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1 Medium Recipes The culture media that CCMP uses (or has used) are listed by family in the menu at left. Clicking on a recipe will bring up the CCMP's prefered method of formulation, as well as any usage notes the CCMP has made. The CCMP can ship media formulation kits for the L1, K, and f/2 families, and also sells these media and their derivatives in premixed form, filtered seawater, etc. You can place orders for these using the CCMP shopping cart. L1 Medium 1

2 Guillard and Hargraves (1993) This enriched seawater medium is based upon f/2 medium (Guillard and Ryther 1962) but has additional trace metals. It is a general purpose marine medium for growing coastal algae. To prepare, begin with 950 ml of filtered natural seawater. Add the quantity of each component as indicated below, and then bring the final volume to 1 liter using filtered natural seawater. The trace element solution and vitamin solutions are given below. Autoclave. Final ph should be 8.0 to 8.2. Component Stock Solution Quantity Molar Concentration in Final Medium NaNO g L -1 dh 2 O 1 ml 8.82 x 10-4 M NaH 2 PO 4 H 2 O 5.00 g L -1 dh 2 O 1 ml 3.62 x 10-5 M Na 2 SiO 3 9 H 2 O g L -1 dh 2 O 1 ml 1.06 x 10-4 M trace element solution (see recipe below) 1 ml --- vitamin solution (see recipe below) 1 ml --- L1 Trace Element Solution To 950 ml dh 2 O add the following components and bring final volume to 1 liter with dh 2 O. Autoclave. Component Stock Solution Quantity Molar Concentration in Final Medium Na 2 EDTA 2H 2 O g 1.17 x 10-5 M 2

3 FeCl 3 6H 2 O g 1.17 x 10-5 M MnCl 2 4 H 2 O g L -1 dh 2 O 1 ml 9.09 x 10-7 M ZnSO 4 7H 2 O g L -1 dh 2 O 1 ml 8.00 x 10-8 M CoCl 2 6H 2 O g L -1 dh 2 O 1 ml 5.00 x 10-8 M CuSO 4 5H 2 O 2.50 g L -1 dh 2 O 1 ml 1.00 x 10-8 M Na 2 MoO 4 2H 2 O 19.9 g L -1 dh 2 O 1 ml 8.22 x 10-8 M H 2 SeO g L -1 dh 2 O 1 ml 1.00 x 10-8 M NiSO 4 6H 2 O 2.63 g L -1 dh 2 O 1 ml 1.00 x 10-8 M Na 3 VO g L -1 dh 2 O 1 ml 1.00 x 10-8 M K 2 CrO g L -1 dh 2 O 1 ml 1.00 x 10-8 M f/2 Vitamin Solution (Guillard and Ryther 1962, Guillard 1975) First, prepare primary stock solutions. To prepare final vitamin solution, begin with 950 ml of dh 2 O, dissolve the thiamine, add 1 ml of the primary stocks and bring final volume to 1 liter with dh 2 O. Filter sterilize. Store in refrigerator or freezer. Component Primary Stock Quantity Molar Concentration in Final 3

4 Solution Medium thiamine HCl (vit. B 1 ) mg 2.96 x 10-7 M biotin (vit. H) 0.5 g L -1 dh 2 O 1 ml 2.05 x 10-9 M cyanocobalamin (vit. B 12 ) 0.5 g L -1 dh 2 O 1 ml 3.69 x M Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Hargraves, P.E Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 32: Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: L1 Derivatives Black Sea Medium: For brackish water organisms (16 psu, half-strength nutrients). Combine 500 ml L1 medium and 500 ml dh 2 O. Autoclave. L1 agar: Prepare 1 liter of L1 medium and dissolve 9g agar (heat and mix). For test tubes, dispense dissolved agar medium into tubes, autoclave, and then cool with tubes slanted at an angle. For Petri plates, autoclave in a flask, cool almost to the gelling point, and then aseptically dispense into sterile Petri plates. Note: The agar concentration can be varied to produce softer or firmer substrates. L1 - Si: Prepare as for L1 medium but omit Na 2 SiO 3 9H 2 O. This is preferred over L1 medium for organisms with no silica requirement because less precipitation forms. 4

