各类藻类培养基.doc

AJS (Acidified JM:SE)Freshwater alga Dunaliella acidophilaMedium Acidified 97:3 mixture of JM and SE2See separate recipes(JM and SE2). For approximately 1litre of final medium, mix 970ml JM with 30ml SE2. Add 10ml of conc H2SO4 to give a pH of approximately 1.5. Autoclave at 15 psi for 15 minutes.ANT (Antias Medium)Marine algae Stock Trace metals stock solution (chelated) per litre EDTANa2.2H2O 3.24 g FeCl3.4H2O 1.08 g MnSO4.4H2O 0.450 g ZnSO4.7H2O 0.230 g Na2MoO4.2H2O 0.097 g CuSO4.5H2O 0.01 g CoSO4.7H2O 0.0056 gMake up to 1 litre with distilled water and adjust pH to 7.6 - 7.8 with dilute HCl or NaOH. Store frozen. Medium per litre KNO3 0.05 g NaH2PO4.2H2O 0.0078 g Tris tris(hydroxymethyl)aminomethane 1.0 g Glycine 0.3 g Trace metals stock solution (chelated) 2.5 ml Thiamine HCl 500.0 g Cyanocobalamin 2.0 g Biotin 1.0 g Filtered natural seawater 800.0 mlMake up to 1 litre with distilled water and autoclave at 15 psi. Final pH should be 7.6 - 7.8. Ref Antia, Cheng & Taylor (1969) ASW (Artificial Seawater)Marine algae Stocks (1) Extra salts per litre NaNO3 30.0 g Na2HPO4 1.2 g K2HPO4 1.0 g (2) Vitamin solution per litre Biotin 0.0002 g Calcium pantothenate 0.02 g Cyanocobalamin 0.004 g Folic acid 0.0004 g Inositol 1.0 g Nicotinic acid 0.02 g Thiamine HCl 0.1 g Thymine 0.6 g May be stored frozen at -20CMedium per litre *Ultramarine Synthetica sea salts 33.6 g Extra salts stock solution (1) 3.75 ml Vitamin stock solution (2) 2.5 ml Soil extract (SE1 - see recipe) 25.0 ml Tricine 0.50 gMake up to 1 litre with distilled water and adjust pH to 7.6 - 7.8 with 1M NaOH or HCl. Autoclave at 15 psi. Supply * Waterlife Research Industries Ltd, 476 Bath Road, Longford, West Drayton, Middlesex UB7 0ED, UK. Tel (01753) 685696 Fax (01753) 685437 ASW + barley (Artificial Seawater + barley grains)Marine dinoflagellate Oxyrrhis MediumFollow the recipe for ASW but add approximately one grain of barley to each 25 ml of prepared medium prior to autoclaving. 2ASW (double strength Artificial Seawater)Marine algaeMediumFollow the recipe for ASW but add 35 g NaCl per litre prior to autoclaving. ASW:BGMarine algaeMedium 1:1 mixtureSee separate recipes. Mix aseptically. ASW:SESMarine algaeMedium 3:1 mixtureSee separate recipes. Mix before autoclaving. ASWP (Artificial Seawater for Protozoa)Marine protozoa Stock Solution (1) per litre NaNO3 5.625 g Na2HPO4 0.225 g K2HPO4 0.188 gMedium per litre *Ultramarine Synthetica sea salts 33.6 g Tricine 0.5 g Stock solution (1) 10.0 ml Soil extract (SE2 - see recipe) 50.0 mlMake up to 1 litre with deionized water and adjust pH to 7.6 - 7.8 with 1M NaOH or HCl. Autoclave at 15 psi for 15 minutes. Supply * as in ASW recipe BB (Bolds Basal Medium)Freshwater algae Stocks per 200 ml (1) NaNO3 5.0 g (2) MgSO4.7H2O 1.5 g (3) NaCl 0.5 g (4) K2HPO4 1.5 g (5) KH2PO4 3.5 g (6) CaCl2.2H2O 0.5 g (7) Trace elements solution: per litre ZnSO4.7H2O 8.82 g MnCl2.4H2O 1.44 g MoO3 0.71 g CuSO4.5H2O 1.57 g Co(NO3)2.6H2O 0.49 g May need autoclaving to dissolve. per 100 ml (8) H3BO3 1.14 g (9) EDTA - KOH solution: EDTANa2 5.0 g KOH 3.1 g per litre (10) FeSO4.7H2O 4.98 g conc H2SO4 1.0 mlMedium Stock solutions 1 - 6 10.0 ml each Stock solutions 7 - 10 1.0 ml eachMake up to 1 litre with deionized water. For agar, add 15.0 g per litre of *Bacteriological Agar (Oxoid L11). Autoclave at 15 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UKBB:MErdsFreshwater/brackish water algae Medium 8:2 mixture or 1:1 mixtureSee separate recipes (BB and MErds). Mix then autoclave at 15 psi for 15 minutes. BG (Blue-Green Medium)Marine cyanobacteria Stocks (1) Extra nutrient salts per 100 ml NaNO3 3.00 g Na2HPO4 0.12 g K2HPO4 0.10 g (2) Trace metal solution per 100 ml H3BO3 0.286 g MnCl2.4H2O 0.022 g Na2MoO4.2H2O 0.039 g CuSO4.5H2O 0.008 g