For undergraduate medical students

1. General properties of fungi
Dr. Pendru Raghunath Reddy
Assistant Professor of Microbiology
Dr. VRK Women’s Medical College

2. Introduction
• Mykes (Greek word) : Mushroom
• Fungi are eukaryotic protista; differ from bacteria
and other prokaryotes.
1. Cell walls containing chitin (rigidity & support), mannan & other
polysaccharides
2. Cytoplasmic membrane contains ergosterols
3. Possess true nuclei with nuclear membrane & paired
chromosomes
4. Cytoplasmic contents include mitochondria and endoplasmic
reticulum
5. Divide asexually, sexually or by both
6. Unicellular or multicellular
7. Most fungi are obligate or facultative aerobes

3. • Cell wall consists of chitin not peptidoglycan like bacteria
• Thus fungi are insensitive to antibiotics as penicillins
• Chitin is a polysaccharide composed of long chain of n-
acetylglucosamine.
• Also the fungal cell wall contain other polysaccharide, β-
glucan, which is the site of action of some antifungal drugs.
• Cell membrane consist of ergosterol rather than cholesterol
like bacterial cell membrane
• Ergosterol is the site of action of antifungal
drugs, amphtericin B & azole group

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6.  Diverse group of heterotrophs.
– Many are ecologically important saprophytes (consume dead and
decaying matter)
– Others are parasites.
 Most are multicellular, but yeasts are unicellular.
 Most are aerobes or facultative anaerobes.
 Cell walls are made up of chitin (polysaccharide).
 Over 100,000 fungal species identified. Only about
100 are human or animal pathogens.
– Most human fungal infections are nosocomial and/or occur in
immunocompromised individuals (opportunistic infections).


7. In addition to those species which are generally
recognized as pathogenic to man it is firmly
established that under unusual circumstances of
abnormal susceptibility of patient, or the
traumatic implantation of the fungus, other fungi
are capable of causing lesions. Those are called
(Opportunistic fungi)

8. Predisposing factors
• Use of Antibiotics,
• Use of steroids,
• A debilitating condition
of the host, as Diabetes.
• A concurrent disease
such as leukaemia
• Immunosuppressive
conditions
8

9. Fungal Morphology
Molds Yeasts
Many pathogenic fungi
are dimorphic, forming
hyphae at ambient
temperatures but yeasts
at body temperature.
7/21/2013 Dr.T.V.Rao MD 9

10. Understanding the Structure of Fungus
• Simplest fungus :- Unicellular budding
yeast
• Hypha :- Elongation of apical cell produces
a tubular, thread like structure called hypha
• Mycelium :- Tangled mass of hyphae is called
mycelium. Fungi producing mycelia are called
molds or filamentous fungi.
• Hyphae may be septate or non-septate
(coenocytic hyphae)

11. Mycelium
• Mass of branching intertwined hyphae
– Vegetative
– Aerial

12. Vegetative types
• Favic chandeliers
• Nodular organs
• Racquet hyphae
• Spiral hyphae

13. Classification of fungi
1.Morphological classification
2.Systematic classification

14. 1. Yeasts
2. Yeast-like fungi
3. Filamentous fungi (molds)
4. Dimorphic fungi
Morphological classification

15. Yeasts
1. These occur in the form of round or oval bodies
which reproduce by the formation of buds known as
blastospores
2. Yeasts colonies resemble bacterial colonies in
appearance and in consistency
3. The only pathogenic yeast in medical mycology is
Cryptococcus neoformans

16. Yeast colonies
Mucoid
colonies

17. Cryptococcus neoformans
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18. Yeast-Like
1.These are fungi which occur in the form of
budding yeast-like cells and as chains of
elongated unbranched filamentous cells which
present the appearance of broad septate
hyphae. these hyphae intertwine to form a
pseudomycelium.
2. The yeast like fungi are grouped together in
the genus Candida.

19. Candida Colonies

20. Candida albicans
SEM

21. Filamentous Fungi
The basic morphological elements of
filamentous fungi are long branching filaments
or hyphae, which intertwine to produce a
mass of filaments or mycelium
Colonies are strongly adherent to the medium
and unlike most bacterial colonies cannot be
emulsified in water
The surface of these colonies may be
velvety, powdery, or may show a cottony
aerial mycelium.

22. mycelium: septate mycelium: non septate

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24. Colony Morphology

25. Dimorphic Fungi
These are fungi which exhibit a yeast form in the
host tissue and in vitro at 370C on enriched media
and mycelial form in vitro at 250C
Examples:
Histoplasma capsulatum
Blastomyces dermatitidis
Coccidioides immitis
Paracoccidoides brasiliesis
Penicillium marneffei
Sporothrix schenckii

26. Histoplasma capsulatum - Dimorphism
• Filamentous mold in environment
– Thin septate hyphae, microconidia, and
tuberculate macroconidia (8-14 µm)
• Budding yeast (2-4 µm) in tissue
– Dimorphic transition is thermally dependent and
reversible (25°C 37°C).
Hyphae, micro- and macroconidia Yeast within histiocyte

