Microbial Culture Collection at the National Institute for Environmental Studies (NIES Collection)　HOME

1 Media for freshwater, terrestrial, hot spring and salt water algae

AAF–6
Acid–CSi/5
AF–6
AF–6+Wheat
AF–6/2
AFAC
Allen
BBM
BG–11
C
C+10% Seawater
C/6G
CA
CAM
Carefoot
CB
CB–V
CC
CSi
CSi + Cu
CSi/5
CT
CYT
DH + Fe
DY–V
HUT
M–11
MA
MAF–6
M–Allen
M–Allen( + U )
MBM
M Chu No. 10
MDM
MG
MGM
Modified acetate medium ( mAC )
Modified M–1 ( mM–1 )
MW
MW/5
N–Free
O
P 35
Pro
SOT
SW
TAP
Tre
URO
URO–H
URO–H+Wheat
URO–T
VT
VTAC
VTYT
W

2 Media for marine and brackish water microalgae

BESM
BESM 2
ESM
ESM2
f/2 + NH₄Cl
f/2
IMK
M–ASP7
MF
mIMR ( modified IMR )
MKM
MNK
PRO–99
WESM

3 Bacteria-free check media for freshwater algae

B–I
B–II
B–III
B–IV
B–V
YT

4 Bacteria-free check media for marine algae

Bf/2
MM 23
STP

5 Trace metals, vitamin mixtures and soil extracts

A2 Fe stock solution
A2 trace element stock solution
A₅ solution
Allen metals
C–Source Mix II
D stock medium
D trace mix
DY trace metal solution
f/2 metals
Fe ( as EDTA; 1:1 molar )
Fe solution
Hutner ’s trace elements
mIMR trace metals
mM–1 Trace elements
mTYGM–9
P II metals
P IV metals
PN metals
PRO–99 trace metals
Soil extract
Trace metal mix A₅ + Co
Vitamin mix S₃

6 Media for protozoa

AF–6 + Wheat
ESM + mTYGM–9 + Rice
f/2 + mTYGM–9 + Rice
f/2 + Wheat
Helio
LE
PYG
SUY
SUY 1/10
SUY 1/10 + mTYGM–9 + Rice
SUY 1/10 + Wheat
URO + Wheat
URO–H + Wheat
UYTS + Rice

7 Media for freshwater red algae

Bold 3N

8 Media for Charales

mSWC–2 ( Modified SWC–2 )
SWC–1
SWCN–1
SWCN–2
SWCN–3
SWCN–4
1/3 Herbst ASW
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1 Media for freshwater, terrestrial, hot spring and salt water algae
AAF–6

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Prepare as for AF–6 medium but adjust to pH 5.5–5.8 .

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Acid–CSi/5

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Dilute CSi ^{1 )} medium with distilled water to one–fifth. Adjust to pH 3 with sulfuric acid.
1 ) See CSi

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1 Media for freshwater, terrestrial, hot spring and salt water algae
AF–6

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NaNO3	14 mg
NH4NO3	2.2 mg
MgSO4 · 7H2O	3 mg
KH2PO4	1 mg
K2HPO4	0.5 mg
CaCl2 · 2H2O	1 mg
CaCO3 1 )	1 mg
Fe–citrate	0.2 mg
Citric acid	0.2 mg
Biotin	0.2 μg
Thiamine HCl	1 μg
Vitamin B6	0.1 μg
Vitamin B12	0.1 μg
Trace metals 1 )	0.5 mL
Distilled water	99.5 mL
pH 6.6 2 )

1 ) In the NIES–Collection, CaCO₃ is removed and PIV metals are used instead of Trace metals.
2 ) In the NIES–Collection, 40 mg MES is added and pH is adjusted to 6.6.

Reference
Kato, S. 1982 Laboratory culture and morphology of Colacium vesiculosum Ehrb. ( Euglenophyceae ). Jpn. J. Phycol., 30, 63-67 ( in Japanese with English summary ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
AF–6/2

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AF–6 ^{1 )} medium is diluted with distilled water to one-half.
1 ) See AF–6

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1 Media for freshwater, terrestrial, hot spring and salt water algae
AFAC

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To 100 mL AF–6 ^{1 )} medium add 20 mg sodium acetate.
1 ) See AF–6

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Allen

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( NH4 )2SO4	132 mg
KH2PO4	27.2 mg
MgSO4 · 7H2O	24.6 mg
CaCl2 · 2H2O	7.4 mg
Allen metals 1 )	0.01 mL
Distilled water	99.9 mL
pH 2.5 2 )

1 ) See Allen metals
2 ) pH is adjusted to 2.5 with 0.5 mol / L H₂SO₄.

Reference
Allen, M. B. 1959 Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Mikrobiol., 32, 270-277.
Starr, R. C., Zeikus, J. A. 1987 UTEX - The culture collection of algae at the University of Texas at Austin. J. Phycol., 23, Suppl. to Sept., 1-106.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
BBM

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NaNO3	25 mg
KH2PO4	17.5 mg
K2HPO4	10 mg
MgSO4 · 7H2O	7.5 mg
CaCl2 · 2H2O	2.5 mg
NaCl	2.5 mg
KOH	3.1 mg
FeSO4 · 7H2O	0.498 mg
H3BO3	1.142 mg
ZnSO4 · 7H2O	0.882 mg
MnCl2 · 7H2O	0.144 mg
MoO3	0.071 mg
CuSO4 · 5H2O	0.157 mg
Co( NO3 )2 · 6H2O	0.049 mg
Na2EDTA	5 mg
Distilled water	100 mL

Reference
Bischoff, H. W., Bold, H. C. 1963 Some soil algae from enchanted rock and related algal species. Phycological Studies IV.,Univ. No. 6318, Texas, p. 95.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
BG–11

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NaNO3	150 mg
K2HPO4 · 3H2O	4 mg
MgSO4 · 7H2O	7.5 mg
CaCl2 · 2H2O	3.6 mg
Citric acid	0.6 mg
Ferric ammonium citrate	0.6 mg
Na2EDTA – Mg	0.1 mg
Na2CO3	2 mg
Trace metal mix A5 + Co 1 )	0.1 mg
Agar	1.5 g
Distilled water	99.9 mL
pH 7.4

1 ) See Trace metal mix A₅ + Co

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1 Media for freshwater, terrestrial, hot spring and salt water algae
C

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Ca( NO3 )2 · 4H2O	15 mg
KNO3	10 mg
β–Na2glycerophosphate · 5H2O	5 mg
MgSO4 · 7H2O	4 mg
Vitamin B12	0.01 μg
Biotin	0.01 μg
Thiamine HCl	1 μg
PIV metals 1 )	0.3 mL
Tris ( hydroxymethyl ) aminomethane	50 mg
Distilled water	99.7 mL
pH 7.5

Add 1.5 g agar to 100 mL of medium to give a solid medium.
1 ) See PIV metals

Reference
Ichimura, T. 1971 Sexual cell division and conjugation-papilla formation in sexual reproduction of Closterium strigosum. In Proceedings of the Seventh International Seaweed Symposium, University of Tokyo Press, Tokyo, p. 208-214.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
C+10% Seawater ( N. Tezuka, unpubl. )

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C ^{1 )} mediumwith 10% filtered seawater.
1 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
C/6G

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Mix 1 volume of C ^{1 )} medium and 5 volumes of Lake Nojiri water ( sterilized through GF/F filter, and store at 5°C ).
1 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CA

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Ca( NO3 )2 · 4H2O	2 mg
KNO3	10 mg
NH4NO3	5 mg
β–Na2glycerophosphate · 5H2O	3 mg
MgSO4 · 7H2O	2 mg
Vitamin B12	0.01 μg
Biotin	0.01 μg
Thiamine HCl	1 μg
PIV metals 1 )	0.1 mL
Fe ( as EDTA; 1:1 molar ) 2 )	0.1 mg
HEPES	40 mg
Distilled water	99.9 mL
pH 7.2

