SELECT MEDIA RECIPES --------------- Potato Dextrose Agar (PDA) --------------- 1000 mL Distilled water 4.0 g Potato starch 20.0 g Glucose 15.0 g Agar A general purpose sporulation medium available commercially (Difco). Preparation Suspend 39 g of powdered medium in 1 L water. Autoclave at 121C /15 minutes. --------------- Corn Meal Agar --------------- Cornmeal (yellow) 50 g Agar 15 g Distilled water 1 L A nutritionally deficient medium used to promote sporulation, especially in ascomycetes and some dematiaceous hyphomycetes Preparation: Mix cornmeal in 500 ml water. Heat for one hour in a 52 C waterbath. Filter through cheesecloth (filtrate does not need to be clear). Discard cornmeal residue. Bring volume of filtrate up to 1000 ml. Divide into 2 1L flasks. Add 7.5 g of agar to each flask. Boil to dissolve agar. Autoclave 121C/15 minutes --------------- Malt Extract Agar (MEA) --------------- 1000 mL dH2O 15 g Bacto Agar 2 g Malt Extract 2 g Yeast Extract 0.75 g Potassium Phosphate (Monobasic) Autoclave at 121 C for 20 minutes Allow to cool to below 80 C Add the following antibiotics (filter-sterilized): Streptomycin [30 ug/mL] Kanamycin [30 ug/mL] *Antibiotic stocks are in fridge and freezer, stored at [50 mg/mL] *Add 150 uL of each antibiotic for every 500 mL of media you are making. ------------- Weak Malt Extract Agar (wMEA) ------------- 1000 mL dH2O 15 g Bacto Agar 1 g Yeast Extract 2 g Malt Extract 1 g Dextrose 0.75 g Dibasic Potassium Phosphate Autoclave at 121 C for 20 minutes in loosely-capped jars (leave plenty of headspace to prevent boilover) Allow to cool to below 80 C Add the following antibiotics (filter-sterilized): Streptomycin [30 ug/mL] Kanamycin [50 ug/mL] ------------- V8 wMEA Agar (V8 wMEA) - Useful to induce sexuality/sporulation in various fungi ------------- substitute 180 mL of dH2O from wMEA Agar recipe for 1 small can of regular V8 (180 mL) same as wMEA otherwise ------------- "Leaf Agar" ------------- (Used to include "magical" leaf components to isolate tricky enophytic fungi) 200 g fresh leaf material, preferrably from host of interest wMEA agar ingredients Steep leaves in 500-800 mL of dH2O in a flask set into a hot water bath at 65 C for 1 hour Strain leaf matter and squeeze leaves to extract as much liquid as possible Add dH2O to make 1000 mL and use as liquid for standard wMEA Agar ------------- Minimal Nutrient Media plus Kanamycin/Penicillin (500 mL) ------------- Agar: 6 g Glucose: 5 g Malt extract: 1.5 g KH2PO4: 5 mL MgSO4: 5 mL CaCl2: 5 mL NaCl: 5 mL FeEDTA: 5 mL (NH4)3PO4: 5 mL Thiamine HCl: 50 µl Water: 470 mL *Add these chemicals AFTER the media has been autoclaved! Kanamycin: 25 mg Ampicillin: 25 mg ------------- To make more MNM: ------------- Chemical Amount in 50 mL mL to add to media Final concentration in media KH2PO4 2.5 g 5 mL 500 mg/L MgSO4*7H2O 0.75 g 5 mL 150 mg/L CaCl2*2H2O 0.25 g 5 mL 50 mg/L NaCl 0.125 5 mL 25 mg/L FeEDTA 0.050g 5 mL 20 mg/L (NH4)3PO4 1.25 g 5 mL 250 mg/L Thiamine HCl 0.0001 mg in 1L (!) 50 µL 0.0001 mg/L