Appendix A - recipes for freshwater and seawater media

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Contents
Appendix A—Recipes
for Freshwater and
Seawater Media
Prepared by
R. A. Andersen, J. A. Berges,
P. J. Harrison, and M. M. Watanabe
429
Essentials of Medical Geology
Copyright © 2004 by Academic Press
All rights of reproduction in any form reserved.
2.0. Marine Culture Media, 487
2.1. Artificial Seawater Media, 487
2.2. Enriched Natural Seawater Media, 501
2.3. Enriched Natural Seawater +Organics, 521
2.4. Soil-Water Enriched Natural Seawater Media, 524
2.5. Bacterial-Fungal Test Media for Seawater, 527
The following culture medium recipes and culturing
protocols are an incomplete compilation from the liter-
ature and the contributors to this volume. Although we
attempted to edit this information into a convenient and
consistent format, we did not attempt to either test or
verify individual recipes. Even so, the compilation that
follows should prove to be a useful guide to a variety of
widely used algal culturing media. The culture medium
recipes provide the components and their concentra-
tions; for more preparation details, see Chapters 2 to 4.
The medium recipes are designed for growing estab-
lished algal strains; for isolation or purification of single
organisms, the nutrients may require a reduction by 10-
to 10,000-fold, depending upon the sensitivity of the
alga (see Chapters 6–9). For subculturing of established
strains, see Chapters 10 and 11. Additional recipes can
be found on Web sites (see Table A.1).
1.0. Freshwater Culture Media, 431
1.1. Freshwater Synthetic Culture Media, 431
1.2. Enriched Natural Freshwater Media, 476
1.3. Soil-Water Enriched Freshwater Media, 478
1.4. Freshwater Media Enriched with Organics, 482
1.5. Bacterial-Fungal Test Media for Freshwater Strains, 485
Water quality, including natural seawater, is very
important. The abbreviation dH2O refers generally to
distilled, deionized, distilled/deionized water, Milli-Q
water (Millipore Corp.), etc. As the culture application
becomes more critical, the quality of water also becomes
more critical. Natural seawater (and natural freshwater)
should be obtained from a nonpolluted source. For oli-
gotrophic (open ocean) phytoplankton, natural water
should be obtained from an open ocean site. When
dissolving chemicals, wait for the first component to
dissolve before adding the second. Stirring (and some-
times heat) is often necessary to dissolve chemicals effi-
ciently. In many cases, preparation of stock solutions is
not only convenient but also necessary to avoid errors
from weighing very tiny quantities. Attention should be
given to the pH of the final medium. In most cases, if a
pH adjustment is required, this occurs before steriliza-
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430 Appendix A – Recipes for Freshwater and Seawater Media
tion. Note that autoclaving drives carbon dioxide out of
media lacking carbonate stabilizing buffers, and this
makes the medium very alkaline immediately after
removal from the autoclave. In these cases, wait approx-
imately 24 hours for gaseous equilibrium before inocu-
lating cells into the medium. Natural seawater may
produce precipitates following autoclave sterilization
(see Chapter 3). In general, inorganic stocks should be
autoclaved and handled aseptically. A silicate stock is best
stored in a dark Teflon-lined bottle. If silicate is not
required for growth of the alga, potential precipitation
problems can be avoided by leaving it out of the medium.
Most trace metal solutions are chelated with EDTA (see
Chapter 4). When preparing an EDTA-buffered trace
metal solution, dissolve the EDTA before dissolving the
metals. Stock solutions of common vitamins (thi-
amine·HCl, B1, B12) should be dispensed into small
volumes in sterile, freeze-safe containers and then frozen
(e.g., 1-mL Eppendorf tubes or 10-mL polycarbonate
screw-top tubes). Vitamins retain more potency if filter-
sterilized and added aseptically after autoclaving (see
Chapters 2, 3, and 5). The stock solutions can be auto-
TABLE I. Major Service culture collections with culture medium
recipes on their Internet websites. For additional culture collections,
see: http://wdcm.nig.ac.jp/hpcc.html.
Culture Collection Internet Website URL
CCAP www.ife.ac.uk/ccap
CCMP ccmp.bigelow.org
NIES www.nies.go.jp/biology/mcc/home.htm
PCC www.pasteur.fr/recherche/banques/PCC
SAG www.epsag.uni-goettingen.de/html/sag.html
UTCC www.botany.utoronto.ca/utcc
UTEX www.bio.utexas.edu/research/utex
claved if first acidified. Aliquots from vitamin stock solu-
tions may be added to the final medium and autoclaved,
usually with little damaging effect on algal growth.
However, the vitamins should be the last component
added when preparing a culture medium, and the other
components should be well mixed.
Generally, the recipes have been normalized to
factors of 1,000 (e.g., g, mg, mg; 1mL, 1 L), and stock
solutions are described in 1-liter quantities. However,
by reducing the quantity of the component by 10, a 100-
mL stock solution is easily prepared. We have calculated
the molar concentration of each component in the final
medium for easy comparisons among various media.
Furthermore, the 1-mL quantity used for most stock
solutions allows the use of traditional sterile glass (and
disposable plastic) pipettes as well as 1,000 mL automatic
pipetters with sterile tips.
The media are organized according to the following
outline, and within each heading individual culture
media are arranged alphabetically. Algae that are known
to grow in the media are often listed; occasionally
simple modifications are listed after the recipe.
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Appendix A – Recipes for Freshwater and Seawater Media 431
1.0. Freshwater Culture Media
1.1. Freshwater Synthetic Culture Media
AF6 Medium, Modified
(Kato 1982, Watanabe et al. 2000)
AF-6 medium was developed by Kato (1982) for culturing Colacium (Euglenophyceae) but has widespread applica-
tion for algae requiring slightly acidic medium. It has been modified (Watanabe et al. 2000) by removing the calcium
carbonate (1.00 ¥10-4M), adding MES buffer, and using a different trace metals mixture. It has been used to culture
volvocalean algae (e.g., Carteria, Gonium, Chlorogonium, Pandorina, Paulschalzia, Platydorina, Pleodorina, Pteromonas,
Pseudocarteria, and some species of Volvox), the xanthophytes Botrydiopsis arrhiza Borzi and Botrydium granulatum (L.)
Greville, many cryptophytes, the dinoflagellate Peridnium bipes Stein f. globosum Lindermann, the synurophyte Synura
sphagnicola (Korsh.) Korsh., the euglenoids Phacus agilis Skuja, Euglena clara Skuja and E. mutabilis Schmitz, and the
green ciliate Paramecium bursaria. A half-strength AF-6 medium is used for maintenance of the freshwater dinofla-
gellates Hemidinium nasutum Stein and Peridinium plonicum Woloszynska (Watanabe et al. 2000). Prepare the stock
solutions. Into 950mL of dH2O, first dissolve the MES, Fe citrate, and citrate; then add the stock solutions and
bring the final volume up to 1 liter. The pH is adjusted to 6.6. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
MES buffer — 400mg 2.05 ¥10-3
Fe-citrate 2 1mL 8.17 ¥10-6
Citric acid 2 1mL 1.04 ¥10-5
NaNO3140 1mL 1.65 ¥10-3
NH4NO322 1mL 2.75 ¥10-4
MgSO4·7H2O 30 1mL 1.22 ¥10-4
KH2PO410 1mL 7.35 ¥10-5
K2HPO45 1mL 2.87 ¥10-5
CaCl2·2H2O 10 1mL 6.80 ¥10-5
Trace metals solution (See following recipe) 1mL —
Vitamin solution (See following recipe) 1mL —
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AF6 Vitamin Solution
Prepare the primary stocks. Into 950 mL of dH2O, add and dissolve the thiamine·HCl, and then add 1 mL from
each of the two primary stocks. Filter-sterilize. Refrigerate or freeze.
432 Appendix A – Recipes for Freshwater and Seawater Media
Trace Metals Solution
Prepare the primary stock solutions. Into 950mL of dH2O, dissolve the EDTA, then individually dissolve the metals,
and finally add 1mL of the two primary stocks.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA·2H2O — 5.00g 1.34 ¥10-5
FeCl3·6H2O — 0.98g 3.63 ¥10-6
MnCl2·4H2O — 0.18g 9.10 ¥10-7
ZnSO4·7H2O — 0.11g 3.83 ¥10-7
CoCl2·6H2O 20.0 1 mL 8.41 ¥10-8
Na2MoO4·2H2O 12.5 1mL 5.17 ¥10-8
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 10mg 2.96 ¥10-8
Biotin (vitamin H) 2.0 1mL 8.19 ¥10-9
Pyridoxine·HCl (vitamin B6) 1.0 1mL 5.91 ¥10-9
Cyanocobalamin (vitamin B12) 1.0 1mL 7.38 ¥10-10
1
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Appendix A – Recipes for Freshwater and Seawater Media 433
Allen’s Blue-Green Algal Medium
(M. M. Allen 1968)
See BG-11 Medium.
Allen’s Cyanidium Medium, Modified
(M. B. Allen 1959, Watanabe et al. 2000)
Mary Belle Allen (1959) developed this medium for culturing Cyanidium caldarium (Tilden) Geitler (Rhodophyceae),
and it can be used to grow the related genera Cyanidioshyzon and Galdieria (Watanabe et al. 2000). Our recipe uses
the original macronutrient concentrations; however, the trace metals solution is from Watanabe et al. (2000) because
it includes a chelator. Allen added 10-3M H2SO4to acidify the medium. We recommend adjusting the pH after all
components are added because of the presence of EDTA. For the growth in the dark, 1% glucose is added to the
medium. Into 900 mL of dH20, individually dissolve the following components and add 100mL of the trace metals
solution. Adjust to pH 2.5, using 1 N sulfuric acid. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
(NH4)2SO4— 1.320g 1.00 ¥10-2
KH2PO4— 0.272g 2.00 ¥10-3
MgSO4·7H2O — 0.247g 1.00 ¥10-3
CaCl2— 0.055g 5.00 ¥10-4
Trace metals solution (See following recipe) 1 mL —
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434 Appendix A – Recipes for Freshwater and Seawater Media
Trace Metals Solution
(Watanabe et al. 2000)
Prepare the primary stock solutions. Into 900mL of dH2O, dissolve the Fe-EDTA, then individually add the metal
compounds. Bring final volume to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Fe-Na-EDTA 3H2O — 30.16g 7.16 ¥10-5
H3BO3— 2.86g 4.63 ¥10-5
MnCl2·4H2O — 1.79g 9.04 ¥10-6
(NH4)6Mo7O24·4H2O — 0.13g 1.05 ¥10-7
ZnSO4·7H2O — 0.22 g 7.65 ¥10-7
CuSO4·5H2O — 0.079g 3.16 ¥10-7
NH4VO3— 0.023 g 1.97 ¥10-7
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Appendix A – Recipes for Freshwater and Seawater Media 435
BG-11 Medium, Modified
(Allen 1968, Allen and Stanier 1968, Watanabe et al. 2000)
BG-11 medium (Allen and Stanier 1968) was derived from medium no.11 (Hughes et al. 1958) for the culture of
freshwater, soil, thermal, and marine cyanobacteria (see Stanier and Cohen-Bazire 1977, M. M. Allen 1968). The
major changes from medium no.11 are a threefold increase of NaNO3(nitrate and phosphate levels are exception-
ally high in this medium) and the replacement of Gaffron’s minor trace elements with a modified A5 trace elements
solution. However, the nitrate value can be lowered considerably, and N2-fixing cyanobacteria have been cultured
routinely in a modified BG-110medium that omits nitrate entirely. Note that this recipe separates the EDTA from
the trace metals solution. It would be more sensible to add the EDTA to the trace metals solution and, conversely,
add the boron with other macronutrients. The MgNa2EDTA·H2O may be replaced by the molar equivalent of
Na2EDTA·2H2O (1.04g). The ferric citrate and the citric acid may be prepared as a combined stock solution.
Na2SiO3·9H2O can be added (2.04 ¥10-4M final concentration) (Allen 1968). Allen (1968) found that when the
medium and agar were autoclaved together, the algae didn’t grow. She prepared a 2¥concentration of the medium
and a 2¥concentration of the agar (1%–2.5% final concentration), autoclaved them separately, and then aseptically
mixed them when the agar was approximately 58°C. She also prepared the ferric citrate and manganese stocks
separately, adding them aseptically to the sterilized medium. Prepare the ferric ammonium citrate stock solution
(dissolve citrate and ferric citrate in 1 liter dH2O) and other stock solutions. Into 900mL of dH2O, add 1mL of the
Fe citrate solution, and then add the remaining components. Autoclave. Final pH should be 7.4 after cooling and
CO2equilibration.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Fe Citrate solution 1mL
Citric acid 6 1mL 3.12 ¥10-5
Ferric ammonium citrate 6 1 mL ~3 ¥10-5
NaNO3— 1.5g 1.76 ¥10-2
K2HPO4·3H2O 40 1mL 1.75 ¥10-4
MgSO4·7H2O 75 1mL 3.04 ¥10-4
CaCl2·2H2O 36 1mL 2.45 ¥10-4
Na2CO320 1mL 1.89 ¥10-4
MgNa2EDTA·H2O 1.0 1mL 2.79 ¥10-6
Trace metals solution (See following recipe) 1 mL —
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Trace Metals Solution
This is also known as A5 +Co trace metals solution. To 950mL of dH2O, add the EDTA and other components;
bring final volume to 1 liter.
436 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
H3BO3— 2.860g 4.63 ¥10-5
MnCl2·4H2O — 1.810g 9.15 ¥10-6
ZnSO4·7H2O — 0.220g 7.65 ¥10-7
CuSO4·5H2O 79.0 1 mL 3.16 ¥10-7
Na2MoO4·2H2O 39.0 1mL 1.61 ¥10-7
Co(NO3)2·6H2O 49.4 1mL 1.70 ¥10-7
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Appendix A – Recipes for Freshwater and Seawater Media 437
Bold’s Basal Medium (BBM)
(Bold 1949, Bischoff and Bold 1963)
This was derived from a modified version of Bristol’s solution (see Bold 1949). The medium lacks vitamins, and some
trace metal concentrations are high. This is a useful medium for many algae, especially chlorococcalean algae, volvo-
calean algae (e.g., Chlamydomonas), the filamentous green alga Klebsormidium flaccidum (Kützing) S. Mattox et Black-
well (Floyd et al. 1972), the xanthophycean alga Heterococcos endolithicus Darling et Friedmann (Darling et al. 1987),
euglenoid Colacium vesiculosum Ehr. (Rosowski and Kugrens 1973), and the cyanobacterium Microcystis aeruginosa
Kützing (Cole and Wynne, 1974). However, the formulation is unsuitable for algae with vitamin requirements.
Several modified media have been developed (see descriptions beneath table). Into 936 mL of dH2O, add 10 mL of
the first six stock solutions. Add 1mL each of the alkaline EDTA, acidified iron, boron, and trace metals solutions.
Autoclave. The final pH should be 6.6.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Macronutrients
NaNO325.00 10mL 2.94 ¥10-3
CaCl2·2H2O 2.50 10 mL 1.70 ¥10-4
MgSO4·7H2O 7.50 10 mL 3.04 ¥10-4
K2HPO47.50 10 mL 4.31 ¥10-4
KH2PO417.50 10mL 1.29 ¥10-3
NaCl 2.50 10 mL 4.28 ¥10-4
Alkaline EDTA Solution 1mL
EDTA 50.00 1.71 ¥10-4
KOH 31.00 5.53 ¥10-4
Acidified Iron Solution 1mL
FeSO4·7H2O 4.98 1.79 ¥10-5
H2SO41mL
Boron Solution 1mL
H3BO311.42 1.85 ¥10-4
Trace Metals Solution 1mL
ZnSO4·7H2O 8.82 3.07 ¥10-5
MnCl2·4H2O 1.44 7.28 ¥10-6
MoO30.71 4.93 ¥10-6
CuSO4·5H2O 1.57 6.29 ¥10-6
Co(NO3)2·6H20 0.49 1.68 ¥10-6
KBBM medium (BBM +0.25% sucrose +1.0% proteose peptone) was developed for a Chlorella-like strain that lives
exosymbiotically with Paramecium bursaria Focke (Schuster et al. 1990).
BBM +GA medium (BBM +1% glucose +0.01M amino acid hydrolysates [e.g., acid hydrolysates of casein] or
0.01M amino acids [e.g., proline, glutamine, or arginine]) was developed to grow the lichenaceous algae Trebouxia
(Fox 1967).
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3NBBM medium (BBM enriched with three times the nitrate) was developed to grow Anabaena flos-aquae
Brébisson ex Bornet et Flahault and Anacystis nidulans Gardner (Thomas and Montes 1978).
3NBBM +vitamins medium (BBM with three times the nitrate plus the addition of three vitamins) was developed
to culture for Batrachospermum (Aghajanian 1979). Vitamins: add 0.1525mg·L-1thiamine·HCl (4.52 ¥10-10 M, final
concentration); 0.125mg·L-1biotin (5.12 ¥10-10 M, final concentration); 0.125mg·L-1cyanocobalamin (9.22 ¥10-11
M, final concentration).
Double-strength BBM +a sterilized wheat seed. This medium was used to culture the cryptomonads Chilomonas
paramaecium Ehr. and Cyathomonas truncata (Fres.) Fish. (Kugrens et al. 1987).
438 Appendix A – Recipes for Freshwater and Seawater Media
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Appendix A – Recipes for Freshwater and Seawater Media 439
C Medium, Modified
(Ichimura 1971, Watanabe et al. 2000)
C (Closterium) medium, a derivative of Volvox medium (Provasoli and Pintner 1960), was developed by Ichimura
(1971) for the culture of desmids, especially Closterium species, from eutrophicated waters. The recipe uses a slightly
modified version of the PIV trace metals solution described by Provasoli and Pintner (1960), and vitamins are added
(Watanabe et al. 2000). C medium also sustains growth for most chlorococcalean algae, some volvocalean algae (e.g.,
Chlamydomonas, Carteria, Chloromonas, Hafniomonas, and Haematococcus), some other desmids (e.g., Cosmarium, Cylin-
drocystis, Gonatozygon, and Mesotaenium), some filamentous green algae (e.g., Ulothrix, Uronema, Draparnaldia,
Hyalotheca, Hydrodictyon, and Stigoeclonium), a glaucophycean alga Cyanophora pradoxa, and the synurophycean algae
Synura petersenii Korsh. and S. spinosa Korsh. (Watanabe et al. 2000). Several modified versions are listed below. Into
900mL dH2O, dissolve the TRIS buffer, then add the remaining components. Bring to 1 liter with dH2O, and
autoclave. Final pH should be 7.5.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Tris base — 0.50g 4.13 ¥10-3
KNO3— 0.10g 9.89 ¥10-4
Ca(NO3)2·4H2O — 0.15g 6.35 ¥10-4
Na2b- 50 1mL 1.63 ¥10-4
glycerophosphate·5H2O
MgSO4·7H2O 40 1mL 1.62 ¥10-4
Trace metals solution (See following recipe) 3mL —
Vitamins solution (See following recipe) 1 mL —
Modified PIV Trace Metals Solution
(Provasoli and Pintner 1960, Watanabe et al. 2000)
PIV used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), but we have substituted Na2EDTA (=13.98g
HOEDTA). Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring to 1 liter
with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA — 1.000g 8.06 ¥10-6
FeCl3·6H2O — 0.194g 2.15 ¥10-6
MnCl2·4H2O 36.00 1mL 5.46 ¥10-7
ZnCl210.44 1mL 2.30 ¥10-7
Na2MoO4·2H2O 12.62 1mL 1.56 ¥10-7
CoCl2·6H2O 4.04 1mL 5.09 ¥10-8
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Vitamins Solution
(Watanabe et al. 2000)
Into 950mL of dH2O, dissolve the thiamine·HCl and then add 1mL of each primary stock. Bring to 1 liter with
dH2O. Filter-sterilize and store frozen.
440 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 10mg 2.97 ¥10-8
(vitamin B1)
Biotin (vitamin H) 0.1 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.1 1mL 7.38 ¥10-11
CB medium (C medium with pH adjusted to 9.0, buffered with bicine substituted for Tris). This has been used to
culture strains of the water-bloom-forming cyanobacterium species Microcystis (Watanabe et al. 2000).
Csi medium (C medium with pH adjusted to 7.0, buffered with 500mg HEPES in place of Tris +100 mg·L-1
Na2SiO3·9H2O; 3.52 ¥10-4M final concentration). This has been used successfully to culture diatoms (Otsuki et al.
1987, Watanabe et al. 1987, Soma 1993), various kinds of freshwater benthic algae (Takamura et al. 1988, 1989) and
two glaucophytes, Cyanophora paradoxa Korshikov and C. tetracyanea Korshikov (Watanabe 2000).
CT medium (C medium with pH adjusted to 8.2, buffered with 400mg TAPS substituted for Tris). The medium
has been used to grow water-bloom-forming Anabaena strains (Li et al. 1998) and oscillatorioid cyanobacteria
(Suda et al. 2002).
CYT medium (1liter C medium +1g yeast +2g tryptone). This medium provides good growth for the colorless
cryptomonad Chilomonas paramecium Ehr. (Erata and Chihara 1987).
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Appendix A – Recipes for Freshwater and Seawater Media 441
CA Medium, Modified
(Ichimura and Watanabe 1974, Watanabe et al. 2000)
CA medium was developed for culturing oligotrophic desmids, and it contains both KNO3and NH4NO3as nitro-
gen sources. It is modified here by adding vitamins and using a slightly different trace metals solution. Closterium
aciculare T. West var. subpronum W. et G. S. West, which forms blooms in mesotrophic lakes and reservoirs (and
sometimes is abundant in eutrophic waters), can be grown in a CA medium because C. aciculare can use only ammo-
nium as the nitrogen source (Coesel 1991). One modification is described below. C medium (see previous recipe)
was also developed for eutrophic desmids (e.g., Closterium species), and the choice among CA medium, C medium,
and their modifications should be made after considering the habitat and environmental conditions of the collection
site. Into 900mL dH2O, dissolve the HEPES buffer, then add the remaining components. Bring to 1 liter with
dH2O, and autoclave. Final pH should be 7.2.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
HEPES — 0.40g 1.68 ¥10-3
KNO3— 0.10g 9.89 ¥10-4
Ca(NO3)2·4H2O 20.0 1 mL 8.47 ¥10-5
NH4NO350.0 1 mL 6.25 ¥10-4
Na2b- 30.0 1mL 9.80 ¥10-5
glycerophosphate·5H2O
MgSO4·7H2O 20.0 1 mL 8.11 ¥10-5
Iron–EDTA solution (See following recipe) 1 mL —
PIV trace metals solution (See following recipe) 3mL —
Vitamins solution (See following recipe) 1 mL —
Iron–EDTA Solution
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA·2H2O — 0.372g 1.00 ¥10-6
FeCl3·6H2O — 0.270g 1.00 ¥10-6
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Modified PIV Trace Metals Solution
(Provasoli and Pintner 1960, Watanabe et al. 2000)
PIV used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), but we have substituted Na2EDTA (=13.98g
HOEDTA). Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring to 1 liter
with dH2O.
442 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA — 1.000g 8.06 ¥10-6
FeCl3·6H2O — 0.194g 2.15 ¥10-6
MnCl2·4H2O 36.00 1mL 5.46 ¥10-7
ZnCl210.44 1mL 2.30 ¥10-7
Na2MoO4·2H2O 12.62 1 mL 1.56 ¥10-7
CoCl2·6H2O 4.04 1 mL 5.09 ¥10-8
Vitamins Solution
(Watanabe et al. 2000)
Into 950mL of dH2O, dissolve the thiamine·HCl and then add 1mL of each primary stock. Bring to 1 liter with
dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (B1) — 10mg 2.97 ¥10-8
Biotin (vitamin H) 0.1 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.1 1mL 7.38 ¥10-11
CAM medium (CA medium, pH 6.5, buffered with MES instead of HEPES); designed for desmids living in
oligotrophic and acidic waters (Watanabe et al. 2000).
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Appendix A – Recipes for Freshwater and Seawater Media 443
Carefoot’s Medium
(Carefoot 1968)
This was developed to grow the freshwater dinoflagellate Peridinium cincutum f. ovoplanum Lindeman. It was derived
from a modified Bold’s Basal Medium (Starr 1964) by adding Provasoli’s PIV trace metal solution (Provasoli and
Pintner 1960). Carefoot (1968) tested many organic compounds and varied the medium pH. He found that malo-
nate, pyruvate, and certain sugars enhanced growth. Watanabe et al. (2000) provide a modified recipe that includes
vitamins, which are necessary for many freshwater dinoflagellates. However, even with vitamins, it is difficult to main-
tain freshwater dinoflagellates in a stable condition for the long term. Carefoot provides both weight addition and
final molarity in his recipe, but because of rounding errors they vary slightly; we have used his weight addition and
provided more precise molarities. Into 950 ml dH2O, dissolve the NaNO3and add the appropriate volume of the
stock solutions. Bring to 1 liter with dH2O. Adjust pH to 7.5 and autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 0.25g 2.94 ¥10-3
K2HPO49.7 1 mL 5.57 ¥10-5
KH2PO4 22.7 1 mL 1.67 ¥10-4
MgSO4·7H2O 4.9 1mL 1.99 ¥10-5
CaCl2·2H2O 16.5 1mL 1.49 ¥10-4
NaCl 16.5 1 mL 2.82 ¥10-4
PIV trace metals soln. (See following recipe) 5 mL —
AACAP 10/6/04 06:44 PM Page 443