5 L1 + Se: Extra silicon and selenium are beneficial to several diatom strains. Prepare 1 L of L1 medium but use 2 ml of silicate stock, then add 1.0 ml of selenium stock solution (1.29 mg H 2 SeO 3 /L distilled H 2 O). Autoclave. L1 at 11 psu: For brackish water organisms. Mix 650 ml distilled H 2 O and 350 ml filtered seawater. Add L1 medium nutrients and autoclave. L/10-Si: Autoclave 1 L of filtered seawater. When cool, aseptically add L1-Si nutrients at one tenth concentration (i.e., 100?L). L1-Si + CCMP1320 as food: Prepare L1 and aseptically add 50?L of healthy, moderately dense culture of CCMP1320. L1m: To 1L of L1 medium, add 1 g methylamine HCl, mix until dissolved and autoclave. This medium is used to test for contamination by methylaminotrophic bacteria. L1p: To 1 L of L1 medium, add 1 g Bacto-peptone, mix until dissolves and autoclave. This medium is used to test for contamination by non- methylaminotrophic bacteria and fungi. L1pm: To 1L of L1 medium add 1 g Bacto-peptone and 1 g methylamine HCl, mix until dissolved and autoclave. This general medium is used to test for contamination by bacteria and fungi. L1 + NPM: Add L1 nutrients to 900 ml of seawater and autoclave. After cooling, aseptically add 100 ml of the following organic stock solution. Dispense aseptically into test tubes. Organics Stock Solution (modified from Guillard 1960) To 900 ml dh 2 O add: Quantity Compound 5

6 1 g sodium acetate 6 g glucose 3 g (di-) sodium succinate 6H 2 O 4 g neopeptone 1 g Bacto-tryptone 100 mg yeast extract Bring up to 1 L with dh 2 O. Dispense in small aliquots and autoclave. References Guillard, R.R.L A mutant of Chlamydomonas moewusii lacking contractile vacuoles. J. Protozool. 7: Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith, W.L. and Chanle,y M.H. (eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: f/2 Medium (Guillard and Ryther 1962, Guillard 1975) 6

7 This is a common and widely used general enriched seawater medium designed for growing coastal marine algae, especially diatoms. The concentration of the original formulation, termed "f Medium" (Guillard and Ryther 1962), has been reduced by half. To prepare, begin with 950 ml of filtered natural seawater and add the following components. The trace element and vitamin solutions are provided below. Bring the final volume to 1 liter with filtered natural seawater. If the alga to be grown does not require silica, then it is recommended that the silica be omitted because it enhances precipitation. Autoclave. Component Stock Solution Quantity Molar Concentration in Final Medium NaNO 3 75 g/l dh 2 O 1 ml 8.82 x 10-4 M NaH 2 PO 4 H 2 O 5 g/l dh 2 O 1 ml 3.62 x 10-5 M Na 2 SiO 3 9H 2 O 30 g/l dh 2 O 1 ml 1.06 x 10-4 M trace metal solution (see recipe below) 1 ml --- vitamin solution (see recipe below) 0.5 ml --- f/2 Trace Metal Solution To prepare, begin with 950 ml of dh 2 O, add the components and bring final volume to 1 liter with dh 2 O. Autoclave. Note that the original medium (Guillard and Ryther 1962) used ferric sequestrene; we have substituted Na 2 EDTA 2H 2 O and FeCl 3 6 H 2 O. 7

8 Component Primary S tock Solution Quantity Molar Concentration in Final Medium FeCl 3 6H 2 O g 1.17 x 10-5 M Na 2 EDTA 2H 2 O g 1.17 x 10-5 M CuSO 4 5H 2 O 9.8 g/l dh 2 O 1 ml 3.93 x 10-8 M Na 2 MoO 4 2H 2 O 6.3 g/l dh 2 O 1 ml 2.60 x 10-8 M ZnSO4 7H 2 O 22.0 g/l dh 2 O 1 ml 7.65 x 10-8 M CoCl 2 6H 2 O 10.0 g/l dh 2 O 1 ml 4.20 x 10-8 M MnCl 4H O g/l dh O 2 1 ml 9.10 x 10-7 M f/2 Vitamin Solution First, prepare primary stock solutions. To prepare final vitamin solution, begin with 950 ml of dh 2 O, dissolve the thiamine, add 1 ml of the primary stocks and bring final volume to 1 liter with dh 2 O. Filter sterilize. Store in refrigerator or freezer. Component Primary Stock Quantity Molar Concentration in Final Solution Medium 8