1 ) See PIV metals
2 ) See Fe ( as EDTA; 1:1 molar )

Reference
Ichimura, T., Watanabe, M. 1974 The Closterium calosporum complex from the Ryukyu Islands - Variation and taxonomical problems. Mem. Natn. Sci. Mus. Tokyo, 7, 89-102, pls. 13-14.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CAM

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CA ^{1 )} medium with pH adjusted to 6.5 by buffering with MES instead of HEPES.
1 ) See CA

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Carefoot

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NaNO3	24.7 mg
CaCl2 · 2H2O	1.1 mg
MgSO4 · 7H2O	4.7 mg
K2HPO4	0.9 mg
KH2PO4	2.3 mg
NaCl	1.5 mg
PIV metals 1 )	0.5 mL
Distilled water	99.5 mL
pH 7.5

In the NIES–Collection, 0.02 μg Vitamin B₁₂, 0.02 μg biotin and 2 μg thiamine HCl are added to this medium.
1 ) See PIV metals

Reference
Carefoot, J. R. 1968 Culture and heterotrophy of the freshwater dinoflagellate, Peridinium cinctum fa. ovoplanum Lindeman. J. Phycol., 4, 129-131.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CB

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To C ^{1 )} medium add Bicine instead of Tris ( hydroxymethyl ) aminomethane, and adjust pH to 9.0.
1 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CB–V

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Make B–V ^{1 )} medium with C ^{2 )} medium.
1 ) See B–V
2 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CC

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C ^{1 )} medium with pH adjusted to 3.0 by buffering with 1,2,3,4–cyclopentane tetracarboxylic acid instead of Tris ( hydroxymethyl ) aminomethane.
1 ) See C

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [ Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CSi

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C ^{1 )} medium with pH adjusted to 7.0 by buffering with 50mg HEPES instead of Tris ( hydroxymethyl ) aminomethane. Thereafter, 10 mg Na₂SiO₃ · 9H₂O is added.
1 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CSi + Cu

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To 100 mL CSi ^{1 )} medium add 0.25 mg CuSO₄ · 5H₂O and 100 mg agar.
1 ) See CSi

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CSi/5

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Dilute CSi ^{1 )} medium with distilled water to 1/5.
1 ) See CSi

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CT

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C ^{1 )} medium with pH adjusted to 8.2 by buffering with 40mg TAPS instead of Tris ( hydroxymethyl ) aminomethane.
1 ) See C

Reference
Watanabe, M. M., Ichimura, T. 1977 Fresh- and salt-water forms of Spirulina platensis in axenic cultures. Bull. Jpn. Soc. Phycol., 25, Suppl. ( Mem. Iss. Yamada ), 371-377.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
CYT

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To 100 mL C ^{1 )} medium add 100 mg yeast extract and 200 mg tryptone.
1 ) See C

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1 Media for freshwater, terrestrial, hot spring and salt water algae
DH + Fe ( I.I. Brown, unpubl. )

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D stock medium 1 )	5 mL
HEPES	0.12 mg
FeCl3 · 6H2O	11.4 mg
Distilled water	95 mL
pH 8.24 – 8.26

After autoclaving, keep in room temperature overnight. Next day, adjust pH to 7.5–7.6 and add 1.5 g agar.
1 ) See D stock medium

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1 Media for freshwater, terrestrial, hot spring and salt water algae
DY–V

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MES	20 mg
MgSO4 · 7H2O	5 mg
KCl	0.3 mg
NH4Cl	0.27 mg
NaNO3	2 mg
β–Na2glycerophosphate · 5H2O	0.22 mg
H3BO3	0.08 mg
Na2EDTA · 2H2O	0.8 mg
Na2SiO3 · 9H2O	1.4 mg
FeCl3 · 6H2O	0.1 mg
CaCl2 · 2H2O	7.5 mg
Vitamin B12	0.05 μg
Biotin	0.05 μg
Thiamine HCl	10 μg
DY trace metal solution 1 )	0.1 mL
Distilled water	99.9 mL
pH 6.8

1 ) See DY trace metal solution

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1 Media for freshwater, terrestrial, hot spring and salt water algae
HUT

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KH2PO4	2 mg
MgSO4 · 7H2O	2.5 mg
Sodium acetate	40 mg
Potassium citrate	4 mg
Polypeptone	60 mg
Yeast extract	40 mg
Vitamin B12	0.05 μg
Thiamine HCl	0.04 mg
Distilled water	100 mL
pH 6.4

Add 150 mg agar to 100 mL of medium to give a semi-solid medium.

Reference
Hutner, S. H., Zahalsky, A. C., Aaronson, S., Baker, H., Frank, O. 1966 Culture media for Euglena gracilis. In Methods in Cell Physiology. Vol. 2, Ed. by Prescott, D. M., Academic Press, New York, p. 217-228.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
M–11

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NaNO3	10 mg
K2HPO4	1 mg
MgSO4 · 7H2O	7.5 mg
CaCl2 · 2H2O	4 mg
Na2CO3	3 mg
FeSO4 · 7H2O	0.1 mg
Na2EDTA · 2H2O	0.1 mg
Distilled water	100 mL
pH 8.0

Reference
Hagiwara, T., Yagi, O., Takamura, Y., Sudo, R. 1984 Isolation of bacteria-free Microcystis aeruginosa from Lake Kasumigaura. Jpn. J. Water Poll. Res., 7, 437-442 ( in Japanese with English summary ).
Yagi, O., Okada, M., Sudo, R. 1979 Cultivation of Microcystis and red-tide-organisms. Res. Rep. Natl. Inst. Environ. Stud., No. 6, 223-229 ( in Japanese with English summary ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MA

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Ca( NO3 )2 · 4H2O	5 mg
KNO3	10 mg
NaNO3	5 mg
Na2SO4	4 mg
MgCl2 · 6H2O	5 mg
β–Na2glycerophosphate · 5H2O	10 mg
Na2EDTA · 2H2O	0.5 mg
FeCl3 · 6H2O	0.05 mg
MnCl2 · 4H2O	0.5 mg
ZnCl2	0.05 mg
CoCll2 · 6H2O	0.5 mg
Na2MoO4 · 2H2O	0.08 mg
H3BO3	2 mg
Bicine	50 mg
Distilled water	100 mL
pH 8.6

Reference
Ichimura, T. 1979 2. Isolation and culture methods of algae. 2.5.B. Freshwater algae [2. Sôrui no bunri to baiyôhô. 2.5.B. Tansui sôrui]. In Methods in Phycological Studies [Sôrui Kenkyûhô], Eds. by Nishizawa, K. & Chihara, M., Kyoritsu Shuppan, Tokyo, p. 294-305 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MAF–6

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To 100 mL of AF–6 ^{1 )} medium add 10 mg glucose and 10 mg sodium acetate.
1 ) See AF–6

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1 Media for freshwater, terrestrial, hot spring and salt water algae
M–Allen

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( NH4 )2SO4	262 mg
KH2PO4	54 mg
MgSO4 · 7H2O	50 mg
CaCl2 · 2H2O	14 mg
A2 trace element stock solution 1 )	0.2 mL
Distilled water	99.4 mL
pH 2.5 2 )

After autoclaving, add 0.4 mL of A2 Fe stock solution ^{3 )} ( filter-sterilized ).
1 ) See A2 trace element stock solution
2 ) pH is adjusted to 2.5 with 0.5 mol / L H₂SO₄.
3 ) See A2 Fe stock solution Indicated as “MA” medium in reference.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
M–Allen( + U )

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To 100 mL M–Allen ^{1 )} medium add 50 mg uracil.
1 ) See M–Allen

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MBM

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KNO3	25 mg
MgSO4 · 7H2O	7.5 mg
K2HPO4	7.5 mg
KH2PO4	17.5 mg
NaCl	2.5 mg
CaCl2 · 2H2O	1 mg
Fe solution 1 )	0.1 mL
A5 solution 2 )	0.1 mL
Agar	1.5 g
Distilled water	99.8 mL
pH 6.0

1 ) See Fe solution.
2 ) See A₅ solution. Indicated as “Modified Bristol medium” in reference.

