Appendix: Media for Growth of Euglena A. The medium for Euglena gracilis Z will serve for other highly heterotrophic strains of E. gracilis, such as var. bacillaris. COMPOUND GRAMS/LITER MOLES/LITER K3P04 0.20 (as K) 0.003 MgS04"7H2O 0.10 0.0004 MgC03 0.50 0.006 CaC03 0.10 0.001 DL-Malic acid l.50 0.011 L-Glutamic acid l.50 0.010 Glucose 10.00 0.055 Urea l.00 0.017 Succinic acid 0040 0.003 Glycine l.00 0.013 DL-Aspartic acid l.50 0.011 DL-Lactic acid 0.60 0.007 Thiamine HCl (Vitamin B1) 5.0 X 10-4 Cyanocobalamin (Vitamin Bd 2.0 X 10-6 "Metals 47" 0.56 174 Appendix: Media for ,Growth of Euglena 175 METALS 47 GRAMS/LITER Zn as Zn(S04)2'7H20 2.64 Mn as MnS04,H20 1.24 Fe as Fe(NH4MS04)2,6H20 1.40 Co as CoS04'7H20 0.24 Cu as CuS04'5H20 0.04 Mo as (NH4)6Mo7024,4H20 0.018 Vas Na3V04,16H20 0.018 Bas HaBOa 0.057 All the ingredients of this medium may be stored and dispensed as a dry mix, except for the lactic acid, which is added separately. For use, the ingredients are dissolved in distilled water with the aid of gentle warming, and the medium is sterilized. One millimeter of the metals solution is added per 100 ml of medium. The pH of the final medium is 3.3 to 3.6 (See Hutner et aI., 1958). B. Hutner et ai. (I966) have devised several new media which give more rapid growth as well as greener and denser cultures. Heterotrophic Acidic Medium COMPOUND GRAMS / LITER MOLES/LITER KH2P04 0.40 (as K) 0.002 MgS04'3H2O 0.10 (as Mg) 0.0006 MgCOa 0.40 (as Mg) 0.005 CaCOa 0.10 (as Ca) 0.001 DL-Malic acid 5.00 0.037 L-Glutamic acid 5.00 0.034 Glucose (anhydrous) 10.00 0.055 Urea 0.40 0.007 Na2 succinate-6H2O 0.10 (as succinate) 0.0004 Glycine 2.50 0.033 DL-Aspartic acid 2.00 0.015 "Metals 60A" 0.20 Thiamine HCI (Vitamin B1) 6.0 X 10-4 Vitamin B12 5.0 X 10-7 pH = 3.1-3.4 176 Euglena Metals 60A GRAMS PER 1,000 MILLIGRAMS METAL LITERS FINAL PER 100 ML COMPOUND MEDIUM FINAL MEDIUM Fe(NH4h(S04)206H20 42.0 Fe 0.6 MnS04oH 2O 15.5 Mn 0.5 ZnS0407H2O 22.0 Zn 0.5 (NH4)6M07024 04 H 2O 3.6 Mo 0.2 CuS04 (anhydrous) 1.0 Cu 0.04 Na3V04016H2O 3.7 V 0.04 CoS0 07H 0 ,. 0048, 1:10 trit. Co om 4 ,. 2 H 3 B03 0.57, 1: 10 trit. B 0.01 ,. To ensure even distribution, the Co and B salts are dispensed as 1:10 triturates with pentaerythritol, e.g., a 1: 10 triturate of CoS0407H20 is prepared by grinding together 1 g of the salt and 9 g of pentaerythritol. Low-pH "A uta trophic" Growth Medium COMPOUND GRAMS/LITER MOLES/LITER KH2P04 0.15 0.001 MgS0403H20 0.20 0.001 MgC03 0.3 0.004 CaC03 0.02 0.0002 K3 citra teo H 20 0040 0.001 Citric acidoH 20 4.00 0.020 NH4HC03 0.50 0.006 L-Histidine HCloH20 1.00 0.005 Thiamine HCl (Vitamin B1) 0.001 Vitamin B12 2.0 X 10-5 HEDTA 0.20 "Metals 60A" 0.18 pH adjusts to 3.2-3.5 Appendix: Media for Growth of Euglena 177 Neutral Medium COMPOUND GRAMS/LITER MOLES/LITER MgS04'7H20 0.50 0.002 Kg citrate' H 20 1.00 0.003 Na acetate'3H20 1.00 0.007 Glycine ethyl ester HCI 1.00 0.007 L-Glutamic acid y-ethyl ester HCI 1.00 0.005 L-Asparagine-H 20 1.50 0.010 N a2glycerophosphate'5H20 0.50 0.002 (NH4)2S04 0.02 0.001 Thiamine HCI (Vitamin B1) 0.01 Vitamin B12 4.0 X 10-6 In addition, trace metals are required (in milligrams): Fe, 1.0; Mn, 0.8; Zn, 0.5; Mo, 0.05; Cu, 0.05; Co, 0.05; B, 0.01; V, 0.005; I, 0.004; Se, 0.002. pH = 6.8. Alkaline Medium