9 thiamine HCl (vit. B 1 ) mg 2.96 x 10-7 M biotin (vit. H) 1.0 g/l dh 2 O 1 ml 2.05 x 10-9 M cyanocobalamin (vit. B 12 ) 1.0 g/l dh 2 O 1 ml 3.69 x M Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: f/2 Medium Derivatives Black Sea Medium: For brackish water organisms (16 psu, half-strength nutrients). Combine 500 ml f/2 medium and 500 ml dh2o. Autoclave. f/2 agar: Prepare 1 liter of f/2 medium and dissolve 9g Bacto-agar (heat and mix). For test tubes, dispense dissolved agar medium into tubes, autoclave, and then cool with tubes slanted at an angle. For Petri plates, autoclave in a flask, cool almost to the gelling point, and then aseptically dispense into sterile Petri plates. Note: Agar can be added to other media (e.g., f/50 agar), and agar concentration can be varied to produce softer or firmer substrates. f/2-si: Prepare as for f/2 medium but omit Na2SiO 3 9H2O. This is preferred over f/2 medium for organisms with no silica requirement because less precipitation forms. f/2 + Se: Extra silicon and selenium are beneficial to several diatom strains. Prepare 1 L of f/2 medium but use 2 ml of silicate stock, then add 1.0 ml of selenium stock solution (1.29 mg H2SeO 3 /L distilled H2O). Autoclave. f/2 (11 psu): For brackish water organisms. Mix 650 ml distilled H2O and 350 ml filtered seawater. Add f/2 medium nutrients and autoclave. 9

10 f/2-si (24 psu): Mix 750 ml distilled H2O and 250 ml filtered seawater. Prepare as for f/2 medium but omit Na2SiO 3 9H2O. f/4: Add 500 ml f/2 medium to 500 ml filtered seawater, then autoclave. f/4-si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-si nutrients at half concentration (i.e., 0.5 ml). f/20-si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-si nutrients at one tenth concentration (i.e., 100?L). f/50-si: This is more than a 1/25 dilution of f/2-si medium. We autoclave 1 L of seawater in a Teflon-lined bottle. Wait for the autoclaved seawater to cool to room temperature (important). Aseptically add 40?L of sterile f/2 nutrients (20?L of vitamins). f/50-si + CCMP1320 as food: Prepare f/50 and aseptically add 50?L of healthy, moderately dense culture of CCMP1320. f/2m: To 1L f/2 medium add 1 g methylamine HCl, mix until dissolved and autoclave. This medium is used to test for contamination by methylaminotrophic bacteria. f/2p: To 1 L f/2 medium, add 1 g Bacto-peptone, mix until dissolves and autoclave. This medium is used to test for contamination by non- methylaminotrophic bacteria and fungi. f/2pm: To 1L f/2 medium add 1 g Bacto-peptone and 1 g methylamine HCl, mix until dissolved and autoclave. This general medium is used to test for contamination by bacteria and fungi. f/2 + NPM: Add f/2 nutrients to 900 ml of seawater and autoclave. After cooling, aseptically add 100 ml of the following organic stock solution. Dispense aseptically into test tubes. Organics Stock Solution (modified from Guillard 1960) 10

11 To 900 ml dh 2 O add: Quantity Compound 1 g sodium acetate 6 g glucose 3 g (di-) sodium succinate 6H 2 O 4 g neopeptone 1 g Bacto-tryptone 100 mg yeast extract Bring up to 1 L with dh 2 O. Dispense in small aliquots and autoclave. References Guillard, R.R.L A mutant of Chlamydomonas moewusii lacking contractile vacuoles. J. Protozool. 7: Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith, W.L. and Chanle,y M.H. (eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: K Medium 11

