WEEK 3: MYCOLOGY together form a mesh-like structure called

MYCELIUM.
I. GENERAL CHARACTERISTICS OF o 2 TYPES OF HYPHAE
FUNGI a. Coenocytic- non-septate hyphae
 Are eukaryotic and most are b. Septate- with cross walls or
obligate aerobes and some are partition
facultative anaerobes 6. MYCELIUM- also called mat of intertwined
 Are saprophytes that can exist hyphae or collection or mass of hyphae
in 2 basic forms: Yeast or o Portions: Vegetative or thallus-
Molds grows in or on a substrate and absorbs
water and nutrients
YEAST MOLD Reproductive or aerial-contains
Unicellular Multicellular fruiting bodies that produce reproductive
Moist colony Dry colony structures (conidia or spores).
Temp: 35- Temp: 250C o Conidia- are asexual spores which are
0
37 C produced singly or multiply in long
CM: BHIA CM: SDA chains or clusters
 Exist as dimorphic or o Spores- are sexual spores which are
monomorphic produced and contained in special
 Has chitin cell wall and sterol sexual structures
membrane
 Has spores III. FUNGAL REPRODUCTION
 All are gram positive; slow A. SEXUAL- union of 2 nuclei; has three
grower and non-motile phases of development which include:
 They lack chlorophyll and  Plasmogamy- two compatible haploid
absorb nutrients nuclei are brought together in the
 Heterotrophic, thallophytic and same cell
chemotrophic organisms  Karyogamy- fusion of the two nuclei
to form a diploid nucleus; may or may
II. FUNGAL STRUCTURES not follow plasmogamy
1. Cell wall- rigid structure (80-90% CHO)  Meiosis- final step; yield four haploid
o Activate complement leading to progeny nuclei.
inflammation; mediates attachment to o Meiosis (reduction of two fertile cells)
host cells followed by cell merging and nuclear
o DEMATIACEOUS FUNGI- classified fusion occur.
by dark pigmented hyphae (brown or o Perfect fungi: exhibits a sexual phase of
black color or gray to black) arising reproduction
from their melanized cell walls.
2. Capsule or Slime layer- mainly TYPES OF SEXUAL SPORES
polysaccharides and responsible for fungal  Basidiospores- contained in a club-
growth. shaped Basidium
3. Cell Membrane- bilayer consists of  Ascospores- contained in a sac like
phospholipids and sterols ASCUS
o For protection and regulate intake and -Cleistothecium- large, round,
secretion of solutes multicellular structure that surrounds
4. Others: mitochondria, endoplasmic the asci until it ruptures, releasing
reticulum, Golgi bodies, vacuoles, nucleus ascospores
and nucleoli and other organelles found in the  Zygospores- fusion of two identical
cytoplasm. cells arising from the same hypha
5. Hyphae- microscopic unit of fungi; fungal  Oospores- fusion of cells from two
structure that are long, branching filaments or separate non identical hyphae
thread-like structures that when come

