VIII. List of Media
1
Potato Sucrose Agar (PSA)
Potato
Sucrose
Distilled water
Agar
5
200
20
1
20
g
g
L
g
pH 5.6
Wash potatoes with tap water, peel and cut into 1 cm
cubes. Rinse with tap water quickly and boil 200 g of
potato cubes with 1 L of distilled water for 20 min. Mash
and squeeze through a muslin bag. Add sucrose and stir
till dissolved. Adjust pH. Add agar. Make up to 1 L.
Autoclave to sterilize.
2
Malt Extract Agar
Malt extract
Glucose
Peptone
Distilled water
Agar
6
20
20
1
1
20
g
g
g
L
g
pH 6.0
10
40
1
20
g
g
L
g
pH 6.0
5
20
1
20
g
g
L
g
Sabouraud's Agar
Peptone
Glucose
Distilled water
Agar
Potato Carrot Agar (PCA)
Potato
Carrot
Distilled water
Agar
20
20
1
20
g
g
L
g
pH 6.0
7
Ebios (tablets)*
Glucose
Tap water
Agar
Peel and slice potatoes and carrots, boil for 20 min with 1
L of distilled water. Mash and strain through a muslin bag.
Adjust pH. Add agar. Make up to 1 L. Autoclave to
sterilize.
* Manufactured by Asahi Food & Health Care Co. Ltd.,
Tokyo, Japan. Distributed by Mitsubishi Tanabe Pharma
Co., Osaka, Japan.
8
3
Potato
Glucose
NaCl
Distilled water
Agar
200
120
50
1
20
50
g
1
L
20
g
pH unadjusted
Boil powdered oatmeal with distilled water on a water
bath at 80℃, stirring occasionally, for 1 h. Squeeze
through a muslin bag, then make up to 1 L. Add agar and
boil till melted. Autoclave to sterilize.
9
4
200
2
20
1
20
Oatmeal Agar
Oatmeal
Distilled water
Agar
g
g
g
L
g
pH 5.6
Wash potatoes with tap water, peel and cut into 1 cm
cubes. Rinse with tap water quickly and boil 200 g of
potato cubes with 1 L of distilled water for 20 min. Mash
and squeeze through a muslin bag. Add agar and boil till
melted. Add glucose and NaCl, and stir till dissolved.
Make up to 1 L. Adjust pH. Autoclave to sterilize.
Potato
Yeast extract
Glucose
Distilled water
Agar
Matsutake Medium
g
g
g
L
g
pH 5.6
NaNO3
K2HPO4
MgSO4·7H2O
KCl
FeSO4·7H2O
Sucrose
Distilled water
Agar
3
g
1
g
0.5
g
0.5
g
0.01 g
200
g
1
L
20
g
pH 6.0
NaNO3
K2HPO4
MgSO4·7H2O
KCl
FeSO4·7H2O
Sucrose
Distilled water
Agar
3
g
1
g
0.5
g
0.5
g
0.01 g
400
g
1
L
20
g
pH 6.0
10
Wash potatoes with tap water, peel and cut into 1 cm
cubes. Rinse with tap water quickly and boil 200 g of
potato cubes with 1 L of distilled water for 20 min. Mash
and squeeze through a muslin bag. Add agar and boil till
melted. Add yeast extract and glucose, and stir till
dissolved. Make up to 1 L. Adjust pH. Autoclave to
sterilize.
939
Media
13
Seawater Starch Agar (SWS)
Soytone
Soluble starch
Seawater (2% Salinity)*
Agar
20
1
10
1
15
g
g
L
g
pH 8.2
* Dilute artificial seawater or natural seawater with distilled
water.
14
22
20
1
20
g
L
g
pH 6.5
24
200
3
800
15
ml
g
ml
g
pH 7.0
25
Cornmeal Seawater Agar (CMSWA)
Cornmeal extract
Seawater (2% Salinity)
Agar
17
2
1
20
Glucose
Peptone
Yeast extract
Malt extract
Olive oil
Distilled water
Agar
10
g
0.4
g
0.15 g
0.15 g
0.06 g
1
L
pH 7.0
Glucose
Peptone
Yeast extract
Malt extract
CaCO3
Distilled water
Agar
Rust Fungi Medium
Murashige & Skoog's plant salt mixture*
Peptic Pepton (USB)
Lab-Lemco Broth (OXOID)
Sucrose
Distilled water make up to
Bacto Agar (Difco)
2
20
1
15
g
g
L
g
pH 5.0
10
5
3
3
10
1
15
g
g
g
g
g
L
g
pH 5.6
10
5
3
3
10
1
15
g
g
g
g
g
L
g
pH 5.6
104
* Whatman No. 1 filter paper pieces, 5 mm square.
19
g
g
g
L
g
103
g
L
g
pH 6.0
Petri-Cellulose Medium
Cellulose*
Ca(NO3)2·4H2O
K2HPO4
MgSO4·7H2O
KCl
Distilled water
1
1
1
1
15
Mycorrhiza Medium
Yeast extract
Glucose
Distilled water
Agar
Cornmeal Agar
Cornmeal extract
Distilled water
Agar
18
26
2
g
1
L
15
g
pH 7.0 - 7.5
2
g
1
g
1
L
15
g
pH 7.0 - 7.5
PYG Agar
Proteose peptone
Yeast extract
Glucose
Distilled water
Agar
*Add CaCO3 (3 g) to V-8 juice (ca. 300 ml) and stir it for 2 h.
After centrifuging, add seawater and agar to 200 ml of the
supernatant. Adjust pH to 7.0.
16
g
g
g
L
Cornmeal Yeast Extract Seawater Agar
(CMYSWA)
Cornmeal extract
Yeast extract
Seawater (2% salinity)
Agar
Vegetable Juice Seawater Agar (V-8 SWA)
Vegetable juice*
CaCO3*
Seawater (2% salinity)
Agar
0.1
1
12
1
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Boil 20 g of the mixture in ca. 1 L water for 1 h. After
filtering the solution, adjust it to 1 L with water and add 20
g of agar.
15
0.4
g
4
g
1
L
15
g
pH 5.0
Trypticase Yeast Extract Agar (TYS)
Trypticase Peptone (BBL) or Polypepton*
Yeast extract
Agar
Seawater (2% salinity)
Corn and Fish Meal Agar
Corn and fish meal mixture (feed for birds)
Distilled water
Agar
1/5 Mycorrhiza Medium
Yeast extract
Glucose
Distilled water
Agar
1.15 g
2
g
2
g
40
g
1
L
20
g
pH 5.8
* Wako Pure Chemicals Ind. Ltd., Osaka, Japan.
940
Media
107
Glucose
Peptone
Yeast extract
Malt extract
NaCl
Distilled water
Agar
108
109
10
5
3
3
1
15
g
g
g
g
L
g
10
20
20
40
1
15
112
119
g
g
g
mg
L
g
10
20
20
1
15
250
10
20
1
g
g
g
L
20
1
15
g
L
g
40
1
15
g
L
g
3
10
1
15
10
g
g
L
g
ml
200
30
25
5
10
5
15
1
15
g
g
g
g
g
g
g
L
g
pH 7.0
MA4
YEP Alcohol
Yeast extract
Peptone
Distilled water
Agar
Ethyl alcohol
201
Potato*
Press yeast
Liver infusion from*
Meat extract
Thioglycolate Medium Dehydrated**
Glucose
Glycerol
Distilled water make up to
Agar (if needed)
g
g
mg
mg
L
g
YPD Medium
Yeast extract
Peptone
Glucose
Distilled water
Agar
* Gently boil sliced potatoes in 500 ml of water (or sliced
liver in 150 ml of water) for 30 min, and remove solids by
filtration through cloth.
g
g
g
L
g
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
202
113
Potato*
Press yeast
Liver infusion from*
Meat extract
Thioglycolate Medium Dehydrated**
Glucose
Glycerol
CaCO3
Distilled water, make up to
Agar (if needed)
Bacto-Yeast Nitrogen Base w/o Amino Acids and Ammonium
Sulfate (Difco)
1.7
g
Methanol
10
ml
NH4Cl
2
g
L-Histidine
10
mg
DL-Methionine
20
mg
DL-Tryptophan
20
mg
Distilled water
1
L
Agar
15
g
114
MA2
Malt extract
Distilled water
Agar
111
Bacto-Yeast Nitrogen Base w/o Amino acids (Difco) 6.7
Glucose
20
L-Tryptophan
20
L-Histidine
20
Distilled water
1
Agar
15
117
118
YPAD Medium
Yeast extract
Peptone
Glucose
Adenine sulfate
Distilled water
Agar
25% Glucose-Yeast Extract-Peptone
Malt extract
Distilled water
Agar
YM Agar
Glucose
Peptone
Yeast extract
Malt extract
Distilled water
Agar
116
Glucose
Yeast extract
Peptone
Distilled water
10
g
5
g
3
g
3
g
100
g
1
L
15
g
pH 5.6 - 6.0
Medium for the 2μ Mapping Strains
Bacto-Yeast Nitrogen Base w/o Amino acids (Difco) 6.7
Glucose
20
L-Leucine
60
L-Histidine
20
L-Tryptophan
20
L-Methionine
20
Distilled water
1
Agar
15
200
30
25
5
10
5
15
15
1
15
g
g
g
g
g
g
g
g
L
g
pH 7.0
* Gently boil sliced potatoes in 500 ml of water (or sliced
liver in 150 ml of water) for 30 min and remove solids by
filtration through cloth.
g
g
mg
mg
mg
mg
L
g
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
941
Media
207
220
Mannitol
K2HPO4
MgSO4·7H2O
NaCl
CaSO4·2H2O
CaCO3
Distilled water
Agar (if needed)
15
g
0.2
g
0.2
g
0.2
g
0.1
g
5
g
1
L
15
g
pH 7.2
Yeast extract
Bacto Tryptone (Difco)
Sodium acetate
Soil extract*
Distilled water
Agar (if needed)
50
100
900
221
g
ml
ml
Uric acid
Yeast extract
K2HPO4
MgSO4·7H2O
FeSO4·7H2O
CaCl2·2H2O
Mercaptoacetic acid (80% solution)
Distilled water
Agar (if needed)
* Nippon Jozo Kyokai, 6-30, Takinogawa 2 chome, Kita-ku,
Tokyo 114.
