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University of Alberta Microfungus Collection & Herbarium (UAMH) 1 Mixed Cereal baby food (Pablum or Heinz) 25 g Used for promoting sporulation in many fungi including dark molds, dermatophytes and other hyphomycetes and for maintaining stock cultures. Not commercially available. Also known as Weitzman-Silva Hutner Agar. A Add cold water to dry ingredients in a 1 l flask. Mix medium to promote the development of the meiotic well and autoclave at º 121C/20 minutes. states of Onygenales and other ascomycetes. Cautions: 1) Because this medium is thick and the mixed cereal often contains spore-bearing bacilli, Pulse oatmeal in blender a few times to break flakes CER should be prepared in small amounts using a into smaller pieces. Babyfood oatmeal may be used. large container and autoclaved for the full time. 2) Caution: 1) This medium boils over easily. Divide Use mixed cereal not oatmeal cereal. 3) For routine recipe into 2 one litre flasks. Adjust pH to 5.6. Auto- culture, this formulation is more suitable than a dilute form of the medium with added salts (Medium B:5, see Padhye et al. 1973). 4) Antibiotics may be added but are not recommended if the medium is to be used Non-keratinous agar media as substrates for the ascigerous state in certain members of the Gymnoascaceae pathogenic for man and animals. Padhye, A.A. et al. 1973. Ascocarp production by Nannizzia and Arthroderma on keratinous and Padhye, A.A. et al. 1973. Ascocarp production by non-keratinous media. Sabouraudia 11:109-114. Nannizzia and Arthroderma on keratinous and non-keratinous media. Sabouraudia 11:109-114. A nutritionally deficient medium used to promote A general purpose sporulation medium available sporulation, especially in ascomycetes and some Suspend 39 g of powdered medium in 1 L water. Mix cornmeal in 500 ml water. Heat for one hour in a 52 C waterbath or autoclave for 10 minutes at 121 C. Filter through cheesecloth (filtrate does not need to be clear) . Discard cornmeal residue. Bring volume of filtrate up to 1000 ml. Divide filtrate evenly into two 1 The media recipes included here are those in common use at UAMH for enhancing sporulation. Recipes for these media as well as additional media and some techniques are described also in Kane, J., R.C. Summerbell, L. Sigler, S. Krajden, G. Land. 1997. Laboratory handbook of dermatophytes. A clinical guide and laboratory manual of dermatophytes and other filamentous fungi from skin, hair and nails. Star Publishing Co., Belmont, CA. 2 University of Alberta Microfungus Collection & Herbarium (UAMH) flasks. Add 7.5 g of agar to each flask. Boil to dissolve agar. Autoclave 121C/15 minutes. Note: To promote sporulation in ascomycetes & McGinnis, M. 1980. Laboratory handbook of medical Mix ingredients and heat to dissolve agar. Autoclave mycology. Academic Press, Harcourt Brace at 121C/15 min. Dispense into plates or tubes. Malloch, D. 1981. Moulds. Their isolation, cultiva- tion and identification. Univ. of Toronto Press. p. 26. To promote sporulation in dark-colored fungi, yeasts and some coelomycetes and ascomycetes. Dissolve and sterilize 121C/15 minutes. Caution: Use fresh V-8 juice and store plates at 5C in dark. Available commercially from BBL. A similar medium is also available as Mycobiotic agar (Difco). Uchida, J.Y. et al. 1986. Basidiospore formation by Ceratobasidium sp. on agar. Mycologia 78:587-592. A selective medium for isolation of most pathogenic fungi that are tolerant of cycloheximide. Mix, heat with agitation until boiling. Sterilize 15 min. @ 118 C. Caution: Avoid overheating. Georg, L.K. et al. 1954. Use of cycloheximide in the selective isolation of fungi pathogenic to man. J. Lab. To promote the sporulation of Onygenales and other ascomycetes. Dissolve