MYCOLOGY LABORTATORY

DIAGNOSIS  Proper collection for sputum and respiratory

toxic creation should be a screw cap wide mouth
SPECIMEN COLLECTION bottle either glass or plastic and properly labeled
Diagnosis of fungal infection depends entirely on
selection, collection, transport, and processing of CEREBROSPINAL FLUID
clinical specimens
o Selection- in terms of appropriate clinical • Filtered through a 0.4 um membrane filter
sample attached to a sterile syringe
▪ Fungi are classified based on their site o Filter removed and place in Culture
of infection example: Media (CM)
▪ Superficial mycosis → o Examined daily
superficial infection, fungi that • If less than 1 ml submitted – centrifuge and 1
will cause skin infection, sample drop aliquots placed on several areas of cm
will either be through swab or o Processed promptly- if not store at RT or
script at 30 degrees Celsius
▪ Systemic mycosis → can cause o Should not contain antibacterial and
systemic infection samples antifungal agents
such as CSF and blood  redAlert: CSF specimen should never be
refrigerated.
RESPIRATORY TRACT INFECTION
BLOOD
• Sputum, induced sputum, BW, BAL, tracheal
aspiration • Blood culture system
→ Respiratory tract secretion: one of the • Lysis centrifugation system (for heavy incidence
most common clinical samples for culture of dimorphic fungi)
that we receive in microbiology for fungal o Effective in isolation of H. capsulatum
identification. Contains normal flora that o H. capsulatum optimum recovery: 10-14
normally reside in the Upper Respiratory days
that will try to contaminate the sample o Temp. 30 degrees Celsius for 21 days
Processing consideration: before reporting negative (PARA SURE!)
o redAlert: Specimens may be stored at • The optimal temperature fungal blood culture is
room temperature if processing is 30 degrees Celsius, and the suggested
completed within 2 hours incubation time is 21 days
o redAlert: if processing is delayed,
specimens should be refrigerated at 4 URINE
degrees Celsius
 if the sample process beyond 2 hours the • Processed immediately after collection
integrity of the sample will be compromised • 24-hour sample- unacceptable
which means that if the sample contains • Centrifuge and sediment cultured using loop
bacteria, the bacteria will try to multiply that can o Centrifuge using sediment not
hinder the replication of fungi result to a supernatant
negative result.
HAIR, SKIN, AND NAIL SCRAPINGS
How can we control the bacteria? • Obtained by scraping the skin with
• Antibacterial antibiotic in the battery of media scalpel/microscope Slide
to be used. • Hairs- plucking with forceps
o Cycloheximide (concentration: 0.5 ml) o Temp. 30 degrees for 21 days before
in at least one of the culture media reporting negative
(prevents overgrowth of molds) o Mycosel agar with chloramphenicol and
 Problem in Respiratory tract secretion- the fungi cycloheximide
is too concentrated which will result to the
overgrowth of molds that will make us hard to
identify EYE (CORNEAL SCRAPING OR VITREOUS
Why do we add antibiotics in culture media? HUMOR)
- Because the usual sample are those sample • Corneal scrapings collected by a physician
that are not sterile esp. with respiratory tract should be placed directly onto microscope slides
except for lower respiratory tract because it is and inoculated onto non-inhibitory media
sterile. It is to prevent normal flora bacteria to
hinder the result. MYCOLOGY MICROSCOPIC METHODS
 3. Fontana-Masson Stain
WET PREPARATIONS
• Demonstration of melanin or melanin-like
• Most common substances in the lightly pigmented
• Saline Mount- direct observation of fungal agents of phaeohyphomycosis
elements (have keratin mixed to the sample)
• 10% KOH preparation- dissolved keratin in skin, GERM TUBE
hair, or nails
• Calcofluor White Stain (brightening agent) • For presumptive identification of
or ultraviolet woodslap C.albicans
o Enhances visibility of fungal elements
• Uses “rabbit serum” for at 37 degrees celsius;
▪ Hair is examined for endothrix
or ectothrix infections 1-3 hours
▪ Endotrix- inside the hair shaft  Usually used in the lab is HUMAN serum
(will not fluoresce in ultraviolet because rabbit serum is expensive
woodslap)
▪ Exotrix- remains confined to the MYCOLOGY CULTURE MEDIA
hair surface (fluoresces bright
or yellow green in an ultraviolet HOT TO READ?
woodslap)
Surface texture:
NEGATIVE STAINING
1. Cottony or wooly
1. India Ink or nigrosine (add 10% KOH or 2. Granular
diiodized water) 3. Chalky
