Preparation of culture media for fungi : Preparation of culture media for fungi Nuzhath fatima.
Department of Microbiology
Faculty of Applied Medical Sciences.
Jazan University, Jazan,KSA Slide 2: Principle: Clinical specimens are processed promptly and plated to isolation media as a means to recover fungi that may be causing disease.
A variety of media are available for the primary inoculation and recovery of fungi and yeast from clinical specimens Slide 3: No one specific medium or combination of media is adequate for all specimens.
Media and incubation temperatures must be carefully selected based on specimen type and fungal suspected agents. Isolation medias for fungi : Isolation medias for fungi Sabouraud Dextrose Agar (SAB): It is
the standard medium for recovery and maintenance of a wide variety of fungi commonly isolated in the clinical laboratory.
SAB + Chloramphenicol + Cycloheximide - Mycosel (BBL) and Mycobiotic (Difco) agars are commercially prepared media containing SAB agar, 1% glucose, chloramphenicol, and cycloheximide.
These media are used for the selective recovery of dimorphic fungi and dermatophytes Slide 5: Potato dextrose agar (PDA):contains potato infusion and dextrose Common organisms that can be cultured on PDA are yeasts such as Candida albicans and Saccharomyces cerevisiae and molds such as Aspergillus niger.
Brain-Heart Infusion Agar (BHI) :BHI is an enriched medium that enhances the recovery of Cryptococcus neoformans from sterile specimens such as CSF
Inhibitory Mould Agar (IMA) :IMA is an enriched medium with inorganic salts, chloramphenicol, and gentamicin. It inhibits growth of bacteria and promotes the growth of fungi. Slide 6: Sterile Bread :Sterile bread without preservatives is recommended for the recovery of zygomycetes from clinical specimens. Bread is often superior to other media used in the Clinical Mycology laboratory for recovering group of opportunistic pathogens. A piece of bread is sterilized in a humidified Petri dish. Specimens from non-contaminated sites can be directly inoculated on bread. Slide 7: Malt Extract Agar: Malt agar is a useful alternative to bread for recovery of zygomycetes
Yeast Extract-Phosphate Medium (YEP): YEP medium was developed for the enhanced recovery of Blastomyces dermatitidis and Histoplasma capsulatum from contaminated specimens. Slide 8: Dermatophyte Test Medium (DTM)
DTM is used to recover dermatophytes from heavily contaminated clinical specimens and to presumptively indicate the presence of a dermatophyte. Its most valuable application is in veterinary mycology.
Dermatophytes, as well as a few other fungi and bacteria turn the medium from pink to red. CAUTION - Observe universal precautions at all times!! : CAUTION - Observe universal precautions at all times!! Media must be carefully selected based on specimen type and fungal suspected agents. Media is dispensed into containers such as 25 x 150 mm screw cap tubes or 100 mm Petri dishes.
Petri plates offer the advantage of a large surface area for isolation and dilution of inhibitory substances in the specimens, but must be poured thick with at least 25 ml of medium to resist dehydration during incubation. Slide 10: Because plates are vented, they are more likely to become contaminated during incubation. to avoid this Plates may be placed in gas permeable bags or sealed with gas permeable tape to offset this disadvantage.
Each Petri plate must be labeled on the bottom, and the lid at least must be taped at two
points to prevent accidental opening of the plate.
All inoculated media should be read every 2 days following incubation and twice weekly thereafter. Slide 11: Media in tubes have a smaller surface area but offer maximum safety and resistance to dehydration and contamination
If the specimen is from a contaminated site, it is important to include media that contain inhibitory substances such as chloramphenicol, gentamicin,Chloramphenicol or gentamicin will inhibit most bacterial contaminants,
Specimens from normally sterile sites can be inoculated to media without inhibitory substances.
Culture Plates must be opened only within a biological safety cabinet to prevent contamination of the plate and exposure of personnel to potentially dangerous fungi Preparation of sabouraud’s media : Preparation of sabouraud’s media Sabauroud’s Dextrose Agar contains
Dextrose- 4 gm%
Neopeptone- 1 gm%
Agar- 1.5 gm%
Distilled water- 100 ml
•Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4
Autoclave and dispense 20 ml amount in test tubes or petri plates.
Use: For the cultivation of Fungi Slide 13: Thank