PIV Trace Metals Solution
(Provasoli and Pintner 1960)
PIV used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), but we have substituted Na2EDTA·2H2O (=13.98
g HOEDTA). Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring to 1 liter
with dH2O.
444 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA·2H2O — 15.117g 2.03 ¥10-5
FeCl3·6H2O — 1.936g 3.58 ¥10-6
MnCl2·4H2O — 0.360g 9.10 ¥10-7
ZnCl2— 0.104g 3.82 ¥10-7
Na2MoO4·2H2O — 0.126g 2.61 ¥10-7
CoCl2·6H2O 36.39 1mL 7.65 ¥10-8
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Appendix A – Recipes for Freshwater and Seawater Media 445
Chu #10 Medium
(Chu 1942)
Chu #10 medium was the most popular of 17 media that Chu (1942) described. It is a synthetic medium designed
to mimic lake water, but it lacks a chelator, vitamins, and trace metals (except for iron). It has been extensively used
for a variety of algae, including green algae, diatoms, and cyanobacteria (Chu 1942, Nalewajiko et al. 1995) and the
glaucophycean alga Glaucocystis nostochilearum Itzigsohn (Hall and Claus 1967). Many synthetic freshwater media are
derived from Chu #10, and several modified Chu #10 media have been developed. We list two modifications below.
Note: Chu used anhydrous silicate, which is very difficult to dissolve. You can substitute 0.0583 g Na2SiO3·9H2O for
the anhydrous silicate. Chu gave two possible phosphate concentrations (0.005, 0.01); we used the smaller amount.
Into 950mL of dH2O, individually dissolve each component. Bring the final volume to 1 liter and autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Ca(NO3)240 1mL 2.44 ¥10-4
K2HPO45 1 mL 2.87 ¥10-5
MgSO4·7H2O 25 1mL 1.01 ¥10-4
Na2CO320 1mL 1.89 ¥10-4
Na2SiO325 1mL 2.05 ¥10-4
FeCl30.8 1mL 4.93 ¥10-6
Chu-10 +0.1mg·L-1vitamin B12 medium was used to culture Cyclotella meneghiniana Kützing and C. cryptica
Reimann (Schultz and Trainor 1968).
AACAP 10/6/04 06:44 PM Page 445

Trace Metals Solution
Note: the solution includes boron as a metal and lacks a chelator. Into 950mL of dH2O, individually dissolve the
components. Bring to 1 liter with dH2O.
Half-Strength Chu #10 Medium
(Nalewajko and O’Mahony 1989)
The original Chu #10 medium lacked both trace metals and vitamins, and the nutrient concentrations were
somewhat high for sensitive oligotrophic organisms. Nalewajko and O’Mahony (1989) corrected all three problems
with this half-strength medium. The medium was used for growing Chlamydomonas vernalis Skuja, Nitzschia sp., and
Oscillatoria utermoehlii (Utermöhl) J. de Toni isolated from Plastic Lake, south-central Ontario, Canada (Nalewajko
and O’Mahony 1989). See Chu #10 Medium for hydrated silicate substitution. Into 950mL of dH2O, individually
dissolve each component. Bring the final volume to 1 liter and autoclave.
446 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Ca(NO3)220 1mL 1.22 ¥10-4
K2HPO42.5 1 mL 1.44 ¥10-5
MgSO4·7H2O 12.5 1 mL 5.07 ¥10-5
Na2CO310 1mL 9.43 ¥10-5
Na2SiO312.5 1 mL 1.02 ¥10-4
FeCl30.4 1mL 2.47 ¥10-6
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1mL —
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
H3BO32.48 1mL 4.01 ¥10-8
MnSO4·H2O 1.47 1mL 8.70 ¥10-9
ZnSO4·7H2O 0.23 1mL 8.00 ¥10-10
CuSO4·5H2O 0.10 1mL 4.01 ¥10-10
(NH4)6Mo7O24·4H2O 0.07 1 mL 5.66 ¥10-11
Co(NO3)2·6H2O 0.14 1mL 4.81 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 447
Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl and then add 1mL of each primary stock. Bring to 1 liter with
dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 50 mg 1.48 ¥10-7
(vitamin B1)
Biotin (vitamin H) 2.5 1mL 1.02 ¥10-8
Cyanocobalamin (vitamin B12) 2.5 1 mL 1.84 ¥10-9
AACAP 10/6/04 06:44 PM Page 447

COMBO Medium
(Kilham et al. 1998)
COMBO medium was derived from Guillard and Lorenzen’s (1972) WC medium (e.g., removing the glycylglycine
or Tris buffer, modifying the trace elements) and adding 1mL of animal trace metals. COMBO medium was used
to support robust growth of cyanobacteria, cryptophytes, green algae, and diatoms that served as food for many clado-
cerans, planaria, and rotifers. COMBO medium supports rates of algal growth statistically comparable to those in
WC medium because of its similarity to WC medium in major salt and nutrient compositions and concentrations.
It also supports fecundity of zooplankton in a range similar to that in natural lake water. COMBO medium is com-
pletely defined, readily manipulated for nutrition research, and superior to other artificial zooplankton media. Into
900mL of dH2O, individually add 1mL of each stock solution. Bring the final volume to 1 liter with dH2O. Adjust
the pH to 7. 8 and filter-sterilize.
448 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO385.01 1mL 1.00 ¥10-3
CaCl2·2H2O 36.76 1 mL 2.50 ¥10-4
MgSO4·7H2O 36.97 1mL 1.66 ¥10-4
NaHCO312.60 1mL 1.50 ¥10-4
Na2SiO3·9H2O 28.42 1 mL 1.00 ¥10-4
K2HPO48.71 1mL 5.00 ¥10-5
H3BO324.00 1mL 3.88 ¥10-4
KCl 7.45 1mL 1.00 ¥10-4
Algal trace elements solution (See following recipe) 1 mL —
Animal trace elements solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1mL —
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Appendix A – Recipes for Freshwater and Seawater Media 449
Algal Trace Elements Solution
Into 950mL of dH2O, first dissolve the EDTA and then add the components. Bring final volume to 1 liter with
dH2O. Autoclave.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA·2H2O — 4.36g 1.17 ¥10-5
FeCl3·6H2O — 1.00g 3.70 ¥10-6
CuSO4·5H2O 1.0 1mL 4.01 ¥10-9
ZnSO4·7H2O 22.0 1mL 7.65 ¥10-8
CoCl2·6H2O 12.0 1 mL 5.04 ¥10-8
MnCl2·4H2O 180.0 1mL 9.10 ¥10-7
Na2MoO4·2H2O 22.0 1 mL 9.09 ¥10-8
H2SeO31.6 1mL 1.24 ¥10-8
Na3VO41.8 1 mL 9.79 ¥10-9
Animal Trace Elements
(Elendt and Bias 1990)
Note: these trace elements are necessary for animals (e.g., Cladocera) but not for algae. Into 950mL of dH2O, first
dissolve the EDTA and then add the components. Bring final volume to 1 liter with dH2O. Autoclave.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
LiCl — 0.31 g 7.31 ¥10-6
RbCl — 0.07g 5.79 ¥10-7
SrCl2·6H2O — 0.15 g 5.63 ¥10-7
NaBr 16 1 mL 1.55 ¥10-7
KI 3.3 1mL 1.99 ¥10-8
AACAP 10/6/04 06:44 PM Page 449

Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, add 1mL of the primary stocks, and bring final volume to 1 liter
with dH2O. Filter-sterilize and store frozen.
450 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (B1) — 100mg 2.96 ¥10-7
Biotin (vitamin H) 0.50 1mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.55 1 mL 4.06 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 451
D Medium
(Sheridan 1966)
This was designed by Sheridan (1966) for growing thermophilic cyanobacteria from hot springs. It has ~1.2 g·L-1
total dissolved solids, a typical amount for many hot springs. Although it contains rather high levels of nitrate and
phosphate, the molar ratio is ~12. Many modifications of this medium have been made and applied to a variety of
cyanobacteria, including thermal and nonthermal (Halfen 1973, Pichel and Castenholtz 1991) and even Chroomonas
sp. (Cryptophyceae; Schmek et al. 1994). The natures of the variants are shown in detail in Castenholtz (1969, 1988).
Into 950mL of dH20, dissolve the nitrilotriacetic acid (NTA), add the other components, and bring the final volume
to 1 liter using dH2O. Adjust pH to 6.8. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Nitrilotriacetic acid — 0.100g 5.23 ¥10-4
KNO3— 0.103 g 1.02 ¥10-3
NaNO3— 0.689 g 8.11 ¥10-3
Na2HPO4— 0.111 g 7.82 ¥10-4
MgSO4·7H2O — 0.100 g 4.06 ¥10-4
CaSO4·2H2O 60.00 1mL 3.48 ¥10-4
NaCl 8.00 1mL 1.37 ¥10-4
FeCl30.29 1mL 1.79 ¥10-6
Trace metals solution — 0.5 mL —
Trace Metals Solution
First, add 0.5mL concentrated H2SO4to 950mL dH2O, then dissolve or add the other components and bring the
final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
H2SO4(concentrated) — 0.5mL —
MnSO4·H2O — 2.28g 5.11 ¥10-6
ZnSO4·7H2O — 0.50g 8.69 ¥10-7
H3BO3— 0.50g 4.04 ¥10-6
CuSO4·5H2O 25 1 mL 5.00 ¥10-8
Na2MoO4·6H2O 25 1mL 5.17 ¥10-8
CoCl2·6H2O 45 1mL 9.46 ¥10-8
AACAP 10/6/04 06:44 PM Page 451

