mycology & viro_05

7/25/2019 Mycology & Viro_05

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Corn Meal Agar

Infusion from Corn Meal is the source of carbon, nitrogen, andvitamins required for organism growth in Corn Meal Agar. Agaris the solidifying agent.

Composition

Suspend appropriate amount in distilled water. Heat to boilingto dissolve the medium completely. If desired add !polysorbate "#. Sterili$e by autoclaving at % lbs pressure& ' (C) for % minutes. Mi* well and pour into sterile +etriplates.

Ingredients g/l

Corn meal infusion %#.###Agar %.###

inal pH & at '%(C) -.# #.'


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Principle

- Chlamydospore production is an accepted criterion for theidenti/cation of Candida species. Corn Meal Agar is a wellestablished mycological medium used for the cultivationof fungi and to study chlamydospores production ofCandida species. Corn Meal Agar is a general purposemedium used for the cultivation of fungi and for the study

of Candida species for chlamydospore production.- 0al1er and Huppert modi/ed this medium by adding

polysorbate "#, which then stimulated faster and plenty ofchlamydospore formation of Candida species.

- 2his is a very simple formulation containing only cornmealinfusion and agar. However this infusion has enoughnutrients to enhance the growth of fungi. Polysorbate 80is a mi*ture of oleic esters, which activates the productionof chlamydospore by Candida albicans , Candidastellatoides and Candida tropicalis .


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- Some Candida species lose their ability of chlamydospore

formation by repeated sub culturing. +ic1 a suspected colonyfrom Sabouraud 3e*trose Agar using a straight wire, and ma1e adeep cut in the Corn Meal Agar plate. 4epeat for each colony.+lace a 5amed sterile coverslip over the line of inoculum. Afterincubation for '676" hours at '%78#(C, the strea1s are e*aminedmicroscopically, through the coverslip, using low and high power

ob9ectives. C. albicans produces mycelium bearing ball7li1eclusters of budding cells and characteristics thic1 walled roundchlamydospores.

- 2he addition of glucose .'! w:v) to Corn Meal Agar enhancesthe chromogenesis of some speciesof Trichophyton e.g. Trichophyton rubrum .

- 2he addition of


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RICE EXTRACT AGAR

4ice >*tract Agar is recommended for di?erentiation of yeasts bymeans of their typical chlamydospores and on basis of

micromorphological criteria.

Composition:

- It was developed by 2aschd9ian to aid in the identi/cation ofchlamydospore producing species of Candida . 2his medium canbe used for culturing yeasts and di?erentiating them on basis ofmicromorphological characteristics particularly for di?erentiationof C. albicans and C. stellatoidea on basis of formation ofchlamydospsores.

- 4ieth had demonstrated that this medium can be used formycological diagnostic procedures. 4ice e*tract in the mediumserves as sole nutrient source. A small inoculum of suspectedCandida colony can be inoculated by strea1ing &very thinly) on

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Ingredients g/l

Concentrated ricee*tract

#.@##

Agar 6.8##inal pH & at '%(C) %." #.'


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!ngal str!ct!res !ngiChlamydospores &diameter -7 ' m,thic1, refractile cell wall with doublecontours), pseudomycelium,blastospores &diameter 87- m)

C.albicans Beryoccasionally C.stellatoidea

+seudomycelium, some times also truemycelium, usually balstospores. o

chlamydospores

Dther Candida species,

Arthrospores, blastospores, truemycelium, occasionally alsopseudomycelium

Trichospore species

Elastospores, no chlamydospores, nopseudomycelium

Dther yeast species

Ascospores in the asci +erfect yeasts

- If the specimen is heavily infected with Candida it can bestrea1ed directly on agar. Dn incubation for appro*imately F-hours at ''7'%GC culture can be directly e*amined undermicroscope through cover glass. Culture may be con/rmedfurther as suggested by A9ello et al . 2ypical morphologies can berevealed as under


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- 2he lac1 of nutrients together with the o*ygen7de/cient cultureconditions create an environment which induces the formationof speci/c morphological forms &chlamydospores andpseudomycelia in particular) in some yeasts. Dbtain o*ygen7de/cient culture conditions by covering the inoculated plateswith a cover glass.