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

1
B. ASEXUAL- requires formation of asexual
spores from the mycelium. It involves only
mitosis with nuclear and cytoplasmic division.
o Imperfect fungi/Fungi imperfecti:
exhibits an asexual phase of reproduction
TYPES OF CONIDIA FORMATION
(conidiogenesis)
 Blastic conidiogenesis: parent cell
enlarges, a septum forms, and the
enlarged portion splits off to form a
daughter cell
 Thallic conidiogenesis: the septum
forms first, and new growth beyond the
septum becomes the daughter cell
TYPES OF ASEXUAL SPORES
 Conidia (conidiophore)- rise from the side
of mycelium. It may be in two different
sizes.
a. macroconidia- large, usually
septate, and sometimes exhibits oval,
club, or spindle shaped
- it may be thick or thin walled and may
have smooth or spiny (echinulate)
surface
b. microconidia- small and unicellular
with round, elliptical, or pyriform or pear
shape.
b.1 Sessile microconidia- borne
directly on the hyphae
b.2 Pedunculate microconidia-
borne on the end of a short
conidiophore
 Blastoconidia (blastospores)- develops
as the daughter cells buds off from the
mother cell and is pinched off eventually
 Chlamydoconidia (chlamydospores)-
thick walled, resistant, resting spores
produced by rounding up and
enlargement of the terminal hyphal cell
Types:
1. Terminal- forms at the hyphal tip
2. Sessile- forms at the hyphal sides
3. Intercalary- within hyphal strand
 Arthroconidia (arthrospores)- results
from simple fragmentation of the
mycelium at the septum
-Cylinder shaped, cask-shaped spores,
rectangular or barrel shaped spores
-DISJUNCTOR CELLS- empty spaces
appear between each arthrospores
HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)
 Sporangiospores- contained in a sac like
structure sporangia that are produced
terminally.
IV. MYCOSES (FUNGAL INFECTIONS)
1. Superficial Mycoses- no cellular
response by host
 Ptyriasis/tinea versicolor –
uneven pigmentation of skin
caused by Malassezia furfur –
olive oil loving (spaghetti &
meatballs appearance)
 Black piedra- brown to black crust
outside the hair shaft. (Piedra
hortae)
 White piedra- Light brown soft
nodules on beard and mustache
caused by (Trichosporon beigelii
or T. cutaneum)
 Tinea nigra palmaris- black to
brown scaly patches on palms of
the hand caused by Exophiala
werneckii or Hortae werneckii
2. Cutaneous Mycoses(Dermatomycoses)
- Keratinized tissue – skin, hair, nails
- Causes Tinea or ringworm
TINEA INFECTIONS (RINGWORM)
T. capitis – scalp
 Gray patch ringworm: M. canis
and M. audouinii (children)
 Black dot ringworm: T. tonsurans
(endothrix infection)
 Inflammatory. T. mentagrophytes
ectothrix infection)
T. barbae – beard (T. mentagrophytes)
T. corporis – body
T. cruris – groin (“jock itch”) E. floccosum
T. pedis – foot (athlete’s foot)
T. unguinum –nails
1. Microsporum – skin & hair
infections
- Macroconidia: large, spindle-shaped
(fusiform), thick-walled multi-septate
 M.canis (zoophilic)
 M.gypseum (geophilic)
 M.audouinii (anthrophilic)
- Rice medium and Fluoresce under
wood’s lamp
2
 2. Epidermophyton- skin & nails -Actinomyces –do not stain w/ fungal
- Macroconidia: numerous, club- stain
shaped smooth-walled -Nocardia
- No microconidia C. Phaeohyphomycosis- rare infection
3. Trichophyton – skin, hair & nails caused by dematiaceous saprobes which
-Macroconidia: smooth, pencil- invade organs (skin, lungs, brain) of
shaped/cigar shaped, thin-walled immunosuppressed hosts excluding
 T. rubrum (-) hair baiting and (-) chromoblastomycosis and mycetoma
urease (Cherry red pigment)  Exophiala (former Phialophora)
 T. mentagrophytes (+) hair jeanselmei
baiting and (+) urease  Phialophora
 T. tonsurans – balloon forms  Wangiella (former Fonsecaea)
aged microconidia dermatitidis
 T. shoenleinii- favic chandelier,  Cladosporium trichoides
favus type of T. capitis D. Sporotrichosis/ Rose Gardener’s
-Alopecia, mousy odor of Disease
scalp  from prick of thorn in plant ex. rose
-Culture: DTM  caused by Sporothrix schenckii
(Dermatophyte Test Med.)  Dimorphic fungus
with indicator phenol red,  RT (mold phase): Floweret
usually red colonies conidia, (conidia in cluster) or
 T. verrucosum- requires thiamine daisy like conidia. Older
or inositol for growth (T3 base characteristic- conidia on side of
medium) hyphae (sleeve)
- clavate or pyriform  370C (yeast phase): Cigar-
microconidia shaped yeast cells that appears
3. Subcutaneous M ycoses – tissues & intracellular (Asteroid body)
muscles Asteroid body in tissue –central halo round
degenerating yeast; concentric radiating
A. Chromoblastomycosis eosinophilic materials caused by Ag-Ab
 Dematiaceous fungi (dark, slow- reaction (Splendore-Hoeppli phenomenon)
growing fungi) 4. Systemic Mycoses – Tissue & organs;
 Microscopic tissue appearance:  Dimorphic fungi
sclerotic bodies or muriform bodies o North American Blastomycosis/
a. Fonsecaea – mixed sporulation; (short Gilchrist’s disease caused by
chain type of sporulation) Blastomyces dermatitidis
TYPES:  RT (mold phase): – Lollipop-shaped
Acrotheca – conidia inside conidia
Cladosporium – conidia in chain  370C (yeast phase/tissue)- thick walled
Phialophora – conidia in duster yeast cells with single broad based
b. Phialophora – only phialophora budding
sporulation (vase-like type of sporulation)  Exoantigen test: specific A band
c. Cladosporium – only cladosporium o South American Blastomycosis/Lutz
sporulation (long chain type of Splendore-Almeida disease caused by
sporulation) Paracoccidioides brasiliensis
B. Mycetoma- granulomatous tumor of  Dimorphic fungi
subcutaneous tissue  RT (mold phase): Chlamydoconidia
a. Eumycotic (true fungi)  370C (yeast phase/tissue): thick walled
-Exophiala yeast cells with multiple buds
-Pseudoallescheria boydii (most which attach to mother cells
common cause) (MARINER’s WHEEL)
b. Actinomycotic (fungus-like bacteria)

