Lab: 21 Practical Microbiology

Mycology
- Diagnosis of fungal infection
Steps for Diagnosis:

 Collecting the appropriate specimen is the key step for an accurate

diagnosis.

Types of Specimens for Fungal Diagnosis

1. Hair: Pull affected hair, place in a sterile petri dish, cut into

pieces—some for culture, some for direct microscopy.

2. Skin: Scrape from the outer edge after cleaning with 70% alcohol,

then examine directly or culture.

3. Nail: Scrape or cut, then process the same way as skin samples.

4. Blood & Bone Marrow: Centrifuge and use for culture.

5. CSF: Centrifuge, then use for culture and direct examination.

6. Abscess/Wound Exudates & Tissue: Crush exudate granules into

culture media and examine directly (for subcutaneous mycoses).

7. Respiratory Samples: Use sputum or transbronchial aspirates for

culture (for pulmonary mycoses).

1
-Direct Microscopy for Fungal Diagnosis

1. KOH Preparation (10-20%): Used for detecting fungal infections

in hair, skin, and nails. Scraped sample is placed on a slide, mixed

with KOH, and gently heated. KOH dissolves keratin, making
fungal elements more visible.
2. KOH + Calcofluor White: A fluorescent dye is added to enhance

fungal visualization under a fluorescent microscope.

3. India Ink Staining: Used to detect Cryptococcus in cerebrospinal

fluid (CSF). The ink creates a clear halo around encapsulated yeast
cells.
4. Tissue Staining: Giemsa stain is commonly used for identifying

fungi in tissue samples.

2
Fungal Isolation and Culture Media

1. Sabouraud Dextrose Agar (SDA): Commonly used for fungal

isolation. Cycloheximide and chloramphenicol are added to

selectively isolate dermatophytes.
2. Brain Heart Infusion (BHI) Agar.

3. Potato Flake Agar.

Incubation of Fungi

-Incubation:

1. Molds: Incubated at 25-30°C.

2. Yeasts: Incubated at 37°C.

Aspergillus spp.

 Saprophytic fungi with hyaline, septate mycelium.

 Colony appearance: Growth rate and color vary; texture ranges
from velvety to cottony.
 Microscopic features: Unbranched conidiophores with conidia.
 Pathogenicity: Aspergillus fumigatus is a potential pathogen,
especially in pulmonary infections.

3
Penicillium spp.

 Saprophytic fungi with hyaline, septate mycelium.

 Colony appearance: Rapid growth; white to bluish-green.
 Microscopic features: Conidiophores form a brush-like structure,
with flask-shaped sterigmata.

Cladosporium spp. is a type of saprophytic fungus that grows quickly.

Its colonies are green on the top and black underneath. The fungus has
segmented, dark-colored mycelium. The conidia (spores) are produced in
chains.

Rhizopus spp. is a type of saprophytic fungus that does not have septa in
its mycelium. It has root-like structures called rhizoids that help anchor it
to surfaces. The sporangiophores, which are structures that produce
spores, originate from the nodes (points where branches or structures
grow).

4
 Dermatophytes
Three main genera:
1-Genus Microsporum
Genus Microsporum is a group of fungi that infect the skin and hair.
Some examples include:

 M. canis (from animals)

 M. gypsum (from soil)
 M. audouinii (from humans)

Culture of Microsporum:

 Colonies grow quickly and have a cottony, wooly, or powdery

appearance.
 The color of the colonies ranges from white to different shades of
brown.
 The reverse side of the colony is yellow to orange-brown.

5
Microscopy of Microsporum:

 Macroconidia are large, thick-walled, rough, and spindle-shaped.

They are also multicellular, with M. canis having 6 to 15 cells.
 There are fewer microconidia, which are smaller.
 The hyphae (the fungal threads) are shaped like racquets.

Microsporum spp.

2-Genus Trichophyton
is a group of fungi that infect the skin, hair, and nails. Some examples
include:

 T. rubrum (from humans)

 T. mentagrophytes (from soil and animals)
 T. verrucosum (from animals)

6
Culture of T. verrucosum:

 The colonies are velvety and waxy in texture.

 The color of the colonies is white, and sometimes yellow.
 The reverse side is colorless and the fungus penetrates deeply into
the media.

Microscopy:

 The hyphae are thin and have septa (divisions).

 There are many microconidia (small spores) and rare
macroconidia (large spores).
 The type of hair invasion caused by T. verrucosum is ectothrix,
meaning the fungus invades the outside of the hair shaft.

Trichophyton

3-Genus Epidermophyton
has only one species, E. floccosum, which attacks the skin and nails but
not the hair.

Culture:

 The colonies have a velvety texture with central radiating furrows

(lines).

7
  The color of the colonies is yellow to green.
 The reverse side of the colony is yellow to tan.

Microscopy of Epidermophyton floccosum:

 Macroconidia are large, with multiple septa (2 to 6 cells), and

have smooth, rounded walls.
 Racquet hyphae are commonly seen.
 No microconidia are formed.

Epidermophyton

Unicellular Fungi: Candida

Specimens:

1. For skin and nail infections, scrape the affected area and mount the

sample in 10% KOH (potassium hydroxide).

2. For sputum, spread it into a thin film on a slide, then cover it with a

cover glass. Gram stain can also be used.

3. For stools, spinal fluid, or bronchial washings, centrifuge the

sample and examine it in the same way as sputum.

8
Culture:

Colonies of Candida albicans on Sabouraud's agar appear within 2 to 4

days at 37°C. They are creamy-colored, smooth, and have a yeasty odor.

A pure culture of Candida albicans in Petri dish. Sabouraud agar.

Microscopic Appearance of Candida:

1. A lactophenol slide mount shows oval, budding yeast cells. In

older cultures, you may also see some pseudohyphae (chains of

connected cells).
2. Gram stain shows Gram-positive yeast cells, which are large and

may be arranged in clusters.

Microscopic Appearance of Candida

9
Identification of Candida species:

Chlamydospore Formation:

1. Use corn meal agar with Tween 80 to promote the

production of chlamydospores (thick-walled asexual

spores).
2. After inoculating the medium, incubate for 3 days, then

examine the culture.

Germ Tube Method (Serum Tube Method):

1. Inoculate a low concentration of Candida albicans cells into

0.5 ml of human or sheep serum.

2. Incubate at 37°C for 2-3 days.

3. Prepare a slide, mount it, and examine under the microscope

for germ tube formation.

Sugar Fermentation & Sugar Assimilation (for other Candida

species):

This test helps identify Candida species by checking their

ability to ferment or assimilate different sugars.

Budding yeast / germ tube

10
 Cryptococcus
• C.neoformansis the pathogenic spp.
• It is a yeast fungi & can be differentiated from candida by 2 main
feature
1-Capsulated
2-Urease positive

11