PRELIM

MYV – VITALES, GIENES

FUNGI
 Eukaryotic microorganisms
 Ubiquitous in nature – seen everywhere
 Spore producing
 Achlorophyllous
 Heterotrophic
 Sexual and asexual reproduction (TELEOMORPH (sexual form) %
ANAMORPH (asexual form))
 Filamentous
 Unicellular
 Dimorphism – they can exist in other form.
 Saprophytes – dead bodies for their nutrition
 Slightly acidic to neutral pH
 Mahilig sila sa warm temperature.

CHARACTERISTICS OF FUNGI

A. NUTRITION – Heterotrophic ; feeds by absorption rather than

ingestion.
B. CELL WALL – typically present; usually based on glucans, chitin,
and mannans.
- 90% carbohydrates
C. NUCLEAR STATUS – Eukaryotic, uni- or multinucleate
D. REPRODUCTION – sexual via nuclear fusion and meiosis and/ or
asexual via purely mitotic nuclear division.
E. PROPAGULES – these are microscopically small spores produced
in high numbers.
F. SPOROCARPS – Fruiting body.
- Microscopic or macroscopic and showing characteristic
shapes but only limited tissue differentiation.
G. HABITAT – Ubiquitous in terrestrial and freshwater habitat, less
so in the marine environment.
H. ECOLOGY – functions as saprotrophs, mutualistic symbionts,
parasites or hyperparasites.
I. DISTRIBUTION – Cosmopolitan
J. POLYMORPHIC FUNGI – have both yeast and molds forms in the
same culture.
- Usually seen in Exophiala spp.
REPRODUCTION

 SEXUAL – “Teleomorph”; perfect reproduction; homothallic

(structure for both male & female reproduction) structures.
- Sexual spores through meiosis; hypha releases
pheromones with compatible species then are
stimulated to undergo plasmogamy, nuclear fusion and
meiosis.
 ASEXUAL – “Anamorph”;imperfect reproduction; heterothallic
structures
- We have 2 types of asexual spores (for
reproduction):
 conidia and in the Zygomycetes,
 the sporangiospores.
- If more than one anamorph is present for the same
teleomorph, the anamorphic strains are termed
Synanamorphs.
- The best example of this phenomenon is:
 the teleomorph,
 Pseudallescheria boydii, which has 2 anamorphs,
A. Scedosporium boydii
B. Graphium spp.
 In others, conidia are produced by a conidiogenous cell, such as
a phialide, which itself may be attached to a specialized hypha
called a conidiophore
 Two common conidiogenous cells are
 the phialides
 annellides
 Phialides - are vase like structures that produce phialoconidia
 Annellides - are ringed structures that produce annelloconidia
 In the Zygomycetes, sporangiospores result from mitotic
replication and spore production within a sac-like structure
called a sporangium, which is supported by a sporangiophore.
 Another type of conidia is the arthroconidia; formed by
fragmentation of fertile hyphae as opposed to being formed by
conidiogenous cells.
MOULDS / MOLDS
 Multicellular mycelium
 Grown at room temperature (22-25) in ambient air conditions
 Hypha/Hyphae: hyaline - nonpigmented :
 Fontana-Masson stains → pink to red
 Phaeoid (dermaticious) - darkly pigmented due to
melanin
 Gomori methylene → black
 Fontana Masson → brown black
 Mycelia/Mycelium - aerial (taas) or vegetative (ilalim)
 Septa: septate and aseptate/nonseptate/coenocytic/sparsely
septate
 Rigid Cell Wall: chitin, mannan and glucan
 Protoplasm: organelles
 Conidia: asexual reproductive spores
 Apical Growth – glide branch
MORPHOLOGY OF FUNGI

YEASTS
 Unicellular; budding; pseudohyphae
 Yeast colonies are usually soft, opaque, 1-3 mm in size, and
cream-colored
 Yeast species are identified on the basis of physiologic tests and
a few key morphologic differences
 Budding involves maturation of the bud to an independent
blastoconidoum.
 This process involves lysis of the yeast cell wall so that a
blastoconidium can form.
 As this structure enlarges, the nucleus of the parent cell
undergoes mitosis.
 Once the new nucleus is passed onto the daughter cell, a
septum forms and the daughter cell breaks free.
 Grown at 37 degree Centigrade with increased CO2.
HOST RESPONSE

 Cell wall polysaccharides may activate complement cascade and

provoke an inflammatory reaction; they are poorly degraded by
the host and can be detected with special stains.
 Cell walls release immunodominant antigens that may elicit
cellular immune responses and diagnostic antibodies.
 Most causative agents of clinical infections are found in four
groups of fungi.
 They consist of the phyla Glomeromycota (Zygomycota),
Ascomycota, Basidiomycota, and the form division Fungi
Imperfecti (Deuteromycota).