** Sterilize by filtration and add to SI medium sterilized by
autoclaving.
210
Glucose
Yeast extract
Peptone
CaCO3
Distilled water
Agar (if needed)
30
5
5
30
1
15
g
g
g
g
L
g
pH 6.8
10
15
7.5
2.6
0.25
0.01
0.5
2.5
1
1
980
15
Sodium lactate
Yeast extract
NH4Cl
K2HPO4
MgSO4·7H2O
CaSO4·2H2O
Fe2(SO4)3·(NH4)2SO4·24H2O
Distilled water
g
ml
g
g
g
g
g
ml
ml
ml
ml
g
3.5
g
1
g
0.5
g
1
g
2
g
1
g
0.5
g
1
L
pH 7.2 - 7.4
223
Peptone
Beef extract
Yeast extract
Glucose
MgSO4·7H2O
Na2SO4
Fe2(SO4)3·(NH4)2SO4·24H2O
Distilled water
* Sterilize separately by filtration.
5
g
3
g
0.2
g
5
g
1.5
g
1.5
g
0.1
g
1
L
pH 7.0 - 7.2
224
214
Skim milk
Distilled water
216
2
g
1
g
1
g
35
mg
35
mg
4.2 mg
2
ml
1
L
15
g
pH 7.0
222
213
Yeast extract
Ethyl alcohol (99.5%)*
Sodium acetate
(NH4)2SO4
MgSO4·7H2O
CaCl2·2H2O
Sodium thioglycolate
FeSO4 (0.02 M)
Na2MoO4 (0.01 M)
MnSO4 (0.01 M)
Potassium phosphate buffer (0.02 M, pH 7.0)
Agar (if needed)
g
g
g
ml
ml
g
pH 7.4
* Autoclave 400 g of farm soil suspended in 1 L of distilled
water for 30 min, and remove solids by filtration.
209
SI Medium Dehydrated*
Ethyl alcohol**
Distilled water
2
1
1
50
950
15
100
1
g
L
40
1
g
L
20
1
1
15
g
g
L
g
(NH4)2SO4
KNO3
KH2PO4
MgCl2·6H2O
CaCl2·2H2O
FeSO4·7H2O
Na2S2O3·5H2O
Na2MoO4·2H2O
Yeast extract
Sulfur (powder)*
Bromophenol blue
Distilled water
Heart Infusion Agar
Bacto Heart Infusion Agar (Difco)
Distilled water
219
Bacto Casitone (Difco)
MgSO4·7H2O
Potassium phosphate buffer (0.01 M, pH 7.2)
Agar (if needed)
2
g
3
g
3
g
0.5
g
0.25 g
0.01 g
5
g
0.3 mg
0.1
g
1
g
10
mg
1
L
pH 4.0 - 4.6
* Sterilize separately by autoclaving for 60 min at 105℃ on
each of 3 successive days and add after inoculation.
942
Media
225
232
Table wine
Yeast extract
Calcium lactate
CaCO3
Distilled water
Agar (if needed)
500
ml
20
g
10
g
20
g
500
ml
15
g
pH 6.6 - 6.8
226
5
g
20
g
1
L
pH 6.8 - 7.0
Methanol*
(NH4)2HPO4
NaCl
MgSO4·7H2O
FeSO4·7H2O
MnSO4·nH2O
Vitamin mixture**
Tap water
Agar (if needed)***
Yeast Extract-Malt Extract Agar (ISP
Medium No.2)
Yeast extract
Malt extract
Glucose
Distilled water
Agar
228
4
10
4
1
20
g
g
g
L
g
pH 7.3
1
1
2
10
1
20
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
g
g
g
g
L
g
pH 7.3
229
Yeast extract
Glycerol
CaCO3
Distilled water
Agar (if needed)
5
50
1
1
20
g
g
g
L
g
pH 7.3
30
1
20
g
L
g
pH 7.3
1
1
2
10
1
20
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
234
(NH4)2SO4
KCl
K2HPO4
MgSO4·7H2O
Polypepton*
Glucose**
Distilled water
Agar (if needed)
2
g
0.1
g
0.5
g
0.5
g
0.1
g
1
g
1
L
15
g
Adjust pH to 3.0 with 6 N H2SO4
Glucose and Polypepton are autoclaved separately as
10% solution, and add to other autoclaved ingredients
after cooling.
Maltose-Bennett's Agar
Yeast extract
Beef extract
NZ Amine, Type A*
Maltose
Distilled water
Agar
0.2
40
0.2
200
40
20
40
40
20
100
2
1
*** Sterilize separately.
Trypticase Soy Agar
Trypticase Soy (BBL)
Distilled water
Agar
10
ml
3
g
1
g
0.2
g
10
mg
5
mg
5
ml
1
L
20
g
pH 7.0
** Vitamin mixture
* Sheffield Chemical Co., San Ramon, CA 94583, USA or
Wako Pure Chemical Ind., Ltd., Osaka, Japan.
231
g
g
g
g
L
g
pH 7.3
* Sterilize by filtration and add to other autoclaved
ingredients after cooling.
Bennett's Agar
Yeast extract
Beef extract
NZ Amine, Type A*
Glucose
Distilled water
Agar
230
4
10
4
200
1
20
233
Yeast extract
Glucose
Distilled water
227
Halophilic Actinomycetes
Bacto Yeast Extract (Difco)
Bacto Malt Extract (Difco)
Glucose
NaCl
Distilled water
Agar (if needed)
g
g
g
g
L
g
pH 7.3
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Sterilize separately.
235
(NH4)2SO4
K2HPO4
MgSO4·7H2O
KCl
Ca(NO3)2
FeSO4·7H2O
Distilled water
* Sheffield Chemical Co., San Ramon, CA 94583, USA or
Wako Pure Chemical Ind., Ltd., Osaka, Japan.
943
2
0.5
0.5
0.1
0.01
40
1
g
g
g
g
g
g
L
Adjust pH to 3.0 with 6 N H2SO4 and sterilize by filtration.
Media
239
248
NaNO2
NaCl
K2HPO4
MgSO4·7H2O
MnSO4·nH2O
Fe2(SO4)3
Distilled water
1
g
0.3
g
0.5
g
0.5
g
0.002 g
0.005 g
1
L
pH 7.5
Yeast extract
Malt extract
Glucose
Na2CO3*
Distilled water
Agar (if needed)
* Na2CO3 is sterilized separately.
240
(NH4)2SO4
NaCl
K2HPO4
MgSO4·7H2O
FeSO4·7H2O
CaCO3*
Distilled water
249
0.5
g
0.3
g
1
g
0.3
g
0.03 g
7.5
g
1
L
pH 8.0
Na2HPO4·12H2O
KH2PO4
Na2CO3
MgCl2·6H2O
NH4Cl
Yeast extract
Bacto Tryptone (Difco)
Na2WO4·2H2O
Wolfe's modified mineral solution*
VFA**
Distilled water
* CaCO3 is practically insoluble.
243
Oatmeal
KNO3
K2HPO4
MgSO4·7H2O
Distilled water
Agar (if needed)
3
g
0.2
g
0.5
g
0.2
g
1
L
20
g
pH 7.0
245
4
10
4
750
250
20
0.1% Resazurin solution
myo-Inositol
Vitamin mixture***
Cellobiose
Cellulose****
Cysteine
Na2S·9H2O
Agar (if needed)
20
1
1
18
g
ml
L
g
pH 7.2
0.5
0.5
15
g
g
g
* Wolfe's modified mineral solution
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
NaCl
MnSO4·H2O
FeSO4·7H2O
Co(NO3)2·6H2O
CaCl2
ZnSO4·7H2O
CuSO4·5H2O
Boric acid
Na2MoO4·2H2O
Na2SeO3
0.1
0.1
0.1
100
g
g
g
ml
21
200
100
700
g
ml
ml
ml
246
Bacto PPLO Broth (Difco)
Active horse serum
Fresh baker's yeast extract (25% solution)*
Distilled water
ml
mg
ml
g
ml
Under anaerobic condition, the medium is autoclaved at
100℃ for 10 min.
* Trace salts solution
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
1
1
10
0.2
200
Heat the above mixed solution at 60℃ for 5 to 10 min
and then bubble with N2 gas. And then add the following
ingredients.
g
g
g
ml
ml
g
pH 7.6
Oatmeal Agar (ISP Medium No.3)
Oatmeal
Trace salts solution*
Distilled water
Agar
4.2
g
1.5
g
4
g
0.4
g
0.6
g
1
g
1
g
0.4 mg
50
ml
1
ml
738
ml
pH 6.5
Adjust pH to 6.5 with 10 N KOH and add the following
medium.
244
Yeast extract
Malt extract
Glucose
Seawater
Distilled water
Agar (if needed)
4
g
10
g
4
g
10
g
1
L
20
g
pH 9.0 - 10.0
1.5
3
1
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.001
g
g
g
g
g
g
g
g
g
g
g
g
Nitrilotriacetic acid is suspended in 500 ml water, and
dissolved by titrating with 2 N KOH until its pH stabilize at
6.5. The rest of the ingredients are added and dissolved.
Finally total volume is adjusted to 1 L.
Horse serum and yeast extract are sterilized by filtration
and added to PPLO broth autoclaved and cooled.
** VFA
* Boil 250 g of dried baker's yeast suspended in 1 L of
distilled water for 30 min, remove yeast cells by
centrifugation, and sterilize by filtration.
944
Media
Acetic acid
Propionic acid
Butyric acid
iso-Butyric acid
n-Valeric acid
iso-Valeric acid
DL-α-Methylbutyric acid
17
6
4
1
1
1
1
ml
ml
ml
ml
ml
ml
ml
0.2
40
0.2
200
40
20
40
40
20
100
2
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
255
Bacto Casamino Acids (Difco)
Yeast extract
Trisodium citrate
KCl
MgSO4·7H2O
FeSO4·7H2O
MnSO4·nH2O
NaCl
Distilled water, make up to
Agar (if needed)*
*** Vitamin mixture
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
* Dissolve agar by heating before adding NaCl.
