not be refrigerated
37 deg C, it might kill any fungal elements present in the specimen
Almost all body fluids are abundant except for
synovial fluid
Both clinical specimens may be processed as blood culture
BONE MARROW AND STERILE BODY FLUIDS
ideal urine specimens
Midstream
clean-catch
Early morning
how many swabs are required for vaginal and cervical discharges, and what are those for
2 swabs: KOH test and culture
urine specimens must be processed by how many hours
2hrs (at RT) or refrigerate
subcutaneous samples
Lesions
abscesses
tissues
what techniques must be done to collect subcutaneous specimens
Needle aspiration or biopsy
machine used to macerate or mash the tissue
Stomacher
where can you get muco-cutaneous specimens
tongue
throat
how many swabs should be used for muco-cutaneous and what are those for
2 swabs: culture and smear
most common clinical specimen
CUTANEOUS
suspected organisms for cutaneous
dermatophytes
allows the visualization of fluorescence which could aid in collecting the correct hair specimen
Woods lamp
culture media perfect for recovery of dermatophytes
Mycosel agar
incubation period of skin
30 deg C (21 days)
culture media must include
Amino acids or urea
Glucose
antimicrobials
Cycloheximide + Chloramphenicol
Gentamicin + Chloramphenicol
Ciprofloxacin
TYPES OF CULTURE MEDIA
Plated and Tubed
permit the growth of virtually all clinically relevant fungi
Non-selective
tailored to isolate specific pathogenic fungi of interest
Selective
- Recovery of (1) saprobic & (2) pathogenic fungi
- Sufficient for recovery of dermatophytes
- Initially without antibiotics to isolate C. neoformans,
candida & aspergillus - they are sensitive to Cycloheximide & chloramphenicol
SABOURAUD DEXTROSE AGAR
- (contains 2% dextrose)
- enhances typical sporulation
- provides more characteristic colony morphology
Emmons modification
- For the primary recovery of (1) saprophytic and (2)
dimorphic fungi
BRAIN-HEART INFUSION AGAR
for tissue specimen w/ dimorphic fungi
BHI with sheep blood + antibiotics
widely used as an INITIAL CULTURE media from collection
BHI broth
Primary recovery/ isolation of (1) saprophytic and
(2) dimorphic fungi, particularly fastidious strains.
SABOURAUD HEART INFUSION
- Usually used for research
- Primary recovery of dimorphic pathogenic fungi
INHIBITORY MOLD AGAR
- General purpose BASAL MEDIUM for the cultivation of yeasts & molds
- Stimulates sporulation and pigmentation
- Aids in cultivating and differentiating pathogenic and non-pathogenic fungi
- Commonly used in finding out fungal organisms from food and beverages
- To control the growth of the contaminating bacterial
agent
POTATO DEXTROSE AGAR
PDA: serves as growth stimulant and carbohydrate source
Dextrose
PDA: provides a nutrient base for luxuriant growth of fungi
Potato infusion
PDA: ___ it may be added to lower the pH to about ___
10% sterile tartaric acid
pH 3.5
- Primary recovery of (1) saprophytic and (2) dimorphic fungi, particularly fastidious and slow-growing strains
- Cultivation, isolation, & induction of sporulation
(Spore formation) of fungi from clinical specimens
POTATO FLAKE AGAR
Recommended for the cultivation of yeasts and
molds
V-8 AGAR
V-8 AGAR: provides essential growth nutrients
Yeast extract
V-8 AGAR: Source of amino acids
L-asparagine
V-8 AGAR: Source of energy
Glucose
V-8 AGAR: supplies the trace ingredients needed to
stimulate the growth of fungal elements
that are found in the specimen.
V-8 juice
- Highly selective for dermatophytes
- For isolation of pathogenic fungi from non-sterile
materials
MYCOSEL/MYCOBIOTIC AGAR
Recommended for selective isolation of pathogenic
fungi (dermatophytes) from cutaneous sources
DERMATOPHYTE TEST MEDIUM
For use in the cultivation of fungi and for the inducement of chlamydospore formation by
Candida albicans
CORN MEAL AGAR w/ TWEEN 80
Allows the growth of the organism vertically so it would lessen the surface tension on the agar; very helpful for candida so that from the base of the agar
they will go out on the surface.
Tween 80
Chlamydospore formation is best seen when the
organisms are incubated
25 deg C
C. neoformans are able to release the enzyme
phenoloxidase
ROUTINE CULTURE MEDIA for fungi but is perfect
for aspergillus
CZAPEK AGAR
NS: serves as a substrate for the detection of
phenoloxidase
Caffeic acid
Identification of C. neoformans
NIGER SEED/ BIRD SEED AGAR
NS: enhances melanization of some strains of
C. neoformans
Creatinine
The action of phenoloxidase to caffeic acid results in the production of
MELANIN
- Conversion of Blastomyces dermatitidis from mold
to yeast form - Used for dimorphic fungi so that we could
see their other form
COTTONSEED CONVERSION AGAR
For differentiation of yeasts by means of their typical chlamydospores and on basis of micromorphological criteria
RICE EXTRACT AGAR
- For isolation and differentiation of major clinically
significant Candida species - allows each Candida spp. to produce its characteristic colonies and pigmentation.
