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Media list

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

AAF–6	Acid–CSi/5	AF–6
AF–6/2	AF–6/3	AFAC
Allen	BBM	BG–11
C	C+10% Seawater	C/6
C/6G	C/10	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 + Glucose
M–Allen + Uracil	MBM	M Chu No. 10
MDM	MG	MGM
Modified acetate medium (mAC)	Modified BG–11₀	Modified Johnson's medium
Modified M–1 (mM–1)	MW	MW/5
N–Free	O	P 35
Pro	SOT	SW
TAP	Tre	URO
URO–H	URO + Soil	URO–T
VT	VTAC	VTYT
W	WC	9:1 agar medium

2. Media for marine and brackish water microalgae

BESM	BESM 2	ESM	ESM + CB–V	ESM2	f/2	f/2 + NH₄Cl
IMK	IMK + NaCl	IMK + Soil Extract	IMK/2	K	K/2	K/2ET
M–ASP7	MB	MF	MKM	MNK	Modified IMR ( mIMR )	Modified SWM–3 (mSWM–3)
PRO–99	WESM

3. Bacteria-free check media for freshwater algae

4. Bacteria-free check media for marine algae

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
Fe ( as EDTA; 1:1 molar )	Fe solution	f/2 metals	Hutner ’s trace elements
K metals	K/2 metals	mIMR trace metals	mM–1 Trace elements
mTYGM–9	P–1 metals	P II metals	P IV metals
PN metals	PRO–99 trace metals	Soil extract	Trace metal mix A₅ + Co
Vitamin mix S₃	Waris EDTA	Waris P-2	WC trace metals

7. Media for freshwater red algae

8. Media for Charales

mSWC–2 ( Modified SWC–2 )

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

AAF–6

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

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

Acid–CSi/5

Print

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

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

AF–6

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

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

AF–6/2

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

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

AF–6/3

Mix 1 volume of AF–6^{1 )} medium and 2 volumes of distilled water.
1 ) See AF–6

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

AFAC

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

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

Allen

( 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(s)
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.

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

BBM

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 · 4H₂O	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

Add 1.2 g agar to 100 mL of medium to give a solid medium.

Reference(s)
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.

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

BG–11

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 mL
Distilled water	99.9 mL
pH 7.4

Add 1.5 g agar to 100 mL of medium to give a solid medium.
1 ) See Trace metal mix A₅ + Co

Reference(s)
Waterbury, J. B., Sranier, R. Y. 1981 Isolation and growth of cyanobacteria from marine and hypersaline environments. In The Prokaryotes, Springer Berlin Heidelberg, New York, pp. 221-223.

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

C

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(s)
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.

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

C+10% Seawater ( N. Tezuka, unpubl. )

Print

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

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

C/6

Mix 1 volume of C^{1 )} medium and 5 volumes of distilled water.
1 ) See C

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

C/6G

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

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

C/10

Mix 1 volume of C^{1 )} medium and 9 volumes of distilled water.
1 ) See C

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

CA

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 mL
HEPES	40 mg
Distilled water	99.9 mL
pH 7.2

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

Reference(s)
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.

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

CAM

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

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

Carefoot

Print

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

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

CB

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

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

CB–V

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

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

CC

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(s)
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 ).

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

CSi

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

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

CSi + Cu

Print

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

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

CSi/5

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

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

CT

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

Reference(s)
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.

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

CYT

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

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

DH + Fe ( I.I. Brown, unpubl. )

D stock medium^{1 )}	5 mL
HEPES	0.12 mg
FeCl₃ · 6H₂O	1.14 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

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

DY–V

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

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

HUT

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(s)
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. 301 (in Japanese without English title).

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

M–11

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(s)
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 ).

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

MA

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
CoCl₂ · 6H₂O	0.5 mg
Na₂MoO₄ · 2H₂O	0.08 mg
H₃BO₃	2 mg
Bicine	50 mg
Distilled water	100 mL
pH 8.6

Reference(s)
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 ).

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

MAF–6

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

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

M–Allen

Print

( 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.