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
M Chu No. 10

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Ca( NO3 )2 · 4H2O	2.0 mg
KH2PO4	0.62 mg
MgSO4 · 7H2O	2.5 mg
Na2CO3	2 mg
Na2SiO3 · 9H2O	2.5 mg
HCl ( 1 mol / L ) 1 )	0.025 mL
Na2EDTA · 2H2O	0.2 mg
FeCl3 · 6H2O	0.1 mg
H3BO3	0.248 mg
MnCl2 · 4H2O	0.139 mg
( NH4 )6Mo7O24 · 4H2O	0.1 mg
Vitamin B12	1 μg
Thiamine HCl	0.1 μg
Biotin	0.1 μg
Distilled water	100 mL

1 ) In the NIES–Collection, pH is adjusted to 7.6 with 1 mol / L HCl.

Reference
Chu, S. P. 1942 The influence of the mineral composition of the medium on the growth of planktonic algae. Part I. Methods and culture media. J. Ecol., 30, 284-325.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MDM

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KNO3	100 mg
MgSO4 · 7H2O	25 mg
K2HPO4	25 mg
NaCl	10 mg
CaCl2 · 2H2O	1 mg
Fe solution 1 )	0.1 mL
A5 solution 2 )	0.1 mL
Agar	1.5 g
Distilled water	99.8 mL
pH 8.0

1 ) See Fe solution
2 ) See A₅ solution

Reference
Watanabe, A. 1960 List of algal strains in collection at the Institute of Applied Micro-biology, University of Tokyo. J. Gen. Appl. Microbiol., 6, 283-292.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MG

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Ca ( NO3 )2·4H2O	2 mg
KNO3	10 mg
β–Na2 glycerophosphate·5H2O	3 mg
MgSO4 · 7H2O	2 mg
Vitamin B12	0.01 µg
Biotin	0.01 µg
Thiamine HCl	1 µg
PIV metals 1 )	0.1  mL
Fe ( as EDTA; 1:1 molar ) 2 )	0.1  mL
HEPES	40 mg
Distilled water	99.8  mL
pH 7.2

1 ) See PIV metals
2 ) See Fe ( as EDTA; 1:1 molar ) 　

Reference
Ichimura, T. 1973 The life cycle and its control in some species of Closterium, with special reference to the biological species problems. Thesis D. Sci., University of Tokyo, 69 pp., 11 tables, 40 figs.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MGM

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MG ^{1 )} medium with pH adjusted to 6.5 by buffering with MES instead of HEPES.
1 ) See MG

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Modified acetate medium ( mAC )

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To 100 mL AF–6 ^{1 )} medium, add 40mg glucose, yeast extract, tryptone, and sodium acetate.
1 ) See AF–6

Reference
Nozaki, H., Wanababe, M. M. & Aizawa, K. 1995 Morphology and paedogamous sexual reproduction in Chlorogonium capillatum sp. nov. ( Volvocales, Chlorophyta ). J. Phycol., 31, 655-663.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Modified M–1 ( mM–1 )

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CaCl2 · 2H2O	0.5 mg
NaNO3	2.5 mg
NH4Cl	0.5 mg
CaSO4 · 2H2O	0.4 mg
MgSO4 · 7H2O	0.5 mg
Na2SiO3 · 9H2O	0.2 mg
Fe ( as EDTA; 1:1 molar ) 1 )	25 mL
mM–1 Trace elements 2 )	0.1 mL
K2HPO4	6.96 mg
KH2PO4	266.5 mg
Distilled water	74.9 mL
pH 5.1 – 5.3

1 ) See Fe ( as EDTA; 1:1 molar )
2 ) See mM–1 Trace elements

Reference
Hoham, R. W., Berman, J. D., Rogers, H. S., Felio, J. H., Ryba, J. B., Miller, P. R. 2006 Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. ( Volvocales, Chlorophyceae ) and the effects of light on their life cycle development. Phycologia, 45, 319-330.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MW

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Urea	0.85 mg
NaNO3	0.17 mg
NH4Cl	0.042 mg
Ca( NO3 )2 · 4H2O	10 mg
CaCO3	1 mg
CaCl2 · 2H2O	1.4 mg
KNO3	1 mg
KHCO3	0.9 mg
β–Na2glycerophosphate · 5H2O	2 mg
MgSO4 · 7H2O	1.5 mg
PIV metals 1 )	0.05 mL
Vitamin B12	0.02 μg
Thiamine HCl	2 μg
Biotin	0.02 μg
Glycylglycine	10 mg
Distilled water	100 mL
pH 7.2

1 ) See PIV metals

Reference
Sako, Y., Ishida, Y., Kadota, H., Hata, Y. 1984 Sexual reproduction and cyst formation in the freshwater dinoflagellate Peridinium cunningtonii. Bull. Jpn. Soc. Sci. Fish., 50, 743-750.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
MW/5

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MW ^{1 )} medium is diluted with distilled water to 1/5.
1 ) See MW

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1 Media for freshwater, terrestrial, hot spring and salt water algae
N–Free

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NaCl	7 mg
MgSO4 · 7H2O	38 mg
CaCl2 · 2H2O	10.6 mg
K2HPO4	60 mg
Fe2( SO4 )3 · 6H2O	1 mg
Na2EDTA · 2H2O	2.7 mg
H3BO3	0.3 mg
MnSO4 · 4H2O 1 )	0.2 mg
Na2MoO4 · 2H2O	0.8 mg
ZnSO4 · 7H2O	0.03 mg
CuSO4 · 5H2O	8 μg
CoCll2 · 6H2O	3.7 μg
Agar	1.5 g
Distilled water	100 mL
pH 7.5

1 ) In the NIES–Colletion, 0.2 mg MnSO₄ · 4H₂O is replaced by 0.22 mg MnSO₄ · 5H₂O.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
O

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Glucose	100 mg
Tryptone	100 mg
Yeast extract	100 mg
Beef extract 1 )	50 mg
Agar	150 mg
Distilled water	100 ml

1 ) In the NIES-Collection, beef extract is removed. Indicated as “Ochomonas medium” in reference.

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).
Starr, R. C. 1964 The culture collection of algae at Indiana University. Amer. J. Bot., 51, 1013-1044.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
P 35

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NH4NO3	10 mg
MgSO4 · 7H2O	4 mg
KCl	5 mg
CaCl2 · 2H2O	7.4 mg
β–Na2glycerophosphate · 5H2O	5 mg
Sodium acetate	100 mg
Vitamin B12	0.01 μg
Biotin	0.01 μg
Thiamine HCl	1 μg
PIV metals 1 )	0.3 mL
Tris ( hydroxymethyl ) aminomethane	50 mg
Distilled water	99.7 mL
pH 8.0

1 ) See PIV metals

Reference
Ichimura, T. 1979 2. Isolation and culture methods of algae. 2.5.B. Freshwater algae [2. Sôrui no bunri to baiyôhô. 2.5.B. Tansui sôrui]. In Methods in Phycological Studies [Sôrui Kenkyûhô ], Eds. by Nishizawa, K. & Chihara, M., Kyoritsu Shuppan, Tokyo, p. 294-305 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Pro

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To 100 mL MBM ^{1 )} medium add 100 mg proteose peptone.
1 ) See MBM Indicated as “Proteose medium” in reference.

