MICRO

Specimen Collection and Transport • Sputum should be the result of a deep cough (not saliva)
or induced by aqueous alcohol.
General Guidelines • Collect 5-10 mL in sterile container.
• Aseptic collection
o Skin and nails: 70% alcohol 5. Urine
§ Remove bacterial contaminants • A clean catch, midstream urine sample should be collected
§ True pathogens are not easily cleansed when aspiration or cystoscopy cannot be performed.
• Placed in sterile, leak-proof containers o Urine specimen most suitable for diagnosis of
o Humidified: preservation of moisture mycoses of the urinary tract is a catheterized
§ Except dermatological specimens (should be placed in specimen.
a dry container/paper envelope) o Fungal normal flora is present on skin of patients à
• Hair comes in contact with urine
• Skin • Early morning specimens are aseptically collected in sterile
• Nail clippings containers.
• Immediate delivery • Twenty-four hour collections have no value.
o Processed within 2 hours after collection o Overgrowth of contaminants
• Preservation • If a delay in processing beyond 2 hours is anticipated, the
o Refrigerated temperature: 4°C urine should be placed at 4°C for up to 12-24 hours to
o Dermatologic samples: 15-30°C inhibit the overgrowth of rapidly growing bacteria.
o Blood and CSF: 30-37°C
• Swabs are not encouraged 6. Blood
o Easily dries out • Blood is collected aseptically to avoid microbial
o Easily contaminated contamination.
o Exception: specimens in anatomical sites that are collected o The collection site is cleansed with a disinfectant at
only by swabbing the time of collection.
§ Throat • Use sodium polyanethol sulfonate (SPS, Liquoid) as an
§ Vagina anticoagulant.
§ Ears o Has antiphagocytic and anti-complement activity
o Abscess, wound samples, pus: needle aspiration • Collect 8 mL blood in a yellow vacutainer tube (contains
1.7 mL of 0.35% SPS; final concentration with blood is
Specific Guidelines 0.05%).
1. Skin and interspaces o This can be used to inoculate a vented biphasic blood
• Wipe lesions and interspaces between the toes with 70% culture bottle containing either trypticase soy or
alcohol to remove surface bacterial contaminants brain/heart infusion agar and broth.
• Using the edge of a glass slide or a blunt scalpel blade, § A ratio of 1 part blood to 10 parts broths is used.
firmly scrape the lesion, particularly at the growing margin. • The Lysis Centrifugation Isolator System is ideal for fungal
• If multiple lesions are present, choose the most recent for blood cultures.
scrapings as old loose scale is often not satisfactory. o 10 mL of blood is directly collected in an Isolator tube.
• Place scrapings between two glass slides or place in a clean
envelope labelled with the patient’s data. 7. Pus and exudates
o One to scrape, one to catch • The skin over pustular lesions should be disinfected.
• Using a sterile needle and syringe, aspirate material from
2. Nail undrained abscesses.
• Clean nail with 70% alcohol. • Place the material in a sterile container.
• Dorsal plate: Scrape outer surface and discard; scrape the o The syringe also serves as a transport container if the
deeper portion. needle is capped.
• Remove a portion of debris from under the nail with a
scalpel. 8. Vaginal material
• Collect whole nail or nail clippings à place all material in a • Using several sterile swabs, collect material from the
clean envelope labelled with the patient’s data. vagina.
• Insert swabs into a sterile tube.
3. Hair
• No cleaning of scalp is needed 9. Tissue
• Infected area: scaling or alopecia • Tissue is aseptically collected from the center and edge of
• Forceps à epilate at least 10 hairs the lesion.
• For hairs broken off at the scalp level, use a scalpel or a • Place between moist gauze squares, add a small amount of
blade knife sterile water or 0.85% NaCl to keep tissue from drying out,
• Place hairs between two clean glass slides or in a clean and send immediately to the laboratory.
envelope labelled with the patient’s data. o Should never be placed on a fixative
• Keep refrigerated not exceeding 8-10 hours at 4°C until
4. Sputum processed.
• Fresh and collected in the early morning (concentrated)
• Have patient remove dentures and rinse mouth vigorously
with water.

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10. Cerebrospinal fluid o Nail clippings

• As much spinal fluid as possible is collected and placed in a § Minced with scalpel à pulverized with mortar
sterile container. and pestle
o Done by physicians o Thick and mucoid specimens
o Lumbar tap: intervertebral space between L3 and L4 § Sputum, gastric aspirates
§ Children: spinal cord may extend up to L3 and L4 § Digested with mucolytic agent à concentrated
à intervertebral space between L4 and L5 • 2% NaOH + NALC
• Collect depending on opening pressure after insertion of o 2% NaOH: disinfectant
syringe o N-acetyl-L-cysteine (NALC): mucolytic
o If normal: 90-180 mmH2O à 10-20 mL CSF
o Children: 10-100 mmH2O à 10-20 mL CSF Methods:
a. Wet preparation (Wet mount)
o If > 200 mmH2O à ≤ 2mL CSF
• Material suspended in fluid on a glass slide + cover
o If sudden drop in opening pressure after collecting
slip à microscopic exam
few mL of CSF, stop collection process.