Reference(s)
Minoda, A., Sakagami, R., Yagisawa, F., Kuroiwa, T., Tanaka, K. 2004 Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol., 45, 667-671.

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

M–Allen + Glucose

Print

To 100 mL M–Allen^{1 )} medium add 0.5 –1 g Glucose.
1 ) See M–Allen

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

M–Allen + Uracil

Print

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

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

MBM

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(s)
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 ).

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

M Chu No. 10

Print

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(s)
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.

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

MDM

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(s)
Watanabe, A. 1960 List of algal strains in collection at the Institute of Applied Microbiology, University of Tokyo. J. Gen. Appl. Microbiol., 6, 283-292.

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

MG

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(s)
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.

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

MGM

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

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

Modified acetate medium ( mAC )

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

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

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

Modified BG–11₀

1M HEPES–NaOH^{1 )}	2 mL
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 ^{2 )}	0.1 mL
Vitamin B₁₂	0.1 µg
Biotin	0.1 µg
Thiamine HCl	200µg
Agar	1.2 g
Distilled water	97.9 mL

1 ) To 60 mL of Distilled water add 23.8 g HEPES, and pH is adjusted to 7.5. Bring to 100 mL with distilled water.v
2 ) See Trace metal mix A₅ + Co

Reference(s)
Castenholz, R. W. 1988 Cultureing methods for cyanobacteria. Methods Enzymol., 167, 68-93.

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

Modified Johnson's medium

NaCl	3 mg
MgCl₂ · 6H₂O	150 mg
KNO₃	100 mg
MgSO₄ · 7H₂O	50 mg
CaCl₂ · 2H₂O¹⁾	26.5 mg
KCl	20 mg
NaHCO₃	4.3 mg
KH₂PO₄	3.5 mg
Tris ( hydroxymethyl ) aminomethane	245 mg
Distilled water	100 mL
pH 8.5

1 ) In the NIES-Collection, 20 mg CaCl₂ is replaced by 26.5 mg CaCl₂ · 2H₂O.

Reference(s)
Johnson, M. K., Johnson, E. J., MacElroy, R. D., Speer, H. L., Bruff, B. S. 1968 Effects of salts on the halophilic alga Dunaliella viridis. J. Bacteriol., 95, 1461-1468.
Utsunomiya, A., Watanuki, T., Matsushita, K., Tomita, I. 1997 Toxic effects of linear alkylbenzene sulfonate, quaternaryalkylammonium chloride and their complexes on Dunaliellasp. and Chlorella pyrenoidosa. Environ. Toxicol. Chem., 16, 1247-1254.
Mihirogi, M., Kikuchi, M., Sawai, J. 2012 Development of screening algal growth inhibition test with Dunaliella sp. Jpn. J. Environ. Toxicol., 15, 11-16 (in Japanese with English summary).

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

Modified M–1 ( mM–1 )

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(s)
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.

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

MW

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(s)
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.

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

MW/5

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

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

N–Free

Print

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
CoCl₂ · 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.

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

O

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(s)
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.

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

P 35

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(s)
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 ).

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

Pro

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

Reference(s)
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.

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

SOT

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(s)
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.

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

SW

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

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

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

TAP

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

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

Tre

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(s)
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.

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

URO

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(s)
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 ).

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

URO–H

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

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

URO + Soil

Print

Prepare as for 100 mL URO^{1 )} medium with 89.9 mL instead of 99.9 mL distilled water, add 10 mL Soil extract^{2 )} and adjust pH 7.5.
1 ) See URO
2 ) See Soil extract

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

URO–T

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

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

VT

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(s)
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.

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

VTAC

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

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

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

VTYT

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

Reference(s)
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 ).

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

W

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

Reference(s)
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 ).