256
Yeast extract
Bacto Casamino Acids (Difco)
KH2PO4
KCl
NH4Cl
MgSO4·7H2O
CaSO4·2H2O
Sodium glutamate
Na2CO3
NaCl
Distilled water, make up to
Agar (if needed)*
**** 3% Cellulose suspension which is ball-milled for 3 days.
250
Yeast extract
Na2S2O3·5H2O
KH2PO4
Na2HPO4
MgSO4·7H2O
NH4Cl
Distilled water
Agar (if needed)
5
g
5
g
1.5
g
4.5
g
0.1
g
0.3
g
1
L
15
g
pH 6.0
g
g
g
g
g
g
g
g
g
g
L
g
* Dissolve agar by heating before adding NaCl.
257
Yeast extract
Na2S2O3·5H2O
KH2PO4
Na2HPO4
MgSO4·7H2O
NH4Cl
Distilled water
Agar (if needed)
NaCl
MgCl2·6H2O
MgSO4·7H2O
CaCl2·2H2O
KCl
NaHCO3
KBr
Yeast extract
Glucose
Distilled water
Agar (if needed)
5
g
5
g
1.5
g
4.5
g
0.1
g
0.3
g
1
L
15
g
pH 7.5
Brain Heart Infusion Agar
Bacto Brain Heart Infusion (Difco)
Distilled water
Agar (if needed)
254
5
5
1
1
1
0.24
0.17
1
5
250
1
20
Adjust pH to 6.5 before autoclaving. Sterilize Na2CO3 in
solution separately, and add to other autoclaved
ingredients. Check the final pH is between 9.0 - 9.5.
251
253
7.5
g
10
g
3
g
2
g
20
g
0.05 g
0.2 mg
250
g
1
L
20
g
pH 7.4
37
1
15
g
L
g
40
1
g
L
258
NaCl
MgCl2·6H2O
CaCl2·2H2O
K2SO4
Polypepton*
Yeast extract
Soluble starch
Distilled water
Agar (if needed)
Blood Agar
Bacto Blood Agar Base (Difco)
Distilled water
156
g
13
g
20
g
1
g
4
g
0.2
g
0.5
g
5
g
1
g
1
L
20
g
pH 7.0
Sterilize by autoclaving and cool to 45-50℃ and add to it
50 ml of sterile defibrillated blood.
125
g
160
g
0.13 g
5
g
1
g
1
g
2
g
1
L
15
g
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
945
Media
260
Yeast extract
Na2S2O3·5H2O
KH2PO4
Na2HPO4
MgSO4·7H2O
NH4Cl
Sodium aspartate
Distilled water
Agar (if needed)
Neutralize the nitrilotriacetic acid with KOH. Dissolve the
remaining ingredients and adjust the pH to 7.2 with KOH.
1
g
5
g
1.5
g
4.5
g
0.1
g
0.3
g
1
g
1
L
15
g
pH 6.0
** PS solution
K2HPO4
KH2PO4
Distilled water
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
5
g
5
g
5.74 g
1.15 g
0.06 g
60
mg
1
L
15
g
pH 7.0
5
1.5
5
1.5
1.5
0.1
5.03
1
15
2Na-EDTA
ZnSO4·7H2O
FeSO4·7H2O
MnSO4·nH2O
CuSO4·5H2O
CoCl2·6H2O
Na2B4O7·10H2O
Distilled water
g
g
g
g
g
g
g
L
g
Peptone
Yeast extract
Malt extract
Bacto Casamino Acids (Difco)
Beef extract
Glycerol
Tween 80
MgSO4·7H2O
Distilled water
Agar (if needed)
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Na2CO3 is sterilized separately by autoclaving.
263
100
g
5
g
9.5
g
5
g
0.2
g
0.1
g
0.1
g
5
g
1
g
20
ml
20
ml
10
ml
960
ml
15
g
pH 8.0
265
g
g
g
g
g
g
g
L
10
g
5
g
5
g
5
g
2
g
2
g
0.05 g
1
g
1
L
15
g
pH 7.0
Glycerol-Asparagine Agar (ISP Medium
No.5)
L-Asparagine
Glycerol
K2HPO4
Distilled water
Trace salts solution*
Agar
1
g
10
g
1
g
1
L
1
ml
20
g
pH 7.0 - 7.4
* Trace salts solution
* HMSS solution
10
29.7
3.3
12.7
99
50
1
2.5
10.95
5
1.54
0.392
0.203
0.177
1
264
The final pH of the freshly prepared medium is between
10.5 and 11.0.
Nitrilotriacetic acid
MgSO4·7H2O
CaCl2·2H2O
Na2MoO4·2H2O
FeSO4·7H2O
Metal 44 solution****
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
**** Metal 44 solution
262
NaCl
MgCl2·6H2O
MgSO4·7H2O
KCl
CaCl2·2H2O
(NH4)2SO4
KNO3
Bacto Casamino Acids (Difco)
Yeast extract
HMSS solution*
PS solution**
Vitamin mixture***
Distilled water
Agar (if needed)
0.2
40
0.2
200
40
20
40
40
20
100
2
1
*** Vitamin mixture
* Sterilize separately by filtration.
Polypepton*
Yeast extract
Glucose
Na2HPO4·12H2O
NaCl
MgCl2·6H2O
Na2CO3**
Distilled water
Agar (if needed)
g
g
L
Sterilize separately, and add after autoclaving.
261
Yeast extract
Malt extract
K2HPO4
NH4H2PO4
MgSO4·7H2O
Catalase*
Distilled water
Agar (if needed)
2.5
2.5
1
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
g
g
g
mg
mg
ml
L
946
0.1
0.1
0.1
100
g
g
g
ml
Media
266
Yeast Extract-Starch Agar
Yeast extract
Soluble starch
Distilled water
Agar
267
272
2
10
1
15
g
g
L
g
pH 7.3
25
700
15
g
ml
g
pH 7.8
Bacto Casitone (Difco)
Yeast extract
CaCl2·2H2O
Distilled water
Agar (if needed)
Modified Hayflick Medium
Bacto Heart Infusion Broth (Difco)
Distilled water
Bacto Agar (Difco, if needed)
273
Peptone
Meat extract
NaCl
Distilled water
Agar (if needed)
Sterilized by autoclaving (121℃ for 20 min).
Aseptically add 200 ml of horse serum (heat inactivated,
56℃ for 30 minutes), 100 ml of fresh yeast extract (25%
solution)*, 12 ml of calf thymus DNA (Sigma, Type I)
(0.2% (w/v) solution), 10 ml of thallium acetate (1% (w/v)
solution), 25 ml of Penicillin G (20,000 IU/ml).
Bacto Tryptone (Difco)
Yeast extract
Beef extract
Sodium acetate
Distilled water
Agar (if needed)
Inorganic Salts Starch Agar (ISP Medium
No.4)
Soluble starch
K2HPO4 (anhydrous base)
MgSO4·7H2O
NaCl
(NH4)2SO4
CaCO3
Trace salts solution*
Bacto Agar (Difco)
Distilled water
10
1
1
1
2
2
1
20
1
275
g
g
g
g
g
g
ml
g
L
270
Bacto Casitone (Difco)
CaCl2·2H2O
Yeast extract
Cellobiose
Distilled water
Agar (if needed)
g
g
g
ml
Bacto Casitone (Difco)
Yeast extract
MgSO4·7H2O
Distilled water
Agar (if needed)
1
g
1
g
2
g
10
g
0.02 g
1
L
20
g
pH 7.3
* Sheffield Chemical Co., San Ramon, CA 94583, USA or
Wako Pure Chemical Ind., Ltd., Osaka, Japan.
271
Yeast-Krainsky's Agar
Glucose
L-Asparagine
K2HPO4
Yeast extract
Distilled water
Agar
10
5
5
1
15
g
g
g
L
g
pH 7.0
3
g
1.36 g
1
g
5
g
1
L
15
g
pH 7.2
277
Hickey & Tresner Agar
Yeast extract
Beef extract
NZ Amine, Type A*
Dextrin
CoCl2·6H2O
Distilled water
Agar
0.5
g
0.5
g
0.2
g
0.2
g
1
L
15
g
pH 7.2
276
* Trace salts solution
0.1
0.1
0.1
100
g
g
g
L
g
pH 7.0
LB Medium
Peptone
Yeast extract
NaCl
Distilled water
Agar (if needed)
The unadjusted pH should be 7.0 - 7.4.
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
5
3
60
1
15
274
* Boil 250 g of dried baker's yeast suspended in 1 L of
distilled water for 30 min, remove yeast cells by
centrifugation, and sterilize by filtration.
268
3
g
1
g
1.36 g
1
L
15
g
pH 7.2
10
g
1
g
0.5
g
2
g
1
L
17
g
pH 7.3
947
5
5
1
1
15
g
g
g
L
g
pH 7.2
Media
278
For solid medium, equal volumes of separately
autoclaved double-strength liquid medium (pH 2.0) and
1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are
mixed, after cooling to 45℃.
Tris (hydroxymethyl) aminomethane
1.2
g
Bacto Casamino Acids (Difco)
1
g
Glucose
1
g
MgSO4·7H2O
0.1
g
KNO3
0.1
g
CaCl2·2H2O
0.1
g
Sodium glycerophosphate
0.1
g
MnCl2·4H2O
1.8 mg
FeSO4·7H2O
1.4 mg
H3BO3
2.8 mg
ZnCl2·2H2O
0.1 mg
Na2MoO4·2H2O
0.1 mg
Vitamin mixture*
5
ml
Distilled water
1
L
Adjust pH to 7.5 with 1 N HCl
282
Glucose
K2HPO4
NH4NO3
MgSO4·7H2O
MnSO4·7H2O
FeSO4·7H2O
CaCl2·2H2O
Yeast extract
Polypepton*
Resazurin
Distilled water
Agar (if needed)
* Vitamin mixture
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
279
0.2
40
0.2
200
40
20
40
40
20
100
2
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
3
5
5
1
20
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
283
Sucrose
Bacto Casitone (Difco)
Yeast extract
Polypepton*
Vitamin mixture**
Distilled water
Agar (if needed)
g
g
g
L
g
pH 7.0
** Vitamin mixture
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
3
g
0.5
g
0.25 g
1
g
10
g
2
g
1
L
Adjust pH to 2.0 with 10 N H2SO4
Glucose is autoclaved separately as 40% solution, and
added to other autoclaved ingredients after cooling. For
solid medium, equal volumes of separately autoclaved
double-strength liquid medium (pH 2.0) and 1.6% Gelrite
(Kelco, Division of Merck & Co, Inc.) are mixed, after
cooling to 45℃.
