culture of fungus

VIVEK.M.T2 nd sem MSc MLTSHS PALAYAD

CULTURE OF FUNGUS

INTRODUCTIONCULTURE MEDIASCULTURE METHODS

CONTENTS

Molds and yeasts are widely distributed in air, dust, fomites and normal flora.

Humans are relatively resistant.Fungi are relatively nonpathogenic

INTRODUCTION

A)BASAL MEDIA1)SABOURAUDS DEXTROSE AGAR2)NEUTRAL SABOURAUDS DEXTROSE AGAR3)SDA WITH ANTIBIOTICS

B)NUTRITIONALLY DEFICIENT MEDIA1)CORN MEAL AGAR2)RICE STARCH AGAR

C)ENRICHED/SELECTIVE MEDIA1)BRAIN HEART INFUSION AGAR2)BI PHASIC MEDIUM3)CYSTEIN HEART AND Hb AGAR4)BLOOD AGAR5)BIRD SEED AGAR6)LJ MEDIUM7)DERMATOPHYTE TEST MEDIUM

CULTURE MEDIA

8)CZAPEK`S DOX AGARD)MEDIA FOR STIMULATION OF ASCOSPORE OF PERFECT FUNGI

1)ALPHACEL-YEAST EXTRACT AGAR2)SOIL EXTRACT AGAR

E)MEDIA USED FOR BIOCHEMICAL TESTS

1)TETRAZOLIUM REDUCTION MEDIUM2)CARBOHYDRATE FERMENTATION MEDIA 3) CARBOHYDRATE ASSIMILATION MEDIA4)UREASE MEDIUM5)DIAZONIUM BLUE B REACTION

PEPTONE -10 gmAGAR -20 gmDEXTROSE -40 gmDISTILLED WATER -1000mlAutoclave at 121*c for 15 min.Adjust pH to 5.5Saprobic fungi may overgrowIt obscuring real pathogen

SDA MEDIA

Low in nutrientsSupress vegetative growthStimulate sporulationAlso used to grow germ tube,negative

yeast for identification

Nutritionally deficient media

INGREDIENTSCORN MEAL -8 gmAGAR -4 gmDISTILLED WATER -200 mlTWEEN 80 -2 gm

CORN MEAL AGAR

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

METHOD

Corn Meal Agar

Used for growing fastidious pathogenic fungi such as

-Histoplasma capsulatum-blastomyces dermatitis

INGREDIENTSBrain heart infusion agar -37 gmGlucose -20 gmL cysteine hydrochloride -1gmAgar -20gmDistilled water -

900gm

BRAIN HEART INFUSION AGAR

Dissolve ingredients by boiling.Dispense into screw capped bottles.Autoclave at 121*c for 15 minCool in slanted position with one inch

buttpH adjusted to 6.7Store in refrigerator

preparation

For cryptococcus neoformansCan utilise creatinine as a source of

nitrogenColonies are brown to black due

phenoloxidase produced by organism.

Bird seed agar

Niger seed ectract -200mlGlucose -1 gmChloromphenicol -400gmGentamicin -25 mgDipheny solution -10mlAgar -20gmDistilled water -800mlAutoclave at 121*c for 15 min.Dispense into plates

Ingredients

Used forHistoplasma capsulatumBlastomyces dermatitidisCryptococcus neoformans

IngredientsBlood agar base -40gmSheep blood -50mlDistilled water -1000ml

Blood agar

CULTURAL METHODS

.

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

INCUBATIONMost fungi and moulds grow at room temperature (25-

30*c)Some at body temperature (35-37*C)Some are dimorfhic fungiAll 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

BYETHANKS

BIRD SEED AGAR