D11 Medium
(Graham et al. 1982)
This medium was developed for culturing the freshwater Cladophora (see also Hoffman and Graham 1984). The
medium has an inordinate number of trace elements, many of which are probably unnecessary. Furthermore, the
concentration of some metals (e.g., copper) is very high. Rather than prepare all three trace element solutions, it may
be worth trying an addition of the most likely elements (Se, Va, Co, Ni) to the first trace elements solution. The
recipe calls for selenic acid (selenate, Na2SeO4), but selenious acid (selenite, Na2SeO3) is often the only form used
by algae; therefore, selenite should be used (1.09g·L-1dH2O Na2SeO3gives the same molar concentration of Se).
The sodium bicarbonate stock should be filter-sterilized, not autoclaved. D11 medium has also been used, with
modifications, for Spirogyra and Coleochaete (Graham et al. 1986, 1995). For nonaxenic cultures of Spirogyra, three
changes were made: glass-distilled water was used, B7 and C13 trace elements were omitted because they were detri-
mental to Spirogyra growth, and the pH was adjusted to 7 with 1N HCl after addition of stock vitamins and bicar-
bonate (Graham et al. 1995). Into 900 mL dH2O, dissolve the magnesium sulfate and calcium nitrate, then add 1
mL of each stock solution except the sodium bicarbonate and vitamins. Bring the final volume to 1 liter with dH2O.
Adjust to pH 6.0. Autoclave and cool. Aseptically add 1mL of sodium bicarbonate solution and 1mL of vitamins
solution. The final pH should be approximately 7.5.
452 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
MgSO4·7H2O — 100mg 4.06 ¥10-4
Ca(NO3)2·4H2O — 150mg 6.35 ¥10-4
NaHCO3100 1mL 1.19 ¥10-3
KCl 30 1mL 4.02 ¥10-4
Na2SiO3·9H2O 60 1 mL 2.11 ¥10-4
K2HPO415 1mL 8.61 ¥10-5
D11 Trace elements solution (See following recipe) 1mL —
B7 Trace elements solution (See following recipe) 1mL —
C13 Trace elements solution (See following recipe) 1mL —
Vitamins stock solution (See following recipe) 1mL —
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Appendix A – Recipes for Freshwater and Seawater Media 453
D11 Trace Metals Solution
Into 900mL dH2O, dissolve the EDTA, and then individually add the remaining components. Bring to 1 liter with
dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA· 2H2O — 12.5g 3.36 ¥10-5
FeSO4·7H2O — 5.0 g 1.80 ¥10-5
H3BO3— 1.0g 1.62 ¥10-5
ZnSO4·7H2O — 0.1 g 3.48 ¥10-7
MnSO4·H2O — 0.2 g 1.18 ¥10-6
Na2MoO4·2H2O 25.0 1mL 1.03 ¥10-7
CuSO4·5H2O 23.0 1mL 9.21 ¥10-8
C13 Trace Elements Solution
(Arnon 1938)
Note: we recommend substituting selenite for selenate (see previous discussion). First, dissolve As2O3in 2 or 3 drops
of concentrated HCl and dilute with dH2O to 1 liter. Next, heat PbCl2in dH2O to dissolve; bring final volume to 1
liter. To prepare the C13 trace elements solution, begin with 900mL dH2O and add 1mL of each stock solution.
Bring the final volume to 1 liter with dH2O. Adjust the pH to 4.0 to 5.0, using 1N NaOH.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Al2(SO4)·18H2O 6.175 1 mL 9.27 ¥10-9
As2O30.660 1mL 3.34 ¥10-9
CdCl20.820 1mL 4.47 ¥10-9
SrCl21.520 1mL 9.59 ¥10-9
HgCl20.680 1mL 2.50 ¥10-9
PbCl20.670 1mL 2.41 ¥10-9
LiCl 3.060 1 mL 7.22 ¥10-8
RbCl 0.710 1 mL 5.87 ¥10-9
KBr 0.740 1mL 6.22 ¥10-9
KI 0.650 1 mL 3.91 ¥10-9
NaF 1.100 1mL 2.62 ¥10-8
Na2SeO41.190 1mL 6.30 ¥10-9
BeSO4·4H2O 9.820 1 mL 5.54 ¥10-8
AACAP 10/6/04 06:44 PM Page 453

B7 Trace Metals Solution
(Arnon 1938)
First prepare the primary stock solutions. Dissolve the NH4VO3in 1 mL of 1:1 concentrated nitric acid:dH2O, and
then bring final volume to 1 liter. Dissolve the SnCl2·2H2O in a couple drops of HCl and then dilute to 1 liter with
dH2O. To 800mL of dH2O, add 1mL of each stock solution. Adjust to a pH of 3 to 4, using 1 N NaOH.
454 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NH4VO32.30 1 mL 1.97 ¥10-8
K[Cr(SO4) 2]·12H2O 9.60 1 mL 1.92 ¥10-8
NiSO4·6H2O 4.50 1mL 1.71 ¥10-8
CoCl2·6H2O 4.00 1mL 1.68 ¥10-8
Na2WO4·2H2O 1.80 1mL 5.46 ¥10-9
K2TiO(C2O4)2·2H2O 7.39 1mL 2.20 ¥10-8
SnCl2·2H2O 1.90 1 mL 8.42 ¥10-9
Vitamins Stock Solution
Into 900mL, dissolve the thiamine·HCl and then add 1mL of each primary stock solution. Bring final volume to 1
liter. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) 10.0 1mL 2.96 ¥10-8
Biotin (vitamin H) 10.0 1mL 4.09 ¥10-8
Cyanocobalamin (vitamin B12) 0.1 1 mL 7.38 ¥10-11
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Appendix A – Recipes for Freshwater and Seawater Media 455
DyIII Medium
(Lehman 1976)
This was based on concentrations of anions and cations present in a pond during exponential or luxurious growth
of Dinobryon (Lehman 1976). The medium contains both nitrate and ammonium as nitrogen sources, and it has a
low level of potassium because of sensitivity of Dinobryon to potassium. In the original recipe, iron wire was dissolved
in a small amount of concentrated HCl. Alternatively, a ferric chloride (FeCl3·6H2O) separate stock solution can be
prepared by adding 3.389 g to 1 liter dH2O, and this will provide the same amount of iron; the additional chloride
(5 ¥10-6M in final medium) is probably insignificant. The medium provides acceptable growth for some chryso-
phytes, but the growth of other species is not supported, apparently because of the lack of certain trace elements (see
DY-V medium). Into 950mL of dH20, dissolve the MES, add the other components, and bring the final volume to
1 liter, using dH2O. Adjust pH to 6.8. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
MES buffer — 200mg 1.02 ¥10-3
CaCl2·2H2O 75.00 1mL 5.10 ¥10-4
Na2SiO3·9H2O 15.00 1mL 5.28 ¥10-5
MgSO4·7H2O 50.00 1 mL 2.03 ¥10-4
NH4NO35.00 1mL 6.25 ¥10-5
NaNO320.00 1mL 2.35 ¥10-4
KCl 3.00 1mL 4.02 ¥10-5
Na2b-glycerophosphate 10.00 1mL 4.63 ¥10-5
H3BO34.58 1mL 7.40 ¥10-5
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1mL —
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456 Appendix A – Recipes for Freshwater and Seawater Media
Trace Metals Solution
The iron wire is dissolved in a small amount of concentrated HCl and added to dH2O. (A ferric chloride [FeCl3·6H2O]
primary stock solution can be prepared by adding 3.389 g to 1 liter dH2O.) Into 950 mL dH2O, add 1mL of each
stock solution and bring the final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA (anhydrous) — 8.000g 2.74 ¥10-5
Fe (iron wire) 0.700g 1.25 ¥10-5
MnCl2·4H2O 0.720g 3.64 ¥10-6
ZnSO4·7H2O 0.176g 6.12 ¥10-7
Na2MoO4·6H2O 50 1mL 2.08 ¥10-7
CoCl2·6H2O 29 1 mL 1.22 ¥10-7
Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, then add 1 mL of the primary stocks and bring the final volume
to 1 liter with dH2O. Filter-sterilize; store in refrigerator or freezer.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 200 mg 5.93 ¥10-7
Biotin (vitamin H) 0.5 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.5 1 mL 3.69 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 457
DY-V Medium
(CCMP)
DY-V Medium was derived, via DY-IV (see Andersen et al. 1997), from Dy III Medium (Lehman 1976). DY IV
medium contained lower concentrations of nitrate and ammonium than those of DY III, but it was supplemented
with vanadium and selenium (other trace metals lower than Dy III). The nitrogen and phosphorus concentrations
were increased for DY-V. It supports the growth of a wide range of heterokont algae, cryptophytes, and other algae
that require slightly acidic to circumneutral pH conditions (see Andersen et al. 1997). Into 950 mL of dH20, dis-
solve the MES and add the other components. Bring the final volume to 1 liter, using dH2O. Adjust pH to 6.8 with
NaOH. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
MES — 200mg 1.02 ¥10-3
MgSO4·7H2O 50.99 1 mL 2.03 ¥10-4
KCl 3.00 1mL 4.02 ¥10-5
NH4Cl 2.68 1mL 5.01 ¥10-5
NaNO320.00 1 mL 2.35 ¥10-4
Na2b-glycerophosphate 2.16 1mL 1.00 ¥10-5
H3BO30.80 1 mL 1.29 ¥10-5
Na2SiO3·9H2O 14.00 1mL 4.93 ¥10-5
CaCl275.00 1 mL 6.76 ¥10-4
Trace elements solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1mL —
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458 Appendix A – Recipes for Freshwater and Seawater Media
Trace Element Solution
Into 900mL dH2O, dissolve the EDTA, and then add the remaining components. Bring the final volume to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA·2H2O — 8.0 g 2.15 ¥10-5
FeCl3·6H2O — 1.0g 3.70 ¥10-6
MnCl2·4H2O — 200mg 1.01 ¥10-6
ZnSO4·7H2O — 40mg 1.39 ¥10-7
CoCl2·6H2O 8.0 1mL 3.36 ¥10-8
Na2MoO4·6H2O 20.0 1mL 8.27 ¥10-8
Na3VO4·10H2O 2.0 1mL 5.49 ¥10-9
H2SeO34 1mL 2.31 ¥10-8
Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, add 1mL of the primary stocks, and bring final volume to 1 liter
with dH2O. Filter-sterilize; store in refrigerator or freezer.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 100 mg 2.96 ¥10-7
Biotin (vitamin H) 0.5 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.5 1 mL 3.69 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 459
Forsberg’s Medium II
(Forsberg 1968)
This was derived from a modified Chu #10 medium (Gerloff et al. 1950). It is suitable for the growth of Chara
globularis Thuillier, C. aspera Willdenow, and C. zeylanica Willdenow and probably other charalean species. Nitella
flexilis (L.) C. Agardh was also cultured with a 1: 1 mixture of 1.8% soil extract and this medium (Foissner 1989).
The anhydrous silicate should be dissolved at high pH; alternatively, substitute 23.28g of Na2SiO3·9H2O. Into
950mL of dH2O, first dissolve the Tris and NTA, then add other components or stock solutions and bring the final
volume up to 1 liter. Adjust the pH to 7.0; autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Tris base — 0.5 g 4.13 ¥10-3
Nitrilotriacetic acid — 20 mg 1.05 ¥10-4
MgSO4·7H2O — 0.1 g 4.06 ¥10-4
Na2CO320.00 1mL 1.89 ¥10-4
Ca(NO3)2·4H2O 80.00 1 mL 3.39 ¥10-3
KCl 30.00 1 mL 4.02 ¥10-4
Na2SiO3(anhydrous) 10.00 1 mL 8.19 ¥10-5
K2HPO40.56 1mL 3.22 ¥10-6
H3BO32.29 1mL 3.70 ¥10-5
FeCl3·6H2O 1.94 1mL 7.18 ¥10-6
ZnCl20.21 1 mL 1.54 ¥10-6
Na2MoO4·2H2O 0.25 1 mL 1.03 ¥10-6
CuCl20.000846 1mL 6.29 ¥10-9
MnCl2·4H2O 0.00072 1 mL 3.64 ¥10-9
CoCl2·6H2O 0.000807 1mL 3.39 ¥10-9
AACAP 10/6/04 06:44 PM Page 459

Fraquil Medium
(Morel et al. 1975)
This was designed for study of trace metal interactions with freshwater phytoplankton (Morel et al. 1975); for a modi-
fication named Fraquil* medium, see Chapter 4. The major salts are those of WC medium, but nitrate, phosphate,
and silicate concentrations were lowered. The mixed trace metals solution was described by Morel et al. (1975), and
the vitamins are identical to those of f/2 medium (Guillard and Ryther 1962). The six stock solutions (CaCl2·2H2O,
MgSO47H2O, NaHCO3, Na2SiO3·9H2O, NaNO3, and KH2PO4) are each passed through a resin Chelex 100 column
(Bio-Rad Laboratories) to remove trace metal impurities (see Chapter 4 for details). The free ion activities and the
speciation of the components can be calculated on the basis of thermodynamic equilibrium in the system with use
of the computer program MINEQL (Westall et al. 1976). Fraquil medium was used for examining the effects of
metals on growth, nutrient uptakes, photosynthetic activity, or morphology of Scenedesmus quadricauda (Turpin)
Brébisson (Rueter and Ades 1987), Anabaena (Rueter 1988), Asterionella ralfsii var. americana Körner (Genesemer
1990, Genesmer et al. 1993), and Selenastrum capricornutum Printz (Errécalde and Campbell 2000). Into 900mL of
salt solution, add 1 mL each of nutrient solution, 0.5mL of mixed vitamin solution, and 1 mL of mixed trace metal
solution. Bring final volume to 1 liter.
460 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
CaCl2·2H2O 36.80 1 mL 2.50 ¥10-4
MgSO4·7H2O 37.00 1mL 1.50 ¥10-4
NaHCO312.60 1 mL 1.50 ¥10-4
Na2SiO3·9H2O 3.55 1 mL 1.25 ¥10-5
NaNO38.50 1 mL 1.00 ¥10-4
K2HPO41.74 1 mL 1.00 ¥10-5
Vitamin solution (See following recipe) 0.5 mL —
Trace metals solution (See following recipe) 1mL —
AACAP 10/6/04 06:44 PM Page 460

Appendix A – Recipes for Freshwater and Seawater Media 461
Vitamin Solution
Into 950mL of dH2O, dissolve the thiamine·HCl and then add 1 mL of each primary stock solution. Bring final
volume to 1 liter. Filter-sterilize. Store in refrigerator or freezer.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 200 mg 2.96 ¥10-7
Biotin (vitamin H) 1.0 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 1.1 1 mL 4.06 ¥10-10
Trace Metals Solution
Into 950mL of dH2O, add 1mL each of primary stock solution, followed by EDTA and FeCl3·6H2O. It is impor-
tant to add Na2EDTA before FeCl3in order to prevent precipitation of ferric hydroxide. Bring final volume to
1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
CuSO4·5H2O 0.249 1mL 9.97 ¥10-10
(NH4)6Mo7O24 ·4H2O 0.265 1mL 2.14 ¥10-10
CoCl2·6H2O 0.595 1mL 2.50 ¥10-9
MnCl2·4H2O 4.550 1mL 2.30 ¥10-8
ZnSO4·7H2O 1.150 1 mL 4.00 ¥10-9
Na2EDTA · 2H2O — 1.860 g 5.00 ¥10-6
FeCl3·6H2O — 0.122 g 4.51 ¥10-7
AACAP 10/6/04 06:44 PM Page 461

MA Medium
(Ichimura 1979)
This was developed for culturing bloom-forming Microcystis strains. It is characterized by a high pH, a bicine buffer,
and high amounts of trace elements except for Fe. Compared with the trace elements of BG-11 medium, there is
approximately a sevenfold increase of boron, threefold increase of Mn, twofold increase of Zn, 20-fold increase of
Mo, and 100-fold increase of Co. Conversely, the medium omits Cu and has a 10-fold lower concentration of Fe
than does BG-11 medium. The molar ratio of the cationic metals (e.g., Fe, Mn, Zn, and Co) to EDTA is approxi-
mately 4 : 1, and therefore it is underchelated by EDTA, but note that the medium contains a significant amount of
bicine. Orthophosphates, such as K2HPO4and Na2HPO4, cause precipitation by complexing with divalent cations
(e.g., Mg++ and Ca++). Therefore, Na2DL-b-glycerophosphate·5H2O is substituted, and it is used by algae with
alkaline phosphatase activity (e.g., most cyanobacteria, including Microcystis). MA medium has been widely used by
Japanese researchers for culturing Microcystis (Kusumi et al. 1987; Ooi et al. 1989; Kaya and Watanabe 1990; Otsuka
et al. 1998a,b, 1999a,b, 2000, 2001; Kato et al. 1991). The medium yields a high cell density for Microcystis, up to
109cells·mL-1. Into 900 mL dH2O, dissolve the bicine and then add 1mL of the stock solutions. Adjust to pH 8.6
(1N NaOH) and then autoclave.
462 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
bicine 0.5g 3.06 ¥10-3
NaNO350.0 1mL 5.88 ¥10-4
KNO3100.0 1mL 9.89 ¥10-4
Ca(NO3)2·4H2O 50.0 1mL 2.12 ¥10-4
Na2b-glycerophosphate·5H2O 50.0 1mL 1.63 ¥10-4
Na2SO440.0 1mL 2.82 ¥10-4
MgCl2·6H2O 50.0 1 mL 2.46 ¥10-4
H3BO320.0 1mL 3.23 ¥10-4
Na2EDTA · 2H2O 5.0 1mL 1.34 ¥10-5
FeCl3·6H2O 0.5 1 mL 1.85 ¥10-6
MnCl2·4H2O 5.0 1mL 2.53 ¥10-5
CoCl2·6H2O 5.0 1mL 2.10 ¥10-5
ZnCl20.5 1mL 3.67 ¥10-6
Na2MoO4·2H2O 0.8 1mL 3.31 ¥10-6
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Appendix A – Recipes for Freshwater and Seawater Media 463
MW Medium
(Sako et al. 1984)
This was derived from W Medium (Watanabe 1983) by adding NH4Cl, CaCO3, CaCl2·2H2O, KHCO3, and a 10-
fold increase in vitamins; W medium was derived from Volvox Medium by adding urea. W Medium contained a low
level of potassium because of the potassium sensitivity of dinoflagellate Peridinium. MW Medium is organically
buffered (glycylglycine), and it is also rich in inorganic and other organic nitrogen sources. MW medium supports
excellent growth of freshwater bloom-forming dinoflagellates such as Peridinium (Sako et al. 1984, 1985, 1987).
However, it is difficult to maintain stable long-term growth of Peridinium in this medium. MW Medium provides
much better short-term growth with a high yield when compared with Carefoot’s Medium. Into 900mL dH2O, dis-
solve the glycylglycine and calcium nitrate, then add the remaining ingredients. Bring final volume to 1 liter with
dH2O and adjust the pH to 7.2 before autoclaving.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Glycylglycine — 0.1g 7.57 ¥10-4
Ca(NO3)2·4H2O — 0.1 g 4.23 ¥10-4
Urea 8.50 1 mL 1.42 ¥10-4
CaCl214.00 1 mL 1.26 ¥10-4
CaCO310.00 1 mL 1.00 ¥10-4
KHCO39.00 1 mL 8.99 ¥10-5
MgSO4·7H2O 15.00 1mL 6.09 ¥10-5
KNO310.00 1 mL 9.89 ¥10-5
NaNO31.700 1 mL 2.00 ¥10-5
Na2b-glycerophosphate·5H2O 20.00 1mL 6.53 ¥10-5
NH4NO30.42 1 mL 5.25 ¥10-6
Trace metals solution (See following recipe) 500mL —
Vitamins solution (See following recipe) 1mL —
AACAP 10/6/04 06:44 PM Page 463

