FACULTY OF MEDICINE

BACHELOR OF MEDICINE & BACHELOR

OF
SURGERY (MBBS 240)

SESSION 2022/2023
Year 1 Semester 1

MEDICAL MICROBIOLOGY & PARASITOLOGY

(MMP)
GENERAL MODULE

PRACTICAL 2 (Part B)
BASIC DIAGNOSTIC TECHNIQUES (MYCOLOGY)

Date: 10.11.2022
Time: 10 am- 1pm
PRACTICAL 2: BASIC DIAGNOSTIC TECHNIQUES

Specific learning outcomes

At the end of the practical session, students should be able to:

• understand and describe the basic techniques in identification of medically
important fungi.
• identify certain medically important fungi based on the provided exhibits

This practical session consists of THREE parts: A, B, and C

Part A Introduction/Theory/Video Presentation and Principle of Test
Part B Exhibits and discussion

PART A: Introduction/Theory/Video Presentation and Principle of Test

Activity: There are a total of 6 video presentations in this section. You may discuss
about the techniques shown in the videos with your respective lecturers during the
practical session.

1. Type of specimens & container

a. Specimens:
• Skin and nail scraping, hair, sputum, tissue, caseous materials,
urine, blood
b. sterile container containing samples.

2. Basic laboratory techniques:

a. Direct smear of specimen & microscopy
• 20% KOH (Video M1): causes the keratin cells to swell and reveal
the fungal elements in the skin or nails:
• https://www.youtube.com/watch?v=PlBENGGAqr4
• Lactophenol cotton blue (Video M2):
https://www.youtube.com/watch?v=qc1F1zym00g
• Gram stain
• India ink for the capsule of Cryptococcus neoformans (Video M3):
https://www.youtube.com/watch?v=0JGw9KUubD8
• Gomori’s Methenamine silver nitrate stain (GMS): the cell wall
appeared dark due to deposit silver stain.
b. Culture
• Sabouraud’s dextrose agar
• Brain heart infusion agar
• Slide culture technique (video M4):
https://youtu.be/e3bN3wpKMHc

c. non-culturable techniques (Ab detection, Ag detection, ELISA)

• API 20C AUX
• Carbohydrate assimilation
• Latex agglutination (video M5):
https://www.youtube.com/embed/8kBYjfpaIF8
• Germ tube (video M6):
https://www.youtube.com/watch?v=4QTt7EOrKl0

d. Histopathology
e. Molecular techniques
PART B: EXHIBITS & DISCUSSION
Activity: Study the following exhibits, fill in the blanks and discuss with your respective
lecturers during the practical session.

B1. MYCOLOGY

1. For culture of the fungi, you are provided with the following:
a. Sabouraud’s Dextrose Agar (SDA) (EXHIBIT M1-M3)
• contains 2% of dextrose, antibiotics (gentamicin, chloramphenicol) and
cycloheximide
• cycloheximide is not used when Cryptococcus, Aspergillus or Penicillium are
suspected.
EXHIBIT M1 Candida albicans on SDA & Gram stain (microscopy)
EXHIBIT M2 Cryptococcus neoformans on SDA & India Ink
(microscopy)
EXHIBIT M3 Penicillium spp on SDA & lactophenol cotton blue Stain
(microscopy)

b. Brain Heart Infusion Agar

• used to cultivate dimorphic and other fastidious fungi.

EXHIBIT M4 Brain Heart Infusion Agar (BHIA)

2. Other exhibits

EXHIBIT M5 API 20C AUX

EXHIBIT M6 Latex agglutination test for detection of Cryptococcal
antigen
EXHIBIT M7 Germ tube test (microscopy)
 EXHIBIT M1: Candida albicans on SDA & Gram stain (microscopy)

Q: Based on Exhibit MI, briefly describe the colonial morphology and microscopic
appearance of the Candida albicans.
a. Colonial Morphology: ___________________________________________
b. Microscopic appearance: ___________________________________________

EXHIBIT M2: Cryptococcus neoformans on SDA agar & India Ink (microscopy)

Q: Based on Exhibit M2, briefly describe the colonial morphology and microscopic
appearance of the Cryptococcus neoformans.
a. Colonial Morphology: ___________________________________________
b. Microscopic appearance: ___________________________________________
EXHIBIT M3: Penicillium spp on SDA & lactophenol cotton blue Stain (microscopy)

Colonial Morphology: ___________________________________________

Microscopic appearance: ___________________________________________

 EXHIBIT M4: Brain Heart Infusion Agar (BHIA)

EXHIBIT M5: API 20C AUX

API 20 C AUX is a system for the precise identification of the most frequently
encountered yeasts. The API 20 C AUX strip consists of 20 cupules containing
dehydrated substrates which enable the performance of 19 assimilation tests. The
cupules are inoculated with a semi-solid minimal medium. The yeasts will only grow if
they are capable of utilizing each substrate as the sole carbon source. The reactions
are read by comparing them to growth controls. Identification is obtained by referring
to the Analytical Profile Index or using the identification software. For example, in this
image showing API identification for Candida tropicalis.
 EXHIBIT M6: Latex agglutination test for detection of Cryptococcal antigen

Latex bead coated with specific antibody are agglutinated in the presence of
Cryptococcal antigen. Agglutination indicates a positive test and interpreted as
suggestive of cryptococcus neoformans meningitis. This is NOT a confirmatory test.

EXHIBIT M7: Germ tube test (microscopy)

This is a positive germ tube test. Candida albicans produce short cylindrical extension
called germ tube (pseudohyphae) when incubate inserum for 2-3 hours at 370C.
Other Candida species are germ tube negative and need to be identified by sugar
fermentation and assimilation test.