CHROMAGAR CANDIDA
- Differential media used in the presumptive
identification of Trichophyton species based on
nutritional requirements
TRICHOPHYTON AGARS
- Detection of C. neoformans
- Differentiates T. mentagrophytes vs T. rubrum
- Detection of Trichosporon spp.
UREA AGAR
This method allows the medical technologists to
obtain information regarding the morphology of the
organism
DIRECT MICROSCOPIC METHOD
- Directly gather sample from the infected sample site
- useful for cutaneous specimens
WET MOUNT
destroy keratin layer particularly the hydroxide component
KOH (10%)
- Not a direct method; sample used is the one that is
used in the culture - a.k.a. AMAN’S MEDIUM
LACTOPHENOL COTTON BLUE
LPCB: serve as a preservative
Lactic acid
LPCB: antimicrobial agent; can kill any live organisms in the specimen
Phenol
method used in LPCB
Basic Tease Mount
LPCB: a stain with affinity to CHITIN and fungal
cell walls
Cotton blue
- Can be used by itself, but it is more effective with
KOH - Uses a non-specific fluorochrome that binds to
CHITIN and CELLULOSE in cell wall of organisms
CALCOFLUOR WHITE + KOH
method for rapid detection of yeast, fungal elements and parasites
Fluorescent staining
- Negative staining method
- Perfect for encapsulated organisms
- Capsules appear as clear halo against dark background
INDIA INK
swabs when collecting the sample.
dacron/rayon swabs
result in CALCOFLUOR WHITE + KOH
apple-green/bluish-white: fungi and parasites
reddish-orange: other materials
may intensely fluorescent in Calcofluor white
Cotton fibers
stain used in Mycelia and Conidia
Gridley’s
stain used in Black Cell Wall
Gomori’s/ Grocott Methenamine Silver
stain used in stains hyphae of molds & some yeast
Periodic Acid Schiff
stain used in Fungi are weakly G (+) but poorly stained and Useful for Candida
- Kinyoun’s AFS
- Hucker’s Gram Stain
- H&E
stain used in Better demo of B. dermatitidis than wet mounts
Papanicolau
stain used in detecting intracellular H. capsulatum in blood smears.
Giemsa and Wright’s stain
stain used in Dematiaceous hyphae
Fontana-Masson
incubation period
yeast:
molds:
30 d Celsius and 22-25 d Celsius for 21 days
Allows the medical technologists to see actual
colonies growing in the culture media and take
note the microscopic details that would help in
identifying the organism
CULTURE METHOD
added to liquefy highly viscous samples & facilitate plating on agar media
Mucolytic agents
If delayed, the specimen can be refrigerated for a
short time except
blood
CSF
skin
hair
nails
what level: Inoculations & manipulation of mold
colonies
BSL 2
desired humidity to prevent the agar from drying out
40-50%
what level: dimorphic fungi
BSL 3
Clean surfaces of BSCs and incubators regularly
use 70% ethanol or bleach
rate of growth of yeast
24hrs to 4 days
rate of growth of molds
4 days to several weeks
- Most accurate method to preserve & observe
fungi - Fungi are preserved in original state
- Requires more skill & time than tease mount
- NOT FOR DIMORPHIC FUNGI because it is too
open and because the MOT for this is respiratory
except for Sporothrix
SLIDE CULTURE
- Used to differentiate between fungal or bacterial
organisms - Its procedure is similar to COAGULASE TEST
- To differentiate Candida albicans from other
Candida spp
GERM TUBE TEST
- test to identify T. mentagrophytes
- Can also utilize soil
hair baiting
hair baiting incubation period
25 deg C for 10 to 14 days
CHO Assimilation Test For Yeast
BIOCHEMICAL TEST
positive results in REDUCTION OF NITRATE TO NITRITE
change in color (red) = positive
If negative add Zinc dust
No change in color = positive
For PRELIMINARY ID of C. neoformans
RAPID UREASE TEST
- Provides enough information to name the
species of the fungal organism - Provide key elements that needed to improve
management of invasive fungal infections
SEROLOGICAL TEST
- Time consuming; mold cultures grow after
several weeks - It has low sensitivity due to the low concentration of the organism in the samples infested.
Culture methods
- Detection of specific host immune responses to fungal antigens using immunological reagents
- Detection and quantitation of specific fungal metabolite byproducts
Non-culture methods
- Good for Cryptococcosis
- Detect polysaccharide antigen
- Qualitative & semi-quantitative
LATEX AGGLUTINATION
Look for antibodies of specific antigens
IMMUNODIFFUSION
Precipitation Band in Active infection
M and H
Precipitation Band in Early/Chronic Infection
M only
- Microbroth dilution breakpoint test
- Allows us to identify which concentration of drugs is susceptible
FungiTest