§ Sudden drop may indicate herniation
Techniques:
• Divide to 4 tubes:
1. Potassium hydroxide (KOH) mount
o Tube 1: chemistry and serology
• Detection of fungal elements
o Tube 2 is generally used for microbiological
o Keratinous material (i.e. hair, nail)
processing.
§ KOH dissolves keratin
§ First few mL may be contaminated with skin
o Thick, mucoid specimens
normal flora
§ KOH is mucolytic
o Tube 3: cell count
o Tube 4: determination of occurrence of tubercular • KOH solutions
meningitis o 10-20% (w/v) KOH in distilled H2O
§ Skin scraping: 10% KOH
• If processing is to be delayed, samples should be stored at
§ Nails and hair: 20% KOH
30-37°C and not be refrigerated as CSF is an adequate fluid
o 20% (v/v) glycerol in distilled H2O
culture in which fungal elements can survive until
§ Glycerol prevents crystallization of
subcultured.
KOH
• If volume collected is inadequate, physician is responsible
• 40% dimethylsulfoxide (DMSO) in distilled H2O
for prioritizing the tests to be performed using the CSF
o DMSO: enhances penetration of KOH
sample
o General rule: prioritize microbiology • Method:
o Material on glass slide
11. Bone marrow o Apply 1 drop KOH
o Cover with cover slip
• Aspirate approximately 3-5 mL of bone marrow and place
o Prepare a wet house chamber (to prevent
it in a sterile container.
drying of set up while waiting for KOH to
• SPS or heparin can be added as an anticoagulant.
clear sample)
o SPS is recommended
§ Clearing process: may be facilitated by
• The pediatric isolator blood culture system may be used.
application of heat

o Microscopic examination
12. Body fluids (pleural, synovial, and peritoneal)
§ LPO à HPO
• Specimens are collected aseptically.
o Visualization of fungal elements
• Place in sterile containers. § Spore
§ Spore + hyphae
Specimen Processing and Examination
• Malassezia furfur – pityriasis

versicolor: hypopigmentation
1. Microscopic Examination
o Spaghetti and meatballs
• Significance: appearance
o Generate preliminary report o Short hyphae; spherical
§ Clue in validating tentative diagnosis
spores
o Contribute to early therapy (especially if fungal
o Color to fungal elements
elements are distinctive) § 10-20% KOH + Parker Super Quick Ink
§ Fungal culture: minimum averaging 2 weeks to (Black)
culture § 1 part ink + 9 parts KOH
o Selection of media for primary culture

o Detection of non-viable fungal elements 2. India ink preparation
§ Ex. Rhinosporidium seeberi
• India ink: fine carbon particles

• Negative staining
• Preparation of specimen
• Visualization of capsules of Cryptococcus
o Clear fluids
neoformans
§ CSF, urine, peritoneal fluid, pleural, synovial fluid
• Method:
§ Concentrated: centrifugation à sediments: used
o Material on a glass slide
o Tissues
o Apply India ink (1 drop
§ Minced with scalpel
o Cover with cover slip

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o Microscopic examination 2. Macroscopic Examination

