Alternaria Broth For toxin production and DNA extraction ddH2O 1 liter 2 liters 3 liters DL Asparagine 1.2 g 2.4 g 3.6 g NaCl 0.1 g 0.2 g 0.3 g K2HPO4_3H2O 1.2 g 2.4 g 3.6 g MgSO4 (anhydrous) ** 0.25 g 0.5 g 0.75 g Yeast Extract 0.5 g 1.0 g 1.5 g CaCl2 (not necessary) 0.13 g D-Glucose (anhydrous) 20.7 g 41.4 g 62.1 g (=Dextrose) Heat at moderate temperature to get into solution Dispense 100ml into 250ml flasks, cap flasks with a cotton foil lid and autoclave. Grow fungi on PDA until plate is populated sufficiently, app. 4-5 days. Using a sterile 10 ml pipette, aseptically cover the culture with 5ml sterile ddH2O. Gently rub the mycelia with the tip of the pipette to loosen the spores. Tip the petri dish to stir up the spores. Using good sterile technique, transfer 2mls of the ddH2O-fungal suspension into the 250 ml. flasks. Shake at 115 rpm at RT for 5-10 days. ** MgSO4*7H2O 0.5 g PDA 39g Potato Dextrose Agar 1000 ml ddH2O Autoclave Acidified PDA Make the PDA medium Autoclave, let cool to 60OC Add 1.25 ml of 20% lactic acid per liter (wear gloves) Weak PDA 1.2g Potato Dextrose Broth 16 g Bacto-Agar 1000ml ddH2O Autoclave Skim Milk 15g Skim Milk 1000ml ddH2O Dispense 4ml to each test tube. Autoclave. Cool, store in the media refrigerator. LB Media (Luria-Bertani Medium) 10g. Bacto-Tryptone 5g. Bacto-Yeast Extract 10g NaCl 950 ml ddH2O Stir until the solutes dissolve. Adjust the pH to 7.0 with 5 N NaOH (app. 0.2 ml). Adjust the volume of the solution to 1 liter with ddH2O. Autoclave LB Media 10g. Bacto-Tryptone 5g. Bacto-Yeast Extract 5g NaCl 950 ml ddH2O Stir until the solutes dissolve. Adjust the pH to 7.0 with 5 N NaOH (app. 0.2 ml). Adjust the volume of the solution to 1 liter with ddH2O. Autoclave 2 % Water Agar 20g. Difco Agar, granulated 1000ml ddH2O Autoclave 0.2 % Water Agar, for Dilution Plating 2g. Difco Agar, granulated 1000ml ddH2O Boil to get into solution. Dispense at 40ml/milk dilution bottle. Cap. Autoclave V-8 Juice Medium 87.5ml V-8 juice 1.5g. CaCO3 10g. Bacto Agar 500ml. ddH2O Autoclave Recipe for V-8 Slants For 1000 ml. For 500 ml. 175 ml. V-8 88 ml. V-8 3g. CaCO3 1.5g. CaCO3 20g. Bacto Agar 10g. Bacto Agar 800ml. ddH2O 400ml. ddH2O Heat until agar dissolves. Use aspirating syringe to dispense into test tubes: use 6mls/tube. Rinse syringe with hot soapy water as soon as you are finished dispensing, then distilled water. Autoclave. When the tubes come out of the autoclave, lean tubes against a crisper lid to create the slant. Potato Carrot Agar 20g. Carrot 20g. Potato (exclude the skin) 20g. Bacto Agar Dice the vegetables into small pieces. Place pieces into a beaker with aprox. 200ml ddH2O. Boil on stir/hot plate until carrot is soft, aprox. 