- Baginal smears obtained using cotton wool swabs can bedirectly smeared on the surface of the culture medium.

- 2he addition of polysorbate "# further stimulateschlamydospore formation due to its content of oleic acids.

- 2he Addition of 2ween "# solution enhances the formation ofchlamydospores in most Candida species.

- 4ice >*tract Agar with '! de*trose may be used to promotechromogenesis &pigment formation) and, therefore, is helpful indistinguishing Trichophyton rubrum from Trichophytonmentagrophytes.


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"imitations:

. A germ tube is not constricted at the point of origin with theblastoconidium.

2. Candida dubliniensis also produces germ tubes and chlamydospores.rowth at elevated temperature and morphology on di?erent media

has been shown to facilitate di?erentiation of C. albicans and C.dubliniensis .

8. 2he test is only part of overall scheme for identi/cation of yeasts.urther tesing is required for de/nitive identi/cation.


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#irdseed Agar or $taibs Medi!m :

Eird Seed Agar is used for selective isolation and di?erentiationof Cryptococcus neoformans from other Cryptococcus speciesand yeasts.

Composition %&iMedia':

Ingredients g/l

Guizotia abyssinicaseeds

@#.###

Creatinine #.@"#

3e*trose #.###Chloramphenicol #.#%#

Agar '#.###inal pH & at '%(C) -.@ #.'


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(irections:

Suspend #.#" grams in FF ml distilled water. Heat to boiling to

dissolve the medium completely. Sterili$e by autoclaving at % lbspressure & ' (C) for % minutes. Cool to 6%(C and add ## mcgdiphenyl per ml of medium & ml of sterile ! w:v aqueoussolution of dipehnyl). Mi* well and pour into sterile +etri plates.

Principle:

Cryptococcus neoformans is an encapsulated yeast7li1e fungusthat can live in both plants and animals. 2his species, also 1nownby its teleomorph name, Filobasidiella neoformans , belongs to thebroad class of organisms called Jclub fungiK or divisionEasidiomycota. C. neoformans usually grows as a yeast&unicellular) and replicates by budding.

2he seeds of Guizotia abyssinica contains ca?eic acid which servesas a substrate for detection phenolo*idase, and en$yme producedby Cryptococcus neoformans. 2he action of phenolo*idase resultsin the production of melanin which is absorbed by the yeast cellwall forming a tan or dar1 brown pigment. Chloram phenicol isadded to inhibit the rowth bacteria and fast rowin fun i.


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Ingredients:Guizotia abyssinica &niger seed) %# g

lucose gLH' +D6 &potassium dihydrogen orthophosphate) g

Creatinine gAgar % g3istilled water ### mlAdditives to each %## ml bottle.+enicillin &'# units:ml) #.% ml

entamicin &6# mg:ml) #.% ml

Met)od:. rind seeds of Guizotia abyssinica as /nely as possible with an

electric mi*er and add to ### ml distilled water in a stainless steel 9ug.'. Eoil for 8# minutes, pass through /lter paper and ad9ust volume to

### ml.8. Add remaining ingredients e*cept Agar to /ltrate and dissolve.6. If required Cool to room temperature and pH to %.%.%. 3ispense into %## ml bottles.-. Add @.% g Agar to each %## ml reagent bottle.@. Autoclave at ' GC for '# minutes.". Cool to 6"GC and add #.% ml +enicillin and #.% ml entamicin toeach %## ml of Eird Seed A ar.


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Interpretation o* t)e test

+ositive test rowth with tan to golden brown pigmentedcolonies

egative test o pigmentation of colonies

"imitations:

- It has been reported that no single medium supportspigmentation of C. neoformans within a @' hrs incubationperiod. alse negative reactions may occur.

- Drganisms that produce pigmented colonies on Eirdseed agarshould be compared with growth of the same organism oranother medium such as S3A to determine if organism isnaturally pigmented.

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