Modified PIV Trace Metals Solution
(Provasoli and Pintner 1960, Watanabe et al. 2000)
PIV used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), but we have substituted Na2EDTA (=13.98g
HOEDTA). Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring to 1 liter
with dH2O.
464 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA — 15.120g 1.34 ¥10-6
FeCl3·6H2O — 1.936g 3.59 ¥10-7
MnCl2·4H2O — 0.370g 9.10 ¥10-8
ZnCl2— 0.104g 3.83 ¥10-8
Na2MoO4·2H2O — 0.126 g 2.61 ¥10-8
CoCl2·6H2O 36.39 1 mL 8.49 ¥10-9
Vitamins Solution
Into 950mL of dH2O, first dissolve the thiamine·HCl and then add 1 mL of each primary stock. Bring to 1 liter
with dH2O, filter-sterilize, and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 20 mg 5.93 ¥10-8
Biotin (vitamin H) 0.2 1mL 8.19 ¥10-10
Cyanocobalamin (vitamin B12) 0.2 1 mL 1.48 ¥10-10
AACAP 10/6/04 06:44 PM Page 464

Appendix A – Recipes for Freshwater and Seawater Media 465
N-HS-Ca Medium
(Schlegel et al. 2000)
This was modified from N-HS Medium (Hepperle and Krienitz 1997) and was used to culture and investigate Pha-
cotus lenticularis (Ehr.) Diesing, particularly the production of its calcified lorica. The medium contains extremely
high calcium and magnesium concentrations compared with other common culture media. The original N-HS
medium was based on the natural conditions of Lake Haussee and Lake Stechlin, Germany, and that first formula-
tion was also used to study calcification in P. lenticularis (Hepperle and Krienitz 1997). The N-HS-Ca medium pro-
vides several advantages over N-HS medium. In contrast to N-HS medium, N-HS-Ca medium is easily prepared
and can be stored in the refrigerator for at least 9 months without detectable alterations. Compared with the
N-HS medium, N-HS-Ca medium contains a 100-fold higher concentration of free carbonic acid, which contributes
to the stabilization of pH (9.0) for the medium. Nutrient limitation is minimized with N-HS-Ca medium because it
has relatively high amounts of nitrogen and phosphorus. N-HS-Ca medium may be useful for investigating the
behavior of other organisms tolerating highly alkaline conditions. Note: sodium metasilicate pentahydrate is used,
but this is a high-pH medium that enhances dissolution. Into 950mL dH2O, add the calcium sulfate and
magnesium carbonate and stir overnight. Dissolve the sodium bicarbonate and then add 1 mL of each stock solution.
Filter-sterilize.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
CaSO4·2H2O — 137.80mg 8.00 ¥10-4
(MgCO3)4Mg(OH)2·5H2O — 38.86mg 8.00 ¥10-4
NaHCO3— 360.40mg 4.29 ¥10-3
Ca(NO3)2·4H2O 177.10 1mL 7.50 ¥10-4
K2HPO48.70 1 mL 5.00 ¥10-5
CaCl 47.80 1mL 4.3 ¥10-4
Na2SiO3·5H2O 21.21 1 mL 1.00 ¥10-4
Trace metals solution (See following recipe) 1 mL —
Vitamins stock solution (See following recipe) 1mL —
AACAP 10/6/04 06:44 PM Page 465

Trace Metals Solution
This is a very dilute trace metals solution. The copper sulfate primary stock is 1,000 times more concentrated than
the other stocks; use 1mL. Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring
to 1 liter with dH2O.
466 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
EDTA 4 1mL 1.37 ¥10-8
FeSO4·7H2O 3.5 1mL 1.26 ¥10-8
H3BO30.05 1mL 8.09 ¥10-10
MgSO4·7H2O 0.01 1 mL 4.06 ¥10-11
ZnSO4·7H2O 0.05 1 mL 1.74 ¥10-11
Co(NO3)2·6H2O 0.05 1 mL 1.72 ¥10-11
Na2MoO4·2H2O 0.05 1mL 2.07 ¥10-11
CuSO4·5H2O 0.25 1mL 1.00 ¥10-13
Vitamin Stock Solution
Into 950mL of dH2O, first dissolve the thiamine·HCl and then add 1 mL of each primary stock. Bring to 1 liter
with dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) 100mg 2.96 ¥10-7
Biotin (vitamin H) 1.0 1mL 4.09 ¥10-9
Cyanocobalamin (vitamin B12) 0.2 1mL 1.48 ¥10-10
Nicotinic acid (niacin) 0.1 1 mL 8.19 ¥10-10
AACAP 10/6/04 06:44 PM Page 466

Appendix A – Recipes for Freshwater and Seawater Media 467
Spirulina Medium, Modified
(Aiba and Ogawa 1977, Schlösser 1994)
This medium was slightly modified from Spirulina platensis Medium of Ogawa and Terui (1970) to prevent the for-
mation of precipitation (Aiba and Ogawa 1977, Schlösser 1994). Schlösser (1994) recommends making two trace
metals solutions: (1) 0.4g EDTA and 0.7 g FeSO4·7H2O in 100mL dH2O, and (2) 0.4g EDTA and remaining
elements in 900mL dH2O. The two solutions should be autoclaved separately and combined aseptically when cool.
Prepare Solutions I and II; Solution II includes 1mL of the trace metals stock solution but not the vitamin stock
solution. Autoclave Solutions I and II separately and cool; aseptically combine the two solutions. Aseptically add 1
mL of the cyanocobalamin (B12) solution.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Solution I 500mL — —
NaHCO3— 13.61g 1.62 ¥10-4
Na2CO3— 4.03 g 3.80 ¥10-5
K2HPO4— 0.50 g 2.87 ¥10-6
Solution II 500 mL — —
NaNO3— 2.5 g 2.94 ¥10-5
K2SO4— 1.0 g 5.74 ¥10-6
NaCl — 1.0g 1.71 ¥10-5
MgSO4·7H2O — 0.2 g 8.11 ¥10-7
CaCl2·2H2O — 0.04g 2.72 ¥10-7
FeSO4·7H2O — 0.01g 3.60 ¥10-8
Na2EDTA · 2H2O — 0.08 g 2.15 ¥10-7
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1 mL —
AACAP 10/6/04 06:44 PM Page 467

Trace Metals Solution
Into 900mL of dH2O, dissolve the EDTA and then dissolve each remaining component; bring the final volume to
1 liter.
468 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 0.8g 2.15 ¥10-6
FeSO4·7H2O — 0.7g 2.52 ¥10-6
ZnSO4·7H2O 1.0 1mL 3.48 ¥10-9
MnSO4·7H2O 2.0 1 mL 8.97 ¥10-9
H3BO310.0 1mL 1.62 ¥10-7
Co(NO3) 2·6H2O 1.0 1 mL 3.44 ¥10-9
Na2MoO4·2H2O 1.0 1mL 4.13 ¥10-9
CuSO4·5H2O 0.005 1 mL 2.00 ¥10-11
Cyanocobalamin Stock Solution
Dissolve the cyanocobalamin in 1 liter of dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Cyanocobalamin (vitamin B12) — 5mg 3.69 ¥10-9
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Appendix A – Recipes for Freshwater and Seawater Media 469
URO Medium
(Nakahara 1981)
URO (Uroglena) medium was developed by Nakahara (1981) on the basis of concentrations of nutrients in Lake Biwa,
Japan, during the seasonal blooms of Uroglena (=Uroglenopsis). The recipe presented here has been modified slightly
by Watanabe et al. (2000). Since this medium contains very low concentrations of nutrients, it is suitable for inves-
tigating the morphology and life cycle of Uroglena. It is also available for maintenance culturing of red-tide-bloom-
forming Peridinium species, although it provides only a low yield. Kimura and Ishida (1986) studied phagotrophy in
Uroglena, and Kurata (1986) investigated the bacterial production of vitamins that are necessary for growth, corre-
lating vitamin availability and Uroglena blooms. Into 950mL dH2O, add the stock solution quantities, and bring final
volume to 1 liter. Adjust pH to 7.5 and then autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NH4NO35.0 1 mL 6.25 ¥10-5
Na2b-glycerophosphate·5H2O 4.0 1 mL 1.31 ¥10-5
MgSO4·7H2O 10.0 1 mL 4.06 ¥10-5
KCl 1.0 1mL 1.34 ¥10-5
CaCl210.0 1 mL 9.01 ¥10-5
Fe-Na-EDTA 3H20 0.5 1 mL 1.19 ¥10-6
Trace metals solution (See following recipe) 1 mL —
Vitamins solution (See following recipe) 1mL —
AACAP 10/6/04 06:44 PM Page 469

Modified PIV Trace Metals Solution
(Provasoli and Pintner 1960, Watanabe et al. 2000)
PIV used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), but we have substituted Na2EDTA (=13.98g
HOEDTA). Into 950mL of dH2O, dissolve the EDTA and then individually dissolve the metals. Bring to 1 liter
with dH2O.
470 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA — 1g 2.69 ¥10-6
FeCl3·6H2O — 0.194g 7.18 ¥10-7
MnCl2·4H2O 36 1mL 1.82 ¥10-7
ZnCl210.44 1mL 7.66 ¥10-7
Na2MoO4·2H2O 12.62 1 mL 5.22 ¥10-8
CoCl2·6H2O 4.04 1 mL 1.70 ¥10-8
Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl and then add 1mL of each primary stock. Bring to 1 liter with
dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 10mg 2.96 ¥10-8
Biotin (vitamin H) 0.1 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.1 1 mL 7.38 ¥10-11
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Appendix A – Recipes for Freshwater and Seawater Media 471
Volvox Medium
(Provasoli and Pintner 1960)
Volvox medium was designed for culturing Volvox globator L. emend. Ehr. and V. tertius Meyer. When formulated in
1960, Volvox medium was a major advance because glycylglycine was first used as a pH buffer. According to Prova-
soli and Pintner (1960), when Tris or TEA buffer was used in the alkaline range, they created toxic effects for the
two Volvox species. The minor variations in pH from 7.0 to 7.6 with buffering by TEA were immediately reflected
in a change in availability of trace metals, particularly Fe. However, when glycylglycine was used as a pH buffer in
place of TEA, minor pH changes no longer affected metal availability. Thus, Volvox showed good growth with the
same metal mixture (PIV metals) when buffered with glycylglycine. Volvox medium is suitable for most axenic strains
of Volvox and some strains of Eudorina, Pandorina, and Gonium. However, it is not suitable for xenic cultures because
many bacteria metabolize glycylglycine. Volvox medium is also suitable for culturing axenic freshwater Cryptomonas
species (Watanabe et al. 2000). There are many modifications of Volvox medium, and we list some below. Several
publications have varied the trace metals solution (e.g., Starr and Zeikus 1993, Watanabe et al. 2000); see C Medium
for an alternative trace metals solution. Into 900 mL dH2O, dissolve the calcium nitrate and glycylglycine, add the
other stock solution quantities, and after thorough mixing, add the vitamins. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Glycylglycine — 500.0mg 2.46 ¥10-3
Ca(NO3)2·4H2O — 117.8mg 5.00 ¥10-4
Na2b-glycerophosphate·5H2O 50.0 1mL 1.63 ¥10-4
MgSO4·7H2O 40.0 1mL 1.62 ¥10-4
KCl 50.0 1mL 6.71 ¥10-4
PIV trace metals solution (See following recipe) 300mL—
Biotin (vitamin H) 0.0001 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.0001 1 mL 7.38 ¥10-11
AACAP 10/6/04 06:44 PM Page 471

PIV Trace Metals Solution
(Provasoli and Pintner 1960)
The original recipe used HOEDTA (hydroxyethyl ethylenediamine triacetic acid), which is difficult to obtain; to use
Na2EDTA·2H2O, substitute 15.12g. Prepare the primary stock solution. Into 950mL of dH2O, dissolve the EDTA
and then individually add and dissolve the metals. Bring to 1 liter with dH2O.
472 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
HOEDTA — 13.98 g 1.22 ¥10-5
FeCl3·6H2O — 1.936g 2.15 ¥10-6
MnCl2·4H2O — 0.360g 5.46 ¥10-7
ZnCl2— 0.104g 2.29 ¥10-7
Na2MoO4·2H2O — 0.126 g 1.56 ¥10-7
CoCl2·6H2O 4.04 1 mL 5.09 ¥10-8
HEPES-Volvox medium: 0.94 g HEPES replaces glycylglycine; the pH is adjusted to 8.2 with 1 N NaOH. It is suit-
able for both axenic and xenic cultures, especially Euglena, Pithophora, and Glaucosphaera vacuolata Korsh. (McCracken
et al. 1980).
S Volvox medium (SVM): Volvox medium +20 mg · L-1Na2CO3+30 mg·L-1urea; either glycylglycine or
600mg·L-1HEPES can be used as buffer. It is especially suitable for growing Volvox carteri Stein (Kirk and Kirk
1983, Kobl et al. 1998).
Volvox TAC medium: Volvox medium +200 mg · L-1sodium acetate. It has been a successful medium for culturing
Astrephomene gubernaculifera Pocock, A. perforata Nozaki, Gonium pectorale Müller, Yamagishiella unicocca (Raybarn et
Starr) Nozaki, and Volvulina steinii Playfair (Watanabe et al. 2000).
AACAP 10/6/04 06:44 PM Page 472

Appendix A – Recipes for Freshwater and Seawater Media 473
MES Volvox Medium
(Starr and Zeikus 1993)
MES Volvox Medium, described by Starr and Zeikus (1993), has perhaps the broadest applicability of the many mod-
ifications of the original Volvox Medium (Provasoli and Pintner 1960). MES buffer is used in place of glycylglycine,
and ammonium chloride is added as a second nitrogen source. Starr and Zeikus (1993) refer to their trace metal
solution as PIV trace metals solution, but it differs slightly from the original PIV trace metals (see Volvox Medium)
(Provasoli and Pintner 1960). MES Volvox Medium grows a wide range of freshwater algae (Starr and Zeikus 1993).
Into 900mL dH2O, dissolve the MES and calcium nitrate, and then add the other components. Adjust pH to 6.7
with NaOH. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
MES — 1.95 g 1.00 ¥10-2
Ca(NO3)2·4H2O — 117.8mg 5.00 ¥10-4
Na2b-glycerophosphate·5H2O 60.0 1mL 1.96 ¥10-4
MgSO4·7H2O 40.0 1 mL 1.62 ¥10-4
KCl 50.0 1 mL 6.71 ¥10-4
NH4Cl 26.7 1mL 5.00 ¥10-4
Trace metals solution (See following recipe) 6 mL —
Biotin (vitamin H) 0.0025 1mL 1.02 ¥10-8
Cyanocobalamin (vitamin B12) 0.0015 1mL 1.11 ¥10-9
Trace Metals Solution
Into 950mL of dH2O, dissolve the EDTA and then individually add and dissolve the metals. Bring to 1 liter with
dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA — 750mg 1.54 ¥10-5
FeCl3·6H2O — 97mg 2.15 ¥10-6
MnCl2·4H2O — 41mg 1.24 ¥10-6
ZnCl25.0 1mL 2.20 ¥10-7
Na2MoO4·2H2O 4.0 1 mL 9.92 ¥10-8
CoCl2·6H2O 2.0 1 mL 5.04 ¥10-8
AACAP 10/6/04 06:44 PM Page 473

