Sie sind auf Seite 1von 15

FUNGAL CULTURE

Fungal culture is frequently performed for isolation and correct


identification of fungi. Following types of culture media are used:
- Sabourauds dextrose agar (SDA): Most commonly used
media in diagnostic mycology. It contains peptone (1%),
dextrose (4%) and has a pH of 5.6. It may not support some
pathogenic fungi.
- Neutral SDA (Emmons modifications): It differs from
original SDA in having neopeptone (1%) and dextrose (2%)
and pH of 7.2.
- Corn meal agar and rice starch agar: They are the
nutritionally deficient media used for stimulation of
chlamydospore production.
- Brain heart infusion agar and blood agar: They are
enriched media, used for growing fastidious fungi like
Cryptococcus and Histoplasma.
- Niger seed agar and bird seed agar: Used for selective

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.

Sabouraud Dextrose Agar (SDA):


- It is a selective medium primarily used for the isolation of
dermatophytes, other fungi and yeasts but can also grow
filamentous bacteria such as Nocardia. The acidic pH of this
medium(pH about 5.6) inhibits the growth of bacteria but
permits the growth of yeasts and most filamentous fungi.
Antibacterial agents can also be added to augment the
antibacterial effect.
- This medium is also employed to determine mycological
evaluation of food, contamination in cosmetics and clinically to
aid in the diagnosis of yeast and fungal infections.
- Antibiotics like chloramphenicol, gentamicin, and tetracycline
can be added as selective agents to inhibit bacterial overgrowth
of competing microorganisms while permitting the successful
isolation of fungi and yeasts. Various other modifications are
also reported by using cycloheximide, penicillin, streptomycin,
neomycin depending upon the intended use.

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

Final pH 5.6 +/- 0.2 at 25C.

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.

Potato Dextrose Agar (PDA)


Potato Dextrose Agar (PDA) is a general purpose medium for
yeasts and molds that can be supplementedwith acid or
antibiotics to inhibit bacterial growth. It is recommended for plate
count
methods
for
foods,
dairyproductsand
testing
cosmetics.PDA can be used for growing clinically significant yeast
and
molds.Thenutritionally
rich
base
(potato
infusion)
encourages mold sporulation and pigment production in
somedermatophytes.
Potato Dextrose Agar is composed of dehydrated Potato Infusion
and Dextrose that encourage luxuriantfungal growth. Agar is
added as the solidifying agent. Many standard procedures use a
specified amount ofsterile tartaric acid (10%) to lower the pH of
this
medium
to
3.5
+/0.1,
inhibiting
bacterial
growth.Chloramphenicol acts as a selective agent to inhibit
bacterial overgrowth of competing microorganisms from mixed
specimens, while permitting the selective isolation of fungi.

Composition of Potato Dextrose Agar (PDA)


- Preparing Ourself
Potato infusion

200 gm

Dextrose

20 gm

Agar

20 gm

Distilled water

1 liter

Note: 200 gm of potato infusion is equivalent


to4.0 gm of potato extract.
- Preparing from Commercial Medium Powder
Commercial PDA Powder (20
gm dextrose, 15 gm agar,
and 4 gm potato starch)

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.

Uses of Potato Dextrose Agar (PDA)


Potato Dextrose Agar is used for the detection of yeasts and
molds in dairy products and prepared foods.
It may also be usedfor the cultivation of yeasts and molds from
clinical specimens.
Potato Dextrose Agar with TA (Tartaric Acid) is recommended for
the microbial examination of food and dairy products.
Potato Dextrose Agar with Chlortetracycline is recommended for
the microbial enumeration of yeast and mold from cosmetics.
Potato Dextrose Agar with Chloramphenicol is recommended for
the selective cultivation of fungi from mixed samples.

Das könnte Ihnen auch gefallen