Mycology ppt ii laboratoy techniques for isolation and identification of fungi

Mycology ppt ii laboratoy techniques for isolation and identification of fungi

1. MYCOLOGY-II
Laboratory Techniques for isolation
& Identification of Fungi

3. ISOLATION & CULTIVATION OF FUNGI
IN LABORATORY
Clean the part with 70% alcohol
Collect the material in a sterile paper or a sterile
petridish
•Avoid drying of the specimen
•Reduce bacterial contamination
•Maintain viability
Proper collection of specimen and in adequate
quantity.
Early transport to the lab to avoid overgrowth of
contaminant

4. Culture Medium
•Sabouraud Dextrose Agar (SDA)----contains 2%
dextrose, antibiotics (gentamicin, chloramphenicol)
and cycloheximide
•Selective media
1)Corn meal agar (CMA) – sporulation, chlamydospore
formation
2)Bird seed agar – cryptococcus, forms brown colonies
3)Brain Heart Infusion (BHI) agar – dimorphic & other
fastidious fungi
4)Potato Dextrose Medium (PDA)

6. •Temperature requirement
–Majority of fungi – 37°C
–Superficial mycosis – 30°C
–Dimorphic fungi – 25°C & 37°C
•Incubation time
–At least 4 weeks
–Usually positive cultures are obtained in 7-10
days
–Candida & Aspergillus - 24 to 72 hrs

8. Direct Examination
•Wet mounts
1)Slide & tube KOH mounts – 10 to 20% KOH –
digests protein debris, dissolves keratin.
DMSO can be added to KOH to hasten
clearing in skin scrapings & nail clippings
2)Calcofluor white – fluorescent stain –
excellent morphology of pathogenic fungi
3)India ink – capsulated fungi

9. CFW – yeast form of
Blastomyces
KOH - Aspergillus
India ink -
Cryptococcus

10. •Slide Culture Technique
1)Place a block of Sabouraud’s dextrose agar (about 1cm
2
)
on a sterile slide.
2)Using a straight inoculating needle, inoculate the
specimen to be assayed in center of the block.
3)Gently heat a coverslip by passing it quickly through the
flame of a Bunsen burner and immediately place it
directly on the surface of the inoculated agar block.
4)Place the slide in a petri dish on a slide supporter glass
rods above a moistened filter paper disk, incubate at 28℃
for 3～5 days.
5)The slide can be observed at intervals during cultivation to
observe the dynamics of hyphae and spore growth.

13. Corn Meal Agar
Bird Seed Agar