12 Keller and Guillard 1985, Keller et al This enriched seawater medium was designed specifically for oligotrophic (oceanic) marine phytoplankters that are poisoned by higher levels of trace metals. The medium uses a 10 fold higher EDTA chelation than most common marine media, and a substantial number of trace elements are included. The necessity of Tris is questionable, and it may be omitted. If organisms do not require silica, the silicate solution should be omitted because it enhances precipitation. To prepare, begin with 950 ml of filtered natural seawater, add the following components and then bring the final volume up to 1 liter with filtered natural seawater. Autoclave. Component Stock Solution Quantity Molar Concentration in Final Medium NaNO g L -1 dh2o 1 ml 8.82 x 10-4 M NH 4 Cl 2.67 g L -1 dh 2 O 1 ml 5.00 x 10-5 M Na b-gly 2 cerophosphate 6H 2 O 2.16 g L -1 dh O 2 1 ml 1.00 x 10-5 M Na SiO 9H O g L -1 dh 2 O 1 ml 5.04 x 10-4 M H SeO mg L -1 dh 2 O 1 ml 1.00 x 10-8 M Tris-base (ph 7.2) g L -1 dh 2 O 1 ml 1.00 x 10-3 M trace metal solution (see recipe below) 1 ml

13 vitamin solution (see recipe below) 0.5 ml --- Trace Metal Solution To prepare, dissolve the following components to 950 ml of dh 2 O (heat if necessary) and bring the final volume to 1 liter using dh 2 O. Component Stock Solution Quantity Molar Concentration in Final Medium Na 2 EDTA 2H 2 O g 1.00 x 10-4 M Fe-Na-EDTA 3H g 1.17 x 10-5 M FeCl 3 6 H 2 O g 1.17 x 10-5 M MnCl 2 4H 2 O g 9.00 x 10-7 M ZnSO 4 7H 2 O g L -1 dh 2 O 1 ml 8.00 x 10-8 M CoSO 4 7 H 2 O g L -1 dh 2 O 1 ml 5.00 x 10-8 M Na 2 MoO 4 2H 2 O 7.26 g L -1 dh 2 O 1 ml 3.00 x 10-8 M CuSO 4 5H 2 O g L dh 2 O 1 ml 1.00 x 10-8 M 13

14 f/2 Vitamin Solution (Guillard and Ryther 1962, Guillard 1975) First, prepare primary stock solutions. To prepare final vitamin solution, begin with 950 ml of dh 2 O, dissolve the thiamine, add 1 ml of the primary stocks and bring final volume to 1 liter with dh 2 O. Filter sterilize. Store in refrigerator or freezer. Component PrimaryStock Quantity Molar Concentration in Final Solution Medium thiamine HCl (vit. B ) mg 2.96 x 10-7 M biotin (vit. H) 0.1 g L -1 dh 2 O 1 ml 2.05 x 10-9 M cyanocobalamin (vit. B 12) 1.0 g L -1 dh2o 1 ml 3.69 x M Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: Keller, M.D. and Guillard, R.R.L Factors significant to marine diatom culture. pp In Anderson, D.M., White, A.W. and Baden, D.G. ( eds.) Toxic Dinoflagellates. Elsevier, New York. Keller, M.D., Selvin, R.C., Claus, W. and Guillard, R.R.L Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23: Prov50 Medium (Provasoli & Guillard, unpublished) 14

15 The CCMP uses this seawater medium for organisms that grow better with soil water extract and better with (or require) ammonia. The medium is similar to other soil-water extract media, such as Provasoli's ES medium (Provasoli 1968), Plymouth Laboratory's Erdschreiber Medium (Tompkins et al. 1995), or Sweeney's General Purpose Medium (Sweeney et al. 1959, Loeblich 1975) but has an ammonia addition. The method for preparing the alkaline soil water extract was first published by Provasoli et al. (1957), and he recommends using a higher concentration of the concentrated extract (e.g., 250 to 500 ml of concentrate per liter). The CCMP has slightly reduced the concentration (1 ml of stock = 200 ml of concentrate). To prepare, begin with 950 ml of filtered natural seawater and add the following components. Recipes for the soil extract, trace element and vitamin solutions are provided below. Bring the final volume to 1 liter with filtered natural seawater. Autoclave. Component Stock Solution Quantity Molar Concentration in Final Medium NaNO 3 75 g/l dh 2 O 1 ml 8.82 x 10-4 M NH 4 Cl 2.67 g L -1 dh 2 O 1 ml 5.00 x 10-5 M NaH 2 PO 4 H 2 O 5 g/l dh 2 O 1 ml 3.62 x 10-5 M soil extract solution (see recipe below) 1 ml --- trace metal solution (see recipe below) 1 ml --- vitamin solution (see recipe below) 0.5 ml --- Alkaline Soil Extract (Provasoli et al. 1957) 15