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).
Starr, R. C. 1964 The culture collection of algae at Indiana University. Amer. J. Bot., 51, 1013-1044.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
SOT

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NaHCO3	1.68 g
K2HPO4	50 mg
NaNO3	250 mg
K2SO4	100 mg
NaCl	100 mg
MgSO4 · 7H2O	20 mg
CaCl2 · 2H2O	4 mg
FeSO4 · 7H2O	1 mg
Na2EDTA · 2H2O	8 mg
A5 solution 1 )	0.1 mL
Distilled water	99.9 mL

1 ) See A₅ solution

Reference
Ogawa, T., Terui, G. 1970 Studies on the growth of Spirulina platensis. ( I ) On the pure culture of Spirulina platensis. J. Ferment. Technol., 48, 361-367.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
SW

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Put a small amount of dried soil into a test tube and add 20mL distilled water.

Reference
Pringsheim, E. G. 1946 The biphasic or soil-water culture method for growing algae and flagellata. J. Ecol., 33, 193-204.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
TAP

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NH4Cl	40 mg
CaCl2 · 2H2O	5.1 mg
MgSO4 · 7H2O	10 mg
K2HPO4	11.9 mg
KH2PO4	6.03 mg
Hutner ’s trace elements 1 )	0.1 mL
Acetic acid	0.1 mL
Tris ( hydroxymethyl ) aminomethane	242 mg
Agar	1.5 g
Distilled water	99.8 mL

1 ) See Hutner ’s trace elements

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1 Media for freshwater, terrestrial, hot spring and salt water algae
Tre

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To 100 mL MBM ^{1 )} medium add 1 g proteose peptone and 2 g glucose.
1 ) See MBM Indicated as “Trebouxia medium” in reference.

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).
Starr, R. C. 1964 The culture collection of algae at Indiana University. Amer. J. Bot., 51, 1013-1044.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
URO

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NH4NO3	0.5 mg
β–Na2glycerophosphate · 5H2O	0.4 mg
MgSO4 · 7H2O	1 mg
CaCl2 · 2H2O	1 mg
KCl	0.1 mg
Thiamine HCl	1 μg
Vitamin B12	0.01 μg
Biotin	0.01 μg
Fe-EDTA	0.05 mg
PIV metals 1 )	0.1 mL
Distilled water	99.9 mL
pH 7.5 2 )

1 ) See PIV metals
2 ) pH is adjusted to 7.5 with 0.1 mol / L HCl.

Reference
Kimura, B., Ishida, Y. 1985 Photophagotrophy in Uroglena americana, Chrysophyceae. Jpn. J. Limnol., 46, 315-318.
Nakahara, H., Sako, Y. 1987 2. Life history of freshwater phytoplankton [2. Tansui syokubutsu purankuton no seikatsushi]. In Freshwater red tide [Tansui Akashio ], Ed. by Kadota, H., Kôseisya-Kôseikaku, Tokyo, p. 21-77 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
URO–H

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To 100 mL URO ^{1 )} medium add 40 mg HEPES.
1 ) See URO

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1 Media for freshwater, terrestrial, hot spring and salt water algae
URO–T

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To 100 mL URO ^{1 )} medium add 50 mg Tris ( hydroxymethyl ) aminomethane.
1 ) See URO

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1 Media for freshwater, terrestrial, hot spring and salt water algae
VT

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Ca( NO3 )2 · 4H2O	11.78 mg
β-Na2glycerophosphate · 5H2O	5 mg
MgSO4 · 7H2O	4 mg
KCl	5 mg
Vitamin B12	0.01 μg
Biotin	0.01 μg
Thiamine HCl	1 μg
PIV metals 1 )	0.3 mL
Glycylglycine	50 mg
Distilled water	99.7 mL
pH 7.5

1 ) See PIV metals

Reference
Provasoli, L., Pintner, I. J. 1959 Artificial media for fresh-water algae: problems and suggestions. In The Ecology of Algae. Spec. Pub. No. 2,, Eds. by Tryon, C. A., Jr. & Hartmann, R. T., Pymatuning Laboratory of Field Biology, University of Pittsburgh, Pittsburgh, p. 84-96.
Starr, R. C. 1973 Special methods-dry soil samples. In Handbook of Phycological Methods. Culture Methods and Growth Measurements, Ed. by Stein, J. R., Cambridge University Press, Cambridge, p. 159-167.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
VTAC

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To 100 mL VT ^{1 )} medium add 20 mg sodium acetate.
1 ) See VT

Reference
Nozaki, H., Kuroiwa, H., Mita, T., Kuroiwa, T. 1989 Pleodorina japonica sp. nov. ( Volvocales, Chlorophyta ) with bacteria-like endosymbionts. Phycologia, 28, 252-267.

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1 Media for freshwater, terrestrial, hot spring and salt water algae
VTYT

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To 100 mL VT ^{1 )} medium add 10 mg yeast extract and 20 mg tryptone.
1 ) See VT

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I ) ]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).

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1 Media for freshwater, terrestrial, hot spring and salt water algae
W

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Ca( NO3 )2 · 4H2O	10 mg
KNO3	1 mg
MgSO4 · 7H2O	1.5 mg
β–Na2glycerophosphate · 5H2O	2 mg
Urea	1.7 mg
Thiamine HCl	0.2 μg
Vitamin B12	0.002 μg
Biotin	0.002 μg
PIV metals 1 )	0.05 mL
Glycylglycine	10 mg
Distilled water	99.95 mL
pH 7.5

1 ) See PIV metals 2.2. Media for marine and brackish water microalgae

Reference
Watanabe, M. M. 1983 Growth characteristics of freshwater red tide alga, Peridinium based on axenic culture. Establishment of synthetic culture medium [Junsuibaiyôhô niyoru tansuiakashio Peridinium no zôsyokutokusei no kaiseki-gôseibaichi no kakuritsu ]. Res. Data Natl. Inst. Environ. Stud., No. 24, 111-121 ( in Japanese without English title ).

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2 Media for marine and brackish water microalgae
BESM

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Make diluted seawater by mixing 27.5 mL seawater and 70 mL distilled water. Make ESM ^{1 )} medium by using this diluted seawater instead of original seawater.
1 ) See ESM

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2 Media for marine and brackish water microalgae
BESM 2

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Make diluted seawater by mixing 47.5 mL seawater and 50 mL distilled water. Make ESM ^{1 )} medium by using this diluted seawater instead of original seawater.
1 ) See ESM

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2 Media for marine and brackish water microalgae
ESM

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NaNO3	12 mg
K2HPO4	0.5 mg
Vitamin B12	0.1 μg
Biotin	0.1 μg
Thiamine HCl	10 μg
Fe – EDTA	25.9 μg
Mn – EDTA	33.2 μg
Tris ( hydroxymethyl ) aminomethane	100 mg
Soil extract 1 )	2.5 mL
Seawater	97.5 mL
pH 8.0

The amount of Soil extract depends on the quality of the soil. In the NIES–Collection, Soil extract was reduced from 5 mL to 2.5 mL after 2002. Add 1.5 g agar to 100 mL of medium to give a solid medium.
1 ) See Soil extract

Reference
Okaichi, T., Nishio, S., Imatomi, Y. 1982 Collection and mass culture [Shiryô no saisyu to baiyô]. In Toxic phytoplankton - Occurrence, mode of action, and toxins [Yûdoku Purankuton -Hassei, Sayôkikô, Dokuseibun ], Ed. by Jpn. Fish. Soc., Kôseisya-Kôseikaku, Tokyo, p. 22-34 ( in Japanese without English title ).

2 Media for marine and brackish water microalgae
ESM2

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Prepare as for 100 mL ESM ^{1 )} medium with 95.5mL instead of 97.5 mL seawater and with 5 mL instead of 2.5 mL Soil extract ^{2 )}.
1 ) See ESM
2 ) See Soil extract

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2 Media for marine and brackish water microalgae
f/2

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NaNO3	7.5 mg
NaH2PO4 · 2H2O	0.6 mg
Vitamin B12	0.05 μg
Biotin	0.05 μg
Thiamine HCl	10 μg
Na2SiO3 · 9H2O	1 mg
f/2 metals 1 )	0.1 mL
Seawater	99.9 mL

1 ) See f/2 metals

Reference
Guillard, R. R. L., Ryther, J. H. 1962 Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea ( Cleve ) Gran. Can. J. Microbiol., 8, 229-239.