CLASSIFICATION: KINGDOM FUNGI

CHYTRIDIOMYCOTA - aqueous environment; not clinically
significant.
ZYGOMYCOTA - aseptate or sparsely septate

Asexual spores: sporangiospores: sporangium

Sexual spores: fusion of nuclei: zygospore

ORDER: Entomophthorales
GENERA: Basidiobolus
Conidiobolus

ORDER: Mucorales
GENERA: Absidia
Mucor - found in the Philippines
Rhizopus - found in the Philippines
Rhizomucor - found in the Philippines
ASCOMYCOTA – septate

Asexual: conidia spores contained in conidiophore

Anamorph: Blastomyces, Emmonsia, Histoplasma
and Candida, Microsporum, Trichophyton, Penicillium

Sexual: asci/ascospores contained in ascus: ascoscarp

Teleomorph: Ajellomyces, Pseudallescheria boydii,
Arthroderma
 BASIDIOMYCOTA – septate

Sexual: fusion of conidia by compatible colonies; basidiospores:

outside a basidium; basidia: macroscopic: basidiocarps

Filobasidiella neoformans, the perfect form (teleomorph) of

Cryptococcus neoformans
Malassezia, Trichosporon, Schizophyllum

 Clamp connections occur at the septations in the vegetative

hyphae
 A portion of the hypha on one side of the septation grows out
and connects to the hypha on the other side of the septation,
thereby bypassing the septation.
 FUNGI IMPERFECTI
Causative agents of mycoses, including cutaneous,
subcutaneous and systemic disease.
No mode of sexual reproduction has been identified.
Characteristic asexual reproductive structures.

PART II
SPECIMEN COLLECTION
 Hair
 Skin
 Nail scrapings
 Blood
 Body fluids
 Lesions should be cleaned with 70% ALCOHOL before collection
to avoid bacterial contamination
 Specimens should be collected into folded squares of stiff black
paper for easy visualization and helps to keep the specimen dry.
 In transporting samples, it could be folded and contained in an
envelope.

SKIN
 Collect the advancing edge of the lesion
 Scrape the edge of lesion with blunt edge scalpel
 Toe webs, soggy skin should be peeled away
 10-15mm2
 Skin must be cleaned with 70% isopropyl alcohol before
sampling
 A KOH Wet mount; Fungal culture
SCALP
 Sterile forceps. Pluck hair with root ends attached.
 Brush hair and collect droppings.
 Black dot tinea capitis lesions: scrape with a scalpel
 4-6 pcs. hairs
 A Wood’s lamp: ultraviolet light>365 nm is useful in identifying
infected hairs
 Hairs infected with fungi such as Microsporum audouinii
fluoresce when a Wood’s lamp is shone on the scalp.
 Cut the hairs close to the scalp with sterile scissors
 Hairs are placed directly into a sterile Petri dish
 KOH Wet mount; Fungal culture

NAIL
 Clip through the entire thickness of the nail; Use pincer type nail
clipper
 Nail dystrophy: scrape with a scalpel blade
 20-30 ml
 Nails are cleaned with 70% isopropyl alcohol before the surface
is scraped
 Deeper scrapings are necessary to prepare a KOH preparation
and inoculate media
 Sterile scissors are used to cut complete nails into small thin
strips, which are used to inoculate media.
 20% KOH, 2-4 days
BLOOD AND BONE MARROW
 Uses 10ml of blood
 The lysis centrifugation system, the Isolator Tube is the most
sensitive method for the recovery of some fungi.
 A biphasic system such as the Septi-Check can also be used.
 Heparinized bone marrow specimens should be plated directly
onto media at bedside. Blood culture bottles are not
recommended.
 Candida sp are most commonly recovered in blood samples

CEREBROSPINAL FLUID
 Concentrated by centrifugation before inoculation.
 One drop of the concentrate is used for India Ink preparation of
latex agglutination for Cryptococcus and the remainder is
inoculated onto media.
 If more than 5ml is submitted, the CSF may be filtered through a
membrane filter and portions of the filter placed on media.
 1-5 mL CSF