and autoclave 121C/15 minutes. Reference Takashio, M. 1972. Sexual reproduction of some Arthroderma and Nannizzia on diluted sabouraud agar with or without salts. Mykosen 15:11-17. Commercially available. Similar to Sabouraud glucose Preparation Dissolve and heat to boiling with agitation. Autoclave 121C/15 minutes. Cool and aseptically add antibiotics to give final concentrations of chloramphenicol 50 ug/ml and streptomycin 30 ug/ml. Caution: Caution: Avoid overheating. Note: This modification of Sabouraud glucose agar incorporates soy based University of Alberta Microfungus Collection & Herbarium (UAMH) 3 peptone and yeast extract. The medium may be made selective by addition of cycloheximide (similar medium available commercially as Mycosel agar). Reference Carmichael, J.W. 1962. Chrysosporium and some other aleuriosporic hyphomycetes. Can. J. Bot. Also known as Sabouraud agar, Sabouraud dextrose agar or peptone glucose agar. Available commer- cially. Follow manufacturer's instructions. Purpose Widely used for growth of fungi but many fungi do not sporulate well on this medium. For use as a Mix well using magnetic stir bar. Autoclave 121C /30 primary isolation medium, antibacterial antibiotics are min. Cool to 55C and pour plates, keeping medium Mix ingredients and adjust pH to 7.0. Autoclave at Hutchison, L.J., Mycotaxon 42:389 (387-504) 1991; 121C/15 min. Notes: Peptone source may vary. Some Marx, D.H., Phytopathology 59: 153-163 1969. fungi demonstrate different colonial morphologies when grown on modifications of Sabouraud agar containing different peptones. Preparation of medium with antibiotics Odds, F.C. 1992. Sabouraud('s) agar. J. Med. Vet. Mix ingredients and autoclave 121C for 20 minutes. Note: The calcium carbonate does not dissolve completely, so plates will look cloudy. CaCO3 is not required, but causes the fungus to grow faster. Winder, R.S. Mycological Research 110:612-623, Used as basal support medium in slide culture method and for stimulation of sporulation especially of darkly pigmented fungi. Preparation Mix well. Autoclave 121C/15 min. Pour 20 ml amounts into 100 mm or 10 -12 ml into 60 mm petri dishes. Reference Harris, J. 1986. Modified methods for fungal slide culture. J. Clin. Microbiol. 9:460-461. 4 University of Alberta Microfungus Collection & Herbarium (UAMH) ANTIBIOTIC SOLUTIONS Chloramphenicol Stock Solution Fungicidal, for inhibition of many saprophytic fungi. For inhibition of bacteria in culture media. Many keratinophilic fungi resembling dermatophytes Dissolve contents of capsules in alcohol. Add water to achieve stock concentration of 25 mg/ml. Add 2 ml to Dissolve cycloheximide in acetone. Add to hot 1 l of medium to achieve final concentration of 50 μg/ ml; add 4 ml to 1 l to obtain a final concentration of 100 μg/ml. Note: Use pure chloramphenicol, not Purpose For inhibition of bacteria in culture media. For inhibition of Gram negative bacteria. Dissolve contents of capsules in water to achieve stock concentration of 25 mg/ml. Add 2 ml to 1000 ml Use injectable gentamicin (Garamycin) to achieve of medium to achieve final concentration of 50 μg/ final concentration of 50 μg/ml. Formulation for SAB ml; add 4 ml to 1000 ml to obtain a final concentration is based on pediatric strength of 10 mg/ml gentamicin dispensed in 2 ml vials. The formulation for adult strength injectable Garamycin is 60 mg/ml or 80 Purpose For inhibition of bacteria. Procedure Dilute 1:100 by adding 1 ml of injectable streptomycin to 99 ml sterile distilled water to obtain a stock concentration of 5 mg/ml. Aliquot into 7 ml volumes. Label with concentration and date prepared. Store at -20 C (or -70 C). Add 6 ml to 1 l of medium to achieve a final concentration of 30 μg/ml.


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