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2 Media for marine and brackish water microalgae
f/2 + NH4Cl

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To 100 mL f/2 ^{1 )} medium add 2.67 mg NH₄Cl.
1 ) See f/2

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2 Media for marine and brackish water microalgae
IMK

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Into 100 mL seawater dissolve 21 mg powder medium of Daigo IMK ( Nihon Pharmaceutical Co., Ltd. ). In the NIES–Collection, IMK medium is used after autoclaving ( 121°C, 20 min ).

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2 Media for marine and brackish water microalgae
M–ASP7

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NTA	7 mg
NaCl	2.5 g
MgSO4 · 7H2O	900 mg
KCl	70 mg
CaCl2 · 2H2O	30 mg
NaNO3	5 mg
NaH2PO4 · 2H2O	2 mg
Vitamin B12	0.1 μg
Vitamin mix S3 1 )	1 mL
Na2SiO3 · 9H2O	1 mg
PN metals 2 )	3 mL
Tris ( hydroxymethyl ) aminomethane	100 mg
Distilled water	96 mL
pH 8.0

1 ) See Vitamin mix S₃
2 ) See P_N metals

Reference
Watanabe, M. M., Satake, K. N. ( Eds. ) 1991 NIES-Collection. List of Strains, Third Edition, 1991, Microalgae and Protozoa. Microbial Culture Collection, National Institute for Environmental Studies, Tsukuba, 163 pp.

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2 Media for marine and brackish water microalgae
MF

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f/2 ^{1 )} medium with Na₂SiO₃ · 9H₂O replaced by 1 mL Soil extract ^{2 )} and adjusted to pH 8.0 by buffering with 100 mg Tris ( hydroxymethyl ) aminomethane.
1 ) See f/2
2 ) See Soil extract

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2 Media for marine and brackish water microalgae
mIMR ( modified IMR )

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KNO3	1.26 mg
K2HPO4	0.22 mg
Na2SiO3 1 )	0.61 mg
mIMR trace metals 2 )	0.1 mL
Thiamine	20 μg
Vitamin B12	1 μg
Biotin	0.1 μg
Seawater	100 mL

1 ) In the NIES–Collection, Na₂SiO₃ is replaced by Na₂SiO₃·9H₂O.
2 ) See mIMR trace metals

Reference
Paasche, E., Bruback, S., Skattebol, S., Young, J. R., Green, J. C 1996 Growth and calcification in the coccolithophorid Emiliania huxleyi ( Haptophyceae ) at low salinities. Phycologia, 35, 394-403.

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2 Media for marine and brackish water microalgae
MKM

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KNO3	75 mg
KH2PO4	2.5 mg
MgSO4 · 7H2O	2 mg
Fe–citrate	250 μg
Agar	1.5 g
Seawater	50 mL
Distilled water	50 mL

Reference
Watanabe, A. 1960 List of algal strains in collection at the Institute of Applied Micro-biology, University of Tokyo. J. Gen. Appl. Microbiol., 6, 283-292.

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2 Media for marine and brackish water microalgae
MNK

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NaNO3	2 mg
K2HPO4	0.1 mg
Na2HPO4 · 12H2O	0.028 mg
Vitamin B12	0.015 μg
Biotin	0.015 μg
Thiamine HCl	2 μg
CoSO4 · 7H2O	0.12 μg
ZnSO4 · 7H2O	0.24 μg
MnCl2 · 4H2O	0.9 μg
CuSO4 · 5H2O	0.006 μg
Na2SeO3	0.003 μg
Na2MoO4 · 2H2O	0.07 μg
Na2EDTA · 2H2O	0.37 μg
Fe–EDTA	2.6 μg
Mn–EDTA	3.3 μg
Seawater	100 mL

Vitamins should be added at the end of the preparation. This medium should not be autoclaved but filter–sterilized.

Reference
Noël, M.-H., Kawachi, M., Inouye, I. 2004 Induced dimorphic life cycle of a coccolithophorid, Calyptrosphaera sphaeroidea ( Prymnesiophyceae, Haptophyta ). J. Phycol., 40, 112-129.

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2 Media for marine and brackish water microalgae
PRO–99

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NaH2PO4 · 2H2O	0.779 mg
NH4Cl	4.28 mg
PRO–99 trace metals 1 )	0.01 mL
Seawater	100 mL

1 ) See PRO–99 trace metals

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2 Media for marine and brackish water microalgae
WESM

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Make diluted seawater by mixing 87.5 mL seawater and 10mL distilled water. Make ESM ^{1 )} medium by using this diluted seawater instead of seawater.
1 ) See ESM

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3 Bacteria-free check media for freshwater algae
B–I

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Appropriate medium	100 mL
Proteose peptone	100 mg

Reference
Ichimura, T., Watanabe, M. M. 1977 An axenic clone of Microcystis aeruginosa Kutz. emend. Elenkin from Lake Kasumigaura. Bull. Jpn. Soc. Phycol., 25, 177-181.

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3 Bacteria-free check media for freshwater algae
B–II

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Appropriate medium	100 mL
Yeast extract	500 mg

Reference
Ichimura, T., Watanabe, M. M. 1977 An axenic clone of Microcystis aeruginosa Kutz. emend. Elenkin from Lake Kasumigaura. Bull. Jpn. Soc. Phycol., 25, 177-181.

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3 Bacteria-free check media for freshwater algae
B–III

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Appropriate medium	100 mL
Peptone	500 mg
Beef extract	300 mg

Reference
Ichimura, T., Watanabe, M. M. 1977 An axenic clone of Microcystis aeruginosa Kutz. emend. Elenkin from Lake Kasumigaura. Bull. Jpn. Soc. Phycol., 25, 177-181.

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3 Bacteria-free check media for freshwater algae
B–IV

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Appropriate medium	100 mL
Glucose	100 mg
Peptone	100 mg

Reference
Ichimura, T., Watanabe, M. M. 1977 An axenic clone of Microcystis aeruginosa Kutz. emend. Elenkin from Lake Kasumigaura. Bull. Jpn. Soc. Phycol., 25, 177-181.

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3 Bacteria-free check media for freshwater algae
B–V

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Appropriate medium	100 mL
Sodium acetate	50 mg
Glucose	50 mg
Tryptone	50 mg
Yeast extract	30 mg

Reference
Ichimura, T., Watanabe, M. M. 1977 An axenic clone of Microcystis aeruginosa Kutz. emend. Elenkin from Lake Kasumigaura. Bull. Jpn. Soc. Phycol., 25, 177-181.

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3 Bacteria-free check media for freshwater algae
YT

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Appropriate medium	100 mL
Yeast extract	100 mg
Tryptone	200 mg

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I ) ]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).

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4 Bacteria-free check media for marine algae
Bf/2

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ASP 7 1 )	100 mL
Trypticase	50 mg
Yeast extract	5 mg

1 ) In the NIES-Collection, ASP 7 is replaced by f/2 medium. See f/2.

Reference
Yoshida, Y., Kawaguchi, K. 1983 Buoyancy and phototaxis of Chattonella antiqua ( Hada ) Ono. Bull. Plankton Soc. Jpn., 30, 11-19 ( in Japanese with English summary ).