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

WC

NaNO₃	8.5 mg
CaCl₂ · 2H₂O	3.67 mg
MgSO₄ · 7H₂O	3.69 mg
NaHCO₃	1.26 mg
Na₂SiO₃ · 9H₂O	2.84 mg
K₂HPO₄	0.87 mg
WC trace metals^{1 )}	0.1 mL
Vitamin B₁₂	0.05 μg
Biotin	0.05 μg
Thiamine HCl	10 μg
Distilled water	99.9 mL
pH 7.5

1 ) See WC trace metals

Reference(s)
Guillard, R. R. L., Lorenzen, C. J. 1972 Yellow-green algae with chlorohyllide C. J. Phycol., 8, 10-14.
Andersen, R. A. E, Berges, J. A., Harrison, P. J., Watanabe, M. M. 2005 Appendix A―Recipes for freshwater and seawater media. In Algal culturing techniques, Ed. by Andersen, R. A. E, Elsevier Academic Press, p. 474.

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

9:1 agar medium

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
Na₂EDTA· 2H₂O	5 mg
FeSO₄ · 7H₂O	0.498 mg
H₃BO₃	1.142 mg
ZnSO₄ · 7H₂O	0.882 mg
MnCl₂ · 4H₂O	0.144 mg
MoO₃	0.071 mg
CuSO₄ · 5H₂O	0.157 mg
Co( NO₃ )₂ · 6H₂O	0.049 mg
Vitamin B₁₂	0.01 μg
Biotin	0.01 μg
Thiamine HCl	1 μg
Soil extract^{1 )}	10 mL
Distilled water	100 mL

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

Reference(s)
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.
Watanabe, S., Fučíková, K., Lewis, L. A., Lewis P.O. 2016 Hiding in plain sight: Koshicola spirodelophila gen. et sp. nov. (Chaetopeltidales, Chlorophyceae), a novel green alga associated with the aquatic angiosperm Spirodela polyrhiza. Am. J. Bot., 103, 865-875.

2 Media for marine and brackish water microalgae

BESM

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

2 Media for marine and brackish water microalgae

BESM 2

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

2 Media for marine and brackish water microalgae

ESM

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

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

Reference(s)
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

ESM + CB–V

Print

Mix 100 mL of ESM^{1 )} medium and 0.25 mL of CB–V^{2 )} medium.

1 ) See ESM
2 ) See CB–V

2 Media for marine and brackish water microalgae

ESM2

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

2 Media for marine and brackish water microalgae

f/2

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(s)
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.

2 Media for marine and brackish water microalgae

f/2 + NH4Cl

Print

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

2 Media for marine and brackish water microalgae

IMK

Into 100 mL seawater dissolve 25.2 mg powder medium of Daigo IMK ( Nihon Pharmaceutical Co., Ltd. ). In the NIES–Collection, IMK medium is used after autoclaving ( 121°C, 20 min ).

2 Media for marine and brackish water microalgae

IMK + NaCl

Into 100 mL seawater dissolve 25.2 mg powder medium of Daigo IMK ( Nihon Pharmaceutical Co., Ltd. ). Adjust to salinity 10 percent with NaCl. In the NIES-Collection, IMK + NaCl medium is used after autoclaving ( 121°C, 20 min ).

2 Media for marine and brackish water microalgae

IMK + Soil Extract

Into 100 mL seawater dissolve 25.2 mg powder medium of Daigo IMK ( Nihon Pharmaceutical Co., Ltd. ). Thereafter, 2 mL Soil extract 1 ) is added. In the NIES–Collection, IMK + Soil Extract medium is used after autoclaving ( 121°C, 20 min ).
1 ) See Soil extract

2 Media for marine and brackish water microalgae

IMK/2

Into 100 mL seawater dissolve 12.6 mg powder medium of Daigo IMK ( Nihon Pharmaceutical Co., Ltd. ). In the NIES–Collection, IMK/2 medium is used after autoclaving ( 121°C, 20 min ).

Reference(s)
Takahashi, K., Lum, W. M., Benico, G., Uchida, H., Ozawa, M., Oikawa, H., Suzuki, T., Nguyen, N. V., Ha, D. V., Iwataki, M. 2021 Harmful algal bloomsToxigenic strains of Azadinium poporum (Amphidomataceae, Dinophyceae) from Japan and Vietnam, with first reports of A. poporum (ribotype A) and A. trinitatum in Asian Pacific. J. Phycol., 69, 175-187.

