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Transcript of 56
MYCOLOGYFUNGAL CULTURE
CUTURE METHOD:
All specimens should be inoculated onto a general purpose fungal medium
fungi will grow very well on culture media used to isolate bacteria
Temperature:
Most MOLDS grow best at: 25 – 30 OC
Most YEAST grow best at 35 – 37 OC
Most pathogenic fungi grow best: 30 to 32°C
EXCEPT: Sporothrix schenckii : 25 to 27°C than 30°C
Incubation period: 14 days: general incubation period 7 days: to detect presence of yeast in the mouth, throat, or
vagina 21 days: tissues and sterile body fluids other than blood 28 days: respiratory, bone marrow, blood specimens, and
specimens in which dimorphic fungus are suspected Plates should be checked at least TWICE during the first week,
when rapidly growing isolates may appear, weekly hereafter.
CHROMagar A selective medium for the isolation and presumptive identification of
yeast and filamentous fungi and differentiation of Candida albicans, C. tropicalis and C. krusei.
Due to the differences in morphology and colors of the yeast colonies, this medium facilitates the detection of mixed yeast cultures in specimens.
It may also be used as a selective isolation medium for other yeasts and for filamentous fungi instead of Sabouraud Dextrose Agar or similar media.
CHROMagarFORMULA IN GRAMS PER LITER
Glucose ....................................... 20.00 Peptone …….................................. 10.00 Chloranphenicol ............................ 0.50 Chromogenic Mixture.............. ........ 0.40 Bacteriological Agar ....................... 15.00
Final pH 6.1 ± 0.2 at 25°C
CHROMagarPreparation Suspend 45.9 grams of the medium in one liter of
distilled water. Mix well and heat with frequent agitation until
complete dissolution. Distribute into adequate containers.
CHROMagarApproximate Formula* Per Liter Purified Water Chromopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g Chromagen Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g Chloramphenicol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g
CHROMagar Candida albicans- green Candida tropicalis - steel
blue Candida krusei - rose,
fuzzy
C. krusei
C. tropicalis
C. albicans
CHLAMYDOSPORE AGAR Used for differentiating Candida albicans from other species
of Candida on the basis of chlamydospore formation. Candida albicans always form chlamydospore on this medium. The medium contains trypan blue to visualize the
chlamydospore under microscopic evaluation. Biotin and polysaccharide are growth factors which stimulate
chlamydospore formation. Potassium phosphate function as a buffer in the medium
Components (g/L)Ammonium Sulphate…………………………… 1.00Monopotassium Phosphate…………………… 1.00Purified Polysaccharide………………………… 20.00Trypan Blue………………………………………… 0.10Biotin…………………………………………………. 0.000005Agar…………………………………………………… 15.00
Final pH (at 25°C) 5.1 ± 0.2
CHLAMYDOSPORE AGAR
Organisms Growth Chlamydospores
Candida albicans luxuriant (+) Candida tropicalis luxuriant
(-) Candida krusei luxuriant (-) Candida minosa luxuriant (-)
CHLAMYDOSPORE AGAR
LEVINE’S EMB AGAR For the isolation and differentiation of Escherichia coli and
Enterobacter The dyes contained in this medium inhibit the growth of many
accompanying Gram-positive microorganisms LEVINE EMB Agar can be used to identify Candida albicans in
clinical specimens, if chlorotetracycline hydrochloride is added to inhibit the entire accompanying bacterial flora
LEVINE EMB Agar can also be utilized for the identification of coagulase-positive staphylococci which grow characteristically as colorless "pin-point" colonies and which show good agreement with the results of the coagulase test
LEVINE’S EMB AGARTypical Composition (g/liter) Peptone …………………………………………………………….10.0 Lactose ……………………………………………………………..10.0 Di-potassium hydrogen phosphate ………………………..2.0 Eosin, yellowish …………………………………………………..0.4 Methylene blue …………………………………………………...0.065 Agar-agar …………………………………………………………..13.5 If cultivating Candida, add 100 mg tetracycline hydrochloride/litre after
autoclaving and mix homogeneously. The culture medium then is blue
LEVINE’S EMB AGAR To obtain a primary culture of Candida, incubate the plates containing chlorotetracycline in a 10 % carbon dioxide atmosphereAppearance: "Spidery" or "feathery“ - Candida albicans Yeast-like, round, smooth - Other Candida species; Sometimes Nocardia
SABHI AGAR Used for the cultivation of pathogenic and nonpathogenic
fungi from a variety of clinical and nonclinical sources Sabouraud Dextrose Agar is a general purpose medium
devised by Sabouraud for the cultivation of dermatophytes Brain Heart Infusion (BHI) Agar has proven to be effective in
the cultivation of a wide variety of microorganisms and is recommended for the primary recovery of fungi from clinical specimens
SABHI AGAR SABHI Agar combines the
ingredients of these two formulations to provide a medium which was found to yield greater recovery of pathogenic fungi than either medium individually
It is recommended for the recovery of fungi from clinical specimens
SABHI AGARFormulation: SABHI Agar contains two
peptones and brain heart infusion solids as sources of amino acids, nitrogen, sulfur, carbon and trace ingredients
Dextrose is an energy source for the metabolism of microorganisms. Sodium chloride provides essential electrolytes
SABHI AGARApproximate Formula* Per Liter Purified Water Brain Heart, Infusion from (Solids) . . . . . . . . . . . . . . . . . 4.0 g Peptic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . . . 5.0 g Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . 10.5 g Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.0 g Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g
Sabhi agar with Chloramphenicol and Cycloheximide
Selective medium for use in the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources
Chloramphenicol Gram positive and Gram negative organisms
Cycloheximide Most saprophytic molds
CZAPEK’S AGAR Czapek's Solution Agar is a synthetic medium widely used in
mycological laboratories Many moulds produce very characteristic colonies on it and may also
exude pigmented substances Aerial growth is often suppressed and sporulation may be enhanced Some moulds, however, grow poorly on this medium and may even
fail to sporulate altogether, often because of their inability to synthesize vitamins
As noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one
CZAPEK’S AGARFormulation: Sucrose . . . . . . . . . . . . . . . . 30
g NaNO3 . . . . . . . . . . . . . . . . 3.0
g K2HPO4 . . . . . . . . . . . . . . . 1.0
g MgSO4.7H2O . . . . . . . . . . .0.5
g KCl . . . . . . . . . . . . . . . . . . . . 0.5
g FeSO4.7H2O . . . . . . . . . . . 0.01
g Agar . . . . . . . .. . . . . . . . . . . . 15
g Distilled water. . . . . . . . . . 1
liter
Immunodiffusion
27
Immunoelectrophoresis
28
Complement Fixation
29
Enzyme-linked Immunosorbent Assay
30
Latex Agglutination
31
Radioimmunoassay
32
Immunoblotting
33
ENDGood evening!