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

3
o Darling’s Disease/Ohio Valle y - Assimilator: glucose, maltose, sucrose,
fever/Spelunker’s disease caused by galactose and xylose
Histoplasma capsulatum - Fermenter: glucose, maltose, galactose
 Dimorphic fungi and trehalose
 RT (mold phase): Presence of knobby Confirmatory Test
tuberculate macroconidia and a. Cornmeal agar- formation of
pyriform microconidia Chlamydoconidia after incubation at RT for
 37 C (yeast phase/tissue): small
0 24-48 hours (C. dublinensis (+) double
budding yeast cell within chlamydospores
macroconidia; intracellular to b. EMB agar- colonies with spider-like
mononuclear cells using giemsa or projections
wright stain; requires 10-12 weeks for
growth 2. Torulosis/Torulopsis caused by
 Exoantigen test – H and/or M bands Cryptococcus neoformans
 Differentiation with Leishmania:  Encapsulated yeast cell in bird and bat
 With central nuclear body droppings
 Do not stain with fungal stains  Demonstration of the capsule by India
o San Joaquin Valley Fever / Desert ink stain
Fever/Valle y Fever caused by  Urease and inositol positive
Coccidioides immitis (major biologic  Nitrate negative
hazard to lab personnel)  Cultured on: SDA medium without
 RT (mold phase): barrel-shaped cycloheximide
Arthroconidia with disjunctor cells -Birdseed/ Niger seed/ Staib’s
 370C (yeast phase/tissue): thick-walled produces Brown- black colonies,
spherules with endospores (assimilates creatinine) (phenol
oxidase (+) responsible for colony
OPPORTUNISTIC INFECTIONS color forming melanin
 Latex agglutination test for
1. Candidiasis or Moniliasis- caused by cryptococcal antigen in CSF
Candida albicans 3. Aspergillosis/Farmer’s Lung
 Can be saprophytic in oral activities, Disease/Fungus Ball Infection caused by
GI or vaginal tract Aspergillus fumigatus
 Potential pathogen for  Grows well at 450C
immunosuppressed patients  Dichotomous septate hyphae (hyphae
 (+) Germ tube formation branched at acute angles)
 Infection similar to C. albicans caused  Most common and is associated in
by Geotrichum candidum- optimal invasive aspergillosis
growth at 250C produces fragmented  A. flavus: Aflatoxin
hyphae with rectangular arthroconidia  A. niger-produces brown-black spores
with rounded ends when grow on  A. tenuis- 3rd most common species
cornmeal agar; colonies resembles
round glass LAB TESTS
A. Microscopic
Screening Test a. Saline mount
a. Germ tube test– formation when incubated b. 10% KOH mount Preparation
with sterile serum at 35-37°c for 1-3 hours c. Lactophenol cotton blue
- Hypha extension without constriction d. India ink or nigrosin
- (+) C. albicans, C. dublinensis, and C. e. Calcofluor white stain (fluorescence
stellatoidea microscopy)
- (-) C. tropicalis f. PAS-Hyphae (+) purplish red re-stain
b. CHO assimilation and fermentation of C. H& E Slides
albicans g. Gram stain (Hucker modification)