2 Media for marine and brackish water microalgae

K

Tris-base ( pH 7.2) ^{1 )}	0.1 mL
NaNO₃	7.5 mg
NH₄Cl	0.267 mg
β–Na₂glycerophosphate · 5H₂O	0.216 mg
H₂SeO₃	0.129 μg
Vitamin B₁₂	0.05 μg
Biotin	0.05 μg
Thiamine HCl	0.01 mg
Na₂SiO₃ · 9H₂O^{2 )}	1.535 mg
K metals^{3 )}	0.1 mL
Seawater	99.8 mL

1 ) To 100 mL of Distilled water add 12.11 g Tris-base, and pH is adjusted to 7.2.
2 ) In the NIES-Collection, Na₂SiO₃ · 9H₂O is removed.
3 ) See K metals

Reference(s)
Keller, M. D., Guillard, R.R.L. 1985 Factors significant to marine diatom culture. pp. 113-6. In Anderson, D.M., White, A.W., Baden, D.G. (eds.) Toxic Dinoflagellates. Elsevier, New York.
Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol., 23: 633-638.

2 Media for marine and brackish water microalgae

K/2 (K/2-modified by Ian Probert)

NaNO₃	2.44 mg
NH₄Cl	0.0267 mg
KH₂PO₄	0.244 mg
Fe-EDTA	0.215 mg
Vitamin B₁₂	0.05 μg
Biotin	0.0005 μg
Thiamine HCl	0.01 mg
K/2 metals^{1 )}	0.05 mL
Seawater	99.95 mL
pH 8.2

K medium is diluted half strength and modified by Ian. This medium should not be autoclaved but filter-sterilized.
1 ) See K/2 metals

Reference(s)
Keller, M. D., Guillard, R.R.L. 1985 Factors significant to marine diatom culture. pp. 113-6. In Anderson, D.M., White, A.W., Baden, D.G. (eds.) Toxic Dinoflagellates. Elsevier, New York.
Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol., 23: 633-638.

2 Media for marine and brackish water microalgae

K/2ET

Print

NaNO₃	2.44 mg
NH₄Cl	0.0267 mg
KH₂PO₄	0.244 mg
Fe-EDTA	0.215 mg
Vitamin B₁₂	0.05 μg
Biotin	0.0005 μg
Thiamine HCl	0.01 mg
K/2 metals^{1 )}	0.05 mL
Soil extract^{2 )}	1.5 mL
Seawater	98.45 mL
pH 8.2

K medium is diluted half strength and modified by Ian. This medium should not be autoclaved but filter-sterilized.
1 ) See K/2 metals
2 ) See Soil extract

Reference(s)
Keller, M. D., Guillard, R.R.L. 1985 Factors significant to marine diatom culture. pp. 113-6. In Anderson, D.M., White, A.W., Baden, D.G. (eds.) Toxic Dinoflagellates. Elsevier, New York.
Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol., 23: 633-638.

2 Media for marine and brackish water microalgae

M–ASP7

Print

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
P_N 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(s)
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.k.pdf

2 Media for marine and brackish water microalgae

Marine Broth (MB) medium

Into 100 mL distilled water dissolve 3.74 g powder medium of Difco^TM Marine broth 2216 (Becton, Dickinson and Company) and 25.2 mg powder medium of Daigo IMK (Nihon Pharmaceutical Co., Ltd.). In the NIES-Collection, MB medium is used after autoclaving (121°C, 20 min).

Reference(s)
Ishii, Y., Maruyama, S., Fujimura-Kamada, K., Kutsuna, N., Takahashi, S., Kawata, M., Minagawa, J. 2018 Isolation of uracil auxotroph mutants of coral symbiont alga for symbiosis studies. Sci Rep, 8, 3237.

2 Media for marine and brackish water microalgae

MF

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

2 Media for marine and brackish water microalgae

MKM

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(s)
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.