WC Medium
(Guillard and Lorenzen 1972)
This was derived from Chu #10 and Wright’s (1964) Medium, which Wright used to grow cryptophytes (WC =
Wright’s cryptophyte) (R. R. L. Guillard, personal communication). Wright used ferric citrate, citrate, and ammo-
nium acetate as well as additional trace elements (Al, Br, I, Li, Ni, Sn). WC Medium is slightly alkaline (pH, 7.8).
Glycylglycine is used as a buffer for axenic cultures, and Tris is used as a buffer for xenic cultures. Note the follow-
ing: (1) nitrate and phosphate should be reduced twofold to 10-fold for sensitive organisms; (2) 2.65 to 26.5mg·
L-1NH4Cl should be used for organisms that cannot utilize nitrate; (3) the more complete trace metal solution using
ferric citrate–citric acid described by Wright (1964) can also be used; and (4) ferric citrate–citric acid (3–9 mg· L-1
each) can be used in place of ferric EDTA. WC medium with no added buffer was used to culture the xanthophytes
Tribonema aequale Pascher, Botrydium becherianum Vischer, Vaucheria sessilis (Vaucher) De Candolle, and Ophiocytium
maius Nägeli, the eustigmatophyte Eustigmatos magna Hibberd, the raphidophytes Gonyostmum semen Diesing and
Vacuolaria virescens Cienkowski, and synurophytes Synura species (Guillard and Lorenzen 1972). It was noted that
Gonyostomum grew best when ca. 2/3 distilled water and 1/3 filtered bog water were used and that Synura and Vaucheria
grew better with 1% soil extract added. The medium also grows chlorococcalean and volvocalean algae
(Ankistrodesmus, Chlorella, Chlamydomonas; Ahn et al. 2002), desmids (Spondylosium, Cosmarium, Staurastrum; Freire-
Nordi et al. 1998, Healey and Hendzel 1988), diatoms (Asterionella, Cyclotella, Sellaphora, Stephanodiscus; Tilman &
Kilham 1976, Donk and Kilham 1990, Mann et al. 1999), the cyanobacterium Microcystis (Olsen 1989, Olsen et al.
1989), and the cryptophyte Cryptomonas (Klaveness 1982). In order to culture cryptomonads such as Cryptomonas
rostratiformis Skuja, C. phaseolus Skuja, C. undulata Gervais, C. ovata Ehr., and Chroomonas sp., WC medium was mod-
ified by Gervais (1997) as follows: no boron was added, and the nitrogen source was ammonium chloride instead of
sodium nitrate. Also, because the growth of C. rostratiformis was suppressed by Tris, a phosphate buffer (0.008 mM
KH2PO4+0.042mM Na2HPO4) was substituted. Into 900 mL of dH2O, dissolve one of the two possible buffers.
Individually, add the remaining components and bring the final volume to 1 liter with dH2O. Adjust the pH to 7.6–8.0
and autoclave.
474 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Buffer (use one, not both)
Glycylglycine — 500mg 3.78 ¥10-3
Tris — 500mg 4.13 ¥10-3
NaNO385.01 1mL 1.00 ¥10-3
CaCl2·2H2O 36.76 1 mL 2.50 ¥10-4
MgSO4·7H2O 36.97 1mL 1.50 ¥10-4
NaHCO312.60 1mL 1.50 ¥10-4
Na2SiO3·9H2O 28.42 1 mL 1.00 ¥10-4
K2HPO48.71 1mL 5.00 ¥10-5
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1 mL —
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Appendix A – Recipes for Freshwater and Seawater Media 475
Trace Metals Solution
Into 950mL of dH2O, add components and bring final volume to 1 liter with dH2O. Autoclave.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA ·2H2O — 4.36 g 1.17 ¥10-5
FeCl3·6H2O — 3.15g 1.17 ¥10-5
CuSO4·5H2O 10.0 1 mL 4.01 ¥10-8
ZnSO4·7H2O 22.0 1mL 7.65 ¥10-8
CoCl2·6H2O 10.0 1 mL 4.20 ¥10-8
MnCl2·4H2O 180.0 1mL 9.10 ¥10-7
Na2MoO4·2H2O 6.0 1mL 2.48 ¥10-8
H3BO3— 1.00g 1.62 ¥10-5
Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, add 1 mL of the primary stocks, and bring final volume to 1 liter
with dH2O. Filter-sterilize and store in refrigerator or freezer.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 100mg 2.96 ¥10-7
Biotin (vitamin H) 0.5 1mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.5 1mL 3.69 ¥10-10
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1.2. Enriched Natural Freshwater Media
Alga-Gro Lake-Water Medium
Carolina Biological Supply Co.
This is prepared by adding 40 mL of “Alga-Gro” medium (Carolina Biology Supply Co.) to 960 mL lake water. A
double-strength Bold’s Basal Medium (see previous description) can be substituted. The medium has been success-
fully used to culture the tetrasporalian green alga Octosporiella coloradoensis Kugrens (Kugrens, 1984), freshwater cryp-
tomonads Storeatula rhinosa Kugrens et al., Pyrenomonas ovalis Kugrens et al. (Kugrens et al. 1999), and Cryptomonas
and Chroomonas species (Kugrens et al. 1987)], and it may prove successful for a variety of other freshwater algae.
476 Appendix A – Recipes for Freshwater and Seawater Media
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Appendix A – Recipes for Freshwater and Seawater Media 477
VS Medium
Gargiulo et al. 2001
This medium was developed for culturing and investigating the life cycle of the freshwater red alga Bangia atropur-
purea (Roth) C. Agardh. The medium uses natural river water as its base. Four different concentrations were used
by Gargiulo et al. (2001), and these are referred to as VS, VS 2.5, VS 5, and VS 10 (1, 2.5, 5, and 10¥concentra-
tion, respectively). We have provided a recipe for the basic VS medium, but with simple changes in stock solution
additions (e.g., 2.5 mL for VS 2.5 medium), the other media can easily be prepared. Into 993mL of sterilized fresh-
water (from a clean lake or stream), add the following components. The two vitamins could be prepared as primary
stocks. Autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO342.52 1 mL 5.00 ¥10-4
Na2b-glycerophosphate·5H2O 5.36 1mL 2.48 ¥10-5
Na2EDTA · 2H2O 3.72 1mL 1.00 ¥10-5
FeSO4·7H2O 0.278 1mL 1.00 ¥10-6
MnCl2·4H2O 1.98 1mL 1.00 ¥10-5
Biotin (vitamin H) 0.10 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.0004 1 mL 2.95 ¥10-10
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478 Appendix A – Recipes for Freshwater and Seawater Media
1.3. Soil-Water Enriched Freshwater Media
Audouinella Medium
(Glazer et al. 1997)
Audouinella medium was developed for culture of the freshwater red alga Audouinella. It uses peat extract, natural sea-
water, and an enrichment solution. The authors, referring to Starr and Zeikus (1993), indicate that the enrichment
solution is Provasoli’s (1968) ES enrichment solution. However, the history of ES medium is complicated (see ES
medium below), and Starr and Zeikus reference the wrong original source (e.g., Provasoli 1963). Furthermore, the
trace metals solution is claimed to be Provasoli’s PII metals solution (see Starr and Zeikus 1993), but sulfated man-
ganese, zinc, and cobalt are substituted for Provasoli’s chlorinated metals, and the iron and boron concentrations are
different (see Provasoli 1958, 1963). Glazer et al. (1997) delete the Tris buffer that was used by both Provasoli (1968)
and Starr and Zeikus (1993), which seems sensible to us. Into 950 mL dH2O, add the peat extract, the natural
seawater, and the enrichment solution. Pasteurize and store at 4°C. Final pH should be 5.5, with conductance of
500–600mS·cm-1.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Dry peat moss extract — 30mL —
Natural seawater (30psu) — 10mL —
Enrichment solution — 10mL —
Enrichment Solution
(Starr and Zeikus 1993)
Into 400 mL of dH2O, dissolve the nitrate and phosphate quantities; add 250mL each of the Fe solution and trace
metal solution; and then finally dissolve the vitamins. Bring final volume to 1 liter with dH2O. Autoclave and cool
before using.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 3.50 g 4.12 ¥10-6
Na2-b-glycerophosphate·5H2O — 0.50g 1.63 ¥10-7
Fe solution (see below) 250mL —
Trace metals solution (see below) 250 mL —
Thiamine·HCl (vitamin B1) — 5 mg 1.48 ¥10-9
Biotin (vitamin H) 0.05 1 mL 2.05 ¥10-11
Cyanocobalamin (vitamin B12) 0.1 1 mL 7.38 ¥10-12
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Appendix A – Recipes for Freshwater and Seawater Media 479
Fe Solution
(Starr and Zeikus 1993)
Dissolve EDTA in 900 mL of dH2O, dissolve iron sulfate and then bring to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA anhydrous — 0.6g 5.13 ¥10-7
Fe(NH4)2(SO4)2·6H2O — 0.7 g 4.46 ¥10-7
Trace Metals Solution
(Starr and Zeikus 1993)
Into 950mL dH2O, dissolve the EDTA and then add the quantities for each of the other components. Bring the
final volume to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA ·2H2O — 1.000g 8.56 ¥10-7
H3BO3— 1.140g 4.61 ¥10-6
FeCl3·6H2O — 0.049g 4.53 ¥10-8
MnSO4·4H2O — 0.164 g 1.84 ¥10-7
ZnSO4·7H2O 22.0 1mL 1.91 ¥10-8
CoSO4·7H2O 4.8 1mL 4.27 ¥10-9
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480 Appendix A – Recipes for Freshwater and Seawater Media
Biphasic Soil Water Medium
This old, but reliable, medium provides good growth and normal morphology for many freshwater algae (e.g., Pring-
sheim 1946). The medium can be slightly acidic or alkaline, depending upon the soil source. For details regarding
the soils and culture medium preparation methods, see Chapter 2.
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Appendix A – Recipes for Freshwater and Seawater Media 481
Vitamin Solution
Into 950mL, dissolve the thiamine·HCl and then add 1mL of each stock solution. Bring final volume to 1 liter.
Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 1 g 2.97 ¥10-6
Biotin (vitamin H) — 0.1 g 4.09 ¥10-7
Nicotinic acid (niacin) — 1g 8.12 ¥10-6
Cyanocobalamin (vitamin B12) 1 1mL 7.38 ¥10-10
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Ca(NO3)2·4H2O 70.85 1mL 3.00 ¥10-4
KH2PO454.44 1 mL 4.00 ¥10-4
MgSO4·7H2O 24.65 1 mL 1.00 ¥10-4
Na2SiO3(27% aq. sat. soln.) 20 mL, pH 8.5 5mL ~3.00 ¥10-4
FeSO4·7H2O 0.278 1mL 1.00 ¥10-6
MnCl2·4H2O 0.02 1 mL 1.00 ¥10-7
Soil extract — 50 mL —
Vitamins solution (See following recipe) 1mL —
Diatom Medium, Modified
(Cohn and Pickett-Heaps 1988, Cohn et al. 2003)
This was developed for culturing the freshwater diatom Surirella and has proved useful for other diatom species as
well. It should not be confused with the “Diatom Medium” of Beakes et al. (1988) (see also Tompkins et al. 1995).
The medium described here contains soil extract (see Chapter 2 for details) and a high concentration of phosphate.
A standard Na2SiO3·9H2O solution (e.g., 1mL of 28.42g·L-1dH2O stock solution =1.00 ¥10-4final concentration)
could be substituted for the commercial silica stock solution (Sigma #S1773, Sigma-Aldrich). Cohn (personal com-
munication) recommends adding NaHCO3(1 mL of 8.4g·L-1dH2O stock solution =1.00 ¥10-4final concentra-
tion) after tyndallization. This medium has also been used for growing Craticula cuspidata (Kütz.) Mann (=Navicula
cuspidata Kütz. in Cohn et al. 1989), Nitzschia species, Pinnularia viridis (Nitzsch) Ehr., and Stauroneis species (Cohn
and Disparti 1994, Cohn and Weitzell 1996, Cohn et al. 2003). Into 900mL of dH2O, dissolve all components except
the vitamins (and NaHCO3if added). Bring the final volume to 1 liter, using dH2O. Sterilize the solution by tyn-
dallization (see Chapter 5). Cool and then add the vitamins (and NaHCO3). Adjust pH to 6.75. Filter-sterilize.
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482 Appendix A – Recipes for Freshwater and Seawater Media
1.4. Freshwater Media Enriched with Organics
Polytoma Medium
(Starr 1964)
Polytoma medium was reported by Starr (1964) on the basis of a personal communication from Pringsheim. Pring-
sheim (1955, 1963) demonstrated that this colorless Chlamydomonas-like alga will utilize acetate but not glucose. It
is suitable for all axenic cultures of Polytoma and Astasia and colorless strains of Euglena (Starr and Zeikus 1993). To
prepare, dissolve 2g sodium acetate, 1 g yeast extract, and 1g tryptone in 1 liter of dH2O. Schlösser (1994) sub-
stitutes 30 mL of soil water extract for 30 mL of dH2O. Dispense into tubes or flasks. Autoclave; store at 4° C until
ready for use.
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Appendix A – Recipes for Freshwater and Seawater Media 483
Porphyridium Medium (Modified)
(Sommerfeld and Nichols 1970)
This medium, used to study the morphology and growth characteristics of Porphyridium aerugineum Geitler, was
modified by Sommerfeld and Nichols (1970). The medium apparently originated with Pringsheim. To prepare,
dissolve 1g yeast extract and 1 g tryptone in 900mL of dH2O. Add 100mL of soil water extract (see Chapter 2).
Autoclave.
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484 Appendix A – Recipes for Freshwater and Seawater Media
Prototheca Medium
(Wolff and Kück 1990)
This is a solid (agar) medium containing malt that was developed to study the structural analysis of mitochondria
SSU rRNA of Prototheca wickerhamii Soneda et Tubaki. It has proved suitable for all Prototheca cultures. Schlösser
(1994) substitutes 30 mL of soil water extract for 30mL of dH2O. See also Anderson (1945) and Patni and
Aaronson (1974). Add 45g of malt extract agar to 1 liter of dH2O (see Chapter 2 for preparation of agar). Alterna-
tively, 30 g malt extract and 15g agar can be added to 1 liter of dH2O.
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Appendix A – Recipes for Freshwater and Seawater Media 485
1.5. Bacterial-Fungal Test Media for Freshwater Strains
Bacterial Test Media
(Watanabe et al. 2000)
The following are prepared by adding the organic compounds to 1 liter of algal culture medium (used to grow the
alga that is being tested). For oligotrophic bacteria, reduce the organic concentration substantially. If agar plates are
required, prepare broth, add agar (10–15g agar · L-1) and heat to “dissolve” (see Chapter 2). Once agar is dissolved,
autoclave in a flask and then dispense into petri dishes.
B-1 Test Medium: 1g proteose peptone.
B-II Test Medium: 5g yeast extract.
B-III Test Medium: 5g peptone +3g beef extract.
B-IV Test Medium: 1g glucose +1g peptone.
B-V Test Medium: 0.5g sodium acetate +0.5g glucose +0.5g tryptone +0.3 g yeast extract.
YT Test Medium: 1g yeast extract +2g tryptone.
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486 Appendix A – Recipes for Freshwater and Seawater Media
Freshwater Test Medium
(CCMP)
This is a general purpose test medium to detect the presence of common bacteria and fungi in freshwater cultures.
To prepare a broth medium, dissolve 5 g peptone and 10 g malt extract in 1 liter of dH2O, dispense into test tubes,
and autoclave. If agar plates are required, prepare broth, add agar (e.g., 10g agar·L-1), and heat to “dissolve”
(see Chapter 2). Once the agar is dissolved, autoclave in a flask and then dispense into petri plates.
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Appendix A – Recipes for Freshwater and Seawater Media 487
2.0. Marine Culture Media
2.1. Artificial Seawater Media
Aquil* Medium
(Sunda et al. 2004)
Aquil* is a new version of the original Aquil medium (Morel et al. 1979, Price et al. 1989) that is presented in Chapter
4. The medium, in all three versions, is designed for critical trace metal experimental work. To prepare properly,
solutions are passed through a Chelex column to remove impurities. Details for column purification are thoroughly
described in Chapter 4, and it is imperative that these details are followed when using the medium for critical trace
metal experimental work. To prepare the synthetic ocean water, use 600mL of high-quality dH2O (e.g., Milli-Q
water) and individually dissolve each of the anhydrous salts. Next, use 300mL of high-quality dH2O, and individu-
ally dissolve each of the hydrous salts. Combine the two solutions. Add 1mL of each of the major nutrient stock
solutions, add 1mL of the trace metals solution, and add 1mL of the vitamins solution. Sterilize in a microwave oven
or filter-sterilize to avoid metal contamination from an autoclave (see Chapter 4). Final salinity is 35 psu.
Synthetic Ocean Water (SOW)
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Anhydrous Salts
NaCl — 24.540g 4.20 ¥10-1
Na2SO4— 4.090 g 2.88 ¥10-2
KCl — 0.700g 9.39 ¥10-3
NaHCO3— 0.200 g 2.38 ¥10-3
KBr — 0.100 g 8.40 ¥10-4
H3BO3— 0.003 g 4.85 ¥10-5
NaF — 0.003g 7.15 ¥10-5
Hydrous Salts
MgCl2·6H2O — 11.100g 5.46 ¥10-2
CaCl2·2H2O — 1.540 g 1.05 ¥10-2
SrCl2·6H2O — 0.017g 6.38 ¥10-5
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488 Appendix A – Recipes for Freshwater and Seawater Media
Major Nutrients
Into 900mL of highest quality deionized water, add 1 mL of each stock solution and bring final volume to 1 liter.
Filter-sterilize or sterilize in microwave oven.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaH2PO4·H2O 1.38 1 mL 1.00 ¥10-5
NaNO385.00 1mL 1.00 ¥10-4
Na2SiO3·9H2O 28.40 1 mL 1.00 ¥10-4
Metal/Metalloid Stock Solution
Into 900mL of highest-quality deionized water, dissolve the EDTA (not Na2EDTA) and add 1 mL of each stock
solution. Bring the volume up to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
EDTA (anhydrous) — 29.200 g 1.00 ¥10-5
FeCl3·6H2O — 0.270 g 1.00 ¥10-6
ZnSO4·7H2O — 0.230g 7.97 ¥10-8
MnCl2·4H2O — 0.0240 g 1.21 ¥10-7
CoCl2·6H2O — 0.0120 g 5.03 ¥10-8
Na2MoO4·2H2O — 0.0242g 1.00 ¥10-7
CuSO4·5H2O 4.9 1 mL 1.96 ¥10-8
Na2SeO31.9 1mL 1.00 ¥10-8
Mixed Vitamin Stock Solution
Into 950mL of highest-quality deionized water, dissolve the thiamine · HCL and add 1mL of the stock solutions.
Bring the final volume to 1 liter. Filter-sterilize.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (Vitamin B1) — 100mg 2.97 ¥10-7
Biotin (Vitamin H) 5.0 1mL 2.25 ¥10-9
Cyanocobalamin (vitamin B12) 5.5 1 mL 3.70 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 489
ASP-2 Medium +NTA
(Provasoli et al. 1957)
This is one of several artificial seawater media described by Provasoli et al. (1957); see original publication for other
recipes. This medium is buffered by Tris and nitrilotriacetic acid. Exclude NTA for basic ASP-2 medium. Into 900
mL dH2O, dissolve the components and stock solutions; bring final volume to 1 liter with dH2O. Final pH should
be 7.6 to 7.8.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaCl — 18.00 g 3.08 ¥10-1
MgSO4·7H2O — 5.00g 2.03 ¥10-2
KCl — 0.60 g 8.05 ¥10-3
CaCl2— 0.10 g 9.01 ¥10-4
Tris base — 1.00g 8.25 ¥10-3
Nitrilotriacetic acid — 0.10g 5.23 ¥10-4
Na2EDTA — 0.03g 1.03 ¥10-4
NaNO3— 0.05 g 5.88 ¥10-4
H3BO36.0 1 mL 5.55 ¥10-4
Na2SiO3·9H2O 15.0 1mL 5.28 ¥10-5
K2HPO45.0 1 mL 2.87 ¥10-5
FeCl30.8 1 mL 1.43 ¥10-5
ZnCl20.15 1 mL 2.29 ¥10-6
MnCl2·4H2O 1.2 1 mL 2.18 ¥10-5
CoCl2·6H2O 0.003 1 mL 5.09 ¥10-8
CuCl20.0012 1 mL 1.89 ¥10-8
Cyanocobalamin (vitamin B12) 0.002 1mL 1.48 ¥10-9
S3 vitamins solution (See following recipe) 1mL —
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490 Appendix A – Recipes for Freshwater and Seawater Media
S3 Vitamins Solution
Into 950mL of highest-quality deionized water, dissolve the components or add 1 mL of the primary stock
solutions. Bring the final volume to 1 liter. Filter-sterilize.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Inositol — 500 mg 2.78 ¥10-6
Thymine — 300mg 2.38 ¥10-6
Thiamine·HCl (vitamin B1) — 50mg 1.48 ¥10-7
Nicotinic acid (niacin) — 10mg 8.12 ¥10-8
Ca pantothenate — 10 mg 4.20 ¥10-8
p-Aminobenzoic acid 1.0 1mL 7.29 ¥10-9
Biotin (vitamin H) 0.1 1 mL 4.09 ¥10-10
Folic acid 0.2 1 mL 4.53 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 491
ASP-M Medium
(McLachlan et al. 1964, Goldman and McCarthy 1978)
This is an artificial enriched seawater medium that was designed as a general medium for marine macroalgae and
microalgae. It is derived from the earlier ASP Medium series (see Provasoli et al.1957). The TMS-II trace metals
solutions (McLachlan 1973) are derived from the S1 metals solution of Provasoli and Pintner (1953). The vitamins
solution is complex, and some of the vitamins are unnecessary for common algal species. To prepare, dissolve the
anhydrous salts in 500 mL dH2O and the hydrous salts in 300 mL dH2O, and then combine the solutions. Dissolve
the Tris base and the glycylglycine. Add the indicated quantity of stock solutions and bring the final volume to
1 liter. Autoclave or sterile-filter. The pH should be 7.5 at room temperature.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Anhydrous salts
NaCl — 23.38 g 4.0 ¥10-1
KCl — 0.75g 1.0 ¥10-2
CaCl2— 1.120 g 1.0 ¥10-2
NaHCO3— 0.168 g 2.0 ¥10-3
Hydrous salts
MgSO4·7H2O — 4.930g 2.0 ¥10-2
MgCl2·4H2O — 4.060 g 2.0 ¥10-2
Macronutrients
NaNO385.0 1mL 1.0 ¥10-3
NaH2PO4·H2O 13.8 1mL 1.0 ¥10-4
Na2SiO3·9H2O 56.8 1 mL 1.0 ¥10-4
Other components
Fe-EDTA 84.2 100mL 2.0 ¥10-6
Tris base — 0.606g 5.0 ¥10-3
Glycylglycine — 0.660g 5.0 ¥10-3
TSM-I solution (See following recipe) 1mL —
TSM-II solution (See following recipe) 1mL —
S3 vitamin solution (See following recipe) 1 mL —
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492 Appendix A – Recipes for Freshwater and Seawater Media
Trace Metal Solution: TMS I
(McLachlin 1964)
Into 900mL of dH2O, first dissolve the EDTA and then individually dissolve the metals. Bring the final volume to
1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
EDTA — 14.026 g 4.8 ¥10-5
FeCl3— 0.324g 2.0 ¥10-6
H3BO3— 24.732g 4.0 ¥10-4
MnCl2·4H2O — 1.979g 1.0 ¥10-5
ZnSO4·7H2O — 10.064 g 3.5 ¥10-5
NaMoO4·2H2O — 1.210g 5.0 ¥10-6
CuSO4·5H2O — 0.075g 3.0 ¥10-7
CoCl2·6H2O — 0.071g 3.0 ¥10-7
Trace Metal Solution: TMS-II
(McLachlin 1964)
These are necessary only for certain marine macrophytes. Into 900mL of dH2O, dissolve individually components
and bring the final volume to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
KBr — 51.450g 5.0 ¥10-4
SrCl2 — 26.662g 1.0 ¥10-4
Ru — 0.242g 2.0 ¥10-6
Li — 0.424g 1.0 ¥10-5
I — 0.030g 2.0 ¥10-7
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Appendix A – Recipes for Freshwater and Seawater Media 493
S3 Vitamins Solution
(Provasoli 1963)
Into 900mL of dH2O, dissolve the first four components and then add 1mL of each primary stock solution. Bring
the final volume to 1 liter. Filter-sterilize and freeze.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Inositol — 900.000 mg 5.0 ¥10-6
Thiamine·HCl (vitamin B1) — 168.635 mg 5.0 ¥10-7
Ca pantethenate 23.830mg 1.0 ¥10-7
Nicotinic acid (niacin) — 12.310mg 1.0 ¥10-7
p-Aminobenzoic acid 1.371 1mL 1.0 ¥10-8
Biotin (vitamin H) 0.244 1 mL 1.0 ¥10-9
Folic acid 0.883 1mL 2.0 ¥10-9
Cyanocobalamin (vitamin B12) 1.355 1mL 1.0 ¥10-9
Thymine 0.378 1mL 3.0 ¥10-6
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494 Appendix A – Recipes for Freshwater and Seawater Media
ESAW Medium
(Harrison et al. 1980, Berges et al. 2001)
This medium is designed for coastal and open ocean phytoplankton. The artificial seawater base is enriched with a
modification of the enrichment solutions of Provasoli’s (1968) ES medium. The recipe is from Berges et al. (2001)
and has been modified from the earlier version (Harrison et al. 1980) by adding borate only in the salt solution (not
in trace metals), replacing glycerophosphate with inorganic phosphate, and preparing the silicate stock solution at
half-strength without acidification to facilitate dissolution. Three additional trace elements have been added:
Na2MoO4·2H2O, Na2SeO3, and NiCl2·6H2O. The anhydrous and hydrated salts must be dissolved separately; masses
assume specific gravity =1.021 at 20° C. The iron is now added solely as chloride (to remove ammonium) from a
separate stock with equimolar EDTA. Filter sterilization is recommended (e.g., with a 147-mm Millipore GS filter
[pore size, 0.22mm] with a Gelman A/E prefilter). Autoclaving the final medium often causes precipitates to form.
If autoclaving is necessary, autoclave the two salt solutions separately, and when they are completely cooled, asepti-
cally combine them. The 1- or 2-mL nutrient additions should be added with a 0.2-mm-pore-size sterile filter and a
syringe. The medium should be bubbled with filtered air for 12 hours before use. Dissolve the anhydrous salts in
600mL of dH2O and dissolve the hydrated salts in 300mL dH2O. Combine salt solutions I and II, and add the indi-
cated amounts of each stock solution. Bring the final volume to 1 liter with dH2O. The final pH should be 8.2.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Salt solution I Anhydrous salts
NaCl — 21.194 g 3.63 ¥10-1
Na2SO4— 3.550 g 2.50 ¥10-2
KCl — 0.599g 8.03 ¥10-3
NaHCO3— 0.174 g 2.07 ¥10-3
KBr — 0.0863g 7.25 ¥10-4
H3BO3— 0.0230 g 3.72 ¥10-4
NaF — 0.0028g 6.67 ¥10-5
Salt solution II Hydrated salts —
MgCl2·6H2O — 9.592g 4.71 ¥10-2
CaCl2·2H2O — 1.344 g 9.14 ¥10-3
SrCl2·6H2O — 0.0218g 8.18 ¥10-5
Major Nutrients —
NaNO346.67 1 mL 5.49 ¥10-4
NaH2PO4·H20 3.094 1 mL 2.24 ¥10-5
Na2SiO3·9H2O 15 2mL 1.06 ¥10-4
Iron-EDTA Stock Solution
Na2EDTA · 2H2O — 2.44g 6.56 ¥10-6
FeCl3·6H2O 1.77 1mL 6.55 ¥10-6
Trace metals stock solution II (See following recipe) 1 mL —
Vitamins solution (See following recipe) 1 mL —
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Appendix A – Recipes for Freshwater and Seawater Media 495
Trace Metals Solution II
Into 900mL of dH2O, add the indicated quantities of trace elements; bring the final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 3.09g 8.30 ¥10-6
ZnSO4·7H2O — 0.073 g 2.54 ¥10-7
CoSO4·7H2O — 0.016 g 5.69 ¥10-8
MnSO4·4H2O — 0.54g 2.42 ¥10-6
Na2MoO4·2H2O 1.48 1mL 6.12 ¥10-9
Na2SeO30.173 1mL 1.00 ¥10-9
NiCl2·6H2O 1.49 1 mL 6.27 ¥10-9
Vitamins Stock Solution
Into 900mL dH2O, dissolve the thiamine·HCl, add 1mL of the other two vitamin solutions, and bring the final
volume to 1 liter with dH2O. Filter-sterilize.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 0.1 g 2.96 ¥10-7
Biotin (vitamin H) 1.0 1 mL 4.09 ¥10-9
Cyanocobalamin (vitamin B12) 2.0 1mL 1.48 ¥10-9
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496 Appendix A – Recipes for Freshwater and Seawater Media
CCAP Artificial Seawater
(Tompkins et al. 1995)
This medium is prepared with commercially available Ultramarine Synthetica sea salts (Waterlife Research Indus-
tries). Into 900 mL dH2O, dissolve the Ultramarine Synthetica sea salts, then dissolve the tricine and add the stock
solution quantities. Bring final volume to 1 liter with dH2O. Adjust to pH of 7.6 to 7.8 and then autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Ultramarine sea salts — 33.6g —
Tricine — 0.50g 2.79 ¥10-3
Macronutrients (See following recipe) 1 mL —
Soil water extract (See Chapter 2) 25 mL —
Vitamins stock solution (See following recipe) 1 mL —
Macronutrients Stock Solution
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 112.50g 1.32 ¥10-3
Na2HPO4— 4.50g 2.58 ¥10-5
K2HPO4— 3.75g 2.76 ¥10-5
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Appendix A – Recipes for Freshwater and Seawater Media 497
Vitamins Stock Solution
Into 900mL dH2O, individually dissolve each component or stock solution and bring final volume to 1 liter. Filter-
sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Inositol — 2.5 g 1.39 ¥10-5
Thymine — 1.5g 1.19 ¥10-5
Thiamine·HCl (vitamin B1) — 0.25 g 7.41 ¥10-7
Nicotinic acid (niacin) — 0.05g 4.06 ¥10-7
Ca pantothenate — 0.05g 2.10 ¥10-7
Cyanocobalamin (vitamin B12) — 0.01g 7.38 ¥10-9
Folic acid 1.0 1mL 2.27 ¥10-9
Biotin (vitamin H) 0.5 1 mL 2.05 ¥10-9
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498 Appendix A – Recipes for Freshwater and Seawater Media
ASN-III Medium
(Rippka 1988)
To prepare, begin with 900 mL of dH2O and dissolve the designated quantities for the following components. Bring
final volume to 1 liter. The final medium should have a pH of 7.3 ±0.2 at room temperature (before autoclaving).
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaCl — 25.0 g 4.28 ¥10-1
MgSO4·7H2O — 3.5g 1.42 ¥10-2
MgCl2·6H2O — 2.0g 9.84 ¥10-3
NaNO3— 0.75 g 8.82 ¥10-3
K2HPO4·3H2O — 0.75g 3.29 ¥10-3
CaCl2·2H2O — 0.5g 3.40 ¥10-3
KCl — 0.5 g 6.71 ¥10-3
NaCO3— 0.02 g 2.41 ¥10-4
Citric acid — 3.0mg 1.56 ¥10-5
Ferric ammonium citrate — 3.0mg (approx.) 9 ¥10-6
Mg EDTA — 0.5mg 1.59 ¥10-6
Cyanocobalamin (vitamin B12) — 10.0 mg 7.39 ¥10-9
A-5 +Co trace metals sol. (See following recipe) 1.0 ml —
A5 +Co Trace Metals Solution
In 950 mL of dH2O, dissolve each of the following components, and then bring the final volume up to 1 liter.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
H3BO3— 2.860g 4.60 ¥10-5
MnCl2·4H2O — 1.810g 9.14 ¥10-6
ZnSO4·7H2O — 0.222g 7.72 ¥10-7
NaMoO4·2H2O — 0.390g 1.61 ¥10-3
CuSO4·5H2O — 0.079g 3.16 ¥10-7
Co(NO3)2·6H2O — 49.40 mg 1.70 ¥10-7
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Appendix A – Recipes for Freshwater and Seawater Media 499
YBC-II Medium
(Chen et al. 1996)
This medium was developed to culture nitrogen-fixing Trichodesmium (no nitrogen source in medium) under critical
culture conditions for physiological studies (Chen et al. 1996). It was based upon Ohki’s medium (Ohki et al. 1992),
but it is significantly different. Another more complex medium, YBC-III medium, is presented in Chen et al. (1996),
and it more closely resembles oligotrophic seawater. To prepare, make the necessary stock solutions, using dH2O.
Into 900mL of dH2O, add the following components. Bring the final volume to 1 liter with high-quality dH2O.
Adjust to a pH of 8.15 to 8.2 with NaOH and filter-sterilize; do not autoclave.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Anhydrous salts
NaCl — 24.5500g 4.20 ¥10-1
KCl — 0.7500 g 1.00 ¥10-2
NaHCO3— 0.2100 g 2.5 ¥10-3
H3BO3— 0.0360 g 5.80 ¥10-4
KBr — 0.1157g 9.72 ¥10-4
NaF 2.94 1 mL 7.0 ¥10-5
Hydrous salts
MgCl2·6H2O — 4.067g 2.00 ¥10-2
CaCl2·2H2O — 1.47g 1.00 ¥10-2
MgSO4·7H2O — 6.16 g 2.50 ¥10-2
SrCl2·6H2O 17.33 1mL 6.50 ¥10-8
Macronutrients
NaH2PO4·H2O 6.9 1mL 5.00 ¥10-5
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 1 ml —
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Trace Metals Solution
Into 950mL of high-quality dH2O, add the EDTA and 1mL of each primary stock solution. Filter-sterilize.
500 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 0.745g 2.00 ¥10-6
FeCl3·6H2O 0.11 1 mL 4.07 ¥10-7
MnCl2·4H2O 3.96 1mL 2.00 ¥10-8
ZnSO4·7H2O 1.15 1 mL 4.00 ¥10-9
CoCl2·6H2O 5.95 1mL 2.50 ¥10-9
Na2MoO2·2H2O 2.66 1mL 1.10 ¥10-8
CuSO42.23 1mL 1.00 ¥10-9
Vitamins Solution
Into 950mL of dH2O, add the thiamine·HCl and primary stock solutions, and bring the final volume to 1 liter with
dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 100mg 2.96 ¥10-7
(vitamin B1)
Biotin (vitamin H) 0.5 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.5 1 mL 3.69 ¥10-10
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Appendix A – Recipes for Freshwater and Seawater Media 501
2.2. Enriched Natural Seawater Media
ES Medium
(Provasoli 1968)
ES Medium (enriched natural seawater), in addition to nitrate and phosphate, includes Tris base buffer, trace metals,
and vitamins in place of soil-water extract. The origin and composition of ES medium is confusing; recipes appeared
in two publications, and they are different (D’Angostino and Provasoli 1968, Provasoli 1968). Provasoli himself cited
the Provasoli (1968) publication (e.g., D’Agostino and Provasoli 1970, Provasoli et al. 1970, Provasoli and Pintner
1980). Unfortunately, the Provasoli (1968) publication contains several errors. Further confusion arose when some
authors (e.g., Starr and Zeikus 1993) erroneously attributed ES Medium to Provasoli (1963). 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 (see Provasoli 1958). The molar concentrations differ and he substitutes sulfated Mn, Z, and
Co for the original chlorinated forms. The PII trace metals recipe 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 subse-
quently in Provasoli (1963). See Provasoli (1968) for the version of trace metals used in ES medium described here.
In addition to this recipe and the ES medium by D’Angostino and Provasoli (1968), McLachlin (1973) provides
another (different) ES Medium recipe. McLachlin’s version, based on personal communication from John West, has
6.6 ¥10-5M Tris, 6.6 ¥10-5M nitrate, 2.5 ¥10-6M glycerophosphate, 7.2 ¥10-3M iron–EDTA, and a different for-
mulation of vitamins and trace metals; i.e., everything is different. West and McBride (1999) provide yet another
version of ES Medium (see following recipe), but their version may be the best formulation for general use. To
prepare the enrichment stock solution, begin with 900mL of dH2O, add the following components (vitamins should
be added last, after mixing other ingredients), bring the final volume to 1 liter with dH2O, and pasteurize (do not
autoclave). To prepare the ES Medium, add 20mL of the enrichment stock solution to 980mL of filtered natural
seawater. Pasteurize.
Enrichment Stock Solution
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Tris base — 5.0g 8.26 ¥10-4
NaNO3— 3.5g 8.24 ¥10-4
Na2b-glycerophosphate·H2O — 0.5 g 4.63 ¥10-5
Iron–EDTA Solution (See following recipe) 250 mL —
Trace metals solution (See following recipe) 25 mL —
Thiamine·HCl (vitamin B1) — 0.500mg 2.96 ¥10-8
Biotin (vitamin H) 0.005 1mL 4.09 ¥10-10
Cyanocobalamin (vitamin B12) 0.010 1mL 1.48 ¥10-10
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Iron–EDTA Solution
Into 900mL of dH2O, dissolve the EDTA and then the iron sulfate. Bring the final volume to 1 liter. Pasteurize
and store refrigerated.
502 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 0.841 g 1.13 ¥10-5
Fe(NH4)2(SO4)2·6H2O — 0.702g 8.95 ¥10-6
Trace Metals Solution
(Provasoli 1968)
Into 900mL of dH2O, 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 volume to 1 liter and store
refrigerated.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 12.74g 1.71 ¥10-4
FeCl3·6H2O — 0.484g 8.95 ¥10-6
H3BO3— 11.439g 9.25 ¥10-5
MnSO4·4H2O — 1.624g 3.64 ¥10-5
ZnSO4·7H2O — 0.220g 3.82 ¥10-6
CoSO4·7H2O — 0.048 g 8.48 ¥10-7
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Appendix A – Recipes for Freshwater and Seawater Media 503
West and McBride’s Modified ES Medium
(West and McBride 1999)
This medium is derived from ES Medium (Provasoli 1968) and Modified ES Medium (McLachlan 1973) (see ES
Medium above). Compared with earlier ES media, this recipe removes the Tris buffer and has lower nitrate, phos-
phate, and trace metal levels; it has higher iron–EDTA and vitamin levels. The trace metals solution is referred to
as PII trace metals in West and McBride (1999), but it varies substantially from Provasoli’s original PII trace metal
solution (see Provasoli 1958, 1963) as well as his formulation for ES Medium (Provasoli 1968). West and McBride
(1999) do not specify the amount of enrichment, but the correct addition is 10 mL of enrichment stock for each liter
of natural seawater (John West, personal communication). The stock solution quantities (but not final molar con-
centrations) have been adjusted in our recipe. To prepare the enrichment stock solution, begin with 800 mL of
dH2O, and then add the components and bring the final volume to 1 liter with dH2O. Pasteurize (do not autoclave).
To prepare the final Modified ES Medium, aseptically add 10mL of the enrichment stock solution to 990mL of
filtered natural seawater that has been sterilized by pasteurization.
Enrichment Stock Solution
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 3.85g 4.53 ¥10-4
Na2b-glycerophosphate·H2O — 0.4 g 1.31 ¥10-5
Fe-EDTA Solution (See following recipe) 100 mL —
Trace metal solution (See following recipe) 20mL —
Thiamine·HCl (vitamin B1) 0.5 8.0mL 1.19 ¥10-7
Biotin (vitamin H) 0.05 8.0mL 1.78 ¥10-8
Cyanocobalamin (vitamin B12) 0.025 3.5mL 6.46 ¥10-10
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Iron–EDTA Solution
Into 950mL of dH2O, first dissolve the EDTA and then the iron compound. Bring the final volume to 1 liter with
dH2O and pasteurize; store in a refrigerator.
504 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 6.00 g 1.61 ¥10-4
Fe(NH4)2(SO4)2·6H2O — 7.00g 1.78 ¥10-4
Trace Metals Solution
Into 900mL of dH2O, dissolve the EDTA and then individually add and dissolve the remaining components. Bring
the final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 2.548g 1.37 ¥10-5
H3BO3— 2.240g 7.25 ¥10-5
MnSO4·4H2O — 0.240g 2.15 ¥10-6
ZnSO4·4H2O — 0.044 g 3.06 ¥10-7
CoSO4·7H2O — 0.010 g 7.11 ¥10-8
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Appendix A – Recipes for Freshwater and Seawater Media 505
ESNW Medium
(Harrison et al. 1980, Berges et al. 2001)
This medium is a modification of Provasoli’s (1968) enrichment solution (ES). For an artificial seawater version, see
ESAW Medium. Into 900mL, add the indicated amounts of each stock solution. Bring the final volume to 1 liter
with dH2O. Final pH should be 8.2. Filter-sterilize.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO346.67 1 mL 5.49 ¥10-4
NaH2PO4.·H2O 3.094 1mL 2.24 ¥10-5
Na2SiO3.·9H2O 15 2 mL 1.06 ¥10-4
Iron-EDTA Stock Solution
Na2EDTA · 2H2O — 2.44 g 6.56 ¥10-6
FeCl3·6H2O 1.77 1mL 6.55 ¥10-6
Trace metals stock solution II (See following recipe) 1 mL —
Vitamins solution (See following recipe) 1mL —
Trace Metals Solution II
Into 900mL of dH2O, add the indicated quantities of trace elements; bring the final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 3.09g 8.30 ¥10-6
ZnSO4·7H2O — 0.073 g 2.54 ¥10-7
CoSO4·7H2O — 0.016 g 5.69 ¥10-8
MnSO4·4H2O — 0.54g 2.42 ¥10-6
Na2MoO4·2H2O 1.48 1mL 6.12 ¥10-9
Na2SeO30.173 1mL 1.00 ¥10-9
NiCl2·6H2O 1.49 1 mL 6.27 ¥10-9
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506 Appendix A – Recipes for Freshwater and Seawater Media
Vitamins Stock Solution
Into 900mL dH2O, dissolve the thiamine·HCl, add 1mL of the other two vitamin solutions, and bring the final
volume to 1 liter with dH2O. Filter-sterilize.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 0.1 g 2.96 ¥10-7
Biotin (vitamin H) 1.0 1 mL 4.09 ¥10-9
Cyanocobalamin (vitamin B12) 2.0 1mL 1.48 ¥10-9
f/2 Medium
(Guillard and Ryther 1962, Guillard 1975)
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 (Guillard 1975). The original medium (Guillard and Ryther 1962) used ferric sequestrene;
we have substituted Na2EDTA · 2H2O and FeCl3·6H2O. Into 950 mL of filtered natural seawater, add the follow-
ing components. Bring the final volume to 1 liter with filtered natural seawater. Autoclave. If silicate is not required,
omit to reduce precipitation.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO375 1 mL 8.82 ¥10-4
NaH2PO4·H2O 5 1mL 3.62 ¥10-5
Na2SiO3·9H2O 30 1mL 1.06 ¥10-4
Trace metals solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 0.5 mL —
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Appendix A – Recipes for Freshwater and Seawater Media 507
f/2 Trace Metals Solution
Into 950mL of dH2O, dissolve the EDTA and other components. Bring the final volume to 1 liter with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
FeCl3·6H2O — 3.15g 1.17 ¥10-5
Na2EDTA · 2H2O — 4.36 g 1.17 ¥10-5
MnCl2·4H2O 180.0 1mL 9.10 ¥10-7
ZnSO4·7H2O 22.0 1 mL 7.65 ¥10-8
CoCl2·6H2O 10.0 1 mL 4.20 ¥10-8
CuSO4·5H2O 9.8 1mL 3.93 ¥10-8
Na2MoO4·2H2O 6.3 1mL 2.60 ¥10-8
f/2 Vitamins Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, and add 1mL of the primary stocks. Bring final volume to 1 liter
with dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 200 mg 2.96 ¥10-7
Biotin (vitamin H) 1.0 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 1.0 1 mL 3.69 ¥10-10
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K Medium
(Keller and Guillard 1985, Keller et al.1987)
K medium was designed for oligotrophic (oceanic) marine phytoplankters. It uses a 10-fold higher EDTA chelation
than most common marine media, and this reduces the availability of trace metals, thereby reducing the possibility
of metal toxicity. The macronutrients are perhaps too high for some open ocean organisms. 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. For best results, use natural oligotrophic ocean water rather than coastal seawater for the
base. The original publications also described an artificial seawater version of K medium. Into 950mL of filtered
natural seawater, add the following components and bring the final volume up to 1 liter with filtered natural
seawater. Autoclave.
508 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO375.00 1mL 8.82 ¥10-4
NH4Cl 2.67 1mL 5.00 ¥10-5
Na2b-glycerophosphate 2.16 1mL 1.00 ¥10-5
Na2SiO3·9H2O 15.35 1mL 5.04 ¥10-4
H2SeO30.00129 1 mL 1.00 ¥10-8
Tris-base (pH 7.2) 121.10 1mL 1.00 ¥10-3
Trace metals solution (See following recipe) 1 mL —
Vitamins solution (See following recipe) 0.5mL —
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Appendix A – Recipes for Freshwater and Seawater Media 509
Trace Metals Solution
Into 950mL of dH2O, dissolve the EDTA and then add the components. Bring the final volume to 1 liter, using
dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 37.220g 1.00 ¥10-4
Fe-Na-EDTA ·3H2O — 4.930 g 1.17 ¥10-5
FeCl3·6H2O — 3.150g 1.17 ¥10-5
MnCl2·4H2O — 0.178 g 9.00 ¥10-7
ZnSO4·7H2O 23.00 1mL 8.00 ¥10-8
CoSO4·7H2O 14.05 1mL 5.00 ¥10-8
Na2MoO4·2H2O 7.26 1mL 3.00 ¥10-8
CuSO4·5H2O 2.50 1mL 1.00 ¥10-8
f/2 Vitamins Solution
(Guillard and Ryther 1962, Guillard 1975)
Into 950mL of dH2O, dissolve the thiamine ·HCl. Add 1mL of the primary stocks and then bring the final volume
to 1 liter with dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 200 mg 2.96 ¥10-7
Biotin (vitamin H) 0.1 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 1.0 1 mL 3.69 ¥10-10
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L1 Medium
(Guillard and Hargraves 1993)
L1 medium is another in the alphabet medium series by Guillard (e.g., f, h, and k). It is derived from f/2 medium
(Guillard and Ryther 1962, Guillard 1975) by adding more trace elements. It is a good general-purpose marine
medium for growing coastal algae. Into 950mL of filtered natural seawater, add the components and bring the final
volume to 1 liter, using filtered natural seawater. Autoclave. Final pH should be 8.0 to 8.2.
510 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO375.00 1 mL 8.82 ¥10-4
NaH2PO4·H2O 5.00 1mL 3.62 ¥10-5
Na2SiO3·9H2O 30.00 1 mL 1.06 ¥10-4
Trace elements solution (See following recipe) 1mL —
Vitamins solution (See following recipe) 0.5 mL —
L1 Trace Elements Solution
To 950 mL dH2O add the following components and bring the final volume to 1 liter with dH2O. Autoclave.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 4.36g 1.17 ¥10-5
FeCl3·6H2O — 3.15 g 1.17 ¥10-5
MnCl2·4·H2O 178.10 1mL 9.09 ¥10-7
ZnSO4·7H2O 23.00 1mL 8.00 ¥10-8
CoCl2·6H2O 11.90 1mL 5.00 ¥10-8
CuSO4·5H2O 2.50 1 mL 1.00 ¥10-8
Na2MoO4·2H2O 19.9 1mL 8.22 ¥10-8
H2SeO31.29 1 mL 1.00 ¥10-8
NiSO4·6H2O 2.63 1mL 1.00 ¥10-8
Na3VO41.84 1mL 1.00 ¥10-8
K2CrO41.94 1 mL 1.00 ¥10-8
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Appendix A – Recipes for Freshwater and Seawater Media 511
f/2 Vitamins Solution
(Guillard and Ryther 1962, Guillard 1975)
Into 950mL of dH2O, dissolve the thiamine·HCl, add 1 mL of the primary stocks, and bring final volume to 1 liter
with dH2O. Filter-sterilize.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 100 mg 2.96 ¥10-7
Biotin (vitamin H) 0.5 1 mL 2.05 ¥10-9
Cyanocobalamin (vitamin B12) 0.5 1 mL 3.69 ¥10-10
AACAP 10/6/04 06:44 PM Page 511