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

4
 h. Giemsa, Gridley, PAS, GMS (+) control: C. neoformans
(Gomori methenamine silver) used to locate (-) control: C. albicans
H. capsulatum in RE cells 4. Hair baiting test
i. Acridine orange, green fluorescence V shaped penetration of hair shaft- T.
fungal elements, orange epithelial cells mentagrophytes
-sterile hair is floated on sterile water and
B. Culture yeast extract
1. Sabouraud dextrose agar (SDA) – Acidic T. mentagrophytes (+): (+) urease
pH. T. rubrum (-): (-) urease
- Inhibitor for bacteria: pH 5.6 5. Exoantigen test- serologic confirmation for
2. MYCOSEL or MYCOBIOTIC- contains systemic fungi
SDA with inhibitors including: -Microscopic immunodiffusion test with
2.1 Chloramphenicol inhibits bacteria antisera
2.2 Cycloheximide inhibits saprophytic - A band: B. dermatitidis
fungi - H/M band: H. capsulatum
3. Dermatophyte test medium (DTM)
- Indicator: phenol red OTHERS
4.Cornmeal agar Asteroid bodies- a.k.a Splendore-Hoeppli
-Candida Sp. Chlamydospore phenomenon (in vivo formation of
5. Czapek’s agar; Aspergillus (Farmer’s eosinophilic material around yeast cells; seen
lungs) in tissue biopsy infected by S. schenckii.
6.Birdseed/Niger seed/Staib’s Sclerotic Bodies/Muriform bodies-
medium/Caffeic acid agar resembles stack of coins, copper-colored
- C. neoformans –Brown black colonies septate cells present in chromoblastomycosis
(Phenol oxidase)
7. Cottonseed medium: Blastomyces
dermatitidis Fungicidal Agent
8. Rice medium- (+) M. canis; (-) M. audouinii  Targets ergosterol (cell membrane)
- Incubated at 30°C or RT or 25°C to 30°C  Amphotericin B, Nystatin: Systemic
- Culture held for 30 days/ 4 weeks / 1 Fungi
month  Azole (Fluconazole): Fungi static
9. BHI- isolation of agents of systemic  Griseofulvin: IV Dermatophytes
mycoses
10. EMBA- colonies with spider-like
projections (feathering of C. albicans) I. OVERVIEW & INTRODUCTION
11. Nitrate reduction medium- to confirm (VIROLOGY)
nitrate reduction in C. neoformans
12. Urea agar- detection of urease production 1. General Key Characteristics
by C. neoformans  Obligate intracellular organisms
13. Potato Dextrose Agar- demonstrates  Lack ribosomal RNA (rRNA)
pigment production of T. rubrum  Composed of either DNA or RNA but
14. Casein medium-isolation of Nocardia not both
 Very small size: Ranging from 18nm
C. Tests (Parvovirus) to 300nm (Poxvirus); not
1. Germ tube/Reynolds-Braude phenomenon visible by light microscopy
- Hypha like extensions of yeast cells without  There are currently 21 viral families
constrictions associated with human infection; 14
-incubate with sterile serum for 2-3 hours at are RNA viruses while the rest are
35-37: (+) control C. Albicans (-) C. tropicalis DNA viruses
2. L-DOPA ferric citrate test: phenol oxidase
(+) Black: C. neoformans
3. Urease test

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

5
2. Basic Viral Structure 4. Early mRNA and protein synthesis
 VIRION: complete viral particle 5. Viral genome replication
 CAPSID: protein coating; Helical or 6. Late mRNA and protein synthesis
icosahedral 7. Assembly of virion
Types of Symmetry: 8. Release
a. helical- arranged in a hollow coil or
helix that appears as rod-shaped II. SPECIMEN COLLECTION, HANDLING &
b. icosahedral- capsomeres arranged PROCESSING, TRANSPORT & STORAGE
in 20 triangles
c. complex- neither helical nor 1. General Considerations
icosahedral (non-conforming a. For most viral infection, viral titer is
symmetry) highest during the first 4 days of infection
 CAPSOMERE: makes up the capsid after onset of symptoms EXCEPT:
 NUCLEOCAPSID: capsid and genetic Enterovirus, Adenovirus, Cytomegalovirus
material (CMV) because prolonged shedding of
 VIRAL GENOME: Genetic material Enterovirus & Adenovirus into the stool and
(RNA OR DNA) CMV into the urine occurs.
 ENVELOPE: outer membrane;
Presence or absence; acquired from b. It is best to sample the infected site
nuclear or cytoplasmic membrane directly such as rash or vesicles; throat swab
a. envelope: (Ether-sensitive or Ether for upper respiratory infection. Exception is
labile) certain CNS viral infections which may need
b. naked or non-envelope (Ether to culture stool or throat instead of CSF
resistant or Ether stable)
 VIRAL PROTEINS: capsid, matrix and c. Viral transport medium is highly
envelope proteins recommended for most specimens which
contains buffered saline, a protein stabilizer,
3. Classification & antibiotics (to suppress bacterial or fungal
 All DNA viruses are Double stranded overgrowth). Commercially available viral
EXCEPT for PARVOVIRUS and transport medium includes:
ANELLOVIRUS  Hank’s Balanced Salt Solution
 All Enveloped DNA Viruses are  Veal Infusion Broth
double stranded  Sucrose-Phosphate-Glutamate Broth
 All Naked DNA Viruses are  Leibovitz-Emory Medium
ICOSAHEDRAL d. Collection Syste ms: Viral Culturettes
 ICOSAHEDRAL; All the rest have (BD): swabs with a viral culture medium
helical symmetry e. Cotton, Dacron, Rayon swab with
 All RNA viruses are Single stranded plastic shaft should be used; Never use
EXCEPT for REOVIRUS and wooden shaft because it is toxic to viruses
PICOBIRNAVIRUS f. Calcium Alginate Swabs should be
 All Naked RNA Virus (REOVIRUS) is avoided because it may bind and
Double stranded inactivate the virus.
o All NEGATIVE SENSE RNA Viruses g. Optimum Storage Temperatures: As
are ENVELOPED much as possible transport & process
o All POSITIVE SENSE RNA Viruses- its specimen without delay but if can’t be
RNA can act as mRNA inside the avoided; store as follows:
infected cell.  4°C: less than 24 hours’ delay
 Freezing Temp 0°C: more than 24
VIRAL REPLICATION CYCLE hours’ delay
1. Adsorption  -70°C Deep Freezing: longer delay
2. Penetration (months)
3. Uncoating  NOTE: -20°C is not suitable for