2 Media for marine and brackish water microalgae

MNK

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. The medium is sterilized by filtering, not autoclaving.

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

2 Media for marine and brackish water microalgae

ModifiedIMR ( mIMR )

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(s)
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.

2 Media for marine and brackish water microalgae

Modified SWM–3 ( mSWM–3 )

Print

NaNO₃	17 mg
NaH₂PO₄· 2H₂O	1.56 mg
Na₂SiO₃· 9H₂O	5.68 mg
Na₂EDTA· 2H₂O	1.12 mg
Fe-EDTA	0.084 mg
Na₂SeO₃	0.0346 μg
P–1 metals	1 mL
Vitamin B₁₂	0.2 μg
Vitamin mix S₃	1 mL
Tris ( hydroxymethyl ) aminomethane	50 mg
Seawater	98.8 mL
pH 7.7 – 7.8

1 ) See P–1 metals
2 ) See Vitamin mix S₃

Reference(s)
Imai, I. 2012 High growth rates of the red tide flagellate Chattonella marina (Raphidophyceae) observed in culture. Hokudai Suisan Ihou, 62, 71-74 ( in Japanese with English summary ).

2 Media for marine and brackish water microalgae

PRO–99

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

The medium is sterilized by filtering, not autoclaving.
1 ) See PRO–99 trace metals

2 Media for marine and brackish water microalgae

WESM

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

3 Bacteria-free check media for freshwater algae

B–I

Appropriate medium	100 mL
Proteose peptone	100 mg

Reference(s)
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.

3 Bacteria-free check media for freshwater algae

B–II

Appropriate medium	100 mL
Yeast extract	500 mg

Reference(s)
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.

3 Bacteria-free check media for freshwater algae

B–III

Appropriate medium	100 mL
Peptone	500 mg
Beef extract	300 mg

Reference(s)
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.

3 Bacteria-free check media for freshwater algae

B–IV

Appropriate medium	100 mL
Glucose	100 mg
Peptone	100 mg

Reference(s)
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.

3 Bacteria-free check media for freshwater algae

B–V

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

Reference(s)
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.

3 Bacteria-free check media for freshwater algae

YT

Appropriate medium	100 mL
Yeast extract	100 mg
Tryptone	200 mg

Reference(s)
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 ).

4 Bacteria-free check media for marine algae

Bf/2

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(s)
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 ).

4 Bacteria-free check media for marine algae

MM 23 ( M. Tatewaki, pers. comm. )

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

4 Bacteria-free check media for marine algae

STP

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

5 Trace metals, vitamin mixtures and soil extracts

A2 Fe stock solution

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

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

5 Trace metals, vitamin mixtures and soil extracts

A2 trace element stock solution

Print

H₃BO₃	285 mg
MnCl₂ · 4H₂O	180 mg
ZnCl₂	10.5 mg
Na₂MoO₄ · 2H₂O	39 mg
CoCl₂ · 6H₂O	4 mg
CuCl₂ · 2H₂O	4.3 mg
Distilled water	100 mL

5 Trace metals, vitamin mixtures and soil extracts

A₅ solution

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

5 Trace metals, vitamin mixtures and soil extracts

Allen metals

Print

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

5 Trace metals, vitamin mixtures and soil extracts

C–Source Mix II ( M. Tatewaki, pers. comm. )

Glycine	100 mg
D,L-Alanine	100 mg
L-Asparagine	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.

5 Trace metals, vitamin mixtures and soil extracts

D stock medium

Print

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 replaced by 550 mg Na₂HPO₄· 12H₂O.

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

5 Trace metals, vitamin mixtures and soil extracts

D trace mix

Print

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
CoCl₂ · 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(s)
Castenholz, R. W. 1969 Thermophilic blue-green algae and the thermal environment. Bacteriol. Rev., 33, 476-504.