COMPOUND GRAMS / LITER MOLES/LITER K2CgH7P06 0.40 0.0016 MgS04'7H20 0.25 (0£ Mg) 0.0010 (0£ Mg) NH2CH2COOH (glycine) 2.00 0.026 L-Asparagine· H 20 1.50 0.010 Sodium butyrate 1.00 0.009 (N H4)6Mo7024·4 H 20 2.0 X 10-4 (0£ Mo) 1.1 X 10-6 (0£ Mo) Na3V04'16H20 4.0 X 1;)-5 (0£ V) 5.9 X 10-7 (0£ V) N itriloacetic acid 0.10 Thiamine HCI (Vitamin B1) 5.0 X 10-4 Vitamin B12 4.0 X 10-6 "Metals 44" 0.28 178 Euglena For studies at alkaline pH's: MEDIUM 18 GRAMS/LITER K2C3H7P06 0.40 MgS04'7H20 0.25 NH2CH2COOH 2.00 L-Asparagine 1.50 Sodium butyrate 1.00 (N H4)6Mo7024 ,4 H 20 2.0 X 10-4 Na3V04'16H20 4.0 X 10-5 Nitriloacetic acid 0.10 4 Thiamine HCl (Vitamin B 1) 5.0 X 10- Vitamin B12 4.0 X 10-6 "Metals 44" 0.28 One millimeter of metals solution per 100 ml of medium. METALS 44 GRAMS/LITER EDTA 2.5 ZnS04'7H2 0 17.6 MnS04'H20 9.2 CuS04'5H20 0.25 Fe(NH4)6(S04h,6H20 0.70 H 3B03 0.57 CoS04'7H20 0.04 Adjust pH with KOH. Check stability of pH by pressure cooking (KOH may contain much carbonate, which gives up CO2 on autoclaving, with a rise in pH). Appendix: Media for Growth of Euglena 179 Euglena High-Yield Medium * COMPOUND GRAMS / LITER MOLES/LITER KH2P04 0_27 0_002 MgCOa 0.80 0.010 CaCOa 0.16 0.001 (NH4)2S04 0.27 0.002 NH4 HCOa 0.27 0.003 DL-Malic acid 8.0 0.60 L-Glutamic acid 5.0 0.34 Na2 succinate·6H20 0.14 0.0005 Glycine 0.27 0.004 L-Aspartic acid 0.27 0.002 L-Arginine 0.14 0.008 Thiamine HCI (Vitamin B1) 0.14 (mg) as trit. Cyanocobalamin (Vitamin B12) 7.0 (mg) as trit. "Metals 61" 0.13 Note: These ingredients have been chosen for compatibility as a dry pre­ mix which can be stored at room temperature. The triturates are made up with pentaerythritol. Mannitol can also be used . .. Unpublished results of S. H. Hutner (1966), private communication. 180 Euglena Metals 61 GRAMS 1,000 FINAL CONC. GRAVIMETRIC LITERS FINAL METAL, MG% DRY MIX FACTOR g MEDIUM Fe 0.6 as Fe(NH4MS04ho6 H20 X 7.0 X 10,000 42.0 Mn 0.5 as MnS04oH 2O X 3.1 X 10,000 15.5 Zn 0.5 as ZnS0407H2O X 4.4 X 10,000 22.0 Mo 0.2 as (NH4)6M07024oH20 X 1.8 X 10,000 3.6 Cu 0.04 as CuS04 (anhydrous) X 2.5 X 10,000 1.0 V 0.02 as NH4V03 X 2.3 X 10,000 0.46 Co om as CoS0407H 2O X 4.8 X 10,000 0.48 B 0.01 as H 3B03 X 5.7 X 10,000 0.57 Ni 0.01 as NiS0406H2O X 4.5 X 10,000 0.45 According to our calculations this metals mix should be used at 8.6 mg percent to yield the indicated final concentrations of trace metals; however, we have found that we get highest yield with Euglena when this mix is used at 13.0 mg percent. Therefore, the actual metal concentrations in the final "high-yield" medium are as follows (in mg percent): Fe 1.0 Mn 0.8 Zn 0.8 Mo 0.325 Cu 0.065 V 0.03 Co 0.016 B 0.016 Ni 0.016 References AARONSON, s., and H. BAKER. 1961. Lipid and sterol content of some protozoa. J. Protozool., 8:274. ACKERMAN, E. 1962. 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Carotenoid de-expoxidations in algae ex­ poxidations. Enzymatic conversion of antheraxanthin to zeaxanthin. J. BioI. Chern., 240:467. BARTSCH, R. G., and M. D. KAMEN. 1960. Isolation and properties of two soluble heme proteins in extracts of the photoanaerobe Chromatium. J. BioI. Chern., 235:825. BASSHAM, ]. A., and M. CALVIN. 1957. The Path of Carbon in Photosynthesis, Englewood Cliffs, N. J., Prentice-Hall. BATRA, P., and G.