0.2
40
0.2
200
40
20
40
40
20
100
2
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
284
Bacto Vitamin Assay Casamino Acids (Difco)
Bacto Yeast Extract (Difco)
MgSO4·7H2O
Trisodium citrate·2H2O
KCl
NaCl
FeSO4·7H2O (4.98% solution)
Distilled water
Agar (if needed)
281
Yeast extract
(NH4)2SO4
CaCl2·2H2O
MgSO4·7H2O
KH2PO4
Na2SiO3·9H2O
Na2MoO4·2H2O
FeSO4·7H2O
Distilled water
1
g
0.75 g
0.25 g
0.25 g
10
ml
1
L
15
g
pH 7.1
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
280
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Glucose
(NH4)2SO4
Distilled water
g
g
g
g
g
g
g
g
g
g
L
g
After autoclaving, adjust pH to 10.0 with sterilized 10%
Na2CO3 solution.
Tryptone Yeast Extract Agar (TYG)
Bacto Tryptone (Difco)
Yeast extract
Glucose
Distilled water
Agar
10
1
2
0.2
0.005
0.005
0.1
3
0.3
0.001
1
15
1
g
1.3
g
0.08 g
0.25 g
0.28 g
0.3
g
0.03 g
0.02 g
1
L
Adjust pH to 3.0 with 10N H2SO4
948
7.5
g
10
g
20
g
3
g
2
g
150
g
1
ml
1
L
15
g
pH 7.4
Media
286
* Vitamin mixture
(NH4)2SO4
CaCl2·2H2O
MgSO4·7H2O
KH2PO4
KCl
Ca(NO3)2·4H2O
Yeast extract*
Sulfur (powder)**
Distilled water
3
g
0.08 g
0.25 g
0.28 g
0.1
g
0.01 g
0.2
g
10
g
1
L
Adjust pH to 1.5 to 2.5 with 6 N H2SO4
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamin-HCl
Choline chloride
Cyanocobalamin
Distilled water
* Yeast extract is autoclaved separately as 10% solution.
** Sulfur is sterilized separately by autoclaving for 60 min at
105℃ on each of 3 successive days and added after
inoculation.
FeCl3·6H2O
Quinic acid
Distilled water
1.3
g
0.08 g
0.25 g
0.28 g
0.1
g
0.03 g
0.02 g
1
g
10
g
1
L
Adjust pH to 2.5 with 6 N H2SO4
Nitrilotriacetic acid
MgSO4·7H2O
CaCl2·2H2O
Na2MoO4·2H2O
FeSO4·7H2O
Metal 44 solution****
Distilled water
g
g
L
10
29.7
3.3
12.7
99
50
1
g
g
g
mg
mg
ml
L
Neutralize the nitrilotriacetic acid with KOH. Dissolve the
remaining ingredients and adjust the pH to 7.2 with KOH.
**** Metal 44 solution
** Sulfur is sterilized separately by autoclaving for 60 min at
105℃ on each of 3 successive days and added after
inoculation.
EDTA
ZnSO4·7H2O
FeSO4·7H2O
MnSO4·nH2O
CuSO4·5H2O
CoCl2·6H2O
Na2B4O7·10H2O
Distilled water
288
0.1
g
0.5
g
1
g
0.1
g
0.1
g
0.5
g
0.006 g
1
L
pH 6.8
2.5
10.95
5
1.54
0.392
0.203
0.177
1
g
g
g
g
g
g
g
L
Good growth can be obtained in semi-solid medium (1 g
of agar in 1 liter).
294
Bacto Yeast Extract (Difco)
Distilled water
Good growth can be obtained in semi-solid medium (1 g
of agar in 1 liter).
289
KH2PO4
NaNO3
Sodium thioglycolate
Sodium tartrate
Sodium succinate
Sodium acetate
Vitamin mixture*
Ferric quinate solution**
HMSS solution***
Distilled water
0.45
0.19
1
*** HMSS solution
* Yeast extract is autoclaved separately as 10% solution.
MgSO4·7H2O
K2HPO4
Sodium acetate
NH4Cl
Bacto Yeast Extract (Difco)
Sodium thioglycolate
Ferric citrate
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
** Ferric quinate solution
287
(NH4)2SO4
CaCl2·2H2O
MgSO4·7H2O
KH2PO4
Na2SiO3·9H2O
Na2MoO4·2H2O
FeSO4·7H2O
Yeast extract*
Sulfur (powder)**
Distilled water
0.2
40
0.2
200
40
20
40
40
20
100
2
1
296
0.28 g
0.12 g
0.05 g
0.37 g
0.37 g
0.05 g
5
ml
2
ml
1
ml
1
L
Adjust pH to 6.75 with 1 N NaOH
5
g
1
L
Adjust pH to 5.0 with 1 N HCl
V-8 Juice Tap Water Agar
V-8 Juice*
CaCO3*
Tap water
Agar
200
3
800
15
ml
g
ml
g
* Add CaCO3 (3 g) to V-8 juice (ca 300 ml) and stir it for 2 h.
After centrifuging, adjust the supernatant to 200 ml with
tap water.
949
Media
297
305
Beef extract
Yeast extract
Peptone
NaCl
Distilled water
Agar (if needed)
298
1
2
5
5
1
15
g
g
g
g
L
g
pH 7.0
20
1
1
1
18
g
ml
g
L
g
pH 7.2
Sulfur (powder)*
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7
ZnSO4·7H2O
Na2MoO3·2H2O
Distilled water
OA-Y Agar
Oatmeal
Trace salts solution*
Bacto Yeast Extract (Difco)
Distilled water
Agar
* Sulfur is sterilized separately by autoclaving for 60 min at
105℃ on each of 3 successive days and added after
inoculation.
* Trace salts solution
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
299
0.1
0.1
0.1
100
For solid medium, equal volumes of separately
autoclaved double-strength liquid medium (pH 2.0) and
1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are
mixed, after cooling to 45℃.
g
g
g
ml
306
NaCl
Bacto Casamino Acids (Difco)
Polypepton*
Yeast extract
Sodium citrate
MgSO4·7H2O
K2HPO4
Fe(NH4)2(SO4)2·6H2O
Distilled water
Agar (if needed)
Gauze's Synthetic Medium No.1
Soluble starch
KNO3
NaCl
MgSO4·7H2O
K2HPO4
FeSO4·7H2O
Tap water
Agar (if needed)
20
g
1
g
0.5
g
0.5
g
0.5
g
10
mg
1
L
15
g
pH 7.2 - 7.4
80
g
7.5
g
5
g
1
g
3
g
20
g
0.5
g
0.05 g
1
L
15
g
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
302
Bacto Yeast Extract (Difco)*
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7
ZnSO4·7H2O
Na2MoO3·2H2O
Distilled water
1
g
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
0.018 g
4.5 mg
0.22 mg
0.05 mg
1
L
Adjust pH to 3.5 with 10 N H2SO4
308
309
Oatmeal Agar (B)
3
g
0.2
g
0.5
g
0.2
g
1
L
15
g
pH 7.0
950
1
2
5
5
10
1
15
g
g
g
g
g
L
g
pH 7.0
Rhamnose Salt Medium
K2HPO4
KH2PO4
NH4Cl
MgSO4·7H2O
NaCl
CaCl2·2H2O
FeSO4·7H2O
Yeast extract
Rhamnose
Distilled water
For solid medium, equal volumes of separately
autoclaved double-strength liquid medium (pH 2.0) and
1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are
mixed, after cooling to 45℃.
Oatmeal (powder, Quaker White Oats)
KNO3
K2HPO4
MgSO4·7H2O
Distilled water
Agar
Glucose Nutrient Agar
Beef extract
Yeast extract
Peptone
NaCl
Glucose
Distilled water
Agar
* Sterilize by filtration as 5% solution, and add to other
ingredients sterilized by autoclaving.
304
0.5
g
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
0.018 g
4.5 mg
0.22 mg
0.05 mg
1
L
Adjust pH to 3.0 with 10 N H2SO4
2.9
g
2.1
g
2
g
0.4
g
30
mg
3
mg
1
mg
3
g
10
g
1
L
pH 7.0
Media
310
MRS Medium
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
311
Polypepton*
Yeast extract
MgSO4·7H2O
Na2CO3**
Distilled water
Agar (if needed)
30
1
317
Peptone
Yeast extract
L-Cysteine
Distilled water
g
L
pH 7.3
319
59
1
g
L
pH 7.3
313
18
7
1
50
25
25
5
0.335
0.25
0.14
0.14
0.002
0.001
900
g
g
g
ml
ml
ml
g
g
g
g
g
g
g
ml
10
10
1
1
g
g
g
L
pH 6.8
NZ Amine with Soluble Starch and Glucose
Agar Medium
Glucose
Soluble starch
Yeast extract
NZ Amine, Type A*
CaCO3 (reagent grade)
Distilled water
Agar
* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.
NaCl
MgCl2·6H2O
Bacto Yeast Extract (Difco)
Fructose (20% solution)*
Cysteine-HCl (2% solution)*
Na2S (2% solution)*
NaHCO3
KCl
NH4Cl
CaCl2·2H2O
K2HPO4
Fe(NH4)2(SO4)2·6H2O
Resazurin
Distilled water
g
g
g
g
L
g
** Sterilize separately by autoclaving and add to other
ingredients. The final pH is between 9.0 and 10.0.