16 Combine two parts dh 2 O with one part rich organic garden soil (containing no recent applications of chemical fertilizer or pesticides). Add 2-3 g NaOH/liter. Autoclave for 2 hours, cool and filter. This concentrated extract is then diluted 5:1 with dh2o to make the final working stock. f/2 Trace Metal Solution To prepare, begin with 950 ml of dh 2 O, add the components and bring final volume to 1 liter with dh 2 O. Autoclave. Note that the original medium (Guillard and Ryther 1962) used ferric sequestrene; we have substituted Na 2 EDTA 2H 2 O and FeCl3 6 H2O. Component Primary S tock Solution Quantity Molar Concentration in Final Medium FeCl 3 6H 2 O g 1.17 x 10-5 M Na 2 EDTA 2H 2 O g 1.17 x 10-5 M CuSO 4 5H 2 O 9.8 g/l dh 2 O 1 ml 3.93 x 10-8 M Na 2 MoO 4 2H 2 O 6.3 g/l dh 2 O 1 ml 2.60 x 10-8 M ZnSO4 7H 2 O 22.0 g/l dh 2 O 1 ml 7.65 x 10-8 M CoCl 2 6H 2 O 10.0 g/l dh 2 O 1 ml 4.20 x 10-8 M MnCl 2 4H 2 O g/l dh 2 O 1 ml 9.10 x 10-7 M f/2 Vitamin Solution 16

17 First, prepare primary stock solutions. To prepare final vitamin solution, begin with 950 ml of dh O, dissolve the thiamine, add 1 ml of the primary stocks and bring final volume to 1 liter with dh 2 O. Filter sterilize. Store in refrigerator or freezer. 2 Component Primary Stock Quantity Molar Concentration in Final Solution Medium thiamine HCl (vit. B 1 ) mg 2.96 x 10-7 M biotin (vit. H) 1.0 g/l dh 2 O 1 ml 2.05 x 10-9 M cyanocobalamin (vit. B 12 ) 1.0 g/l dh 2 O 1 ml 3.69 x M References Guillard, R.R.L Culture of phytoplankton for feeding marine invertebrates. pp In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. Guillard, R.R.L. and Ryther, J.H Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: Loeblich, A A seawater medium for dinoflagellates and the nutrition of Cachonina niei. J. Phycol. 11:

18 P rovasoli, L Media and prospects for the cultivation of marine algae. pp In Watanabe, A. and H attori, A. (Eds.) Cultures and Collections of Algae. Proc. U.S.-Japan Conf. Hakone, Japan, September Publ. by the Jap. Soc. Plant Physiol. Provasoli, L., McLaughlin, J.J.A. and Droop, M.R The development of artificial media for marine algae. Arch. Mikrobiol. 25: Sweeney, B., Haxo, F. and Hastings, J Action spectra for two effects of light on luminescence in Gonyaulax polyedra. J. Gen. Physiol. 43: Tompkins, J., DeVille, M.M., Day, J.G. and Turner, M.F Culture Collection of Algae and Protozoa. Catalog of Strains. Ambleside, UK. 204 pp. ES Medium Provasoli (1968) ES Medium is an abbreviated name for enriched natural seawater medium. In addition to nitrate and phosphate, it includes TRIS base buffer, trace metals and vitamins in place of soil water extract. The origin and composition of ES medium is confusing. Recipes for the medium appeared in two 1968 publications (D'Angostino and Provasoli 1968; Provasoli 1968) and the recipes are different. Provasoli himself cited the Provasoli (1968) paper (e.g., D'Agostino and Provasoli 1970; Provasoli et al. 1970; Provasoli and Pintner 1980). Unfortunately, the Provasoli (1968) paper contains several errors. Further confusion arose when some authors (e.g., Starr and Zeikus 1993) attributed ES Medium to Provasoli (1963), but they are incorrect. Provasoli (1963) describes a new enriched natural seawater medium (SWII Medium), derived from Iwasaki's SWI Medium (Iwasaki 1961); however, SWII Medium is not ES Medium. Finally, the PII trace metals solution in ES Medium is not the original formulation (Provasoli 1958). The molar concentrations differ and he substitutes sulfated Mn, Z and Co for the original chlorinated forms. PII trace metals is sometimes traced back to Provasoli et al. (1957) (e.g., by Provasoli himself in D'Angostino and Provasoli 1968), but it first appeared in Provasoli (1958) and subsequently in Provasoli (1963). See Provasoli (1968) for this version of PII trace metals. 18