MNK Medium
(Noël et al. 2004)
This enriched seawater medium was developed to grow oceanic coccolithophores (Noël et al. 2004), but it is also a
good general medium for marine phytoplankton. Noël (personal communication) recommends storing filtered
natural seawater in a cold (4° C) dark environment for at least 3 months before using it. Because open ocean phyto-
plankters are often sensitive, all glassware should be acid-cleaned and rinsed thoroughly with high-quality water
(e.g., Milli-Q water) before use. The medium should be filter-sterilized rather than autoclaved; however, see Chapter
22 regarding viruses. Use natural oligotrophic ocean water. Into 997mL of filtered natural seawater, add 1 mL of
nutrient stock, trace metals stock, and vitamin stock solutions. Filter-sterilize. The final pH is 8.7.
Nutrient Stock Solution
Into 950mL dH2O, dissolve the components and bring the final volume to 1 liter with dH2O. Filter-sterilize
(0.2mm pore size) and store refrigerated or frozen. For convenience, the solution can be distributed as 1-mL quan-
tities into Eppendorf or cryovial tubes.
512 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 20.00g 2.35 ¥10-4
Na2HPO4,·12H2O — 0.28g 7.82 ¥10-7
K2HPO4(anhydrous) — 1.00g 5.74 ¥10-6
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Appendix A – Recipes for Freshwater and Seawater Media 513
Trace Metals Solution
Into 950mL dH2O, add 1mL each of the primary stock solution. Bring the final volume to 1 liter with dH2O. Store
refrigerated or frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Fe-Na-EDTA 3H2O 25.900 1mL 6.15 ¥10-8
Mn-EDTA · 3H2O 33.200 1mL 7.49 ¥10-8
Na2EDTA · 2H2O 3.723 1mL 1.00 ¥10-8
MnCl2·4H2O 9.000 1mL 4.55 ¥10-8
ZnSO4·7H2O 2.400 1 mL 8.35 ¥10-9
CoSO4·7H2O 1.200 1mL 4.27 ¥10-9
Na2MoO4·2H2O 0.720 1 mL 2.98 ¥10-9
CuSO4·5H2O 0.060 1 mL 2.40 ¥10-10
Na2SeO30.030 1 mL 1.73 ¥10-10
Vitamins Solution
Into 950mL of dH2O, add the thiamine·HCl and 1mL of each primary stock solution. Bring the final volume to
1 liter with dH2O. Filter-sterilize and freeze.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 20mg 5.93 ¥10-8
(vitamin B1)
Biotin (vitamin H) 0.15 1 mL 6.14 ¥10-10
Cyanocobalamin (vitamin B12) 0.15 1mL 1.11 ¥10-10
AACAP 10/6/04 06:44 PM Page 513