§ LPO à HPO • Cultivation
o Capsule appear as halo-like • Culture media
§ Capsule: relatively colorless o Primary isolation – isolates pathogen from sample
o Background: dark a. Saboraud dextrose agar (SDA / SAB)
• Most routine media for fungal culture
3. Calcofluor White Preparation (CFW) • High sugar content and low pH (5.6)
• Calcofluor white: textile whitener • Developed by Raymond Jacques Saboraud
• Reagent preparation: • Emmon’s modification
o 0.1% CFW (w/v) in distilled H2O o Lower glucose concentration
o CFW with KOH o pH: 6.8-7.0
• Same method with KOH and India ink o Enhances fungal isolation
• Requires fluorescent microscope o Enables more bacterial growth
o (UV) Short wavelength: < 420 nm o Isolation of:
o Fungi emit a fluorescent light § Dermatophytes – cause
§ Color emitted depends on excitation cutaneous mycoses
filter (blue-white or apple green) • Microsporum
• Trichophyton
b. Dried stained smears • Epidermophyton
• Fix specimen on slide à staining à microscopic § Yeast from vaginal samples
evaluation
• Not routinely used SDA with antimicrobials
1. Mycosel (BBL) / Mycobiotic (Difco)
1. Gram stain • Incorporation of:
• Solutions: o Chloramphenicol – inhibits
o Primary stain: crystal violet bacterial growth
o Mordant: Gram’s iodine o Cycloheximine – inhibits
§ Link between stain and element being saprophytic fungi / saprobes
stained (contaminants)
§ Increase affinity of element to the stain 2. SDA + gentamicin + chloramphenicol
o Decolorizer: 95% ethanol / acetone and 3. SDA + penicillin + streptomycin
95% ethanol
o Secondary stain: Safranin b. Brain/heart infusion agar (BHIA)
• Yeast cells and pseudohyphae: Gram positive • Enriched medium
o Gram positive cocci but larger • Contains:
o Potato infusion
2. Acid fast stain o 1% dextrose
• Solutions:
o Carbol fuchsin BHIA with antimicrobials
§ Primary stain: Basic fuchsin • Same antibiotic combinations with SDA
§ Mordant: Phenol • Diagnosis of infection with dimorphic fungi
o Decolorizer: Acid alcohol (conc. HCl + 95%
ethanol) BHIA with sheep blood
o Secondary stain: methylene blue / • 6-10% sheep blood
malachite green • Diagnosis of systemic mycoses
§ Blue color is less strenuous to the eyes
• Demonstration of Nocardia species c. SABHI
o Partially acid-fast organisms • SDA + BHI
o Once thought as fungi (along with • Ideal medium
Actinomyces) – filamentous bacteria
• Does not allow visualization of fungi o Subculture – enhance pigmentation of colonies and
enhance conidiation: clues for identification
3. Romanowsky stain a. Potato dextrose agar (PDA)
• Bone marrow and blood smears • Useful for slide culture technique
• Yeast form of Histoplasma capsulatum and b. Potato flake agar (PFA)
Penicillium marneffei c. Cottonseed medium
• Selective for Blastomyces dermatitidis
c. Histopathology d. Dermatophyte test medium
• Fixation à dehydration à clearing à impregnation • Differential: due to incorporation of phenol
à embedding à sectioning à staining à mounting red
à microscopic evaluation o Dermatophytes – red coloration of the
• Note: stains agar after incubation à capability to
produce alkaline products

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e. Rice medium o Yeasts

• Differentiation of Mycosporum cannis and § 35-37°C
Mycoscporum audouinii § Cannot survive in RT
o M. audouinni: not capable of growing
in rice medium • Age of culture
f. Birdseed / Nigerseed medium a. Rapid growers
• Growth of Cryptococcus neoformans à b. Intermediate growers
produce brown-black colonies c. Slow growers
o Capable of producing phenol oxidase
• Caffeic acid – phenol oxidase à melanin Group Growth Rate Representative Fungi
(brown-black) (Days)
• (Apollon): brown-black à capability of Rapid growers < 5 Saprobes
assimilating creatinine Opportunists
g. Czapek’s medium Yeast cells
• Selective for Aspergillus spp. Intermediate 6-10 Opportunists
h. Cornmeal – Candida growers Dermatophytes
• For fungal culture, test tube and petri dish can be used Subcutaneous fungi
Slow growers > 11 Systemic fungi
Characteristics Petri Dish Test Tube Subcutaneous fungi
(100 mm) (25x150 mm)
Surface area Large Small Techniques in Mold Identification
Detection of mixed Relatively easy Relatively hard 1. Macroscopic examination
culture a. Pigment production
Oxygen supply Good Poor • Obverse: surface of the culture
Rate of drying Relatively fast Relatively slow • Reverse: underside of the culture
Security of closure Poor Good
Probability of Relatively large Relatively small • Dematiaceous
dissemination o Both obverse and reverse side are colored
o Obverse side has a different color vs reverse side
• Incubation • Hyaline
o Aerobic o Only the obverse side is colored = due to color of
o 25-30°C conidia à only the hyphae is not capable of
§ Dimorphic fungi: producing pigments
st
• 1 plate: 25-30°C
nd b. Texture
• 2 plate: 35-37°C
st
o Or subculture 1 plate for 7-10 days • Length of aerial mycelium
and incubate at 35-37°C • Number of conidia formed
o 30 days
§
st
1 week: check daily 1. Glabrous / waxy, leathery
§
nd th
2 -4 week: 2 times/week • Few / no aerial mycelia
• Hyphae are submerged in the medium
• Cultural Characteristics • Waxy, leathery
1. Type of growth • Blastomyces dermatitidis, Histoplasma
• Mold: filamentous colonies capsulatum
• Yeast: pasty, opaque bacteria-like colonies 2. Velvety / suede
• Few aerial mycelia
2. Conditions of growth • Short aerial hyphae
• Type of medium • Penicillium marneffei
o Cycloheximide resistance 3. Cottony / wooly / floccous
§ Dimorphic fungi & dermatophytes • Long aerial mycelia
• Resistant à growth • “Lid lifters” – aerial mycelia capable of pushing
§ Cryptococcus neoformans & the lid of Petri dish
zygomycetes • Trichophyton mentagrophytes
• Susceptible à no growth 4. Granular
• Numerous conidia
• Temperature • Powdery colonies
o Dimorphic fungi • Microsporum gypseum
§ 25-30°C à molds
§ 35-37°C à yeasts c. Topography
o Saprobes a. Flat
§ < 30°C (RT) • Trichophyton mentagrophytes
§ Not capable of growing under normal b. Folded
body temperature • Trichophyton mentagrophytes