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4 Bacteria-free check media for marine algae
MM 23 ( M. Tatewaki, pers. comm. )

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NaCl	1.8 g
MgSO4 · 7H2O	500 mg
KCl	60 mg
NaNO3	100 mg
CaCl2 · 2H2O	36.7 mg
K2HPO4	6 mg
Sucrose	400 mg
PII metals 1 )	2 mL
FeCl3 · 6H2O	48 μg
Thiamine HCl	10 μg
Biotin	0.1 μg
Vitamin B12	0.2 μg
C–Source Mix II 2 )	1 mL
Tris ( hydroxymethyl ) aminomethane	100 mg
Distilled water	97 mL
pH 8.0

1 ) See PII metals
2 ) See C–Source Mix II

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4 Bacteria-free check media for marine algae
STP

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NaNO3	20 mg
K2HPO4	1 mg
Sodium glutamate	50 mg
Glucose	20 mg
Glycine	10 mg
D,L–Alanine	10 mg
Vitamin mix 8 1 )	0.1 mL
Trypticase	20 mg
Yeast autolysate 2 )	20 mg
Sucrose	100 mg
Soil extract 3 )	5 mL
Seawater	80 mL
Distilled water	15 mL
pH 7.5

1 ) In the NIES–Collection, Vitamin mix 8 is replaced by

Vitamin mix S₃. See Vitamin mix S₃.
2 ) In the NIES–Collection, yeast autolysate is replaced by yeast extract.
3 ) See Soil extract

Reference
Provasoli, L., McLaughlin, J. J. A., Droop, M. R. 1957 The development of artificial media for marine algae. Arch. Mikrobiol., 25, 392-428.

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5 Trace metals, vitamin mixtures and soil extracts
A2 Fe stock solution

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EDTA · 2Na	700 mg
FeCl3 · 6H2O	400 mg
Distilled water	100 mL

Sterilize by passing through a Millipore filter ( 0.22 μm ).

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5 Trace metals, vitamin mixtures and soil extracts
A2 trace element stock solution

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H3BO3	285 mg
MnCl2 · 4H2O	180 mg
ZnCl2	10.5 mg
Na2MoO4 · 2H2O	39 mg
CoCll2 · 6H2O	4 mg
CuCl2 · 2H2O	4.3 mg
Distilled water	100 mL

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5 Trace metals, vitamin mixtures and soil extracts
A5 solution

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H3BO3	286 mg
MnSO4 · 7H2O 1 )	250 mg
ZnSO4 · 7H2O	22.2 mg
CuSO4 · 5H2O	7.9 mg
Na2MoO4 · 2H2O	2.1 mg
Distilled water	100 mL

1 ) In the NIES–Collection, 250 mg MnSO₄ · 7H₂O is replaced by 217 mg MnSO₄ · 5H₂O.

Reference
Holm-Hansen, O., Gerloff, G. C., Skoog, F. 1954 Cobalt as an essential element for blue-green algae. Physiol. Planta., 7, 665-675.

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5 Trace metals, vitamin mixtures and soil extracts
Allen metals

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Fe–EDTA	30.16 g
MnCl2 · 4H2O	1.79 g
H3BO3	2.86 g
ZnSO4 · 7H2O	220 mg
CuSO4 · 5H2O	79 mg
( NH4 )6Mo7O24 · 4H2O	130 mg
NH4VO3	23 mg
Distilled water	100 mL

In the NIES–Collection, Allen metals are used after dilution with distilled water to 1/1,000.

Reference
Allen, M. B. 1959 Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Mikrobiol., 32, 270-277.

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5 Trace metals, vitamin mixtures and soil extracts
C–Source Mix II ( M. Tatewaki, pers. comm. )

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Glycine	100 mg
D,L-Alanine	100 mg
L-Aspargine	100 mg
Sodium acetate · 3H2O 1 )	200 mg
Glucose	200 mg
L-Glutamic acid	200 mg
Distilled water	100 mL

1 ) In the NIES–Collection, 200 mg sodium acetate · 3H₂O is replaced by 120 mg sodium acetate, anhydrous.

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5 Trace metals, vitamin mixtures and soil extracts
D stock medium

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NTA	0.2 g
D trace mix 1 )	1 mL
FeCl3 · 6H2O	0.58 mg
CaSO4 · 2H2O	120 mg
MgSO4 · 7H2O	200 mg
NaCl	16 mg
KNO3	200 mg
NaNO3	1.4 g
Na2HPO4 2 )	220 mg
Distilled water	99 mL

1 ) See D trace mix
2 ) In the NIES-Collection, 220 mg Na₂HPO₄ is replacedy by 550 mg Na₂HPO₄· 12H₂O.

Reference
Castenholz, R. W. 1969 Thermophilic blue-green algae and the thermal environment. Bacteriol. Rev., 33, 476-504.

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5 Trace metals, vitamin mixtures and soil extracts
D trace mix

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conc · H2SO4	0.05 mL
MnSO4 · H2O 1 )	228 mg
ZnSO4 · 7H2O	50 mg
H3BO3	50 mg
CuSO4 · 5H2O	2.5 mg
Na2MoO4 · 2H2O	2.5 mg
CoCll2 · 6H2O	4.5 mg
Distilled water	100 mL

1 ) In the NIES–Collection, 228 mg MnSO₄ · H₂O is replaced by 349 mg MnSO₄ · 5H₂O.

Reference
Castenholz, R. W. 1969 Thermophilic blue-green algae and the thermal environment. Bacteriol. Rev., 33, 476-504.

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5 Trace metals, vitamin mixtures and soil extracts
DY trace metal solution

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MnCl2 · 4H2O	20 mg
ZnSO4 · 7H2O	4 mg
CoCll2 · 6H2O	0.8 mg
Na2MoO4 · 6H2O 1 )	2 mg
Na3VO4	0.2 mg
H2SeO3	0.2 mg
Distilled water	100 mL

1 ) In the NIES–Collection, Na₂MoO₄ · 6H₂O is replaced by Na₂MoO₄ · 2H₂O.

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5 Trace metals, vitamin mixtures and soil extracts
Fe ( as EDTA; 1:1 molar )

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Fe( NH4 )2( SO4 )2 · 6H2O	70.2 mg
Na2EDTA · 2H2O	66 mg
Distilled water	100 mL

1 mL of   this solution contains 0.1 mg Fe.

Reference
Provasoli, L. 1966 Media and prospects for the cultivation of marine algae. In Cultures and Collections of Algae, Eds. by Watanabe, A. & Hattori, A., Proc. U.S.-Japan Conf., Hakone, Sept. 1966., Jpn. Soc. Plant Physiol., p. 63-75.

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5 Trace metals, vitamin mixtures and soil extracts
Fe solution

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FeSO4 · 7H2O	200 mg
Distilled water	100 mL
Conc · H2SO4	0.026　mL 1 )

1 ) 2 drops/500 mL

Reference
Ichimura, T., Itoh, T. 1977 17. Preservation methods of microalgae ( I ) [17. Bisaisôrui no hozonhô ( I )]. In Preservation methods of microorganisms [Biseibutsu Hozonhô], Ed. by Nei, T., University of Tokyo Press, Tokyo, p. 355-373 ( in Japanese without English title ).

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5 Trace metals, vitamin mixtures and soil extracts
f/2 metals

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Na2EDTA · 2H2O	440 mg
FeCl3 · 6H2O	316 mg
CoSO4 · 7H2O	1.2 mg
ZnSO4 · 7H2O	2.1 mg
MnCl2 · 4H2O	18 mg
CuSO4 · 5H2O	0.7 mg
Na2MoO4 · 2H2O	0.7 mg
Distilled water	100 mL

Reference
Guillard, R. R. L., Ryther, J. H. 1962 Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea ( Cleve ) Gran. Can. J. Microbiol., 8, 229-239.

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5 Trace metals, vitamin mixtures and soil extracts
Hutner ’s trace elements

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Na2EDTA · 2H2O	5 g
ZnSO4 · 7H2O	2.2 g
H3BO3	1.14 g
MnCl2 · 4H2O	506 mg
FeSO4 · 7H2O	499 mg
CoCll2 · 6H2O	161 mg
CuSO4· 5H2O	157 mg
( NH4 )6Mo7O24 · 4H2O	110 mg
Distilled water	100 mL

Adjust the pH to 6.5-6.8 with KOH ( ~1.6 g ). Store the solution in a refrigerator ( 5°C ). The solution should have turned to violet color before use. This process takes a while and is necessary.

