INTRODUCTION

CULTURE MEDIAS

CULTURE METHODS

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

INGREDIENTS CORN MEAL -8 gm

AGAR -4 gm

DISTILLED WATER -200 ml

TWEEN 80 -2 gm

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

CULTURAL METHODS

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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

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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

REFERENCE

BIRD SEED AGAR