Асептики лаборатории: Покрытие методы

Materials

1. Yeast Tryptone Agar (YTA) Yeast extract 2.0 g Tryptone 10.0 g Agar 15.0 g Distilled water up to 1000.0 ml pH 7.0 Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55°C then add 2.0 ml of 50 mg/ml cycloheximide. Aseptically dispense 18.0 ml of the melted agar per 18 mm tube then store at 4°C. The agar will solidify and will need to be melted in a steamer or microwave prior to use. 2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source NH4Cl 1.0 g NH2HPO4•2H2O 2.14 g KH2PO4 1.09 g MgSO4•7H2O 0.2 g Carbon source* 1.0 g Trace salts solution** 10.0 ml Agar 15.0 g Distilled water up to 1000.0 ml pH 7.0 Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. * Carbon sources used for experiments presented in Figure 13 include acetamide, lactose, and glycine. ** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121°C for 20 minutes). FeSO4•7H2O 300.0 mg MnCl2•4H2O 180.0 mg Co(NO3)2•6H2O 130.0 mg ZnSO4.7H2O 40.0 mg H2MoO4 20.0 mg CuSO4•5H2O 1.0 mg CaCl2 1000.0 mg HCl (0.1 N) up to 1000.0 ml 3. EHA soft agar (0.65 % w/v) Agar 6.5 g Tryptone 13.0 g NaCl 8.0 g Na Citrate•2H2O 2.0 g Glucose 3.0 g Distilled water up to 1000.0 ml Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. 4. EHA hard agar (1.2% w/v) Agar 12.0 g Tryptone 13.0 g NaCl 8.0 g Na Citrate•2H2O 2.0 g Glucose 3.0 g Distilled water up to 1000.0 ml Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. 5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v) 100 mM CaCl2 stock* 1 ml 7H9 liquid medium: Neat ** 50 ml 2XTA *** 50 ml Melt 50 ml of 2XTA and allow it to cool to ~55°C. Using aseptic technique, add the CaCl2 and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55°C incubator ≤7 days. Cooling MBTA to room temperature or 4°C will cause the CaCl2 to precipitate out of solution. * 100 mM CaCl2. stock must be stored at room temperature to prevent CaCl2 from precipitating out of solution. ** 7H9 liquid medium: Neat 7H9 broth base 4.7 g 40% glycerol stock 5 ml Distilled water up to 900.0 ml Mix the base with water then add the glycerol while stirring. Autoclave at 121°C for 20 minutes to sterilize. Store at 4°C. *** 2X Middlebrook Top Agar (2XTA, 1.0% w/v) 7H9 broth base 4.7 g Agar 1.0 g Distilled water up to 1000.0 ml Autoclave at 121°C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4°C. 6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v) 7H10 agar base 19.0 g 40% glycerol stock 12.5 ml Distilled water 887.5 ml Mix the agar base with water then add the glycerol while stirring. Heat the solution to boiling then stir for one minute to completely dissolve the base powder. Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add the following reagents: AD supplement (pre-warmed to 37°C)* 100 ml 50 mg/ml Carbenicillin ** 1.0 ml 10 mg/ml Cycloheximide ** 1.0 ml * AD supplement NaCl > 17 g > Albumin (Fraction V)> 100 g> Dextrose (D-Glucose)> 40 g> Distilled water> up to 2000.0 ml> Filter-sterilize this solution; do not autoclave. Store at 4°C. ** Filter-sterilize and store these solutions at 4°C for ≤60 days. 7. LB agar (1.5% w/v) + X-Gal (60 μg/ml) Tryptone 10.0 g Yeast extract 5.0 g NaCl 10.0 g Agar 15.0 g Distilled water up to 1000.0 ml pH 7.5 at 25°C Autoclave at 121°C for 20 minutes to sterilize. Allow the agar to cool to ~55°C then aseptically add 3.0 ml of 20 mg/ml X-Gal solution. Freshly prepare X-gal stock by dissolving 400 mg X-Gal in 20 ml dimethylformamide (DMF). Table of specific reagents: Name of the reagent Company Catalogue number Yeast extract Becton Dickenson 212750 Tryptone Becton Dickenson 211705 Agar Becton Dickenson 214030 NH4Cl Acros Organics 123340010 NH2HPO4•2H2O Sigma-Aldrich 30435 KH2PO4 Fisher Scientific BP 303-500 MgSO4•7H2O Sigma-Aldrich 230391 Acetamide Sigma-Aldrich A-0500 Lactose Fisher Scientific L6-500 Glycine Sigma-Aldrich G-7126 FeSO4•7H2O Sigma-Aldrich F8048 MnCl2•4H2O Sigma-Aldrich M-3634 Co(NO3)2•6H2O Sigma-Aldrich 230375 ZnSO4.7H2O Sigma-Aldrich Z4750 H2MoO4 Acros Organics 213621000 CuSO4•5H2O Sigma-Aldrich 209198 CaCl2 Sigma-Aldrich C1016 HCl Fisher Scientific A144-212 Cycloheximide Sigma 038K1561 Carbenicillin Cellgro 46-100-RG NaCl Fisher S271-1 Na Citrate•2H2O Fisher S279-500 Glucose (Dextrose) BD 215530 7H9 broth base BD 271310 7H10 agar base BD 262710 Glycerol Shelton IB15760 Albumin (Fraction V) Fisher S71907 X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) Teknova X1205 Dimethylformamide (DMF) Sigma D4551 Ethanol Fisher CDA19 Chlorine Bleach Chlorox 02490/06644884 CiDecon (disinfectant) Decon Laboratories, Inc. 8504 Table of specific equipment: Name of equipment Company Catalogue number Experiment Metal loops American Educational Products S17352 Streak plating Disposable plastic loops Fisher 22-363-602 Streak plating Wooden sticks Fisher 23-400-104 Streak plating Flat Toothpicks American Educational Products S67859 Streak plating Turn tables Fisher 08-758Q Spread plating Glass rods Bellco Glass NC9004380 Spread plating 4 mm glass beads Fisher 11-312B Spread plating Velveteen cloth Bel-Art Products 09-718-2 Replica plating Cylindrical block Bel-Art Products 09-718-1 Replica plating