19 In addition to this recipe, and the one by D'Angostino and Provasoli (1968), McLachlin (1973) provides an ES Medium recipe that is different. McLachlin's version (via John West) has 6.6 x 10-5 M TRIS, 6.6 x 10-5 M nitrate, 2.5 x 10-6 M glycerophosphate, 7.2 x 10-3 M iron-edta and a different formulation of vitamins and the PII trace metals; i.e., everything is different. West and McBride (1999) provide another version of ES Medium (see below) that is probably the best formulation for general use. First, prepare and then autoclave the necessary stock solutions, filter sterilize and store refrigerated or frozen (vitamins). To prepare the enrichment stock solution, begin with 900 ml of dh 2 O, add the following components (vitamins should be added last after mixing other ingredients), bring the final volume to 1 liter with dh 2 O and pasteurize. To prepare ES Medium, add 20 ml of the enrichment stock solution to 980 ml of filtered natural seawater. Pasteurize. Enrichment Stock Solution Component Stock Solution Quantity Molar Concentration in Final Medium TRIS base g 8.26 x 10-4 M NaNO g x 10-4 M Na b-glycerophosphate H O g x 10-5 M Iron-EDTA Solution (see recipe below) 250 ml --- PII trace metal solution (see below) 25 ml --- Thiamine (vit. B 1 ) mg 2.96 x 10-8 M 19

20 Biotin (vit. H) 5.0 mg L -1 dh 2 O 1 ml 4.09 x M Cyanocobalomin (vit. B 10.0 mg L ) dh 2 O 1 ml 1.48 x M Iron-EDTA Solution The original recipe used anhydrous Na 2 EDTA, but because this is difficult to dissolve, we have used times as much Na 2 EDTA 2H 2 O to achieve the same molarity. Begin with 900 ml of dh 2 O, dissolve the EDTA and then the iron sulfate. Bring the final volume to 1 liter, sterilize and store refrigerated. Component Stock Solution Quantity Molar Concentration in Final Medium Na 2 EDTA 2H 2 O g 1.13 x 10-5 Fe(NH 4 ) 2 (SO 4 ) 2 6H 2 O x 10-6 PII Trace Metals Provasoli 1968 The appropriat e amount of Na 2 EDTA 2H 2 O is substituted for the original anhydrous Na 2 EDTA. Beginning with 900 ml of dh 2 O, dissolve the EDTA and then individually dissolve the following components. (The boron is not necessary for enriching natural seawater and should be left out.) Bring the final vo lume to 1 liter and store refrigerated. 20

21 Component Stock Solution Quantity Molar Concentration in Final Medium Na 2 EDTA 2H 2 O g 1.71 x 10-4 M FeCl 3 6H 2 O g 8.95 x 10-6 M H3BO g 9.25 x 10-5 M MnSO 4 4H 2 O g 3.64 x 10-5 M ZnSO 4 7H 2 O g 3.82 x 10-6 M CoSO 4 7H 2 O g 8.48 x 10-7 M References D'Agostino, A.S. and Provasoli, L Effects of salinity and nutrients on mono- and diaxenic cultures of two strains of Artemia salina. Biol. Bull. 134: D'Agostino, A. and Provasoli, L Dixenic culture of Daphnia magna, Straus. Biol. Bull. 139: Iwasaki, H The life-cycle of Porphyra tenera in vitro. Biol. Bull. 121: McLachlan, J Growth media - marine. pp In Stein, J.R (ed.) Culture Methods & Growth Measurements. Cambridge Univ. Press, Cambridge. Provasoli, L Effect of plant hormones on Ulva. Biol. Bull. 114: Provasoli, L Growing marine seaweeds. pp In De Virville, A.D. and Feldmann, J. (eds.) Proc. 4th Internatl. Seaweed Symp. Pergamon Press, Oxford. 21