Co(NO3)2.6H2O 0.005 gMedium This medium is made up in 2 parts: PART 1 per litre Tricine 0.50 g Soil extract (SE1 - see recipe) 25.00 ml Extra nutrient salts (1) 3.75 mlMake up to 1 litre with filtered natural seawater and adjust pH to 7.6 - 7.8 with 1M NaOH or HCl. PART 2 per litre NaNO3 1.500 g K2HPO4.3H2O 0.040 g MgSO4.7H2O 0.075 g CaCl2.2H2O 0.036 g Citric acid 0.006 g Ammonium ferric citrate green 0.006 g EDTANa2 0.001 g Na2CO3 0.020 g Trace metal solution (2) 1.00 mlMake up to 1 litre with distilled water and adjust pH to 7.4. FinalAutoclave Parts 1 and 2 separately at 15 psi, allow to cool then mix aseptically. For agar plates, add 15 g non-nutrient agar per litre.BG11 (Blue-Green Medium)Freshwater algae and protozoa Stocks per litre (1) NaNO3 15.0 g per 500 ml (2) K2HPO4 2.0 g (3) MgSO4.7H2O 3.75 g (4) CaCl2.2H2O 1.80 g (5) Citric acid 0.30 g(6) Ammonium ferric citrate green(枸橼酸铁铵, 柠檬酸铁铵) 0.30 g (7) EDTANa2 0.05 g (8) Na2CO3 1.00 g (9) Trace metal solution per liter H3BO3 2.86 g MnCl2.4H2O 1.81 g ZnSO4.7H2O 0.22 g Na2MoO4.2H2O 0.39 g CuSO4.5H2O 0.08 g Co(NO3)2.6H2O 0.05 gMedium Stock solution 1 100.0 ml Stock solutions 2 - 8 10.0 ml each Stock solution 9 1.0 mlMake up to 1 litre with deionized water. Adjust pH to 7.1 with 1M NaOH or HCl. For agar add 15.0 g per litre of *Bacteriological Agar (Oxoid L11). Autoclave at 15 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UK Ref Stanier et al, (1971)CH (Chalkleys Medium)Freshwater protozoa Stocks per 100 ml (1) NaCl 2.0 g (2) KCl 0.08 g (3) CaCl2 0.12 gMedium Stock solutions 1 - 3 5.0 ml eachMake up to 1 litre with deionized water. Autoclave at 15 psi for 15 minutes.CHM (Chilomonas Medium)Freshwater protozoa Medium per litre Sodium acetate trihydrate 1.0 g *Lab-Lemco powder (Oxoid L29) 1.0 gAdd the above constituents to 1 litre of deionized water. Autoclave at 15 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UKCMA (Corn Meal Glucose Agar)Freshwater protozoa Medium per litre *Corn meal agar (Oxoid CM103) 17.0 g D-glucose 2.0 g *Yeast extract (Oxoid L21) 1.0 gDisperse the agar in cold deionized water. Apply heat, stirring constantly. During heating add glucose and yeast extract. Bring contents to boil. Transfer molten agar to suitable vessels. Sterilize by pressure cooking at 10 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UKDM (Diatom Medium)Freshwater algae Stocks per 200 ml (1) Ca(NO3)2.4H2O 4.00 g (2) KH2PO4 2.48 g (3) MgSO4.7H2O 5.00 g (4) NaHCO3 3.18 g (5) EDTAFeNa 0.45 g EDTANa2 0.45 g (6) H3BO3 0.496 g MnCl2.4H2O 0.278 g (NH4)6Mo7O24.4H2O 0.20 g (7) Cyanocobalamin 0.008 g Thiamine HCl 0.008 g Biotin 0.008 g *(8) NaSiO3.9H20 (Sigma S4392) 11.4 gMedium Stock solutions 1 - 8 1.0 ml eachMake up to 1 litre with deionized water. Adjust to pH 6.9 with 1M HCl. Dispense to suitable vessels and autoclave at 15 psi for 15 minutes. Supply * Sigma Chemical Co, Fancy Road, Poole, Dorset BH17 7TG, UK Ref Beakes, Canter & Jaworski (l988)EG (Euglena Gracilis Medium)Freshwater algae and protozoa Stock (1) CaCl2 stock solution: per litre CaCl2 1.0 gMedium per litre Sodium acetate trihydrate 1.0 g *Lab-Lemco powder (Oxoid L29) 1.0 g *Tryptone (Oxoid L42) 2.0 g *Yeast extract (Oxoid L21) 2.0 g CaCl2 stock solution (1) 10.0 mlAdd constituents above and make up to 1 litre with deionized water. For agar add 15 g per litre *Bacteriological Agar (Oxoid L11). Autoclave at 15 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UKEG:JMMedium 1:1 mixtureSee separate recipes (EG and JM). Mix then autoclave at 15psi for 15 minutes. E27 (E27 Medium)Marine algae, including sterility testingMedium This medium is made up in 2 parts to avoid precipitation: PART 1 per litre Soil extract (SE1 - see recipe) 25.0 ml KNO3 0.050 g K2HPO4 0.005 g MgSO4.7H20 0.005 g Glucose 0.250 g *Tryptone (Oxoid L4