Media Appendix Aspergillus flavus and parasiticus agar (AFPA) MgSO4Á7H2O5g FeSO Á7H O 0.1 g Peptone, bacteriological 10 g 4 2 Water, distilled 100 ml Yeast extract 20 g Ferric ammonium citrate 0.5 g Czapek concentrate will keep indefinitely without Chloramphenicol 100 mg sterilisation. The precipitate of Fe(OH) which forms Agar 15 g 3 Dichloran 2 mg in time can be resuspended by shaking before use. (0.2% in ethanol, 1.0 ml) Water, distilled 1 l Czapek iprodione dichloran agar (CZID) After addition of all ingredients, sterilise by auto- Sucrose 30 g Yeast extract 5 g claving at 1218C for 15 min. The final pH of this Chloramphenicol 100 mg medium is 6.0–6.5. Dichloran 2 mg (0.2% in ethanol, 1 ml) Creatine sucrose neutral agar (CSN) Czapek concentrate 10 ml CS concentrate 10 ml Trace metal solution 1 ml Sucrose 10 g Agar 15 g Creatine 5.0 g Water, distilled 1 l Iprodione (suspension) 1 ml KH2PO4 1.0 g Bromocresol purple 0.05 g Add iprodione suspension [0.3 g Roval 50 WP Agar 15 g Water, distilled to 1 l (Rhone-Poulenc Agro-Chemie, Lyon, France) in 50 ml sterile water, shaken before addition to med- Creatine sucrose (CS) concentrate ium] after autoclaving. Sterilise by autoclaving at KCl 5 g 1218C for 15 min. This formulation is an adaptation MgSO4Á7H2O5gof the original published formulation (Abildgren FeSO4Á7H2O 0.1 g et al., 1987) made from basic ingredients rather ZnSO Á7H O 0.1 g 4 2 than using commercial Czapek–Dox broth. Chlor- CuSO4Á5H2O 0.05 g Water, distilled to 100 ml amphenicol (100 mg/l) replaces the original combi- nation of chlortetracycline (50 mg) and chloram- Sterilise by autoclaving at 1218C for 15 min. Final phenicol (50 mg). unadjusted pH is approximately 6.8. A pH between 5.5 and 6.8 is satisfactory. For identification of Czapek yeast extract agar (CYA) Penicillium subgenus Penicillium species. K2HPO4 1g Czapek concentrate Czapek concentrate 10 ml Trace metal solution 1 ml NaNO3 30 g Yeast extract, powdered 5 g KCl 5 g Sucrose 30 g 423 424 Media Appendix Agar 15 g Dichloran 2 mg Water, distilled 1 l (0.2% w/v in ethanol, 1 ml) Chloramphenicol 100 mg Refined table grade sucrose is satisfactory for use in Water, distilled 1 l CYA provided it is free from sulphur dioxide. Ster- ilise by autoclaving at 1218C for 15 min. The final To produce this medium, add minor ingredients and pH is 6.7. agar to ca. 800 ml distilled water. Steam to dissolve agar, then make to 1 l with distilled water. Add Czapek yeast extract agar with 20% sucrose glycerol: note that the final concentration is 18% (CY20S) w/w, not w/v. Sterilise by autoclaving at 1218C for 15 min. The final a of this medium is 0.955 and pH K2HPO4 1g w Czapek concentrate 10 ml is in the range 5.5–5.8. Yeast extract 5 g Sucrose 200 g Dichloran rose bengal chloramphenicol agar Agar 15 g (DRBC) Water, distilled 1 l Glucose 10 g Sterilise by autoclaving at 1218C for 15 min. The Peptone, bacteriological 5 g final pH is 5.2. KH2PO4 1g MgSO4Á7H2O 0.5 g Dichloran chloramphenicol malt extract agar Agar 15 g (DCMA) Rose bengal 25 mg (5% w/v in water, 0.5 ml) Malt extract 10 g Dichloran 2 mg Dichloran 2 mg (0.2% w/v in ethanol, 1 ml) (0.2% w/v in ethanol, 1 ml) Chloramphenicol 100 mg Chloramphenicol 0.1 g Water, distilled 1 l Agar 15 g Water, distilled to 1 l After the addition of all ingredients, sterilise by autoclaving at 1218C for 15 min. The final pH is in Sterilise by autoclaving at 1218C for 15 min. the range 5.5–5.8. Store prepared media away from Recommended for identification of Alternaria spe- light; photoproducts of rose bengal are highly inhi- cies and some other dematiaceous Hyphomycetes. bitory to some fungi, especially yeasts. In the dark, The final pH is 5.5–6.0. the medium is stable for at least 1 month at 1–48C. Dichloran chloramphenicol peptone agar (DCPA) The stock solutions of rose bengal and dichloran need no sterilisation, and are also stable for very Peptone 15 g long periods. The chlortetracycline in the original KH2PO4 1g MgSO4Á7H2O 0.5 g formulation of King et al. (1979) has been replaced Chloramphenicol 0.1 g with chloramphenicol, an effective antibiotic origin- Dichloran 2 mg ally recommended for mycological media by Put (0.2% in ethanol, 1 ml) (1974). Media containing chloramphenicol are Agar 15 g Water, distilled l l easier to prepare, are not affected by autoclaving