5 Trace metals, vitamin mixtures and soil extracts

DY trace metal solution

Print

MnCl₂ · 4H₂O	20 mg
ZnSO₄ · 7H₂O	4 mg
CoCl₂ · 6H₂O	0.8 mg
Na₂MoO₄ · 6H₂O ^{1 )}	2 mg
Na₃VO₄ · 10H₂O ^{2 )}	0.2 mg
H₂SeO₃	0.2 mg
Distilled water	100 mL

1 ) In the NIES–Collection, Na₂MoO₄ · 6H₂O is replaced by Na₂MoO₄ · 2H₂O.
2 ) In the NIES-Collection, 0.2 mg Na₃VO₄ · 10H₂O is replaced by 0.1 mg Na₃VO₄.

5 Trace metals, vitamin mixtures and soil extracts

Fe ( as EDTA; 1:1 molar )

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(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

Fe solution

Print

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

1 ) 2 drops/500 mL

Reference(s)
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 ).

5 Trace metals, vitamin mixtures and soil extracts

f/2 metals

Print

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(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

Hutner ’s trace elements

Print

Na₂EDTA · 2H₂O	5 g
ZnSO₄ · 7H₂O	2.2 g
H₃BO₃	1.14 g
MnCl₂ · 4H₂O	506 mg
FeSO₄ · 7H₂O	499 mg
CoCl₂ · 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.

5 Trace metals, vitamin mixtures and soil extracts

K metals

Print

Na₂EDTA · 2H₂O	3.722 g
Fe-EDTA	0.493 g
FeCl₃ · 6H₂O	0.315 g
MnCl₂ · 4H₂O	17.8 mg
ZnSO₄ · 7H₂O	2.3 mg
CoSO₄ · 7H₂O	1.405 mg
Na₂MoO₄ · 2H₂O	0.726 mg
CuSO₄ · 5H₂O	0.25 mg
Distilled water	100 mL

Reference(s)
Keller, M. D., Guillard, R.R.L. 1985 Factors significant to marine diatom culture. pp. 113-6. In Anderson, D.M., White, A.W., Baden, D.G. (eds.) Toxic Dinoflagellates. Elsevier, New York.
Keller, M.D., Selvin, R.C., Claus, W., Guillard, R.R.L. 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol., 23: 633-638.

5 Trace metals, vitamin mixtures and soil extracts

K/2 metals

Print

1 ) In the NIES–Collection, 0.149 mg NiCl₂ · 6H₂O is replaced by 0.812 mg NiCl₂.

Reference(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

mIMR trace metals

Print

FeCl₃ · 6H₂O	100 mg
MnSO₄ · 4H₂O	85.5 mg
ZnSO₄ · 7H₂O	25 mg
Na₂MoO₄ · 2H₂O	13 mg
CoCl₂ · 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.

5 Trace metals, vitamin mixtures and soil extracts

mM–1 Trace elements

Print

CuSO₄ · 5H₂O	10 mg
MnCl₂ · 4H₂O	10 mg
Br ( 1mol / L solution )	0.01 mL
ZnSO₄ · 7H₂O	10 mg
CoCl₂ · 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(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

mTYGM–9

Print

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.

5 Trace metals, vitamin mixtures and soil extracts

P II metals

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

5 Trace metals, vitamin mixtures and soil extracts

P–1 metals

H₃BO₃	618.3 mg
MnCl₂ · 4H₂O	69.25 mg
ZnCl₂	5.45 mg
CoCl₂ · 6H₂O	238 μg
Distilled water	100 mL

1 ) 1) In the NIES-Collection, CuCl₂ · 2H₂O is removed.

Reference(s)
Imai, I. 2012 High growth rates of the red tide flagellate Chattonella marina (Raphidophyceae) observed in culture. Hokudai Suisan Ihou, 62, 71-74 (in Japanese with English summary).

5 Trace metals, vitamin mixtures and soil extracts

P IV metals

Na₂EDTA · 2H₂O	100 mg
FeCl₃ · 6H₂O	19.6 mg
MnCl₂ · 4H₂O	3.6 mg
ZnCl₂^{1 )}	1.04 mg
CoCl₂ · 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(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

P_N metals

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(s)
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.