GAM Medium
Nissui GAM Broth*
Distilled water
10
2
0.5
10
1
15
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Todd Hewitt Medium
Bacto Todd Hewitt Broth (Difco)
Distilled water
312
316
10
g
10
g
5
g
20
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 6.0 - 6.5
10
20
5
5
1
1
15
g
g
g
g
g
L
g
pH 7.3
* Sheffield Chemical Co., San Ramon, CA 94583, USA or
Wako Pure Chemical Ind., Ltd., Osaka, Japan.
323
Solution A*
Solution B**
500
500
ml
ml
* Solution A
Yeast extract
(NH4)2SO4
MgSO4·7H2O
CaCl2·2H2O
KH2PO4
Distilled water
The pH is not adjusted and usually a value of 7.4.
* Sterilize separately and add to other autoclaved
ingredients.
1
g
0.2
g
0.5
g
0.25 g
0.6
g
500
ml
pH 3.0 - 4.0
** Solution B
314
Bacto Brain Heart Infusion (Difco)
Horse serum*
Distilled water
37
50
950
g
ml
ml
30
5
1
g
ml
L
pH 7.3
Glucose
Agar (if needed)
Distilled water
g
g
ml
Solutions A and B are separately sterilized by autoclaving
and mixed.
* Sterilize separately by filtration.
315
Bacto Todd Hewitt Broth (Difco)
Horse serum*
Distilled water
1
20
500
324
(NH4)2SO4
KCl
K2HPO4
MgSO4·7H2O
Glucose*
Yeast extract*
Distilled water
* Sterilize separately by filtration.
2
g
0.1
g
0.5
g
0.5
g
1
g
0.1
g
1
L
Adjust pH to 3.5 with 1 N H2SO4
* Autoclave separately as 20% solution, and added to other
autoclaved ingredients.
951
Media
332
For solid medium, equal volumes of separately
autoclaved double-strength liquid medium (pH 3.5) and
1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) were
mixed, after cooling to 50℃.
325
Polypepton*
Yeast extract
MgSO4·7H2O
Seawater**
Distilled water
Agar (if needed)
10
g
2
g
0.5
g
750
ml
250
ml
15
g
pH 7.2 - 7.4
333
** Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP*).
2
g
0.5
g
0.5
g
0.2
g
15
g
1
L
pH 7.2 - 7.4
M a r i n e C y t op h a g a A g a r
Bacto Tryptone (Difco)
Beef extract
Yeast extract
Sodium acetate
Agar (if needed)
Seawater*
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
2
g
0.5
g
0.5
g
0.2
g
15
g
1
L
pH 7.2 - 7.4
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**).
326
Peptone
Yeast extract
MgSO4·7H2O
NaCl
Distilled water
Agar (if needed)
10
2
1
30
1
15
g
g
g
g
L
g
pH 7.0
334
30
10
100
1
5
900
g
g
ml
g
g
ml
pH 7.2
335
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Glycerol
Keratin
Soil extract*
CaCO3
Yeast extract
Distilled water
329
2
10
50
15
1
g
g
g
g
L
pH 7.3
9
1
15
1
g
g
g
L
pH 7.2
0.5
g
0.1
g
1
ml
1
g
5
g
5
g
15
g
1
L
pH 7.0
* Trace element solution
H3BO4
MnCl2·4H2O
FeSO4·7H2O
CuCl2·2H2O
ZnCl2
CoCl2·6H2O
Na2MoO4·2H2O
Sodium tartrate
Distilled water
SP5 Medium
Bacto Casitone (Difco)
Yeast extract
Agar (if needed)
Seawater*
2
g
0.5
g
0.5
g
0.2
g
10
g
1
L
pH 7.2 - 7.4
Medium for Marine Flexibacteria
KNO3
Sodium glycerophosphate
Trace element solution*
Tris (hydroxymethyl) aminomethane
Bacto Tryptone (Difco)
Yeast extract
Agar (if needed)
Seawater**
* 500 g of soil in 500 ml of water was autoclaved at 121℃
for 30 min and filtered.
Yeast extract
Soluble starch
NaCl
Agar (if needed)
Distilled water
F l a v o b a c t e r iu m c o l u m n ar e A g a r
Bacto Tryptone (Difco)
Beef extract
Yeast extract
Sodium acetate
Agar (if needed)
Distilled water
328
331
Enriched Cytophaga Agar
Bacto Tryptone (Difco)
Beef extract
Yeast extract
Sodium acetate
Agar (if needed)
Distilled water
2.85
1.8
1.36
26.9
20.8
40.4
25.2
1.77
1
g
g
g
mg
mg
mg
mg
g
L
** Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP***).
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**).
*** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
336
Cytophaga (Marine) Medium
Bacto Tryptone (Difco)
Yeast extract
Agar (if needed)
Seawater*
1
1
15
1
g
g
g
L
pH 7.2
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**)
952
Media
350
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
338
Caulobacter Medium
Peptone
Yeast extract
MgSO4·7H2O
Agar (if needed)
Distilled water
339
340
37.4
g
1
L
pH unadjusted
** Sterilize separately by filtration.
351
Polypepton*
Yeast extract
N-Acetyl glucosamine**
MgSO4·7H2O
Agar (if needed)
Distilled water
55.1
g
1
L
pH unadjusted
Medium for Freshwater Flexibacteria
MgSO4·7H2O
KNO3
CaCl2·2H2O
Sodium glycerophosphate
Trace element solution*
Tris (hydroxymethyl) aminomethane
Thiamine
Bacto Casamino Acids (Difco)
Glucose
Cobalamine
Agar (if needed)
Distilled water
0.1
g
0.1
g
0.1
g
0.1
g
1
ml
1
g
1
mg
1
g
1
g
1
μg
15
g
1
L
pH 7.5
H3BO4
MnCl2·4H2O
FeSO4·7H2O
CuCl2·2H2O
ZnCl2
CoCl2·6H2O
Na2MoO4·2H2O
Sodium tartrate
Distilled water
352
10
2
5
1
15
1
g
g
ml
g
g
L
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
2.85
1.8
1.36
26.9
20.8
40.4
25.2
1.77
1
** Sterilize separately by filtration.
g
g
g
mg
mg
mg
mg
g
L
355
R-CW Medium
Peptone
Bacto Tryptone (Difco)
Yeast extract
KH2PO4
Sodium acetate trihydrate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Tween 80
Cheese whey*
5
g
5
g
5
g
5
g
5
g
2
g
0.5
g
0.5
g
1
ml
1
L
pH 6.0
Sucrose-Bennett's Agar
1
1
2
10
1
20
* Add HCl to warmed milk and coagulate. After
centrifuging, use the supernatant.
g
g
g
g
L
g
pH 7.3
356
Polypepton*
Yeast extract
MgSO4·7H2O
N-Acetyl-D-glucosamine
Distilled water
Agar (if needed)
* Sheffield Chemical Co., San Ramon, CA 94583, USA or
Wako Pure Chemical Ind., Ltd., Osaka, Japan.
347
Methylobacterium Medium
Polypepton*
Yeast extract
Methanol**
MgSO4·7H2O
Agar (if needed)
Distilled water
Nissui Plate Sheep Blood Agar G*
Yeast extract
Beef extract
NZ Amine, Type A*
Sucrose
Distilled water
Agar
g
g
mg
g
g
L
pH 7.0
** Sterilize separately by filtration.
* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.
343
10
2
30
1
15
1
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* Trace element solution
342
5
g
5
g
5
g
5
g
1
g
5
ml
15
g
1
L
pH 6.6 - 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Marine Agar 2216
Bacto Marine Agar 2216 (Difco)
Distilled water
341
2
g
1
g
0.2
g
10
g
1
L
pH 7.0
Marine Broth 2216
Bacto Marine Broth 2216 (Difco)
Distilled water
Acetobacter xylinus Medium
Polypepton*
Yeast extract
Glucose
Mannitol
MgSO4·7H2O
Ethanol**
Agar (if needed)
Distilled water
10
2
1
50
1
15
Nissui Plate Sheep Blood Agar*
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.