and have greater long-term stability. After addition of all ingredients, sterilise by auto- claving at 1218C for 15 min. The final pH of this Dichloran rose bengal yeast extract sucrose agar medium is 5.5–6.0. (DRYS) Dichloran 18% glycerol agar (DG18) Yeast extract 20 g Sucrose 150 g Glucose 10 g Dichloran 2 mg Peptone 5 g (0.2% in ethanol, 1 ml) KH2PO4 1g Rose bengal 25 mg MgSO4Á7H2O 0.5 g (5% w/v in water, 0.5 ml) Glycerol, A.R. 220 g Chloramphenicol 50 mg Agar 15 g Agar 20 g Media Appendix 425 Water, distilled to 1 l Agar 10 g Chlortetracycline 50 mg Water, distilled to 500 g (l% in water, filter sterilised, 5 ml) Glucose, A.R. 500 g Sterilise all ingredients except chlortetracycline by Add the minor constituents and agar to ca. 450 ml autoclaving at 1218C for 15 min. Add chlortetracy- distilled water and steam to dissolve the agar. cline after tempering to 508C. In our experience, Immediately make up to 500 g with distilled water. chloramphenicol at twice the concentration speci- While the solution is still hot, add the glucose all at fied (i.e. 100 mg/l) adequately controls bacteria in once and stir rapidly to prevent the formation of most situations, and this avoids the need for a sec- hard lumps of glucose monohydrate. If lumps do ond antibiotic which must be filter sterilised. form, dissolve them by steaming for a few minutes. Sterilise by steaming for 30 min; note that this med- 25% Glycerol nitrate agar (G25N) ium is of a sufficiently low aw not to require auto- K2HPO4 0.75 g claving. Food grade glucose monohydrate (dex- Czapek concentrate 7.5 ml trose) may be used in this medium instead of Yeast extract 3.7 g Glycerol, analytical grade 250 g analytical reagent grade glucose, but allowance Agar 12 g must be made for the additional water present. Water, distilled 750 ml Use 550 g of C6H12O6ÁH2O, and 450 g of the basal Glycerol for G25N should be of high quality, with a medium. As the concentration of water is unaf- low (1%) water content. If a lower grade is used, fected by this procedure, the quantities of the allowance should be made for the additional water. minor ingredients are unaltered. The final aw of Sterilised by autoclaving at 1218C for 15 min. The this medium is 0.89. The final pH is 5.3. final pH is 7.0. Malt extract yeast extract 70% glucose fructose agar Malt acetic agar (MAA) (MY70GF) To 100 ml sterile tempered malt extract agar, asepti- Malt extract 6 g cally add 0.5 ml of glacial acetic acid, giving a final Yeast extract 1.5 g Agar 6 g concentration of 0.5% acetic acid. Mix well before Water, distilled to 300 g pouring. Note that MAA cannot be autoclaved or Glucose, A.R. 350 g reheated as the low pH (approx. 3.2) causes the agar Fructose, A.R. 350 g gel to break down if the medium is subjected to any After steaming to dissolve agar, make the solution further heat treatment after the addition of acetic acid. accurately to 300 g with water and, while it is still There is no need to sterilise the glacial acetic acid. hot, add both sugars. Steam gently for up to 30 min Malt extract agar (MEA) to completely dissolve the sugars. Further sterilisa- tion is unnecessary: contaminant micro-organisms Malt extract, powdered 20 g of any kind are unable to grow on this medium, Peptone l g Glucose 20 g which is about 0.76 aw. MY70GF will take some Agar 20 g hours to gel, because of the low proportion of water Water, distilled l l and agar. If possible, allow 24 h after pouring for Commercial malt extract used for home brewing is the medium to attain gel strength before use. satisfactory for use in MEA, as is bacteriological pep- Malt extract yeast extract 5% (or 10%) salt 12% tone. Sterilise by autoclaving at 1218C for 15 min. Do glucose agar (MY5-12 and MY10-12) not sterilise for longer, as this medium will become soft on prolonged or repeated heating. The final pH is 5.6. Malt extract 20 g Yeast extract 5 g Malt extract yeast extract 50% glucose agar NaCl 50 g (MY50G) (100 g for MY10-12) Glucose 120 g Malt extract 10 g Agar 20 g Yeast extract 2.5 g Water, distilled to 1 l 426 Media Appendix Sterilise MY5-12 by autoclaving at 1218C for Trace metal solution 10 min, and MY10-12 by steaming for 30 min. Over- CuSO Á5H O 0.5 g heating of these media will cause softening. The 4 2 ZnSO4Á7H2O1g final aw of MY5-12 is 0.93 and of MY10-12 is 0.88.