1、AJS (Acidified JM:SE) Freshwater alga Dunaliella acidophila Medium Acidified 97:3 mixture of JM and SE2 See separate recipes(JM and SE2). For approximately 1 litre of final medium, mix 970 ml JM with 30 ml SE2. Add 10 ml of conc H2SO4 to give a pH of approximately 1.5. Autoclave at 15 psi for 15 min
2、utes. 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 g Make up to 1 litre with distilled water and adjust pH to 7.6 - 7.8 with
3、 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 ml Make up to 1 litre wi
4、th 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 Cyanoc
5、obalamin 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 -20C Medium 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)
6、 25.0 ml Tricine 0.50 g Make 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 (Artificia
7、l Seawater + barley grains) Marine dinoflagellate Oxyrrhis Medium Follow 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 algae Medium Follow the recipe for ASW but add 35 g NaCl per
8、litre prior to autoclaving. ASW:BG Marine algae Medium 1:1 mixture See separate recipes. Mix aseptically. ASW:SES Marine algae Medium 3:1 mixture See separate recipes. Mix before autoclaving. ASWP (Artificial Seawater for Protozoa) Marine protozoa Stock Solution (1) per litre NaNO3 5.625 g Na2HPO4 0
9、.225 g K2HPO4 0.188 g Medium 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 ml Make 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 i
10、n 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.
11、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 ml Medium Stock solutions 1 - 6 10.0 ml each Stock solutions 7 - 10 1.0 ml each Make up to 1 litre with deionized water. For agar, add 1
12、5.0 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 BB:MErds Freshwater/brackish water algae Medium 8:2 mixture or 1:1 mixture See separate recipes (BB and MErds). Mix then autoclave at 15 psi for
13、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 g Medium This medium is made u
14、p 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 ml Make 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
15、.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 ml Make up to 1 litre with distilled water and adjust pH to 7.4. Final Autoclave Parts 1 and 2 separately at 15 psi, allow to cool then mix aseptically. For agar plates, add
16、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) Na2
17、CO3 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 g Medium Stock solution 1 100.0 ml Stock solutions 2 - 8 10.0 ml each Stock solution 9 1.0 ml Make up to 1 litre with deionized water. Adjust pH to
18、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) K
19、Cl 0.08 g (3) CaCl2 0.12 g Medium Stock solutions 1 - 3 5.0 ml each Make 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 g Add the above constituen
20、ts to 1 litre of deionized water. Autoclave at 15 psi for 15 minutes. Supply * Unipath Ltd, Wade Road, Basingstoke, Hants RG24 0PW, UK CMA (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 g Disperse the
21、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, UK DM (
22、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) Na
23、SiO3.9H20 (Sigma S4392) 11.4 g Medium Stock solutions 1 - 8 1.0 ml each Make 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 &
24、Jaworski (l988) EG (Euglena Gracilis Medium) Freshwater algae and protozoa Stock (1) CaCl2 stock solution: per litre CaCl2 1.0 g Medium 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
25、) 10.0 ml Add 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, UK EG:JM Medium 1:1 mixture See separate recipes (EG and JM).
26、 Mix then autoclave at 15psi for 15 minutes. E27 (E27 Medium) Marine algae, including sterility testing Medium 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 (
27、Oxoid L42) 0.025 g *Liver digest (Oxoid L27) 0.025 g Cyanocobalamin 100.00 ng Thiamine HCl 50.00 g Make up to 500 ml with distilled water. PART 2 Filtered natural seawater 500.0 ml Final Autoclave parts 1 and 2 separately at 15 psi, allow to cool then mix aseptically. Supply * Unipath Ltd, Wade Road
28、, Basingstoke, Hants RG24 0PW, UK E31 (E31 Medium) Marine algae Medium This medium is made up in 2 parts to avoid precipitation: PART 1 per litre Soil extract (SE1 - see recipe) 50.0 ml KNO3 0.10 g K2HPO4 0.01 g MgSO4.7H20 0.01 g Cyanocobalamin 100.00 ng Thiamine HCl 50.00 g Biotin 100.0 ng Make up to 500 ml with distilled water. PART 2 Filtered natural seawater 500.0 ml Final Autoclave Parts 1 and 2 separately at 15 psi, allow to cool then mix aseptically. E31:ANT Medium 1:1 mixture See separate recipes (E31 and ANT). Autoclave separately. Mix aseptically when cool.