• Identify capsule of the yeast C. 4. Velvety
neoformans in CSF 5. Powdery
• Difficult to interpret 6. Silky
2. Lactophenol Cotton Blue (LPCB) 7. Glabrous (smooth, creamy)
 Usually performed when there are aerial 8. Waxy
mycelia Pigmentation:
• Imparts a blue color to cell wall 1. Observation in the surface (ex. cottony) and
• Used in tease mounts (wet preparation) or reverse plate (flip→booty part then describes any
slide culture coloration based on the pigmentation)
3. Giemsa or Wright Stain
• Detection of intracellular H.capsulatum in Culture and Microscopic characteristics of medically
blood, lymph nodes, lung, liver, or BM important fungi
Periodic Acid Schiff Culture Microscopy
characteristic
• Internal details
4. Gomori’s methanamine silver stain Microcporum Downy white Steril
audouinii to salmon- hyphae,terminal
• Stains viable and non-viable fungi
Disease: pink colony chlamydoconida,
• Both PAS and Gomori are useful in Tinea Reverse: Tan FAVIC CHANDELIERS,
staining fungal elements from deeply Capitis to salmon and pectinate bodies
seated tissues pink
• Periodic Acid Shift (PAS)- internal Microsporum Membranous Thick walled,
canis with feathery spindle shaped,
details
Disease: periphery; multiseptated
Tinea center of rough walled
capitis colony macroconidia
associated is white to
HISTOCHEMICAL STAINS with alopecia buff
over orange
yellow.
1. Mayer’s mucicarmine stain
Reverse:
• Demonstration of the mucoid capsule of Lemon- yellow
C. neoformans or
2. H&E yellow orange
• Determines the hyaline and apron
dematiaceous fungi
Microcporum Cinnamon- Thick walled, ▪ Alternative for SDA same with
gypseum colored, rough, multiseptated nutrient agar
Disease: powdery macroconidia
Tinea capitis colony II. DIFFERENTIAL MEDIA
Reverse: light o Birdseed (niger agar)
tan ▪ C. neoformans, producing phenol
oxidase
CULTURE MEDIA ▪ Resulting to production of melanin
(brown to black color)
• Generally, must contain nitrogen and o Cornmeal agar with tween 80
carbon sources, and vitamins ▪ Chlamydospore production of
o Glucose, fructose and mannose, Candida species
sucrose (table sugar) o Czapek agar and MALT agar
o Peptone, yeast extract, malt extract, ▪ Isolation of Aspergillus species
amino acids, ammonium and NO2 o Malt agar (MA)
compounds ▪ Useful in isolation of Ascomycota
o Salts (Fe, Zn, Mn)- for define d media
MOST COMMON TECHNIQUE USED IN
o Thiamin (B1) and biotin (B12)
MYCOLOGY
I. PRIMARY ISOLATION MEDIA • Slide culture
o Brain- heart infusion agar (BHI) o Optimal examination method for
▪ in bacteriology it is used for preservation of fungal morphology
fastidious/ maaarteng ▪ Example you have prepare SDA
organism and while preparing SDA you’ll
▪ In mycology it is used to also prepare sterile plate then add
isolate Saphhrophytic and glass rod then on the top put slide
pathogenic fungi from sterile then put distilled water inside 1-2
sites ml; the water will provide moister
o BHI with antibiotics later when you incubate
▪ Pathogenic fungi, specimens ▪ When you have culture media-
contaminated with bacteria or using a scalpel cut it vertically and
saprobes fungi horizontally then get a sample
▪ Cycloheximide (0.5 ml) in at (black type) which is yun yung
least one of the culture media ilalagay mo sa taas ng slide
(prevents overgrowth of (mentioned above); sa taas ng
molds) slide using wire needle magkuha
o Dermatophyte test medium (DTM) ka ng arial miscella sa culture
▪ Dermatophytes from then ipahid sa taas ng culture
keratinized specimens (use media after that put the cover slip
10% KOH if keratinized spec. then close in the upper lid of plate
need to be dissolve) then incubate at 24 degrees
▪ Can replace SDA-CC for (mycella); 37 degrees when yeast
recovery of dermatophytes o Advantage:
o Mycosel/Mycobiotic- similar to DTM ▪ No need to remove a portion of
▪ Similar with DTM in terms of the fungus from a culture plate
purpose which is to isolate and transfer it to the slide
dermatophyte but different in ▪ This reduces the chance of
contents damage to the fragile
o Sabouraud dextrose Agar (SDA) reproductive structure of spore-
▪ Saprobic and pathogenic fungi bearing structures fungi
o SDA- (Cyclohexemide)
▪ Pathogenic fungi • Adhesive tape Preparation
▪ Bacteria saprobe fungi –
inhibited
o SDA with oil (Olive oil)
▪ For isolation of Malassezia
o Potato Dextrose Agar (PDA)
▪ Grows wide range of fungi
 o Same with cellophane scotch tape
method

 before ilagay sa slide and scotch tape dapat ang

slide merong LPCB then perform microscopic
characterization