5 Trace metals, vitamin mixtures and soil extracts

PRO–99 trace metals

Print

Na₂EDTA^{1 )}	0.58 g
FeCl₃ · 6H₂O	0.422 g
ZnSO₄ · 7H₂O	2.933 mg
CoCl₂ · 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

1 ) In the NIES–Collection, 0.58 g Na₂EDTA is replaced by 0.637 g Na₂EDTA · 2H₂O.

5 Trace metals, vitamin mixtures and soil extracts

Soil extract

Print

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

5 Trace metals, vitamin mixtures and soil extracts

Trace metal mix A₅ + Co

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

5 Trace metals, vitamin mixtures and soil extracts

Vitamin mix S₃

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

5 Trace metals, vitamin mixtures and soil extracts

Waris EDTA

Print

EDTA	0.522 g
FeSO₄ · 7H₂O	0.498 g
KOH (1 mol/L solution)	5.4 mL
Distilled water	94.6 mL

EDTA and FeSO₄ · 7H₂O is heated for 30 min (100°C ); KOH is added to the cooled mixture.

Reference(s)
McFadden, G. I., Melkonian, M. 1986 Use of Hepes buffer for microalgael culture media and fixation for eledtron microscopy. Phycology, 25, 551-557.

5 Trace metals, vitamin mixtures and soil extracts

Waris P-2

Na₂EDTA· 2H₂O	0.3 g
H₃BO₃	0.114 g
MnCl₂· 4H₂O	14.4 mg
ZnSO₄·7H₂O	2.1 mg
CoCl₂·6H₂O	0.4 mg
Distilled water	100 mL

Reference(s)
McFadden, G. I., Melkonian, M. 1986 Use of Hepes buffer for microalgael culture media and fixation for eledtron microscopy. Phycology, 25, 551-557.

5 Trace metals, vitamin mixtures and soil extracts

WC trace metals

print

Na₂EDTA· 2H₂O	436 mg
FeCl₃ · 6H₂O	315 mg
CuSO₄ · 5H₂O	1 mg
ZnSO₄ · 7H₂O	2.2 mg
CoCl₂ · 6H₂O	1 mg
MnCl₂ · 4H₂O	18 mg
Na₂MoO₄ · 2H₂O	0.6 mg
H₃BO₃	100 mg
Distilled water	100 mL

Reference(s)
Guillard, R. R. L., Lorenzen, C. J. 1972 Yellow-green algae with chlorohyllide C. J. Phycol., 8, 10-14.
Andersen, R. A. E, Berges, J. A., Harrison, P. J., Watanabe, M. M. 2005 Appendix A — Recipes for freshwater and seawater media. In Algal culturing techniques, Ed. by Andersen, R. A. E, Elsevier Academic Press, p. 474.

6 Media for protozoa

AF–6 + Wheat

Print

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

6 Media for protozoa

BLEY medium ( Barley leaf & Egg-yolk extracted liquid )

BL solution: 1 pack ( 3 g ) of Barley leaf ( Trade name: Oomugi Wakaba Funmatu 100%, Yamamoto Kanpou Seiyaku Co., Ltd. ) is added to 1,000 mL distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.
EY solution: 9 g of crushed yolk of hard-boiled egg is added to 1,000 mL distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.
BL and EY Solution are mixed. The mixed solution is dilution with distilled water up to 6,000 mL, and then sterilized by autoclaving ( 121ºC, 15 min ).

6 Media for protozoa

dGTY

Print

Glucose	0.2 g
Tryptone	0.1 g
Yeast extract	0.05 g
Seawater	100 mL

6 Media for protozoa

ESM + mTYGM–9 + Rice

Print

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

6 Media for protozoa

ESM + Rice

Print

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 ESM^{1 )} medium.
1 ) See ESM

6 Media for protozoa

f/2 + mTYGM–9 + Rice

Print

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

6 Media for protozoa

f/2 + Rice

Print

Beforehand, sterilize polished rice 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

6 Media for protozoa

f/2 + Wheat

Print

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

6 Media for protozoa

Helio

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

6 Media for protozoa

Hemi

Into 98.9 mL distilled water 3.6 g powder medium of Daigo's Artificial Seawater SP ( Nihon Pharmaceutical Co., Ltd. ) and 0.1 mL LB broth medium ( BD ). After autoclaving, add aseptically 1 mL horse serum ( Sigma-Aldrich Co. LLC ).