UROGENITAL AND FECAL SPECIMENS

 Urine should be centrifuged and the sediment used to inoculate
media.
 A first-voided morning urine specimen is preferred.
 Approx. 10 ml of urine, Candida spp.
RESPIRATORY SPECIMENS
 collect sa umaga
 Includes lower respiratory tract secretions such as sputum and
also pleural lavage fluids
 Obtain sputa from deep cough shortly after arising in the
morning
 A nebulizer may be used to induce sputum; Use a sterile
screwtop container
 Nonviscous - inoculate directly onto media using sterile pipette
 Viscous - use Dacron Swab to inoculate material onto media, or
specimen can be digested using mucolytic agent such as N-
acetyl-L-cysteine (tunawin para madali mastreak) and
concentrated before inoculation.
 Routine fungal media can be used but selective chromogenic
agars such ash CHROMagar Candida and Candida ID provides a
preliminary identification.
 Nasal sinus specimens obtained surgically can be plated directly
to media containing antimicrobial agents except for
Cycloheximide, which can inhibit some fungi.
 Cycloheximide – hindi pwede gamitin kasi na iinhibit nya si
bacteria pero naiinhibit nya si Fungi.

MICROSCOPIC EXAMINATION

 10% KOH - Digest the host material and reveals the presence of
fungi
 Enhanced and speed up by heating then cooled at 15 minutes
 Modifications of the KOH: Dimethyl Sulfoxide (DMSO) (NO
NEED HEAT) and a stain into the KOH Solution
KOH WITH CALCOFLUOR WHITE
 Calcofluor white binds to polysaccharides present in the chitin
of the fungus or to cellulose.
 They fluoresce APPLE GREEN or BLUE WHITE.
 Any element with a polysaccharide skeleton fluoresces.
 The actual fungal structure must be seen
 before a positive preparation is reported.

INDIA INK
 India Ink or Nigrosin preparations: CSF: Encapsulated yeast: C.
neoformans
 A drop of India ink is mixed with a drop of sediment from a
centrifuged CSF specimen, and the preparation is examined on a
High Power Objective (HPO)
 This is a negative stain
 Budding yeast surrounded by a large clear area against black
background
 Substituted by latex agglutination test for cryptococcal antigen.
TISSUE STAIN
 Periodic Acid Schiff, Gomori Methenamine Silver Nitrate,
Hematoxylin and Eosin, Giemsa and Fontana Masson stains.
 Giemsa - used to primarily detect H. capsulatum in blood or
bone marrow
 PAS - attaches to polysaccharides in the fungal wall and stains
fungi Pink.
 FONTANA MASSON - stains melanin in the cell wall and
identifies the presence of phaeoid fungi.

COLOR OF FUNGAL
STAINS BACKGROUND COLOR
ELEMENT
PAS Magenta Pink or Green
GOMORI Black Green
GIEMSA Purple blue yeast with Pink-purple
clear halo
INDIA INK Yeast with Clear halo Black
KOH Refractile Clear
KOH – Calcofluor Fluorescent Dark
white
Masson -Fontana Brown Pink-purple

FUNGAL CULTURE

CULTURE MEDIA NILALAGYAN NG ANTIBIOTIC

 Sabouraud Dextrose Agar (SDA), SDA with antimicrobial agents,
Potato Dextrose agar (PDA), or the slightly modified potato
flakes agar, and Brain Heart Infusionn (BHI) agar enriched with
blood and antimicrobial agents.
 GENTAMICIN and CHLORAMPENICOL inhibits bacterial growth
 Cycloheximide inhibits bacteria and many of the environmental
fungi typically considered as contaminants.
 Petri dishes must be poured thicker than a standard medium for
bacterial growth.
 Plates can be sealed with tape of parafilm or sealed in
semipermeable bags to minimize dehydration and prevent the
spread of fungal spores.
 Tubed media have the advantage of being safer to handle and
less susceptible to drying.
INCUBATION
 Room temperature or at 30 degree Centigrade
 If the causative agent suspected is a dimorphic fungus, cultures
should also be incubated at 37 degree Centigrade
 Culture last for 4-6 weeks
 Should be examined twice weekly for growth.
 Mucorales, such as Mucor and Rhizopus spp., grow rapidly and
may fill the tube with aerial mycelium within a few days,
whereas more slowly growing organisms such as Fonsecaea and
Phialophora spp. Might require 2 weeks or longer before
growth is seen.