953
g
g
g
mg
L
g
pH 7.0
Media
357
** Trace element solution
Polypepton*
Yeast extract
MgSO4·7H2O
D-Alanine
Distilled water
Agar (if needed)
10
2
1
50
1
15
g
g
g
mg
L
g
pH 7.0
2Na-EDTA
FeCl3·6H2O
ZnCl2
MnCl2·2H2O
H3BO3
CoCl2·6H2O
Na2MoO4·2H2O
CuCl2·2H2O
NiCl2·6H2O
Na2SeO3
Distilled water
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
358
Polypepton*
Yeast extract
MgSO4·7H2O
Thymine
Distilled water
Agar (if needed)
10
2
1
100
1
15
g
g
g
mg
L
g
pH 7.0
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
NaCl
Distilled water
359
10
2
1
50
1
15
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
Distilled water
Agar (if needed)
50
50
30
10
5
5
100
5
5
5
1
1
15
g
g
g
g
L
g
pH 8.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
1
g
1
g
0.5
g
0.6
g
1
g
0.2
g
0.2
g
0.05 g
0.1
g
0.1
g
1
ml
1
ml
1
L
15
g
pH 6.7
365
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
Distilled water
5
5
5
1
1
g
g
g
g
L
pH 5.5
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
366
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
* Vitamin mixture
Thiamine-HCl
Niacine
p-Aminobenzoic acid
Pyridoxal-HCl
Biotin
Vitamin B12
Distilled water
g
g
g
g
g
L
pH 7.5
364
SA Medium
Sodium acetate
Sodium succinate
KH2PO4
K2HPO4
(NH4)2SO4
MgSO4·7H2O
NaCl
CaCl2·2H2O
Na2S2O3·5H2O
Yeast extract
Vitamin mixture*
Trace element solution**
Distilled water
Agar (if needed)
5
5
5
1
80
1
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
g
g
g
mg
L
g
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
360
g
g
g
g
g
g
mg
mg
mg
mg
L
362
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Polypepton*
Yeast extract
MgSO4·7H2O
Cytidine
Distilled water
Agar (if needed)
1
2
0.1
0.1
0.1
0.1
20
10
10
5
1
mg
mg
mg
mg
mg
mg
ml
954
10
g
10
g
5
g
20
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 5.5
Media
367
mjYPGS Medium
NaCl
K2HPO4
CaCl2·2H2O
NH4Cl
NaNO3
MgSO4·7H2O
MgCl2·6H2O
KCl
NiCl2·6H2O
Na2SeO3·5H2O
Ferric citrate
Trace mineral solution*
Na2S2O3·5H2O
Na2SiO3·9H2O
Bacto Yeast Extract (Difco)
Peptone (BBL)
Glucose
Sodium succinate
Distilled water
* Trace element solution SL8
3
g
14
mg
14
mg
12.5 mg
12.5 mg
0.34 g
41.8 mg
33
mg
0.05 mg
0.05 mg
5
mg
1
ml
2
g
1
g
1
g
1
g
0.2
g
0.2
g
1
L
Adjust pH 7.5 - 8.0 with H2SO4
2Na-EDTA
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
Na2MoO4·2H2O
CuCl2·2H2O
NiCl2·6H2O
Distilled water
372
1.5
g
0.5
g
0.5
g
0.18 g
0.01 mg
0.02 g
0.01 g
0.01 mg
1
L
* Solution I
10
g
7.5
g
250
g
40
g
2
g
3
g
36
mg
0.36 mg
1
L
20
g
pH 7.2
** Solution II
(NH4)2SO4
NaCl
MgCl2·6H2O
CaCl2·2H2O
Trace element solution SL8***
Sodium DL-malate
Yeast extract
Na2S2O3·5H2O
Distilled water
20 mM Potassium phosphate buffer
370
2Na-EDTA
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
Na2MoO4·2H2O
CuCl2·2H2O
NiCl2·6H2O
Distilled water
1
g
3
g
2
g
0.5
g
1
L
pH 6.8
1
g
8.2
g
0.2
g
0.05 g
1
ml
3.6
g
1
g
0.5
g
500
ml
500
ml
pH 7.8
5.2
1.5
70
100
62
190
36
17
24
1
g
g
mg
mg
mg
mg
mg
mg
mg
L
373
Nutrient Broth (OXOID)
Distilled water
Agar (if needed)
PYS Medium
KH2PO4
(NH4)2SO4
NaCl
MgCl2·6H2O
CaCl2·2H2O
Trace element solution SL8*
Sodium pyruvate**
Yeast extract
Distilled water
ml
ml
pH 7.8
*** Trace element solution SL8
MYCA Medium
Sodium DL-malate
Bacto Yeast Extract (Difco)
Bacto Casamino Acids (Difco)
(NH4)2SO4
Distilled water
500
500
Separately autoclave Solutions I and II. Mix aseptically
and adjust final pH to 7.7 - 7.8.
* Dissolve agar by heating before adding NaCl.
369
MMYS-II Medium
Solution I*
Solution II**
368
Yeast extract
Casamino acids
NaCl
MgSO4·7H2O
KCl
Trisodium citrate
FeCl3·4H2O
MnCl2·4H2O
Distilled water, make up to
Agar* (if needed)
g
g
mg
mg
mg
mg
mg
mg
mg
L
** Separately sterilize by filtration and aseptically add to the
medium.
* Trace mineral solution
Nitlirotriacetic acid
MnSO4·2H2O
CoSO4·7H2O
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
Distilled water
5.2
1.5
70
100
62
190
36
17
24
1
374
1
g
1
g
0.2
g
0.2
g
0.05 g
1
ml
2.2
g
1
g
1
L
pH 6.8
PPES-II Agar Medium
Peptone
Bacto Proteose Peptone No.3 (Difco)
Soytone
Yeast extract
Fe(III)-EDTA
Agar
Artificial sea water*
955
6.5
g
1
L
15
g
pH unadjusted
2
g
1
g
1
g
1
g
0.1
g
15
g
1
L
pH 7.8
Media
**** Metal salt solution 44
* Artificial sea water
NaCl
KCl
MgCl2·6H2O
MgSO4·7H2O
CaCl2·2H2O
Distilled water
376
30
0.7
10.8
5.4
1
1
Na-EDTA
ZnSO4·7H2O
FeSO4·7H2O
MnSO4·H2O
CuSO4·5H2O
Co(NO3)2·6H2O
Na2B4O7·10H2O
Distilled water
g
g
g
g
g
L
Ekho Lake Strains Medium
Mineral salt solution* ("Hutner/Cohen-Bazire")
Bacto Peptone (Difco)
Bacto Yeast Extract (Difco)
Bacto Agar (Difco)
Glucose solution (2.5%, filter-sterilized)
Vitamin solution**
Artificial sea water***
Distilled water
20
ml
0.25 g
0.25 g
15
g
10
ml
5
ml
250
ml
715
ml
pH 7.5
377
g
g
g
mg
mg
ml
ml
* Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
** Vitamin solution
4
4
20
10
10
10
10
0.2
10
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.025
g
g
g
g
g
g
g
g
g
g
g
g
g
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
*** Artificial sea water
NaCl
Na2SO4
MgCl2·6H2O
CaCl2
NaHCO3
KCl
KBr
H3BO3
SrCl2
NaF
Distilled water
0.75 g
0.78 g
0.36 g
0.1
g
0.54 g
5
g
10
ml
10
ml
2
g
1.7
g
20
g
1
L
pH unadjusted
Mix ingredients, except NaHCO3 and vitamin solution.
Dispense the medium into suitable culture vessels under
a stream of N2 and seal with butyl rubber stoppers. After
autoclaving, add filter-sterile NaHCO3 and vitamin
solutions.
Dissolve NTA first by neutralizing with KOH, then add
other salts. Adjust pH to 7.2 with KOH or H2SO4. Adjust
volume to 1 L with distilled water.
Biotin
Folic acid
Pyridoxine-HCl
Riboflavin
Thiamine-HCl
Nicotinamide
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Distilled water
Oceanithermus Medium
KH2PO4
K2HPO4
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
NaHCO3
Trace element solution*
Vitamin solution**
Yeast extract
NaNO3
NaCl
Distilled water
* Mineral salt solution
10
29.7
3.34
12.67
99
50
900
mg
mg
mg
mg
mg
mg
mg
ml
Dissolve the EDTA and add a few drops of concentrated
H2SO4 to retard precipitation of the heavy metal ions.
Store in the dark and cold (5℃).
Mix ingredients (except glucose and vitamin solutions),
and sterilize 20 min at 121℃. After cooling to 60℃,
aseptically add glucose and vitamin solutions to the
medium. The medium is only weakly buffered; one needs
approximately 10 drops/1 medium of 6 N KOH.
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
CaCl2·2H2O
Na2MoO4·2H2O
FeSO4·7H2O
Metal salt solution 44****
Distilled water
250
1095
500
154
39.2
24.8
17.7
100
23.477 g
3.917 g
4.981 g
1.102 g
192
mg
664
mg
6
mg
26
mg
24
mg
3
mg
1
L
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
956
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
380
NA + 0.5% Yeast Extract
Pepton
Beef extract
Yeast extract
NaCl
Agar (if needed)
Distilled water
383
5
3
5
3
15
1
g
g
g
g
g
L
pH 7.0
382
Yeast extract
Ethanol
Sodium succinate
Diammonium hydrogencitrate
Ferric citrate solution (0.1%)
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Vitamin B12 solution (0.01%)
Trace element solution SL-6*
Sodium malate
Na2S2O3
Distilled water
0.3
g
0.5 ml
1
g
0.5
g
5
ml
0.5
g
0.4
g
0.4
g
0.4
g
0.05 g
0.4 ml
1
ml
1
g
0.5
g
1.05 L
pH 6.8
** Salt solution
MgSO4·7H2O
MnSO4·4H2O
FeSO4·7H2O
Distilled water
384
385
1.5
100
40
2
2
1
g
g
g
L
Flexiligladius Medium
Bacto Heart Infusion Broth (Difco)
Distilled water
Agar (if needed)
2.5
g
1
L
15
g
pH unadjusted
Bifidobacterium Medium
Casein peptone, tryptic digest
Meat extract
Yeast extract
Glucose
K2HPO4
Tween 80
Tap water
10
5
5
10
3
1
1
g
g
g
g
g
ml
L
pH 6.8
After sterilization, aseptically add solutions of sodium
ascorbate and cysteine-HCl to final concentration of 1.0%
and 0.05%, respectively.
g
g
g
g
g
g
g
L
387
PE Medium
Basal salts solution*
Phosphate solution**
Vitamin solution***
(NH4)2SO4
Sodium glutamate
Sodium succinate
Sodium acetate
Yeast extract
Casamino acids
Na2S2O3·5H2O
Distilled water
** Neutralized sulfide solution
Na2S·9H2O
Distilled water
g
g
g
g
g
g
ml
g
L
pH 8.5
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* Trace element solution SL-6
0.1
0.03
0.3
0.2
0.01
0.02
0.03
1
10
5
5
5
25
10
5
15
1
Sterilize by filtration.
Boil the medium under a stream of nitrogen gas for few
minutes and distribute 45 ml medium into 50 ml
screw-capped bottles (already flushed with nitrogen gas).
Bubble each bottle with nitrogen gas and close
immediately with a rubber septum and screw tight.
Autoclave at 121℃ for 15 min. Sterile syringes are used
to inoculate and remove samples. After sterilization
neutralized sterile sodium sulfide solution** is added to a
final concentration of 0.03%. Incubate in the light using a
tungsten lamp.
ZnSO4·7H2O
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
2.5% NaCl GYPFK Agar/Broth
Glucose
Polypepton*
Yeast extract
Extract Bonito*
NaCl
K2HPO4
Salt solution**
Agar (if needed)
Distilled water
g
ml
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min.
under a N2 atmosphere. After cooling to room
temperature the pH is adjusted to about 7.3 by adding of
sterile 2N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
5
ml
5
ml
1
ml
0.5
g
0.5
g
0.5
g
0.5
g
0.5
g
0.5
g
0.5
g
1
L
pH 7.5
Add the vitamin solution aseptically to the cooled
autoclaved medium.