27. Systematic classification
• Based on sexual spores formation:
4 classes
1. Zygomycetes
2. Ascomycetes reproduce sexually
3. Basidiomycetes
4. Deuteromycetes (fungi imperfectii)

28. Zygomycetes
• Lower fungi
• Broad, nonseptate hyphae
• Asexual spores -
Sporangiospores: present
within a swollen sac- like
structure called
Sporangium
• Examples:
Rhizopus, Absidia, Mucor

29. Ascomycetes
• Sexual spores called
ascospores are present
within a sac like
structure called Ascus.
• Each ascus has 4 to 8
ascospores
• Includes both yeasts
and filamentous fungi

30. Ascomycetes
• Narrow, septate hyphae
• Asexual spores are called conidia borne on
conidiophore
• Examples: Penicillium, Aspergillus

31. Basidiomycetes
Sexual fusion results in the formation of a club shaped
organ called base or basidium which bearspores called
basidiospores
Examples: Cryptococcus neoformans, mushrooms

32. Deuteromycetes
or Fungi imperfectii
• Group of fungi whose sexual phases are not
identified
• Grow as molds as well as yeasts
• Most fungi of medical importance belong
to this class
• Examples: Coccidioides
immitis, Paracoccidioides
brasiliensis, Candida albicans

33. Reproduction and sporulation
Types of fungal spores
1.Sexual spores
2.Asexual spores

34. Sexual spores
• Sexual spore is formed by fusion of cells and
meiosis as in all forms of higher life
• Ascospores
– Ascus
– Ascocarp
• Basidiospores
• Zygospores

35. Asexual spores
 These spores are produced by mitosis
1. Vegetative spores
2. Aerial spores

36. Vegetative spores
• Blastospores: These
are formed by budding from
parent cell, as in yeasts
• Arthrospores – formed
by segmentation &
condensation of hyphae
• Chlamydospores –
thick walled resting spores
e.g. C.albicans

37. Aerial spores
1. Conidiospores
Spores borne externally on sides or
tips of hyphae are called
conidiospores or simply conidia
2. Microconidia
3. Macroconidia
4. Sporangiospores

38. • Microconidia -
Small, single celled
• Macroconidia – Large
and septate and are
often multicellular

39. Immunity to fungal diseases
 The defense to fungal infection involves both innate and
aquired immunity
 The passive protection is provided by intact skin and mucosal
surfaces
 The fatty acids like sebum also provide protection because of
their antifungal activity
 The alveolar macrophages are important in engulfing cells in
lungs which are removed by ciliary action and coughing
 A variety of innate defense factors in saliva such as lysozymes
and lactoferrin contribute to mucosal protection

40. Cellular immunity
 For the most of fungi, cellular immunity is mainstay of host
defenses
 Monocyte-macrophage activity, especially in respiratory tract,
appears to be major defence mechanism against most fungi
 Renal transplant recipients have peculiar predilection for
cryptococcal infection
Humoral immunity
 The precise role of humoral immunity in host defense against
fungal infections is difficult to determine

41. Diagnosis of fungal diseases
1. Clinical diagnosis
2. Laboratory diagnosis

42. Clinical diagnosis
 The clinical criteria may give presumptive diagnosis of fungal
infections
 The superficial and subcutaneous mycoses often produce
characteristic lesions that strongly suggest their fungal etiology
 In case of systemic mycoses, there is no sign or symptom that
specifically suggests a fungal disease

43. Laboratory diagnosis
Specimens collection
1. Respiratory specimens
2. CSF
3. Blood
4. Tissue, Bone marrow and body fluids
5. Skin, hair and nail
6. Urine, stool
7. Vaginal secretions
8. Corneal scrapings

44. Direct examination
 The direct demonstration of fungal elements is essential in
establishing diagnosis by detecting presence of causative agent
in the clinical material
 The following procedures are adopted to directly observe fungi
in clinical specimens
1. Wet mounts
2. Histopathology
3. Fluorescent-antibody technique
4. Gram staining

45. Wet mounts
KOH wet mount
Slide KOH
 Most of the specimens can be examined in wet mounts after
partial digestion with 10-20% KOH
 The clinical specimens like skin, hair and nails should be
mounted under cover slip in KOH on slide
 This clears material within 5 – 20 minutes, depending on its
thickness
 A slight warming over a low flame hastens digestion of keratin

46.  KOH can also be supplemented with DMSO to increase clearing
of fungi especially in skin scrapings and nail clippings
 The KOH can be supplemented with a fluorescent dye,
calcofluor white (CFW)
 The CFW supplemented KOH especially in corneal scrapings
can detect even scanty amount of fungal elements
Tube KOH
 The tube KOH is prepared mainly for biopsy specimens, which
take longer time for dissolution
 The homogenized biopsy tissue is dissolved in 10% KOH and
examined after keeping for an overnight in an incubator at 370C