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5 Trace metals, vitamin mixtures and soil extracts
mIMR trace metals

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FeCl3 · 6H2O	100 mg
MnSO4 · 4H2O	85.5 mg
ZnSO4 · 7H2O	25 mg
Na2MoO4 · 2H2O	13 mg
CoCll2 · 6H2O	0.4 mg
CuSO4 · 5H2O	0.4 mg
Na2SeO3	0.173 mg
Na2EDTA 1 )	600 mg
Distilled water	100 mL

1 ) In the NIES–Collection, Na₂EDTA is replaced by Na₂EDTA · 2H₂O.

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5 Trace metals, vitamin mixtures and soil extracts
mM–1 Trace elements

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CuSO4 · 5H2O	10 mg
MnCl2 · 4H2O	10 mg
Br ( 1mol / L solution )	0.01 mL
ZnSO4 · 7H2O	10 mg
CoCll2 · 6H2O	5 mg
BaCl2 · 2H2O	1 mg
H3BO3	10 mg
FeCl3 · 6H2O	10 mg
Na2MoO4 · 2H2O	5 mg
Distilled water	100 mL

Indicated as “Trace elements” in reference.

Reference
Hoham, R. W., Berman, J. D., Rogers, H. S., Felio, J. H., Ryba, J. B., Miller, P. R. 2006 Two new species of green snow algae from Upstate New York, Chloromonas chenangoensis sp. nov. and Chloromonas tughillensis sp. nov. ( Volvocales, Chlorophyceae ) and the effects of light on their life cycle development. Phycologia, 45, 319-330.

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5 Trace metals, vitamin mixtures and soil extracts
mTYGM–9

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Pre–solution

K2HPO4	0.28 g
KH2PO4	40 mg
Casein Digest	0.2 g
Yeast extract	0.1 g
NaCl	0.75 g
Mucin, gastric	0.2 g
Distilled water	97 mL

Sterilize Pre–solution by autoclaving ( 121°C, 15 min ) , add aseptically 3 mL horse serum and 50 μL 10% Tween 80 dissolved in absolute ethanol ( both filter–sterilized ). Keep in a cool place.

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5 Trace metals, vitamin mixtures and soil extracts
P II metals

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Na2EDTA · 2H2O	100 mg
H3BO3	114 mg
FeCl3 · 6H2O	4.9 mg
MnSO4 · 4H2O	16.4 mg
ZnSO4 · 7H2O	2.2 mg
CoSO4 · 7H2O	480 μg
Distilled water	100 mL

Reference
Provasoli, L. 1963 Growing marine seaweeds. In Proceedings of the Fourth International Seaweed Symposium, University of Tokyo Press, Tokyo, p. 9-17.

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5 Trace metals, vitamin mixtures and soil extracts
P IV metals

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Na2EDTA · 2H2O	100 mg
FeCl3 · 6H2O	19.6 mg
MnCl2 · 4H2O	3.6 mg
ZnCl2 1 )	1.04 mg
CoCll2 · 6H2O	0.4 mg
Na2MoO4 · 2H2O	0.25 mg
Distilled water	100 mL

1 ) In the NIES–Collection, 1.04mg ZnCl₂ is replaced by 2.2mg ZnSO₄ · 7H₂O.

Reference
Provasoli, L., Pintner, I. J. 1959 Artificial media for fresh-water algae: problems and suggestions. In The Ecology of Algae. Spec. Pub. No. 2,, Eds. by Tryon, C. A., Jr. & Hartmann, R. T., Pymatuning Laboratory of Field Biology, University of Pittsburgh, Pittsburgh, p. 84-96.

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5 Trace metals, vitamin mixtures and soil extracts
PN metals

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Na2EDTA · 2H2O	100 mg
H3BO3	113 mg
FeCl3 · 6H2O	6.3 mg
CoSO4 · 7H2O	0.093 mg
ZnSO4 · 7H2O	4.66 mg
MnCl2 · 4H2O	3.2 mg
Distilled water	100 mL

Reference
Watanabe, M. M., Satake, K. N. ( Eds. ) 1991 NIES-Collection. List of Strains, Third Edition, 1991, Microalgae and Protozoa. Microbial Culture Collection, National Institute for Environmental Studies, Tsukuba, 163 pp.

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5 Trace metals, vitamin mixtures and soil extracts
PRO–99 trace metals

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Na2EDTA · 2H2O	0.637 g
FeCl3 · 6H2O	0.422 g
ZnSO4 · 7H2O	2.933 mg
CoCll2 · 6H2O	1.332 mg
MnCl2 · 4H2O	24 mg
Na2MoO4 · 2H2O	0.839 mg
Na2SeO3	2.306 mg
NiCl2 · 6H2O	0.37 mg
Distilled water	100 mL

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5 Trace metals, vitamin mixtures and soil extracts
Soil extract

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To 1000 mL distilled water add 200 mL of soil ( soil from undisturbed deciduous woodland is best ) and heat by autoclaving for 1 h at 105°C. When cool, heat by autoclaving for 1 h at 105°C again. Pass the supernatant through a GF/C filter and Celite, and then pass the filtrate through a GF/F filter. Adjust to 1000 mL by adding distilled water. Dispense 10 mL of the final filtrate into each test tube and sterilize by autoclaving for 20min at 121°C. Keep in a cool place.

Reference
Provasoli, L., McLaughlin, J. J. A., Droop, M. R. 1957 The development of artificial media for marine algae. Arch. Mikrobiol., 25, 392-428.

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5 Trace metals, vitamin mixtures and soil extracts
Trace metal mix A5 + Co

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H3BO3	286 mg
MnCl2 · 4H2O	181 mg
ZnSO4 · 7H2O	22.2 mg
Na2MoO4 · 2H2O	39 mg
CuSO4 · 5H2O	7.9 mg
Co( NO3 )2·6H2O	4.9 mg
Distilled water	100 mL

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5 Trace metals, vitamin mixtures and soil extracts
Vitamin mix S3

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Thiamine HCl	5 mg
Nicotinic acid	1 mg
Calcium pantothenate	1 mg
p–Aminobenzoic acid	0.1 mg
Biotin	0.01 mg
Inositol	50 mg
Folic acid	0.02 mg
Thymine	30 mg
Distilled water	100 mL

Reference
Provasoli, L. 1963 Growing marine seaweeds. In Proceedings of the Fourth International Seaweed Symposium, University of Tokyo Press, Tokyo, p. 9-17.

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6 Media for protozoa
AF–6 + Wheat

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Beforehand, sterilize wheat grains by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile wheat to 10 mL AF–6 ^{1 )} medium.
1 ) See AF–6

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6 Media for protozoa
ESM + mTYGM–9 + Rice

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Beforehand, sterilize polished rice by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add 500 μL mTYGM–9 ^{1 )} and a grain of sterile rice to 10 mL ESM ^{2 )} medium.
1 ) See mTYGM–9
2 ) See ESM

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6 Media for protozoa
f/2 + mTYGM–9 + Rice

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Beforehand, sterilize polished rice by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add 500 μL mTYGM–9 ^{1 )} and a grain of sterile rice to 10 mL f/2 ^{2 )} medium.
1 ) See mTYGM–9
2 ) See f/2

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6 Media for protozoa
f/2 + Wheat

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Beforehand, sterilize wheat grains by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile wheat to 10 mL f/2 ^{1 )} medium.
1 ) See f/2

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6 Media for protozoa
Helio

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Sodium acetate	10 mg
Polypeptone	10 mg
Tryptone	20 mg
Yeast extract	20 mg
CaCl2 · 2H2O	0.1 mg
Sea water	10 mL
Distilled water	90 mL

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6 Media for protozoa
LE

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L solution: White part of lettuce is dried at 90ºC for 16–18 h without scorching; 300 mg of the dried lettuce is added to 100 mL boiling water ( 9:1 distilled water to tap water ) and boiled for 30 min, while stirring. The supernatant is passed through cotton wool.
E solution: 300 mg of crushed yolk of hardboiled egg is added to 100 mL water ( 9:1 distilled water to tap water ) and boiled for 30 min, while stirring. The supernatant is passed through cotton wool. Equal quantities of L and E solutions are mixed. The pH is adjusted to 6.8–7.0 with 1 mol / L NaOH, and 100 mL of the solution is dispensed into each 200–mL Erlenmeyer flask and sterilized by autoclaving ( 121ºC, 15 min ).