MACROSCOPIC EXAMINATION OF CULTURES

 Includes gross morphological traits such as color, texture and
growth rate
 Rapidly growing organisms usually appear within 1-3 days,
Intermediate growers takes 5-9 days, and slow growers up to 2
weeks or more.
 Pigment on the reverse side of the colony or in the aerial
mycelium can be noted but is not always helpful.
MICROSCOPIC EXAMINATION OF CULTURES
 Direct mount of the fungal isolate
 A tease mount or cellophane tape mount
 Tease and tape mounts typically disturb conidia, preventing
viewing of how they are formed.
 Slide cultures provide a more intact specimen

Tease Mount
 Use 2 teasing needles to remove portion of the mycelium from
the middle third of the colony
 Mycelia are placed on a drop of LPCB or Lactophenol ___ and
gently teased apart using needles
 Coverslip is added and the slide is examined microscopically at
low and high magnification.
 Hyphae takes up the LPCB but the stain does not work well with
phaeoid fungi because they retain their dark color.
 The major disadvantage of this procedure is the disruption of
conidia during the teasing process.
 ALPCB – Lactic Acid (kills fungi), Phenol (preservative), Cotton
Blue ( Evan’s dye stain)

Cellophane Tape Preparation

 Gently touch a piece of clear tape, sticky side down to the
surface of the colony and then removing it.
 Tape is placed onto a drop of LPCB on a slide and examined.
 Conidial arrangement is retained.
 Potential contamination of the colony and temporary nature of
this preparation.
 Read within 30 mins. then discard.

Slide Culture
 Useful for demonstrating the natural morphology of fungal
structures and for encouraging conidiation in some poorly
fruiting fungi.
 Advantage is that it can be preserved indefinitely.
 Observe early stages of fungi.
Differential Agars
 Ascospore agar - for the detection of ascospore of
Saccharomyces
 Niger seed agar - ID of C. neoformans
 Czapek's agar - Differential of Aspergillus spp
 Nitrate reduction medium - differentiation of nitrate reduction
tests in confirmation of C. neoformans
 Cornmeal agar (with Tween 80 and Trypan Blue) - ID of Candida
albicans
 Cotton seed conversion agar - conversion of dimorphic fungi B.
dermatitidis from mold to yeast form.

PART III
MISCELLANEOUS TEST FOR THE IDENTIFICATION
Hair Perforation Test
 Sterile 5-10 mm hair fragments are floated on sterile water
supplemented with a few drops of 10% yeast extract.
 Conidia or hyphae from the specimen is inoculated onto the
water surface
 Hair shafts are removed and microscopically examined in LPCB
at weekly intervals for up to 1 month.
 T. rubrum usually only causes surface erosion of hair shafts on
this test, whereas T. mentagrophytes typically forms
perpendicular pegs in the hair shafts.
 Used to distinguish penetration-capable Microsporum canis
from M. equinum which does not penetrate hair.
Urease Test
 Test to differentiate T. rubrum from T. mentagrophytes.
 Tubes of Christensen Urea Agar are very lightly inoculated with
the dermatophyte and held for 5 days at room temperature.
 Most isolates of T. mentagrophytes demonstrate urease
production, resulting in a color change of the medium from
peach to bright fuchsia within that period, whereas most T.
rubrum isolates are negative or require more than 5 days to give
a positive reaction.

Thiamine Requirement
 Tubes of media with and without thiamine are inoculated with a
tiny, medium free portion of the colony and observed for
growth after 10-14 days.

Trichophyton Agars
 There are 7 Trichophyton agars numbered 1-7, used to
determine the nutritional requirements of Trichophyton spp.
 After inoculation and incubation, growth is scored on a scale of
1 (lowest) -4 (highest)
Growth on Rice Grains
 Sterile, nonfortified rice is inoculated lightly with a portion of a
colony
 After 10 days of incubation at room temperature, the medium is
observed for growth.
 M. canis and almost all other dermatophytes grow well and
usually form many conidia, whereas M. audouinii does not grow
but turns the rice grain Brown.
 MISCELLANEOUS TESTS FOR THE IDENTIFICATION OF
YEASTS
Germ Tube Production
 Both C. albicans and C. dubliniensis are identified by germ tube
production.
 The SOP requires the use of serum or plasma, such as fetal
bovine serum. Expired FFP from the blood bank can also be
used and can be stored at 4°C almost indefinitely.
 Substrate is inoculated and then incubated at 37°C for 3hrs.
 True germ tube lacks constriction at their bases, where they
attach to mother cells.
 If a constriction is present at the base of a germ tube, the yeast
is not either species.
 Such constricted germ tubes, called pseudo-germ tubes are
more characteristic of Candida tropicalis.
 C. dubliniensis is differentiated from C. albicans by its inability to
grow at 42°C.
 Candida dubliniensis hindi naggo grow sa 42°C
Carbohydrate Assimilation
 Assimilation tests identify which carbohydrates a yeast can use
aerobically as a sole carbon source.
 The API 20C yeast identification system is probably the most
commonly used.
 In this method, a series of freeze-dried sugars are placed into
wells on a plastic strip.
 Yeast isolates are suspended in an agar basal medium, pipetted
into the wells and incubated at 30°C for 72hrs /3days.
 As sugars are assimilated, wells become turbid with growth.
Wells remain clear when the sugars are not assimilated.