Reference(s)
Tashyreva, D., Prokopchuk, G., Yabuki, A., Kaur, B., Faktorová, D., Votýpka, J., Kusaka, C., Fujikura, K., Shiratori, T., Ishida, K., Horák, A., Lukeš, J. 2018 Phylogeny and morphology of new diplonemids from Japan. Protist, 169, 158-179.

6 Media for protozoa

LE

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 ).

6 Media for protozoa

Mizinko

Put 10 - 20 of dried water fleas commercially available for aquarium fish into a test tube and add 10mL distilled water.

Reference(s)
Takahashi, Y., Yoshida, M., Inouye, I., Watanabe, M. M. 2014 Diplophrys mutabilis sp. nov., a new member of Labyrinthulomycetes from freshwater habitats. Protist, 165, 50-65.

6 Media for protozoa

Modified WarisH–Si

Print

HEPES	11.915 mg
KNO₃	5 mg
MgSO₄ · 7H₂O	1 mg
(NH₄)₂HPO₄	1 mg
Ca(NO₃)₂ · 4H₂O	5 mg
Waris P-2	0.05 mL
Waris EDTA	0.05 mL
Vitamin B₁₂	0.01 μg
Biotin	0.05 μg
Thiamine HCl	5 μg
Niacinamide	0.005 μg
Soil extract^{3 )}	0.5 mL
Na₂SiO₃ · 9H₂O	2.842 mg
Distilled water	99.4 mL
pH 7.0

Waris H is diluted half strength and added Na₂SiO₃ · 9H₂O.
1 ) See Waris P-2
2 ) See Waris EDTA
3 ) See Soil extract

Reference(s)
McFadden, G. I., Melkonian, M. 1986 Use of Hepes buffer for microalgael culture media and fixation for eledtron microscopy. Phycology, 25, 551-557.

6 Media for protozoa

PYG

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

PPYG medium is diluted half strength and reduced proteose peptone.

Reference(s)
Mori, K., Yomo, T., Kashiwagi, A. 2011 Single-cell isolation and cloning of tetrahymena thermophila cells with a fluorescence-activated cell sorter. J. Eukaryotic Microbiol., 58(1), 37-42.

6 Media for protozoa

SUY

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(s)
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.

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(s)
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.

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

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

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

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

6 Media for protozoa

URO + YT (1/10) + Wheat

Prepare as for 100 mL URO^{1 )} medium. Adjust to pH 7.5 with 0.1 mol / L HCl,　and add 10 mg yeast extract and 20 mg tryptone (URO + YT (1/10) medium). 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 + YT (1/10) medium.
1 ) See URO

Reference(s)
Yabuki, A., Nakayama, T., Yubuki, N., Hashimoto, T., Ishida, K-I. Inagaki, Y. 2011 Tsukubamonas globosa n. gen., n. sp., a novel excavate flagellate possibly holding a key for the early evolution in "Discoba". J. Eukaryot. Microbiol., 58, 319-331.

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

6 Media for protozoa

YE100ppm

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Into 100 mL distilled water 9 mg powder medium of Daigo's Artificial Seawater SP
( Nihon Pharmaceutical Co., Ltd. ) and 10 mg yeast extract.

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

8 Media for Charales

mSWC–2 ( Modified SWC–2 )

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(s)
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.

8 Media for Charales

SWC–1

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(s)
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.

8 Media for Charales

SWCN–1

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 ).

8 Media for Charales

SWCN–2

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 ).

8 Media for Charales

SWCN–3

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 ).

8 Media for Charales

SWCN–4

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 ).

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(s)
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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