957
Media
* Vitamin solution
* Basal salts solution
3Na-EDTA
FeSO4·7H2O
MgSO4·7H2O
CaCl2·2H2O
NaCl
MnSO4·4H2O
ZnSO4·7H2O
Co(NO3)2·6H2O
CuSO4·5H2O
Na2MoO4·2H2O
H3BO3
Distilled water
4.53
1.11
24.65
2.94
23.4
112
29
29
25
24
31
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
g
g
g
g
g
mg
mg
mg
mg
mg
mg
L
75
78
1
g
g
L
200
100
100
5
50
1
50
50
50
100
mg
mg
mg
mg
mg
mg
mg
mg
mg
ml
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
*** Vitamin solution
3Na-EDTA
Nicotinic acid
Thiamine-HCl
Biotin
p-Aminobenzoic acid
Vitamin B12
Ca-pantothenate
Pyridoxine-HCl
Folic acid
Distilled water
389
Na2SeO3
Na2WO4
4
4
mg
mg
1
1
g
L
**** Resazurin solution
Resazurin
Distilled water
10
g
2
g
1
g
1
g
0.5
g
5
g
1
L
pH 7.0
For vial cultures, dissolve all ingredients except for
glucose, yeast extract , Na2S·9H2O and cysteine-HCl (up
to 900 ml), flush N2/CO2 (80/20) (pH 7), dispense 18 ml
into 50-ml vials, fix with flushing N2/CO2 (80/20) and
autoclave. Add 0.5 ml of each of filter-sterilized
Na2S·9H2O solution (0.6 g Na2S·9H2O/50 ml) and
cysteine-HCl solution (0.6 g cysteine-HCl/ 50ml)
separately. Add 0.5 ml of neutralized and filter sterilized
0.8 M glucose solution and 0.5 ml of autoclaved yeast
extract solution (%). Inoculum size 10%.
Medium for Tepidanaerobacter
Glucose
Yeast extract
KH2PO4
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Vitamin solution*
Trace element solution**
Se/W solution***
Resazurin solution****
NaHCO3
Na2S·9H2O
Cysteine-HCl
Distilled water
g
g
g
mg
g
g
mg
mg
mg
g
L
*** Se/W solution
NPB Medium
Bacto Tryptone (Difco)
Bacto Yeast Extract (Difco)
MgSO4·7H2O
K2HPO4
KH2PO4
D-Glucose
Distilled water
12.8
1.35
0.1
24
0.1
0.1
25
10
24
0.12
1
First dissolve NTA in 200 ml of water and adjust the pH to
6.5 with KOH.
Sterilized by filtration.
388
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace element solution
** Phosphate solution
KH2PO4
K2HPO4
Distilled water
0.5
0.9
0.4
0.7
0.8
0.2
0.5
2.7
0.3
0.4
1
2.2
g
2.3
g
0.14 g
0.2
g
0.15 g
0.54 g
2
ml
1
ml
1
ml
1
ml
2.5
g
0.3
g
0.3
g
1
L
pH7.0
390
Modified SP Medium
Bacto Casitone (Difco)
Yeast extract
Raffinose
Sucrose
Galactose
Soluble starch
MgSO4·7H2O
K2HPO4
Agar (if needed)
Distilled water
958
2.5
g
1
g
1
g
1
g
1
g
5
g
0.5
g
0.25 g
15
g
1
L
pH 7.4
Media
391
T h e r m o d e s ul f o b i u m n a r u g e n s e M e d i u m
K2HPO4
KH2PO4
3Na-EDTA
FeSO4·7H2O
MgSO4·7H2O
CaCl2·2H2O
NaCl
NH4Cl
NaSO4
Sodium acetate
Vitamin solution*
Trace element solution**
Cysteine-HCl
Distilled water
Dissolve ingredients except bicarbonate and sulfide,
bring to boil for few minutes, then cool quickly to room
temperature while gassing with N2/CO2 (80/20), add
sodium bicarbonate and adjust pH to 6.9. Distribute in
serum bottles (20 ml per 100 ml bottle) under N2/CO2,
seal, pressurize bottles up to 2 bar overpressure, then
autoclave. Before use, reduce the medium with sodium
sulfide from anaerobic sterile neutral stock solution.
0.78 g
0.75 g
0.04 g
0.01 g
0.25 g
0.03 g
0.2
g
0.5
g
20
mM
2
mM
10
ml
10
ml
0.25 g
1
L
* Trace element solution
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Na2WO4·H2O
Distilled water
For vial culture, dissolve all ingredients except
cysteine-HCl, flush N2/CO2, dispense 20 ml/50-ml vials,
fix with a stopper with flushing N2/CO2 and autoclave.
After autoclaved, the gas phase is replaced with H2/CO2
(80/20, 150 kPa), and add 0.1 ml of autoclaved
cysteine-HCl solution (50 g/l) per each vial separately.
Inoculum size 5-10%.
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
393
392
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.026
1
g
g
g
g
g
g
g
g
g
g
g
g
L
0.34 g
4
g
3.45 g
0.25 g
0.14 g
0.14 g
18
g
1
g
1
g
2.7
g
0.5
g
2
mg
10
ml
1
mg
0.5
g
1
L
Dissolve ingredients except bicarbonate and sulfide,
bring to boil for few minutes, then cool quickly to room
temperature while gassing with H2/CO2 (80/20), add
sodium bicarbonate and adjust pH to 6.5. Distribute in
serum bottles (20 ml per 100 ml bottle) under H2/CO2,
seal, then autoclave. Before use, reduce the medium with
sodium sulfide from anaerobic sterile neutral stock
solution, and pressurize bottles up to 2 bar overpressure.
Archaeoglobus Medium
KCl
MgCl2·6H2O
MgSO4·7H2O
NH4Cl
CaCl2·2H2O
K2HPO4
NaCl
NaHCO3
Bacto Yeast Extract (Difco)
Sodium lactate
Fe(NH4)2(SO4)2·7H2O
Trace element solution*
Resazurin
Na2S·9H2O
Distilled water
A r c h a e o g l o b u s p r o f u n du s M e d i u m
KCl
MgCl2·6H2O
MgSO4·7H2O
NH4Cl
CaCl2·2H2O
K2HPO4
NaCl
NaHCO3
Sodium acetate
Na2SO4
Bacto Yeast Extract (Difco)
Fe(NH4)2(SO4)2·7H2O
Trace element solution*
Resazurin
Na2S·9H2O
Distilled water
** Trace element solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO3·5H2O
Distilled water
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
4
mg
0.01 g
0.01 g
0.025 g
0.8 mg
0.8 mg
1
L
0.34 g
4
g
3.45 g
0.25 g
0.14 g
0.14 g
18
g
3
g
0.5
g
1.5
g
2
mg
10
ml
1
mg
0.5
g
1
L
959
Media
* Trace element solution
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Distilled water
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
0.02 g
0.01 g
0.01 g
0.025 g
0.3 mg
1
L
*** Sulfur powder
Sulfur is sterilized separately by autoclaving for 60 min at
105℃ on each of 3 successive days.
395
Pyrococcus Medium 2
Salt base solution*
Trace minerals**
Citric acid
Bacto Yeast Extract (Difco)
Bacto Peptone (Difco)
Sulfur (powder)***
Na2S·9H2O
Resazurin
1
10
5
1
5
30
0.5
1
396
L
ml
mg
g
g
g
g
mg
* Salt base solution
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·xH2O
CoSO4·7H2O
Distilled water
13.85 g
3.5
g
2.75 g
0.325 g
0.05 g
15
mg
7.5 mg
0.05 mg
0.75 g
0.5
g
2
mg
1
L
397
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
1.8 mg
4.5 mg
0.22 mg
0.05 mg
0.03 mg
0.03 mg
0.01 mg
1
L
Adjust pH to 3.5 with 10 N H2SO4
VY/2 Agar
Baker's yeast
CaCl2·2H2O
Vitamin B12
Bacto Agar (Difco)
Distilled water
5
g
1.36 g
0.5 mg
15
g
1
L
Sterilized vitamin B12 separately by filtration. Kill yeast
cells by heating before adding other ingredients and pH
adjustment. Adjust pH to 7.2 with KOH before adding
agar.
** Trace minerals
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
1
L
1
g
Adjust pH to 3.5 with 10 N H2SO4
Add yeast extract solution just prior to inoculation.
* Salt base solution
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
Sulfolobus tokodaii Medium
Salt base solution*
Bacto Yeast Extract (Difco)
Mix ingredients except sulfur and Na2S·9H2O, and adjust
pH to 6.5 with H2SO4. Sterilize by filtration under a N2
atmosphere. Prepare Na2S·9H2O as a 5% solution, and
autoclave under a N2 atmosphere. Aseptically and
anaerobically add sterile sulfur to the medium. Prior to
inoculation, add the sterile Na2S·9H2O solution, and
readjust pH to 6.5, if necessary.
NaCl
MgSO4·7H2O
MgCl2·6H2O
KCl
NaBr
H3BO3
SrCl2·6H2O
KI
CaCl2
KH2PO4
(NH4)2Ni(SO4)2·6H2O
Distilled water
1
g
1
g
1
g
1
L
pH 7.0 - 7.2
Use filtrated natural seawater or artificial seawater (e.g.
Aquamarine, Yashima Co.). Separately sterilize a
solution of Na2S2O3·5H2O by filtration, and aseptically add
to the medium.
