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CULTURE CONDITIONS
1. Temperature: Most of the fungi grow well at 25-30C except
the dimorphic fungi that grow both at 25C and 37C.
2. BOD incubators: it is a special incubator used in diagnostic
mycology, which is capable of maintaining low temperature.
3. Incubation time: Culture plates should be incubated for 2-3
weeks.
4. Antibiotics: such as cycloheximide (actidione),
chloramphenicol and gentamicin can be added to the culture
media to inhibit bacterial growth.
Principle :
The SDA media is comprised ofenzymatic digest of caseinand
animal tissues which provide a nutritious source of amino acids and
nitrogenous compounds for the growth of fungi and yeasts.
Dextroseis the fermentable carbohydrate incorporated in high
concentration as a carbon and energy source.Agaris the solidifying
agent. Addition of antibiotics
like Chloramphenicol and/or tetracycline acts as broad spectrum
antimicrobials to inhibit the growth of a wide range of gram-positive
and gram-negative bacteria. Gentamicin is added to further inhibit
the growth of gram-negative bacteria.
Composition of SDA:
Ingredients
In gm/L
Dextrose (Glucose)
40 gm
Peptone
10 gm
Agar
15 gm
Distilled Water
1000 ml
Preparationof SDA
- Combine all ingredients in ~900 ml of deioinized water.
- Adjust to pH 5.6 with hydrochloric acid and adjust final volume
to 1 liter.
- Heat to boiling to dissolve the medium completely.
- Autoclave at 121C for 15 minutes.
Cool to ~45 to 50C and pour into petri dishes or tubes for slants.
Sabouraud agar plates can be inoculated by streaking, as with
standard bacteriological media, or by exposing the medium to
ambient air. Typically, molds are incubated at room temperature
(22 to 25C) and yeasts are incubated at 28 to 30C or 37C if
suspected of being dimorphic fungi. Incubation times will vary,
from approximately 2 days for the growth of yeast colonies such
as Malasezzia, to 2 to 4 weeks for growth of dermatophytes or
dimorphic fungi such as Histoplasma capsulatum. Indeed, the
incubation time required to acquire fungal growth is one
diagnostic indicator used to identify or confirm fungal species.
Limitationsof SDA
- Some strains may be encountered that grow poorly or fail to
grow on this medium.
- Antimicrobial agents added into a medium to inhibit bacteria
may also inhibit certain pathogenic fungi.
- Avoid overheating a medium with an acidic pH, this may result
in a soft medium.
- For identification, organisms must be in pure culture.
- Morphological, biochemical, and/or serological tests should be
performedfor final identification.
- It does not promote conidiation of filamentous fungi.
200 gm
Dextrose
20 gm
Agar
20 gm
Distilled water
1 liter
39 gm
Distilled water
1 liter
Preparing Ourself:
- To prepare potato infusion, boil 200 g sliced, unpeeled
potatoes in 1 liter distilled water for 30 min.
- Filter through cheesecloth, saving effluent, which is potato
infusion (or use commercial dehydrated form).
- Mix with Dextrose, Agar and Waterand boil to dissolve.
- Autoclave 15 min at 121C.
- Dispense 20-25 ml portions into sterile 15 100 mm petri
dishes.
- Final pH, 5.6 0.2.
Preparing from Commercial Medium Powder:
- Add 39 gm of Commercial PDA Powder to 1 Litre ofDistilled
water.
- Boil while mixing to dissolve.