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Media Appendix Media appendix Aspergillus flavus and parasiticus agar Czapek concentrate (AFPA) NaN03 30 g Peptone, bacteriological 10 g EI 5g Yeast extract 20 g MgS04.7H20 5 g Ferric ammonium citrate 0.5 g FeS04.7H20 0.1 g Chloramphenicol 100 mg Water, distilled 100 ml Agar 15 g Czapek concentrate will keep indefinitely Dichloran 2mg without sterilisation. The precipitate of (0.2% in ethanol, 1.0 ml) Fe(OH)3 which forms in time can be resus­ Water, distilled 1 litre pended by shaking before use. After addition of all ingredients, sterilise by autoclaving at 121 DC for 15 min. The final pH of this medium is 6.0-6.5. Czapek iprodione dichloran agar (CZID) Sucrose 30 g Creatine sucrose neutral agar (CSN) Yeast extract 5g CS concentrate 10 ml Chloramphenicol 100 mg Sucrose 10 g Dichloran 2mg Creatine 5.0 g (0.2% in ethanol, 1 mD KH2P04 1.0 g Czapek concentrate 10 ml Bromocresol purple 0.05 g Trace metal solution 1 ml Agar 15 g Agar 15 g Water, distilled to 1 litre Water, distilled 1 litre Iprodione (suspension) 1 ml Creatine sucrose (CS) concentrate Add iprodione suspension [0.3 g Roval 50WP KCI 5 g (Rhone-Poulenc Agro-Chemie, Lyon, France) MgS04.7H2 5 g in 50 ml sterile water, shaken before addi­ FeS04.7H20 0.1 g tion to medium] after autoclaving. Sterilise ZnS04 .7H20 0.1 g by autoclaving at 121 DC for 15 min. This CuS04.5H20 0.05 g formulation is an adaptation of the original Water, distilled to 100 ml published formulation (Abildgren et al., Sterilise by autoclaving at 121 DC for 15 min. 1987), made from basic ingredients rather Final unadjusted pH is approximately 6.8. A than using commercial Czapek-Dox broth. pH between 5.5 and 6.8 is satisfactory. For Chloramphenicol (l00 mg per litre) replaces identification of Penicillium subgenus Peni­ the original combination of chlortetracycline cillium species. (50 mg) and chloramphenicol (50 mg). 509 510 Media appendix Czapek yeast extract agar (CYA) Dichloran 2 mg KzHP04 1 g (0.2% in ethanol, 1 ml) Czapek concentrate 10 ml Agar 15 g Trace metal solution 1 ml Water, distilled I litre Yeast extract, powdered 5 g Mter addition of all ingredients, sterilise by Sucrose 30 g autoclaving at 121 DC for 15 min. The final Agar 15 g pH of this medium is 5.5-6.0. Water, distilled 1 litre Refined table grade sucrose is satisfactory for use in CYA provided it is free from sulphur Dichloran 18% glycerol agar (DG18) dioxide. Sterilise by autoclaving at 121 DC Glucose 10 g for 15 min. Final pH 6.7. Peptone 5 g KH2P04 1 g MgS04.7H20 0.5 g Czapek yeast extract agar with 20% Glycerol, AR. 220 g sucrose (CY20S) Agar 15 g KzHP04 1 g Dichloran 2 mg Czapek concentrate 10 ml (0.2% w/v in ethanol, 1 ml) Yeast extract 5 g Chloramphenicol 100 mg Sucrose 200 g Water, distilled 1 litre Agar 15 g To produce this medium, add minor ingredi­ Water, distilled 1 litre ents and agar to ca 800 ml distilled water. Sterilise by autoclaving at 121 DC for 15 min. Steam to dissolve agar, then make to 1 litre Final pH 5.2. with distilled water. Add glycerol: note that the final concentration is 18% weight in weight, not weight in volume. Sterilise by Dichloran chloramphenicol malt extract autoclaving at 121 DC for 15 min. The final agar (DCMA) 3.w of this medium is 0.955 and pH is in the Malt extract 10 g range 5.5 to 5.8. Dichloran 2 mg (0.2% w/v in ethanol, 1 ml) Chloramphenicol 0.1 g Dichloran rose bengal chloramphenicol Agar 15 g agar (DRBC) Distilled water to 1 litre Glucose 10 g Sterilise by autoclaving at 121 DC for 15 min. Peptone, bacteriological 5 g Recommended for identification ofAlternaria KH2P04 1 g species and some other dematiaceous Hyph­ MgS04.7H20 0.5 g omycetes. Final pH is 5.5-6.0. Agar 15 g Rose bengal 25 mg (5% w/v in water, 0.5 ml) Dichloran chloramphenicol peptone Dichloran 2 mg agar (DCPA) (0.2% w/v in ethanol, 1 ml) Peptone 15 g Chloramphenicol 100 mg KH2P04 19 Water, distilled 1 litre MgS04·7H20 0.5 g Mter the addition of all ingredients, sterilise Chloramphenicol 0.1 g by autoclaving at 121 DC for 15 min. The Media appendix 511 final pH is in the range 5.5 to 5.8. Store Glycerol, analytical grade 250 g prepared media away from light; photo­ Agar 12 g products of rose bengal are highly inhibitory Water, distilled 750 ml to some fungi, especially yeasts. In the dark, Glycerol for G25N should be of high quality, the medium is stable for at least one month with a low (1%) water content. If a