PC Medium
(Keller in Andersen et al. 1997)
PC medium is an enriched natural seawater medium developed by Maureen Keller specifically for growing Prochloro-
coccus, but it can be used for other open ocean phytoplankters. All containers should be acid-cleaned and rinsed with
high-quality dH2O (e.g., Milli-Q water). Seawater should be collected from the oligotrophic open ocean (e.g., Sar-
gasso seawater), taking the usual precautions to avoid contamination. Only ultrapure chemicals and high-quality
dH2O should be used in preparing nutrient stock solutions. A microwave oven is used for sterilization (see Chapter
5). To prepare, filter 1 liter of seawater and sterilize in a microwave oven. After cooling, aseptically add the
following components.
514 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2 b-glycerophosphate 2.16 1mL 1.00 ¥10-5
NH4Cl 2.68 1mL 5.01 ¥10-5
Urea 3 1mL 5.00 ¥10-5
Trace metals solution (See following recipe) 100mL—
F/2 vitamins solution (See following recipe) 50mL—
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Appendix A – Recipes for Freshwater and Seawater Media 515
PC Trace Metals Solution
Into 900mL of high-quality dH2O, dissolve the EDTA and then individually dissolve the metals. Store at 4°C.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 41.60g 1.12 ¥10-4
FeCl3·6H2O — 3.15g 1.17 ¥10-5
MnCl2·4H2O — 0.18 g 9.09 ¥10-8
ZnSO4·7H2O 22.0 1mL 7.65 ¥10-9
CoCl2·6H2O 10.0 1 mL 4.20 ¥10-9
Na2MoO4·2H2O 6.3 1mL 2.60 ¥10-9
H2SeO31.3 1mL 1.01 ¥10-9
NiSO4·6H2O 2.7 1mL 1.03 ¥10-9
f/2 Vitamins Solution
(Guillard and Ryther 1962, Guillard 1975)
Into 950mL of dH2O, dissolve the thiamine ·HCl. Add 1 mL of the primary stocks and bring final volume to 1 liter
with dH2O. Filter-sterilize and store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 200mg 2.96 ¥10-8
(vitamin B1)
Biotin (vitamin H) 1.0 1 mL 2.05 ¥10-10
Cyanocobalamin (vitamin B12) 1.0 1 mL 3.69 ¥10-11
AACAP 10/6/04 06:44 PM Page 515