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c. Rugose o Incubation chamber: aseptically remove cap and

• Radial grooves – originate from central to the set aside cover slip
periphery o Agar block (1x1 cm) à center of slide
• Spokes-like grooves o Colonies from primary culture à agar block
• Penicillium spp. o Inoculate 4 sides of agar block
d. Crateriform o Moisten filer paper with sterile distilled H2O
• Central depressions and raised margins o Cover and incubate
• Penicillium spp. § Aerobic
e. Verrucose § 25-30°C
• Many warts or rough knobs § Up to 30 days
• Blastomyces dermatitidis o Check for presence of mature colonies
f. Cerebriform o Separate cover slip from glass slide à 2 smears
• Convolutions resembling brain surface o 2 smears
• Microsporum canis § Glass slide + LPCB + cover slip à
microscopic evaluation
2. Microscopic examination § Cover slip + (glass slide + LPCB) à
microscopic evaluation
• Mounting medium: Lactophenol cotton blue (LPCB)
o Smear preservation: by rimming all sides of the
o Components:
cover slip with mountant
§ Lactic acid – preservation of fungi morphology

§ Phenol – kills the fungi
Biochemical testing is not used for mold identification
§ Cotton blue / aniline dye – acidic dye for

staining
Techniques in Yeast Identification
§ Glycerol – clearing agent
• Yeasts
Methods utilizing LPCB o Candida
a. Tease mount preparation o Torulopsis
o Cryptococcus
• Steps:
o Saccharomyces
o Drop of LPCB on a glass slide
o Teasing needle à pick up a portion of colony • Yeast-like fungi – initially grow as yeast fungi but later grows
o Transfer to glass slide à Tease colonies apart aerial mycelia
o Cover slip à microscopic examination o Trichosporon
o Geotrichum
• Advantage
o Malassezia
o Easy and quick method
o Rhodotorula
• Disadvantage

o Disrupts morphology
1. Macroscopic characteristics

• Color
b. Scotch tape method
• Shape
• Steps:
o Drop LPCB on a glass slide • Texture
o Cut a small piece of scotch tape
o Loop the scotch tape over a tongue depressor • Yeast and yeast-like fungi – bacteria-like
(adhesive part should project outward) o Entire margin
o Press adhesive side on the colony o Dome/flat-shaped
o Carefully remove scotch tape à flatten on slide o Creamy / butyrous / pasty consistency
• Advantage • Gram-staining
o Easy and quick method o Yeast cells:
• Disadvantages § Gram-positive, cocci-shaped
o Disrupts morphology § Bigger than bacteria
o Hyphae may not be picked
2. Microscopic characteristics
c. Slide culture technique • Germ tube test
• Advantage o Distinguish Candida albicans from other yeasts
o Best in preserving the morphology o Steps:
§ 8x75 mm test tube
• Disadvantage
§ Pipet 0.5 mL serum
o Required incubation period
§ Heavily inoculate with yeast colonies (no definite
• Steps:
number of colonies, serum should be very turbid
o Setting up the incubation chamber
after inoculation)
§ Petri dish
§ Incubate at 35-37°C for 2-4 hours
• Filter paper: fit the bottom of the petri
§ Pasteur pipet: serum culture à glass slide
dish
§ Mount cover slip à microscopic examination
• (2) wooden sticks / bent glass rod
o Positive result:
• Glass slide and cover slip § Germ tube
• Recap petri dish • Filamentous extension
§ Sterilize by autoclaving

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• ½ width of yeast cell; 3-4 x length of yeast

cell
§ Confirms Candida albicans
o Negative result:
§ Absence of germ tube
§ Presence of pseudohyphae
o Germ tube vs pseudophyphae
§ Germ tube: no constrictions at the origin of the
filamentous extension
§ Pseudohyphae: presence of constrictions at the
point of origin
• Rule out Candida albicans

• Yeast morphology test
o Dalmau morphology test
§ Use of Dalmau plate