47. KOH mount
Blastomyces dermatitidisMold (note: septate hyphae)

48. KOH mount
Ectothrix Endothrix

49. Other methods
The wet india ink and nigrosin preparations are also very useful
in making diagnosis of certain capsulated fungi
Histopathology
The histopathological examination of biopsy specimens is an
excellent way to diagnose mycotic infections
The H&E stain is routine procedure in histopathology laboratory
and stains most of fungi

50. H&E stain
Endospores and sporangia
of Rhinosporidium seeberi
Cryptococcus neoformans

51. Blastomyces dermatitidis Rhizopus species
H&E stain

52. The specific fungal stains, such as periodic acid-Schiff (PAS),
Grocott-Gomori’s Methenamine silver (GMS) and Gridely stains
are widely used for demonstrating fungi in tissues
Mayer’s mucicarmine stain can also be used specifically to show
capsular material of Cryptococcus, endospores and sporangia
of Rhinosporidium seeberi

53. PAS stain GMS stain

54. Fluorescent-antibody staining
May used to detect fungal antigen in clinical material such as
pus, blood,CSF, tissue impression smears and in paraffin
sections of formalin fixed tissue
It is less satisfacory for sputum specimens
Detects fungus when there are only few organisms present, as
seen in pus from sporotrichosis
Its use is limited by restricted availability of specific antisera

55. Gram staining
All fungi are gram positive
Gram stained smear of Candida species

56. Fungal culture
The solid media are employed for fungal culture
The medium commonly employed is emmon’s modification of
Sabouraud Dextrose Agar (SDA) [pH – 5.4]
The media may supplemented with antibiotics, such as
gentamicin and chloramphenicol to minimize bacterial
contamination and cycloheximide to inhibit saprophytic fungi
The special media may be used for isolation and to help rapid
identification, when identity of a particular pathogen is strongly
suspected Eg: Bird seed agar for Cryptococcus neoformans
Potato dextrose agar, Brain heart infusion (BHI) agar and
Czapek Dox agar may also be used for isolation of fungi

57. Incubation
Cultures are routinely incubated in parallel at room temperature
(for weeks) and at 370C for days
Many fungi develop relatively slowly and cultures should be
retained for atleast 2-3 weeks (in some cases upto 6 weeks)
before being discarded
Yeasts usually grow within 1-5 days

58. Interpretation of fungal cultures
 The significance of fungal isolate depends on its source and
identity
 The isolation of an established pathogen such as H. capsulatum
or Coccidioides species from any specimen is generally
regarded as an evidence of infection
 In case of commensal or opportunistic fungi following points
should be considered
1. Isolation of same strain in all culture tubes
2. Repeated isolation of same strain in multiple specimens
3. Immune status of patients and
4. Serological evidence to confirm significance of isolate

59. Identification of fungi
Gross or macroscopic examination of cultures
Microscopic examination
1. Tease mount
2. Slide culture

60. Tease mount
For microscopic examination, fragment of fungal culture is
teased out using two teasing needles and placed on a glass
slide in drop of LCB stain
To study the morphology of hyphae, spores and other structures,
tease mounts are prepared in lactophenol cotton blue (LCB)
which contains lactic acid, phenol, glycerol and cotton blue
Microscopic characteristics that should be observed are the
following
1. Sepatate versus sparsely septate hyphae
2. Spores or conidia

62. Slide culture
Is used to study undisturbed morphological details of fungi,
particularly relationship between reproductive structures like
conidia, conidiophore and hyphae
It is indicated when teased mount of LCB is inconclusive in
particular fungal isolate

63. Procedure
Place sterile microscopic slide on bent glass rod at the bottom
of petri dish
A piece of one square centimeter block of cornmeal agar or
potato dextrose agar is put up on the slide
Inoculate the fungal strain under identification at four sides of
agar block
Cover the inoculated block with sterile coverslip and incubate at
250C in BOD incubator

64. Slide culture

65. Add a little sterile distilled water on filter paper to avoid drying of
agar
When growth appears, place drop of LCB on slide and coverslip
from block
Cellophane tape preparation
A piece of tape is gently laid over portion of fungal colony and
slowly lifted and place over a glass slide containing LCB solution
This preparation has come into greater use to overcome
obstacles of time consumption and requirement of extra
equipment to prepare slide culture

66. Aspergillus Penicillium

67. Miscellaneous tests for the identification of molds
1. Hair perforation test
2. Urease test
3. Thiamine requirement
Miscellaneous tests for identification of yeasts
Germ tube production, carbohydrate assimilation, chromogenic
substrates, potassium nitrate assimilation, temperature studies
and urease

68. Serologic tests
Detection of antibodies
1. Immunodiffusion
2. Countercurrent immunoelectrophoresis (CIE)
3. Whole cell agglutination
4. Complement fixation
5. ELISA
Detection of antigens
1. Latex particle agglutination
2. ELISA

69. Molecular techniques
1. PCR
2. DNA probes

70. Mycoses
Infection caused by fungus is known as mycosis
(Plural mycoses)
Classification of mycoses
Classification of fungal disease according to primary sites of
infections
1. Superficial mycoses
2. Cutaneous mycoses
3. Subcutaneous mycoses
4. Systemic mycoses
5. Opportunistic mycoses