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6 Media for protozoa
PYG

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Modified PPYG medium (by Sugai T) is diluted with distilled water to 1/2

Proteose Peptone	0.15 g
Yeast extract	0.1 g
Glucose	0.5 g
Distilled water	100 mL
pH 7.0

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6 Media for protozoa
SUY

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Prepare as for 100 mL URO ^{1 )} medium with seawater instead of distilled water. Add 10 mg yeast extract and 20 mg tryptone. Indicated as " URO–YT " in reference.
1 ) See URO

Reference
Moriya, M., Nakayama, T., Inouye, I. 2000 Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate ( Stramenopiles, incertae sedis ). Protist, 151, 41-55.

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6 Media for protozoa
SUY 1/10

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Prepare as for 100 mL URO ^{1 )} medium with seawater instead of distilled water. Add 1 mg yeast extract and 2 mg tryptone. Indicated as " URO–1/10 YT " in reference.
1 ) See URO

Reference
Moriya, M., Nakayama, T., Inouye, I. 2002 A new class of the Stramenopiles, Placididea Classis nova: description of Placidia cafeteriopsis gen. et sp. nov. Protist, 153, 143-156.

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6 Media for protozoa
SUY 1/10 + mTYGM–9 + Rice

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Beforehand, sterilize polished rice by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add 500 μL mTYGM–9 ^{1 )} and a grain of sterile rice to 10 mL SUY 1/10 ^{2 )} medium.
1 ) See mTYGM–9
2 ) See SUY 1/10

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6 Media for protozoa
SUY 1/10 + Wheat

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Beforehand, sterilize wheat grains by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile wheat to 10 mL SUY 1/10 ^{1 )} medium.
1 ) See SUY 1/10

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6 Media for protozoa
URO + Wheat

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Beforehand, sterilize wheat grains by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile wheat to 10 mL URO ^{1 )} medium.
1 ) See URO

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6 Media for protozoa
URO–H + Wheat

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Beforehand, sterilize wheat grains by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile wheat to 10 mL URO–H ^{1 )} medium.
1 ) See URO–H

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6 Media for protozoa
UYTS + Rice

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Prepare as for 100 mL URO ^{1 )} medium with 99.2 mL instead of 99.9 mL distilled water. Adjust to pH 7.5 with 0.1 mol / L HCl, and add 10 mg yeast extract, 20 mg tryptone and 0.3 ml horse serum ( UYTS medium ). Beforehand, sterilize polished rice by dry heating ( 150ºC, 30 min ). Keep in a cool place. For use, add a grain of sterile rice to 10 mL UYTS medium.
1 ) See URO

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7 Media for freshwater red algae
Bold 3N

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NaNO3	75 mg
CaCl2 · 2H2O	2.5 mg
MgSO4 · 7H2O	7.5 mg
K2HPO4	7.5 mg
KH2PO4 1 )	17.5 mg
NaCl	2.5 mg
Vitamin B12 2 )	0. 015 μg
PIV metals 3 )	0.6 mL
Soil extract 4 )	4 mL
Distilled water	96.4 mL

1 ) In the NIES–Collection, the amount of KH₂PO₄ is reduced from 17.5 mg to 10.5 mg.
2 ) In the NIES–Collection, the amount of Vitamin B₁₂ is increased from 0.015 μg to 0.02 μg.
3 ) See PIV metals
4 ) See Soil extract

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8 Media for Charales
mSWC–2 ( Modified SWC–2 )

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Put leaf mould into a glass vessel to make a thin bottom layer, and add river sand onto the bottom layer up to one–quarter to one–fifth from the bottom. Add a pinch of garden lime to the river sand before use. Dampen the soil with deionized water ( or distilled water ). Cover the glass vessel with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ). In the case of brackish water strains, deionized water is replaced by about one–third–diluted Herbst artificial seawater ( 1/3 Herbst ASW ).

Reference
Sakayama, H., Hara Y., Nozaki, H. 2004 Taxonomic re-examination of six species of Nitella ( Charales, Charophyceae ) from Asia, and phylogenetic relationships within the genus based on rbcL and atpB gene sequences. Phycologia, 43, 91-104.

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8 Media for Charales
SWC–1

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Put leaf mould into a glass vessel to make a thin bottom layer, and add black soil onto the bottom layer up to one-quarter to one–fifth from the bottom. Dampen the soil with deionized water ( or distilled water ). Cover the glass vessel with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ).

Reference
Sakayama, H., Hara Y., Nozaki, H. 2004 Taxonomic re-examination of six species of Nitella ( Charales, Charophyceae ) from Asia, and phylogenetic relationships within the genus based on rbcL and atpB gene sequences. Phycologia, 43, 91-104.

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8 Media for Charales
SWCN–1

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Put bottom mud from a paddy field into a glass vessel up to one–quarter to one–fifth from the bottom. Dampen the mud with deionized water ( or distilled water ). Cover the glass vessel or jar with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ).

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8 Media for Charales
SWCN–2

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Put leaf mould into a glass vessel to make a thin bottom layer, and add a mixture of black soil and river sand onto the bottom layer up to one–quarter to one–fifth from the bottom. Dampen the soil with deionized water ( or distilled water ). Cover the glass vessel or jar with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ). In the case of brackish water strains, deionized water is replaced by about one–third–diluted Herbst artificial seawater ( 1/3 Herbst ASW ).

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8 Media for Charales
SWCN–3

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Put a mixture of black soil and bottom mud from a paddy field into a glass vessel up to one–quarter to one–fifth from the bottom. Dampen the soil with deionized water ( or distilled water ). Cover the glass vessel or jar with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ).

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8 Media for Charales
SWCN–4

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Put a mixture of river sand and bottom mud from a paddy field into a glass vessel up to one–quarter to one–fifth from the bottom. Dampen the soil with deionized water ( or distilled water ). Cover the glass vessel or jar with a plastic cap or aluminum foil, and autoclave it twice with overnight rest in between ( 121ºC, 20 min ). After cooling the mixture to room temperature, pour sterilized deionized water ( or sterilized distilled water ) into it carefully ( so as not to disturb the soil ).

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8 Media for Charales
1/3 Herbst ASW

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NaCl	3.0 mg
KCl 1 )	81.4 mg
CaCl2 1 )	132 mg
MgSO4 1 )	660 mg
NaHCO3 1 )	504 mg
Distilled water	100 mL

1 ) In the NIES–Collection, the amount of KCl is reduced from 81.4 mg to 80.0 mg, 132 mg CaCl₂ is replaced by 172 mg CaCl₂ · 2H₂O, 660 mg MgSO₄ is replaced by 1.35 g MgSO₄ · 7H₂O, and the amount of NaHCO₃ is reduced from 504 mg to 49.5 mg.

Reference
Okazaki, Y., Shimmen, T., Tazawa, M. 1984 Turgor regulation in a brackish charophyte, Lamprothamnium succinctum I. Artificial modification of intracellular osmotic pressure. Plant Cell Physiol., 25, 565-571.

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