lower at 1-4DC. The stock solutions of rose bengal grade is used, allowance should be made for and dichloran need no sterilisation, and are the additional water. Sterilised by auto­ also stable for very long periods. The origi­ claving at 121 DC for 15 min. Final pH 7.0. nal formulation of King et al. (1979) con­ tained chlortetracycline as the antibiotic. Pitt and Hocking (1985) substituted chlor­ Malt acetic agar (MAA) amphenicol, an effective antibiotic originally To 100 ml sterile tempered Malt Extract recommended for mycological media by Put Agar, aseptically add 0.5 ml glacial acetic (1974). Media containing chloramphenicol acid, giving a final concentration of 0.5% are easier to prepare, are not affected by acetic acid. Mix well before pouring. Note autoclaving, and have greater long term that MAA cannot be autoclaved or reheated stability. as the low pH (approx. 3.2) causes the agar gel to break down if the medium is subjected to any further heat treatment after the Dichloran rose bengal yeast extract addition of the acetic acid. There is no need sucrose agar (DRYS) to sterilise the glacial acetic acid. Yeast extract 20 g Sucrose 150 g Dichloran 2mg Malt extract agar (MEA) (0.2% in ethanol, 1 rol) Malt extract, powdered 20 g Rose bengal 25 mg Peptone I g (5% w/v in water, 0.5 rol) Glucose 20 g Chloramphenicol 50 mg Agar 20 g Agar 20 g Water, distilled I litre Water, distilled, to 11itre Commercial malt extract used for home Chlortetracycline 50 mg brewing is satisfactory for use in MEA, as is (1% in water, filter sterilised, 5 rol) bacteriological peptone. Sterilise by auto­ Sterilise all ingredients except chlortetracycl­ claving at 121 DC for 15 min. Do not sterilise ine by autoclaving at 121 DC for 15 min. Add for longer, as this medium will become soft chlortetracycline after tempering to 50DC. In on prolonged or repeated heating. Final pH our experience, chloramphenicol at twice the 5.6. concentration specified (i.e. 100 mg/l), ad­ equately controls bacteria in most situations, and this avoids the need for a second anti­ Malt extract yeast extract 50% glucose biotic which must be filter sterilised. agar (MY50G) Malt extract 10 g Yeast extract 2.5 g 25% Glycerol nitrate agar (G25N) Agar 10 g ~HP04 0.75 g Water, distilled, to 500 g Czapek concentrate 7.5 ml Glucose, A.R. 500 g Yeast extract 3.7 g Add the minor constituents and agar to ca 512 Media appendix 450 ml distilled water and steam to dissolve Malt extract yeast extract 5% (or 10%) the agar. Immediately make up to 500 g salt 12% glucose agar (MY5·12 and with distilled water. While the solution is MY10·12) still hot, add the glucose all at once and stir Malt extract 20 g rapidly to prevent the formation of hard Yeast extract 5g lumps of glucose monohydrate. If lumps do NaCI 50 g form, dissolve them by steaming for a few (100 g for MY10-12) minutes. Sterilise by steaming for 30 min; Glucose 120 g note that this medium is of a sufficiently low Agar 20 g llw not to require autoclaving. Food grade Water, distilled, to 1 litre glucose monohydrate (dextrose) may be used Sterilise MY5-12 by autoclaving at 121 DC for in this medium instead of analytical reagent 10 minutes, and MY10-12 by steaming for 30 grade glucose, but allowance must be made minutes. Overheating of these media will for the additional water present. Use 550 g cause softening. The final aw of MY5-12 is of C6Hlz06.HzO, and 450 g of the basal 0.93 and of MY10-12 is 0.88. medium. As the concentration of water is unaffected by this procedure, the quantities of the minor ingredients are unaltered. The Oxytetracycline glucose yeast extract finalllw of this medium is 0.89. Final pH is agar (OGY) 5.3. Glucose 20 g Yeast extract 5 g Agar 15 g Malt extract yeast extract 70% glucose Water, distilled 1 litre fructose agar (MY70GF) Oxytetracycline 100 mg Malt extract 6 g Sterilise by autoclaving at 121 DC for 15 min. Yeast extract 1.5 g After tempering to 50 DC, add 10 ml of filter Agar 6 g sterilised oxytetracycline (Terramycin, Pfizer; Water, distilled, to 300 g 0.1% aqueous) per 100 ml of medium. The Glucose, A.R. 350 g final pH is 6.8-7.2. Fructose, A.R. 350 g Mter steaming to dissolve agar, make the solution accurately to 300 g with water and, Potato dextrose agar (PDA) while it is still hot, add both sugars. Steam Potatoes 250 g gently for up to 30 minutes to completely Glucose 20 g dissolve the sugars.
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