22 Provasoli, L Media and prospects for the cultivation of marine algae. pp In Watanabe, A. and Hattori, A. (Eds.) Cultures and Collections of Algae. Proc. U.S.-Japan Conf. Hakone, Japan, September Publ. by the Jap. Soc. Plant Physiol. Provasoli, L., Conklin, D.E. and D'Angostino, A.S Factors inducing fertility in aseptic Crustacea. Helgolà nder wiss. Meeresunters. 20: Provasoli, L. and Pintner, I.J Bacteria induced polymorphism in an axenic laboratory strain of Ulva lactuca (Chlorophyceae). J. Phycol. 16: Starr, R.C. and Zeikus, J.A UTEX- the culture collection of algae at the University of Texas at Austin. J. Phycol. 29 (suppl.): West, J. A. & McBride, D. L Long-term and diurnal carpospore discharge patterns in the Ceramiaceae, Rhodomelaceae and Delesseriaceae ( Rhodophyta). Hydrobiologia 298/299: Pro99 Medium (Sally Chisholm, unpublished) This medium was developed specifically for Prochlorococcus, but it can be used for other oceanic species tolerating high ammonia concentrations (e.g, Bolidomonas) and no vitamin requirement. All containers should be acid cleaned and rinsed with high quality H 2 O (e.g., Milli-Q). Seawater should be collected from the oligotrophic open ocean (e.g., Sargasso Sea water), taking the usual precautions to avoid contamination. Ultrapure grade reagents should be used. This recipe was developed in Dr. Penny Chisholm's Lab (MIT), and it used smaller volumes of stock solutions. Good sterile technique is required when growing axenic strains, and a laminar flow hood is recommended. To prepare, filter one liter of oligotrophic open ocean seawater into a Teflon-lined container, autoclave and cool before adding nutrients. Aseptically, add 1 ml each of the NaH 2 PO 4, NH 4 Cl and trace element solutions. The ammonium chloride and sodium phosphate solutions should be prepared by adding the amounts indicated below, and after they are dissolved, the solution should be sterile filtered into a sterile container. The two stocks should be stored in a 4 C refrigerator. 22

23 Component Stock Solution Quantity Molar Concentration in Final Medium NaH 2 PO g L dh2o 1.0 ml 5.0 x 10-5 M NH 4 Cl g L -1 dh 2 O 1.0 ml 8.0 x 10-4 M Trace Elements (see recipe below) 1.0 ml --- PRO99 Trace Element Solution Primary stocks of most metals and selenium are prepared first, as indicated below. To prepare primary stocks, add the indicated amount of the component to 1 liter of high quality water. Next, the trace element solution is prepared by dissolving the EDTA in 1 liter of high quality water, by dissolving the iron, and finally by adding 1 ml of each primary stock. The final trace element solution should be sterile filtered into a clean, sterile container and stored at 4 C in a refrigerator. Component Stock Solution Quantity Molar Concentration in Final Medium Na 2 EDTA 2H 2 O g 1.17 x 10-6 M FeCl 3 6H 2 O g 1.17 x 10-6 M 23

24 ZnSO 4 7H 2 O 2.30 g L -1 dh 2 O 1 ml 8.00 x 10-9 M CoCl 6H O g L -1 dh O 2 1 ml 5.00 x 10-9 M MnCl 2 4H 2 O g L dh2o 1 ml 9.00 x 10-8 M Na 2 MoO 4 2H 2 O 0.73 g L -1 dh 2 O 1 ml 3.00 x 10-9 M Na 2 SeO g L -1 dh 2 O 1 ml 1.00 x 10-8 M NiSO 4 6H 2 O 2.63 g L -1 dh 2 O 1 ml 1.00 x 10-8 M f/2 Methylamine Test Medium This is a specific marine test medium to detect the presence of methylaminotrophic bacteria. To prepare, dissolve 1 g methylamine HCl in 1 liter of f/2 medium (see above for f/2 medium recipe), dissolve, dispense into tubes and autoclave. If agar plates are required, prepare as above, and then dissolve/melt the agar (e.g., 10 g agar L -1 ), autoclave, and dispense into petri plates just before it cools to the gelling point. 24