Pro99 Medium
(Sally Chisholm, unpublished)
This medium was developed specifically for Prochlorococcus (Sally Chisholm, personal communication) and is currently
the best medium available for Prochlorococcus. It can also be used for other oceanic species tolerating high ammonia
concentrations (e.g., Bolidomonas). All containers should be acid-cleaned and rinsed with high-quality H2O (e.g., Milli-
Q). Seawater should be collected from the oligotrophic open ocean (e.g., Sargasso Sea) and only ultrapure-grade
reagents should be used. Good sterile technique is required and a laminar flow hood is recommended. The oli-
gotrophic seawater is autoclaved in a Teflon-lined container and cooled before aseptically adding nutrients. Again,
use only oligotrophic natural seawater, ultrapure-grade reagents, and high-quality H2O (e.g., Milli-Q). To prepare,
filter 1 liter of natural oligotrophic seawater into a Teflon-lined container. Autoclave and cool. Aseptically, add 1mL
each of the NaH2PO4, NH4Cl, and trace element solutions. Note: ammonium chloride and sodium phosphate solu-
tions should be filter-sterilized and stored at 4° C.
516 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaH2PO4·H2O 6.90 1.0mL 5.0 ¥10-5
NH4Cl 42.80 1.0mL 8.0 ¥10-4
Trace elements solution (See following recipe) 1.0mL —
PRO99 Trace Elements Solution
Into 900mL of high-quality dH2O, dissolve EDTA and add other components. Bring final volume to 1 liter with
high-quality dH2O, filter-sterilize, and store at 4°C.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 0.436 g 1.17 ¥10-6
FeCl3·6H2O — 0.316g 1.17 ¥10-6
ZnSO4·7H2O 2.30 1mL 8.00 ¥10-9
CoCl2·6H2O 1.19 1mL 5.00 ¥10-9
MnCl2·4H2O 17.80 1 mL 9.00 ¥10-8
Na2MoO4·2H2O 0.73 1mL 3.00 ¥10-9
Na2SeO31.73 1mL 1.00 ¥10-8
NiSO4·6H2O 2.63 1mL 1.00 ¥10-8
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Appendix A – Recipes for Freshwater and Seawater Media 517
SN Medium
(Waterbury et al. 1986)
SN Medium is the most popular of three media developed by Waterbury et al. (1986) for culturing marine Syn-
chococcus sensu lato (Cyanophyceae). The medium has very high nitrogen, and the trace metals are buffered with citrate
rather than EDTA. If a solid medium (agar) is prepared, standard agar should be washed to remove impurities (see
Chapter 2 and Waterbury et al., 1986). The salinity of the natural seawater is reduced by adding dH2O. To prepare,
autoclave 750mL of filtered natural seawater in a Teflon-lined bottle and separately autoclave 236mL of double-
distilled H2O; cool and aseptically combine the two solutions. Aseptically, add 10 mL of sodium nitrate solution and
1mL of the other five stock solutions. Final salinity should be ~26psu.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO376.5 10 mL 9.0 ¥10-3
K2HPO4, anhydrous 15.68 1mL 9.9 ¥10-5
Na2EDTA · 2H2O 5.58 1mL 1.5 ¥10-5
Na2CO310.70 1 mL 1.0 ¥10-4
Cyanocobalamin (vitamin B12) 0.001 1mL 7.38 ¥10-10
Trace metals solution (See following recipe) 1mL —
Cyano Trace Metals Solution
Into 900mL of dH2O, dissolve the citrates and then add the remaining components. Bring final volume to 1 liter
with dH2O.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Citric Acid·H2O — 6.250g 3.25 ¥10-5
Ferric ammonium citrate — 6.000g —
MnCl2·4H2O — 1.400g 7.08 ¥10-6
Na2MoO4·2H2O — 0.390 g 1.61 ¥10-6
ZnSO4·7H2O — 0.222g 7.72 ¥10-7
Co(NO3)2·6H2O — 0.025 g 8.59 ¥10-8
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von Stosch (Grund) Medium
(Guiry and Cunningham 1984)
This enriched seawater medium was modified from Grund Medium (von Stosch 1963); the Tris buffer was removed
in an effort to reduce bacterial growth. Guiry and Cunningham (1984) used it to grow the red seaweed Gigartina,
and it is suitable for growing many different red seaweeds. To prepare, pasteurize 940 mL of filtered natural
seawater and aseptically add 10 mL each of the following stock solutions. Autoclave.
518 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2b-glycerophosphate 5.36 10mL 2.48 ¥10-4
NaNO342.52 10 mL 5.00 ¥10-3
FeSO4·7H2O 0.28 10mL 1.00 ¥10-5
MnCl2·4H2O 1.96 10 mL 1.00 ¥10-4
Na2EDTA · 2H2O 3.72 10 mL 1.00 ¥10-4
Vitamins stock solution (See following recipe) 10 mL —
Vitamins Stock Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, and add 1mL of each of the two primary stock solutions.
Filter-sterilize and freeze.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 200mg 5.93 ¥10-6
(vitamin B1)
Biotin (vitamin H) 0.1 1 mL 4.09 ¥10-9
Cyanocobalamin (vitamin B12) 0.2 1 mL 1.48 ¥10-9
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Appendix A – Recipes for Freshwater and Seawater Media 519
Walne’s Medium
(Walne 1970)
This enriched seawater medium was designed for mass culture of marine phytoplankton as feed for shellfish. The
medium has a boron addition, which is completely unnecessary when using natural seawater as a base. Sodium metasil-
icate (40mg·L-1) should be added for the growth of diatoms. The vitamins solution is extremely dilute and lacks
biotin. It would be more sensible to move the EDTA, iron, and manganese to the trace metals solution (with 1,000¥
increase in concentrations to compensate for the 1 mL addition). Note that the EDTA concentration is approximately
10¥higher than usual for coastal culture media; it is comparable to the oceanic K Medium (Keller and Guillard 1985,
Keller et al. 1987). To prepare, pasteurize 1 liter of filtered natural seawater; after cooling, aseptically add 1 mL of
the nutrient solution and 100 mL of the vitamins solution.
Nutrient Solution
Into 900 mL of high quality dH2O, dissolve the components. Bring final volume to 1 liter with high-quality dH2O,
filter sterilize, and store at 4°C.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 100.0 g 1.18 ¥10-3
H3BO3— 33.6 g 5.43 ¥10-4
Na2EDTA (anhydrous) — 45g 1.54 ¥10-4
NaH2PO4·H2O — 20.0g 1.28 ¥10-4
FeCl3·6H2O — 1.3g 4.81 ¥10-6
MnCl2·4H2O — 0.36g 1.82 ¥10-6
Trace metals solution (See following recipe) 1mL —
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Trace Metals Solution
Into 900mL of high quality dH2O, dissolve the components. This solution is normally cloudy. Acidify with a few
drops of concentrated HCl to give a clear solution. Bring final volume to 1 liter with high-quality dH2O, filter
sterilize, and store at 4° C.
520 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
ZnCl2— 21.0g 1.54 ¥10-7
CoCl2·6H2O — 20.0g 8.41 ¥10-8
(NH4)6Mo7O24 ·4H2O — 9.0g 7.28 ¥10-9
CuSO4·5H2O — 20.0g 8.01 ¥10-8
Vitamins Solution
Into 950 mL of dH2O, dissolve the thiamine·HCl and cyanocobalamin. Bring final volume to 1 liter, filter sterilize,
and freeze.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 1.0g 2.96 ¥10-10
Cyanocobalamin (vitamin B12) — 50mg 3.69 ¥10-12
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Appendix A – Recipes for Freshwater and Seawater Media 521
2.3. Enriched Natural Seawater +Organics
Antia’s Medium
(Antia et al. 1969)
This medium was designed for heterotrophic growth of a cryptophyte by enriching with glycerol (Antia et al. 1969);
it was the first demonstration of heterotrophic growth in cryptophytes. This is a reduced-salinity medium (~28psu).
The original recipe gives a component weight of 1.125 mg MnSO4·4H2O to equal 5.00 ¥10-6M; a weight of 1.25
mg gives a more precise 5.00 ¥10-6M concentration, and perhaps 1.125mg is a typographical error. The medium
is not recommended for routine maintenance of cryptophytes. Into 800mL of filtered natural seawater, add each
component as indicated below, and bring the final volume to 1 liter, using dH2O. Adjust the Tris·HCl stock
solution to a pH of 6.8 to 6.9 to give a pH of 7.6 to 7.8 after autoclaving. Final salinity should be ~28 psu.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
KNO325 1 mL 2.47 ¥10-3
NaH2PO4·2H2O 34.5 1mL 2.50 ¥10-4
Na2SiO39·H2O 84.1 1mL 2.96 ¥10-4
Tris·HCl (pH 6.8–6.9) 1.31 1 mL 8.31 ¥10-6
Glycerol — 46.1 g 5.00 ¥10-1
Trace metals solution (See following recipe) 2.5mL —
Vitamins solution (See following recipe) 1 mL —
Trace Metals Stock Solution
Into 900mL of dH2O, dissolve the EDTA and add remaining components. Bring final volume to 1 liter with dH2O
and adjust the pH to 7.6–7.8. Store frozen.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Na2EDTA · 2H2O — 8.110 g 2.18 ¥10-5
FeCl3·6H2O — 2.700g 1.00 ¥10-5
MnSO4·4H2O — 1.125g 4.56 ¥10-6
ZnSO4·7H2O — 0.575 g 2.00 ¥10-6
Na2MoO4·2H2O — 0.243g 1.00 ¥10-6
CuSO4·5H2O 24.97 1mL 1.00 ¥10-7
CoSO4·7H2O 14.06 1 mL 5.00 ¥10-8
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Vitamins Stock Solution
Into 950mL of dH2O, dissolve the thiamine·HCl, and add 1mL of each primary stock solution. Bring to 1 liter
with dH2O. Filter-sterilize.
522 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl — 500mg 1.48 ¥10-6
(vitamin B1)
Biotin (vitamin H) 1.00 1 mL 4.09 ¥10-9
Cyanocobalamin (B12) 2.00 1 mL 1.48 ¥10-9
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Appendix A – Recipes for Freshwater and Seawater Media 523
ANT Medium
(Tompkins et al. 1995)
This medium, prepared by the Culture Centre for Algae and Protozoa (Tompkins et al. 1995), is derived from Antia’s
medium (Antia et al. 1969). However, it is highly modified from Antia et al. (1969), especially with the substitution
of glycine for glycerol (possible typographical error; glycerine =glycerol) and Tris base for Tris·HCl. The recipe
does not provide for a vitamin stock solution, and this should be prepared because it is nearly impossible to accu-
rately weigh mg quantities. Into 800mL of natural seawater, add the quantities of each component, and bring up to
1 liter with dH2O. Autoclave. Final pH should be 7.6 to 7.8, and final salinity ~28psu.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Tris base — 1.0 g 8.26 ¥10-3
Glycine — 0.3 g 4.00 ¥10-3
KNO350 1mL 4.95 ¥10-4
NaH2PO4·2H2O 7.8 1 mL 5.65 ¥10-5
Trace metals solution (See following recipe) 2.5mL —
Thiamine·HCl (vitamin B1) — 500.0mg 1.48 ¥10-6
Biotin (vitamin H) — 1.0mg 4.09 ¥10-9
Cyanocobalamin (vitamin B12) — 2.0mg 1.48 ¥10-9
Trace Metals Solution
Into 950mL of dH2O, dissolve the EDTA and other components; bring final volume to 1 liter with dH2O. Adjust
the pH to 7.6–7.8.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
EDTA Na2·2H2O — 3.240g 2.18 ¥10-5
FeCl3·4H2O — 1.080g 1.36 ¥10-5
MnSO4·4H2O — 0.450g 4.56 ¥10-6
ZnSO4·7H2O — 0.230 g 2.00 ¥10-6
Na2MoO4·2H2O 9.7 10 mL 1.00 ¥10-8
CuSO4·5H2O 10.0 10mL 1.00 ¥10-8
CoSO4·7H2O 5.6 1mL 5.00 ¥10-8
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2.4. Soil-Water Enriched Natural Seawater Media
Plymouth Erdschreiber Medium (PE)
(Tompkins et al. 1995)
This medium can be traced back to Schreiber’s Medium and Erd-Schreiber’s Medium (Schreiber 1927, Hämmerling
1931, Føyn 1934; see Chapter 1). The soil-water extract substitutes for micronutrients, a chelator, and trace metals;
for preparation of soil-water extract, see Chapter 2. This is a good medium for maintenance culturing of many marine
algae, but the undefined nature of the soil extract makes it a problematic medium for experimental research. The
CCAP recipe presented here was used extensively by Mary Parke and coworkers at the Plymouth Marine Labora-
tory. Combine 902.5mL of filtered natural seawater and 47.5mL of dH2O to produce a 95% seawater solution.
Autoclave and cool. Aseptically add 1mL of nitrate and phosphate solutions and 50mL of soil-water extract. Final
salinity should be ~32psu.
524 Appendix A – Recipes for Freshwater and Seawater Media
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3200 1mL 2.35 ¥10-3
Na2HPO4·12H2O 20 1 mL 5.58 ¥10-5
Soil-water extract — 50mL —
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Appendix A – Recipes for Freshwater and Seawater Media 525
General Purpose Medium (GPM), Modified
(Sweeney et al. 1959, modified by Loeblich 1975)
This is a reduced-salinity seawater medium that originated with Sweeney et al. (1959) and was then modified by
Loeblich (1975). Essentially, it is Erd-Schreiber’s medium supplemented with trace metals and vitamins. The salin-
ity is reduced to ~28 psu. It will grow many types of marine algae, but its primary application has been for growing
dinoflagellates (e.g., Blackburn et al. 1989). Into 800mL of filtered natural seawater, add 204 mL of dH2O. Add the
components as indicated. Pasteurize or autoclave (autoclaving will cause precipitation, which can be reduced by
rapid cooling). Final pH should be 8.2.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
KNO3— 0.200gK2HPO41.98 ¥10-3
— 0.035 g 2.01 ¥10-4
Soil extract — 15 mL —
Trace metals solution (See following recipe) 30 ml —
Vitamins stock solution (See following recipe) 1 mL —
Trace Metals Solution
Into 900mL of dH2O, dissolve the EDTA and other components. Bring final volume to 1 liter. Adjust pH to 7.5.
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
EDTA Na2·2H2O — 1.2700g 3.41 ¥10-6
FeCl3·6H2O — 0.0484g 1.79 ¥10-7
H3BO3— 1.1400g 1.84 ¥10-5
MnCl2·4H2O — 0.1440g 7.28 ¥10-7
ZnCl210.4 1mL 7.63 ¥10-8
CoCl2·6H2O 4.0 1mL 1.68 ¥10-8
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Vitamins Stock Solution
Into 900mL of dH2O, add the thiamine·HCl and 1mL each of the two primary stocks; bring final volume to 1 liter
and filter-sterilize.
526 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 100 mg 2.96 ¥10-7
Biotin (vitamin H) 1 1 mL 4.09 ¥10-9
Cyanocobalamin (vitamin B12) 2 1mL 1.48 ¥10-9
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Appendix A – Recipes for Freshwater and Seawater Media 527
2.5. Bacterial-Fungal Test Media for Seawater
f/2 Peptone Test Medium
This is a general marine test medium used to detect the presence of nonmethylaminotrophic bacteria when attempt-
ing to establish the axenicity of a strain. Dissolve 1g peptone into 1 liter of f/2 medium (see previous recipe); another
culture medium, marine or freshwater, may be substituted. Dispense in test tubes and autoclave. Agar can be added
to produce a solid medium (see Chapter 2).
AACAP 10/6/04 06:44 PM Page 527

f/2 Methylamine Test Medium
This is a specific marine test medium to detect the presence of methylaminotrophic bacteria when attempting to
establish the axenicity of a strain. Dissolve 1g methylamine·HCl into 1 liter of f/2 medium (see previous recipe);
another culture medium, marine or freshwater, may be substituted. Dispense in test tubes and autoclave. Agar can
be added to produce a solid medium (see Chapter 2).
528 Appendix A – Recipes for Freshwater and Seawater Media
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Appendix A – Recipes for Freshwater and Seawater Media 529
f/2 Peptone-Methylamine Test Medium
This is a broad-spectrum marine test medium to detect the presence of many types of marine bacteria when attempt-
ing to establish the axenicity of a strain. Dissolve 1g peptone and 1 g methylamine·HCl into 1 liter of f/2 medium
(see previous recipe); another culture medium, marine or freshwater, may be substituted. Dispense in test tubes and
autoclave. Agar can be added to produce a solid medium (see Chapter 2).
AACAP 10/6/04 06:44 PM Page 529

Malt Medium in Seawater
This is a good test medium for detecting the presence of marine fungi when attempting to establish the axenicity of
a strain. It uses reduced-salinity seawater as a base. Dissolve 5g of peptone and 10g malt extract in a mixture of 750
mL filtered natural seawater and 250mL dH2O. Final pH should be 7.0 to 7.2 after autoclaving. Dispense in test
tubes and autoclave. Agar can be added to produce a solid medium (see Chapter 2). Final salinity should be ~26psu.
530 Appendix A – Recipes for Freshwater and Seawater Media
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Appendix A – Recipes for Freshwater and Seawater Media 531
STP Test Medium
(Tatewaki and Provasoli 1964)
This is a complex test medium for marine bacteria from coastal habitats (e.g., phytoplankton and seaweed cultures)
(Tatewaki and Provasoli 1964). The V8 vitamins solution was first published by Provasoli et al. (1957), but see also
Hutner et al. (1949) and Hamilton et al. (1952) for similar compilations. Note that the cyanocobalamin in this mix
results in an extremely low concentration in the final medium, and therefore this compound is added twice. For prac-
tical purposes, the cyanocobalamin could be excluded from the 8A vitamins mix without having any significant effect
on the final solution. STP medium is probably too rich, as formulated, for oceanic bacteria, but it may be effective
in growing some “unculturable” bacteria if extremely dilute (e.g., diluted by 106or more). A variant of this medium,
STPM medium, has 1g methylamine·HCl per liter for growth of methylaminotrophic bacteria. Tatewaki and Prova-
soli (1964) provide a second test medium, ST3medium, that contains a rich organic mixture (C-source mix II). To
prepare, mix 800mL of natural seawater and 150mL of dH2O. Add the following components, making sure each is
dissolved before adding the next. Autoclave. Final pH 7.5 to 7.6.
Component Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
NaNO3— 200 mg 2.35 ¥10-3
K2HPO4·H2O 10.0 1mL 7.25 ¥10-5
Yeast autolysate — 200mg —
Sucrose — 1g 2.92 ¥10-5
Na H-glutamate — 500 mg 2.96 ¥10-5
DL-alanine — 100 mg 1.12 ¥10-5
Tripticase (BBL) — 200mg —
Glycine — 100mg 1.33 ¥10-5
8A vitamin solution (See following recipe) 100 mL—
Soil-water extract — 50mL —
AACAP 10/6/04 06:44 PM Page 531

A Vitamin Solution
(Provasoli et al. 1957)
Although not specified in the recipe, stock solutions should be prepared for those components with extremely small
weights. To 900mL of dH2O, add the components and bring final volume to 1 liter with dH2O. Filter-sterilize.
532 Appendix A – Recipes for Freshwater and Seawater Media
Component 1° Stock Solution Quantity Used Concentration in Final
(g·L-1dH2O) Medium (M)
Thiamine·HCl (vitamin B1) — 200 mg 5.39 ¥10-8
Nicotinic acid (niacin) — 100mg 8.12 ¥10-8
Putrecine·HCl — 40mg 2.48 ¥10-8
Ca pantothenate — 100mg 4.20 ¥10-8
Riboflavin (vitamin B2) — 5 mg 1.33 ¥10-9
Pyridoxine·HCl (vitamin B6) — 40 mg 1.95 ¥10-9
Pyridoxamine·2HCl — 20 mg 8.30 ¥10-9
p-Aminobenzoic acid — 10 mg 7.29 ¥10-9
Biotin (vitamin H) — 0.5mg 2.23 ¥10-10
Choline H2citrate — 500mg 1.69 ¥10-7
Inositol — 1 g 5.55 ¥10-7
Thymine — 800mg 6.34 ¥10-7
Orotic acid — 260mg 1.67 ¥10-7
Cyanocobalamin (vitamin B12) — 0.05mg 3.69 ¥10-12
Folic acid — 2.5mg 5.67 ¥10-10
Folinic acid — 0.2 mg 3.91 ¥10-11
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538 Appendix A – Recipes for Freshwater and Seawater Media
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