Chromogenic Substrates
 CHROMagar Candida presumptively identifies C. albicans, C.
tropicalis, and C. krusei.
 ID is based on the different colony colors, depending on the
breakdown of chromogenic substrates by different species.
Potassium Nitrate Assimilation
 Use of the modified KNO3 agar is a fairly rapid, easy and
accurate method to determine the ability of yeast to use nitrate
as the sole source of nitrogen.
 A positive KNO3 result turns the medium blue, and a negative
result turns the medium yellow.
 Control organisms may be used such as Cryptococcus albidus (+)
and Candida albicans (-).

Cornmeal Agar
 Recognition of 4 different types of morphology is an important
clue to yeast identification – blastoconidia, chlamydoconidia,
pseudohyphae and arthroconidia.
 C. albicans produces chlamydoconidia along with hyphae.
 Pseudohyphae are produced when the blastoconidia germinate
to form a filamentous mat.
 The cross walls help determine whether the structures are true
hyphae or pseudohyphae.
 Arthroconidia begin as true hyphae but break apart at the cross
walls with maturity.
 Rectangular fragments of hyphae should be accompanied by
blastoconidia for an isolate to be considered a yeast.
Temperature Studies
 Cryptococcus spp. Have weak growth at 35°C and no growth at
42°C
 C. albicans grows at 42°C, whereas C. dubliniensis does not
grow at 42°C-45°C.

Urease
 Yeast isolates producing the enzyme urease can be isolated
easily with Christensen urea agar.
 Almost all clinically significant encountered Candida spp are
urease negative, whereas all Cryptococcus and Rhodotorula
organisms are urease positive.
 Most strains of Trichosporon spp are urease positive.

(1-3) Beta-D-Glucan Detection

 (1-3) Beta-D-Glucan is present in the cell wall of most yeasts and
molds, including Pneumocystis with the exception of the
mucoraceous molds.
 Fungitell test is available in the US.
 Uses Limulus amoebocyte lysate from the North American
horseshoe crab.
 Positive result produces a yellow color.
 Recommended cutoff for positivity is at 80pg/ml

Immunodiagnosis of Fungi Infection

 The Fungitec Test is an assay method for glucan, a cell wall
component for most fungi except the Family Zygomycetes
(walang glucan).
 The assay is based on the sensitivity of Factor G in the
horseshoe crab to glucan.
 Normal individuals have glucan concentrations less than 10
pg/ml, whereas concentrations greater than 20 pc/ml (positive
for fungal infection) correlate with fungal infection.
Antifungal Susceptibility
 Mostly from 4 drug classes: polyenes, azoles, echinocandins,
and allylamines.
 The primary antifungal agent is amphotericin B.
 The class that has provided the largest number of agents is the
azoles. Including FLU. ITRA, POSA and VORI.
 FLUCANOZOLE is the leading agent for treating yeast infections
but has limited to no activity against molds. Its overuse has led
to development of resistance particularly with C. glabrata
 ITRACONAZOLE - useful for treating aspergilli and phaeoid
infections
 POSACONAZOLE - newest azole and has perhaps the best
activity against most fungal species
 VORICANOZOLE - has shown improved activity against the
aspergilli, in addition to several emerging pathogens.
 CASPOFUNGIN is under the echinocandins group.
 This class targets cell wall synthesis.
 Two other echinocandins are Anidulafungin and micafungin.
 Two US FDA approved allylamines are terbinafine and naftifine.
 They interfere with the synthesis of ergosterol, a principal sterol
in the plasma membrane of many fungi.
 TERBINAFINE is given orally and is active against several groups
of fungi including dermatophytes and phaeoid fungus.
 NAFTIFINE is only used topically.