50-60 min.on low heat (#3-4). Force slurry through a fine sieve (available in Mary Olson’s lab, Marley 719). Add ddH2O to make l liter, then add agar. Mix and autoclave. While pouring plates, occasionally swirl to keep carrot/potato mix suspended. ARSA MEDIA This medium was developed specifically for the isolation and enumeration of A. radicina from carrot seed and field soil. Step I ARSA is prepared in two parts: Part A 16.0g Bacto Agar 1.0g KH2PO4 1.0g KNO3 0.5g KCl 0.5g MgSO4 500ml ddH2O Part B 5.0 g Glucose (normally prepared with Sodium polypectate (Sigma P-1879) 500 ml ddH2O (Mix to get into solution, then remove the stir bar from Part B!!!) Autoclave Parts A and B separately, cool to 50 C, and combine. Use good sterile technique. Step II 1) Prepare Antibiotics: 50mg chlortetracycline HCl (Sigma C-4881) 50mg streptomycin sulfate (Sigma S-6501) Place each into a sterile 15 ml centrifuge tube; add 10 ml sterile ddH2O and vortex to get into solution. 2) Prepare Fungicides: A) Mertect 340-F (contains 106 mg thiabendazole, Merick & Co.) -Mertect has the consistency of sludge: you need to stir vigorously to get it into solution. Use a sterile 10ml pipette, and stir for app. 4-5 min. until you get a consistent mixture. -Make a 10X stock solution by placing 1 ml Mertect with 9 ml sterile ddH2O into a sterile 15 ml centrifuge tube and vortex. B) 2.5 ml 10X Mertect 5mg Botran 75WP (contains 4 mg dicloran, Upjohn Co.) 200mg Bayleton 50WP (contains 100 mg triadimefon, Miles) 7.5mls sterile ddH2O Place each into a 15 ml centrifuge tube and vortex to get into solution. Step III Add the antibiotics and the fungicides to the combined parts A and B. Pour plates. KMB (Kings Medium B Agar) 20g. Proteose Peptone No. 3 (Difco) 1.5g. K2HPO4 0.738g MgSO4 (Anhydrous) or 1.5g MgSO4*7H2O 10ml Glycerol 990ml ddH2O 15g Bacto Agar Mix Proteose Peptone, K2HPO4, MgSO4, and ddH2O till in solution. Add glycerol with a pipette. Rinse the pipette in the solution to remove residual glycerol from the inside of the pipette. Adjust pH to 7.4. Add agar and autoclave. YDC (Yeast Dextrose Carbonate Agar) Solution I 20g. Dextrose 800 ml ddH2O Solution II 10g. Yeast Extract 20g. CaCO3 10 ml Glycerol 800ml ddH2O 15g Bacto Agar Make Solution I and Solution II, remove the stir bar from Solution I only. Autoclave separately. Combine both solutions using good sterile technique. The CaCO3 can easily precipitate, so gently mix media periodically while pouring the media into plates or tubes. Komada’s Medium (1975) 1g. K2HPO4 (or 1.3g K2HPO4*3 H2O) 0.5g. KCl 0.25g MgSO4 Anhydrous (or 0.5g MgSO4*7H2O) 2g L-asparagine 20g D-galactose 10mg Fe-Na-EDTA 20g Bacto-Agar 1000ml ddH2O Autoclave, then add to the warm media (cool to 55*C first): 1g Quintozene (PCNB) 0.5g Oxgall 1g Na2B4O7*10H2O 300mg Streptomycin sulfate Adjust pH to 3.8+0.2 with a solution of 10% phosphoric acid. Isolation from soil: dilution plate method. Keep the dishes in diffuse light for incubation. Under these conditions, the colonies of F. oxysporum are pigmented with a reddish purple color and surmounted by a pinkish white aerial mycelium. Nash and Snyder’s Medium (1962) 15g. Peptone 1g. K2HPO4 0.25g MgSO4 Anhydrous (or 0.5g MgSO4*7H2O) 20g Bacto-Agar 1000ml ddH2O Autoclave, then add to the warm media (cool to 55*C first): 1g Quintozene (PCNB) 100mg Penicillin 300mg Streptomycin sulfate Prepare the petri dishes 5-7 days before proceeding with the isolations. Incubate in dark @24 *C. Light will kill Fusarium. Too moist plates will favor bacteria.