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Molds and yeasts are widely distributed in air, dust, fomites and normal flora.
Humans are relatively resistant.
Fungi are relatively nonpathogenic
A)BASAL MEDIA 1)SABOURAUDS DEXTROSE AGAR
2)NEUTRAL SABOURAUDS DEXTROSE AGAR 3)SDA WITH ANTIBIOTICS
B)NUTRITIONALLY DEFICIENT MEDIA 1)CORN MEAL AGAR 2)RICE STARCH AGAR
C)ENRICHED/SELECTIVE MEDIA 1)BRAIN HEART INFUSION AGAR 2)BI PHASIC MEDIUM 3)CYSTEIN HEART AND Hb AGAR 4)BLOOD AGAR 5)BIRD SEED AGAR 6)LJ MEDIUM 7)DERMATOPHYTE TEST MEDIUM
8)CZAPEK`S DOX AGAR
D)MEDIA FOR STIMULATION OF ASCOSPORE OF PERFECT FUNGI
1)ALPHACEL-YEAST EXTRACT AGAR
2)SOIL EXTRACT AGAR
E)MEDIA USED FOR BIOCHEMICAL TESTS
1)TETRAZOLIUM REDUCTION MEDIUM
2)CARBOHYDRATE FERMENTATION MEDIA
3) CARBOHYDRATE ASSIMILATION MEDIA
4)UREASE MEDIUM
5)DIAZONIUM BLUE B REACTION
PEPTONE -10 gm
AGAR -20 gm
DEXTROSE -40 gm
DISTILLED WATER -1000ml
Autoclave at 121*c for 15 min.
Adjust pH to 5.5
Saprobic fungi may overgrow
It obscuring real pathogen
A Heavy inoculum of yeast is streaked across a plate containing the medium.
Cover slip is placed over it.
Streak should project beyond cover slip.
Examine under low power at edge of cover slip.
It is a sort of junction of aerobic and anerobic condition.
Clamidiospores are best found in this area.
Shows clamidiospores seen in candida albicans after 24-48 hrs incubation at 25*c
Used for growing fastidious pathogenic fungi such as
-Histoplasma capsulatum
-blastomyces dermatitis
INGREDIENTS
Brain heart infusion agar -37 gm
Glucose -20 gm
L cysteine hydrochloride -1gm
Agar -20gm
Distilled water -900gm
Dissolve ingredients by boiling.
Dispense into screw capped bottles.
Autoclave at 121*c for 15 min
Cool in slanted position with one inch butt
pH adjusted to 6.7
Store in refrigerator
For cryptococcus neoformans
Can utilise creatinine as a source of nitrogen
Colonies are brown to black due phenoloxidase produced by organism.
Niger seed ectract -200ml
Glucose -1 gm
Chloromphenicol -400gm
Gentamicin -25 mg
Dipheny solution -10ml
Agar -20gm
Distilled water -800ml
Autoclave at 121*c for 15 min.
Dispense into plates
Used for
Histoplasma capsulatum
Blastomyces dermatitidis
Cryptococcus neoformans
Ingredients Blood agar base -40gm
Sheep blood -50ml
Distilled water -1000ml
.
Sabrourd’s medium is used world-wide and is generally satisfactory
Some workers prefer malt peptone agar
It is claimed that, in the latter from the syrup is slightly more inhibitory to bacteria and produces more rapid growth and sporulation of fungi than sabouraud’s medium. In additional it is often possible to distinguish mixed cultures of yeast species by their colony morphology on malt agar
Two types of containers are used for culture
1. Petri dish
2. Culture tube
.
Petridish Test tube
Surface area Large Small
O2 supply Good Poor
Security of closure Poor Good
Detection of mixed culture Easy Hard
Blood culture
• Same as that of microbiology
• For manual system blood culture bottle with agar and mycological broth or sabouraud broth media may be employed
• Aerat the cultures periodically by shaking and sub culture routinely rather than to wait until the medium is cloudy
TISSUE
Biopsy and other tissue should be reduced in
size 1 -2mm
Put these pieces into agar medium, it should
contain antibiotics if the material is
contaminated with bacteria
SWABS
o Heavily feed swab rotate over the surface of media several times
o Secondary dilution strokes should be made with a sterile loop
CSFo A loop full of spun deposit should be take to inoculate the
agar media in usual manner
Urine Peritoneal fluid
Spread 0.1ml un concentrated urine over the surface of agar containing antibiotics
Centrifuge the specimen and remove a loop full of sediment to another agar plate of the same medium and streak out from the well in the normal way
Process as urine but take an additional sample of 1ml of the neat dialysate and spread over a plate containing mycological medium
.
INCUBATION Most fungi and moulds grow at room temperature (25-
30*c)
Some at body temperature (35-37*C)
Some are dimorfhic fungi
All media are incubated at 25˚ to 30˚C initially and, when a potential dimorphic organism is isolated an attempt is made to convert it to the tissue phase by subeultuning it and incubating the new set of culture at 35˚C
The pathogenic fungi are aerobic organisms. A good supply of oxygen is mandatory if they are to be isolated in primary culture