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

6
 storing specimens for virology CELL LINE SOURCE
Vero Cell Line Kidney cells of an African
III. LABORATORY METHODS Green Monkey
A549 Cells Human lung carcinoma
1.Cytological or Histological Examinations HELA Cell Line Human cervical carcinoma
o Presence of multinucleated giant cells Hep-2 Cell Line Human epithelial cells of
or cytoplasmic or nuclear inclusions larynx carcinoma
may aid in diagnosis KB Human Nasopharyngeal
o CMV may produce characteristics Carcinoma
“OWLS EYE” inclusions ***Cytopathic Effect (CPE): cellular
o Disadvantage: Insensitive & non damage or changes cellular structure which
specific results from viral inoculation and incubation of
2. Electron Microscopy tissue cell lines. CPE includes:
o Sensitive tool but not readily available  Rounding of Cells
in most laboratory  Forming of giant multinucleated cells
o PRINCIPLE: Clinical material is placed  Discoloration of cytoplasm
in a carbon-coated grid then stained  Disintegration of cells
with potassium phosphotungstate or
uranyl acetate. Stain surrounds that 4. Molecular Diagnostic Methods
virus, & the electron beam cannot pass o Detection of viral DNA or RNA in
through the virus; hence virus is seen specimens
as a light structure over a dark o Molecular Methods used in Virology
background.  Restriction Fragment Length
o The only method that can detect Polymorphism (RFLP)
Norwalk Agent, Astroviruses,  Gene Probes in situ
Caliciviruses, & Coronaviruses Hybridization
because they can’t grow in tissue cell  Polymerase Chain Reaction
lines & no available serological tests. (PCR)
3. Viral Tissue Culture  Reverse Transcriptase PCR
o remains to be the “GOLD
 Real Time PCR
STANDARD” for the isolation of many 5. Serological Methods
viruses o Detection of viral antibodies is an
o Three (3) types exist: indirect indicator of infection or
-Traditional cell culture using cell lines exposure to a particular virus
- Shell vial centrifugation-enhanced o useful when the virus will not grow in
(SVCE) cultures cell cultures
- Multi-Well Microplate Cultures o Paired sera are recommended for
o Primary Cell Cultures detection:
-Human Embryonic Kidney (HEK)  Acute Phase Sample:
-Rabbit Kidney (RK) Collected when the clinical
-Primary Monkey Kidney (PMK) signs first appear
-Rhesus Monkey Kidney (RMK)  Convalescent Phase
-Cynomolgus Monkey Kidney (CMK) Sample: Collected 2 to 3
-African Green Monkey Kidney
weeks later, depending on the
(AGMK) virus
o Heteroploid Cell line o Traditionally, a FOUR FOLD
INCREASE in antibody titer indicates a
seropositive reaction and strongly
supports a diagnosis of current
infection

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

7
DNA VIRUSES

RNA VIRUSES

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

8
HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)
9
References:
Delost. Introduction to Diagnostic
Microbiology
Per handbook
Mahon, C. R., Lehman, D.C., & Manuselis,
G., Jr. (2014). Textbook of diagnostic
microbiology. 5th ed. USA: Elsevier.
McPherson, R.A., Pincus, M.R., (2011).
Henry’s clinical diagnosis and management
by laboratory methods. 22nd ed. USA:
Elsevier
Rodriguez, M.T. (2018). Review handbook in
diagnostic bacteriology. 2nd ed.
Forbes, Betty, Daniel Salm and Alice
Weissfield. Bailey & Scott’s Diagnostic
Microbiology 12th ed. USA: Mosby, 2007.
Compiled Review notes by Rañon Jr, N.B.
(2016) and Navarro, A.L.

HJGS. 2022.Medical Technology Assessment Program I (MICROBIOLOGY)

10