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
394
JXT Medium
Bacto Yeast Extract (Difco)
Trypticase Peptone (BBL)
Na2S2O3·5H2O
Sea water
g
g
g
g
g
g
g
g
g
g
g
g
L
960
Media
398
Methanothermobacter Medium
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
NaHCO3
Bacto Yeast Extract (Difco)
Vitamin solution*
Trace element solution**
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
399
0.136 g
0.54 g
0.2
g
0.147 g
2.5
g
0.2
g
10
ml
10
ml
1
mg
0.5
g
0.5
g
1
L
Mix ingredients except vitamin solution, cysteine-HCl and
Na2S·9H2O, and autoclave under a H2/CO2 atmosphere
(80/20). Concentrated cysteine-HCl and sodium sulfide
solutions are sterilized separately under a N2 atmosphere
in tightly closed vessels. Prior to inoculation, add the
filter-sterile vitamin solution, Cysteine-HCl and
Na2S·9H2O. Pressurize the inoculated vessels to 150 kPa
with H2/CO2 (80/20).
* Trace element solution SL-10
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
** Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
0.5
g
0.4
g
0.4
g
0.4
g
0.05 g
0.002 g
1
ml
1
g
1
g
2
g
50
ml
4
g
20
ml
0.001 g
0.5
g
0.5
g
940
ml
Adjust pH to 6.7-7.0
Prepare the medium anaerobically under a atmosphere
H2/CO2 (80/20, 150 kPa).
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
Methanobacterium Medium
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
FeSO4·7H2O
Trace element solution SL-10*
Yeast extract
Sodium acetate
Sodium formate
Sludge fluid**
NaHCO3
Fatty acid mixture***
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
10
1.5
7
100
6
190
2
24
36
990
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
** Sludge fluid
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.025
g
g
g
g
g
g
g
g
g
g
g
g
g
Add 0.4% yeast extract to sludge from an anaerobic
digester, and after gassing with nitrogen gas for a few
minutes incubate it at 37°C for 24 hours. Then centrifuge
the sludge at 13,000 g and autoclave the resulting, clear
supernatant in screw-capped vessels under nitrogen gas.
The sludge fluid can be stored at room temperature in the
dark.
*** Fatty acid mixture
Valeric acid
0.5
g
Isovaleric acid
0.5
g
alpha-Methylbutyric acid
0.5
g
Isobutyric acid
0.5
g
Distilled water
20
ml
Adjust pH to 7.5 with conc. NaOH
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
400
M1D Medium
Bacto PPLO Broth w/o CV (Difco)
Bacto Tryptone (Difco)
Bacto Peptone (Difco)
Glucose
Fructose
Sucrose
Sorbitol
Phenol red
2
1
0.8
0.1
0.1
1
7.5
6
g
g
g
g
g
g
g
mg
Dissolve above in 80 ml of water and adjust to pH 7.8.
Bring to 90 ml with water. Autoclave and cool to room
temperature. Add the following under sterile conditions:
Schneider's Drosophila medium (Invitorogen, Gibco) 160
ml, Fetal bovine serum (heat-inactivated) 50 ml, Penicillin
G (100,000 U/ml) 2.5 ml.
961
Media
402
CYC Agar
** Trace element solution
Czapek-Dox Liquid Medium, Modified (OXOID)
33.4
g
Bacto Yeast Extract (Difco)
2
g
Bacto Vitamin Assay Casamino Acids (Difco)
6
g
Agar
16
g
Distilled water
1
L
Adjust pH to 7.2
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Distilled water
403
K2HPO4
KH2PO4
MgSO4·7H2O
NaCl
CaCO3
Yeast extract
Arabinose
Sodium glutamate
Agar (if needed)
Distilled water
404
0.5
g
0.6
g
0.2
g
0.1
g
3
g
0.6
g
4
g
6
g
15
g
1
L
pH 6.7±0.2
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
405
0.33 g
0.33 g
0.33 g
0.33 g
0.33 g
30
g
2.38 g
0.2
g
1
g
2
g
1
ml
1
ml
1
L
Prepare the medium anaerobically, under N2, omitting the
CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and
sucrose. The pH should be 7.0-7.5. Dispense the medium
into vessels suitable for anaerobic growth (Hungate tubes
or serum bottles) under an atmosphere of nitrogen and
autoclave. To the sterile, cooled medium add, from sterile
stock solutions the CaCl2, MgCl2, KNO3, tryptone, yeast
extract, vitamins and sucrose. The CaCl2, MgCl2, KNO3,
tryptone, yeast extract, and sucrose stock solutions
should be autoclaved, while the vitamin solution is
filter-sterilized.
2
2
10
5
5
5
5
0.1
5
5
1
0.227 g
0.348 g
0.5
g
0.5
g
0.25 g
2.25 g
2
mg
2
g
2
g
10
ml
1
ml
1
ml
0.85 g
10
ml
0.3
g
0.3
g
1
mg
980
ml
Mix ingredients except NaHCO3, methanol, cysteine-HCl
and Na2S·9H2O, and bring to a boil for 5-10 sec. Cool
down under a stream of N2/CO2 (80/20), and add
NaHCO3 and methanol to the medium. Dispense the
medium into suitable culture vessels (e.g. 20 ml of the
medium in 120 ml serum bottles) under a stream of
N2/CO2 (80/20) and seal with butyl rubber stoppers. After
autoclaving, stand the medium overnight. Separately
autoclave cysteine-HCl and Na2S·9H2O as 5% solutions,
respectively, under a N2 atmosphere. Prior to inoculation,
aseptically and anaerobically add the cysteine-HCl and
Na2S·9H2O solutions.
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
Methanosarcina Medium
KH2PO4
K2HPO4
NH4Cl
MgSO4·7H2O
CaCl2·2H2O
NaCl
FeSO4·7H2O
Bacto Yeast Extract (Difco)
Bacto Casitone (Difco)
Trace vitamins*
FeCl2 solution**
Trace element solution***
NaHCO3
Methanol
Cysteine-HCl
Na2S·9H2O
Resazurin
Distilled water
O c e a n i t h e r m u s p r of u n d u s M e d i u m
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
KCl
KNO3
NaCl
HEPES
Yeast extract
Tryptone
Sucrose
Vitamin solution*
Trace element solution**
Distilled water
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
0.02 g
0.01 g
0.01 g
0.025 g
0.3 mg
1
L
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
* Trace vitamins
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
962
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
** Trace vitamins
** FeCl2 solution
25% HCl (7.7 M)
FeCl2·4H2O
Distilled water
10
1.5
990
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Calcium pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
ml
g
ml
*** Trace element solution
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
406
70
100
6
190
2
24
36
1
mg
mg
mg
mg
mg
mg
mg
L
0.3
0.3
0.3
0.6
0.13
2
8
2
2
2.5
2.5
10
10
5
0.5
0.5
1
1
Polypepton*
Yeast extract
MgSO4·7H2O
Distilled water
g
g
g
g
g
mg
mg
g
g
g
g
ml
ml
g
g
g
mg
L
10
2
1
1
g
g
g
L
pH 7.0
* Wako Pure Chemicals Ind. Ltd., Osaka, Japan.
Prepared medium is available from Wako Pure Chemical
Ind., Ltd.
703
YM Liquid Medium
Glucose
Peptone
Yeast extract
Malt extract
Distilled water
704
10
5
3
3
1
g
g
g
g
L
YPD Liquid Medium
Yeast extract
Peptone
Glucose
Distilled water
Mix ingredients except NaHCO3, cysteine-HCl and
Na2S·9H2O, and bring to a boil for 5-10 sec. Cool down
under a stream of H2/CO2 (80/20), and add NaHCO3 to
the medium. Dispense the medium into suitable culture
vessels (e.g. 20 ml of the medium in 120 ml serum
bottles) under a stream of H2/CO2 (80/20) and seal with
butyl rubber stoppers. Separately autoclave cysteine-HCl
and Na2S·9H2O as 5% solutions, respectively, under a N2
atmosphere. Prior to inoculation, aseptically and
anaerobically add the cysteine-HCl and Na2S·9H2O
solutions. Pressurize the inoculated vessels to 200 kPa
with H2/CO2 (80/20).
10
g
20
g
20
g
1
L
pH unadjusted
707
Peptone
Yeast extract
MgSO4·7H2O
Distilled water
5
3
1
1
g
g
g
L
pH 7.0
5
2
1
750
250
g
g
g
ml
ml
pH 7.0
708
* Trace minerals
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H20
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
702
Methanobacterium Medium
KH2PO4
K2HPO4
(NH4)2SO4
NaCl
MgSO2·7H2O
FeSO2·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Trypticase Peptone (BBL)
Sodium acetate
Sodium formate
Trace minerals*
Trace vitamins**
NaHCO3
Na2S·9H2O
Cysteine-HCl
Resazurin
Distilled water
2
2
10
5
5
5
5
0.1
5
5
1
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
Polypepton*
Yeast extract
MgSO4·7H2O
Seawater**
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
L
* Wako Pure Chemicals Ind. Ltd., Osaka, Japan.
** Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP*).
802
Polypepton*
Yeast extract
MgSO4·7H2O
Distilled water
Agar (if needed)
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
963
10
2
1
1
15
g
g
g
L
g
pH 7.0
Media
808
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Prepared medium (Code No. 398-01671) is available
from Wako Pure Chemical Ind., Ltd.
803
Polypepton*
Yeast extract
Glucose
Lactose
Tween 80
MgSO4·7H2O
Distilled water
Agar (if needed)
5
g
5
g
5
g
2
g
0.5
g
1
g
1
L
15
g
pH 6.5 - 6.8
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
804
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
Distilled water
Agar (if needed)
5
g
5
g
5
g
1
g
1
L
15
g
pH 6.6 - 7.0
0.3
0.3
0.3
0.6
0.13
2
8
2
2
2.5
2.5
10
10
5
0.5
0.5
1
1
g
g
g
g
g
mg
mg
g
g
g
g
ml
ml
g
g
g
mg
L
Mix ingredients except NaHCO3, cysteine-HCl and
Na2S·9H2O, and bring to a boil for 5-10 sec. Cool down
under a stream of H2/CO2 (80/20), and add NaHCO3 to
the medium. Separately autoclave cysteine-HCl and
Na2S·9H2O as 5% solutions, respectively, under a N2
atmosphere. Prior to inoculation, aseptically and
anaerobically add the cysteine-HCl and Na2S·9H2O
solutions. Pressurize the inoculated vessels to 200 kPa
with H2/CO2 (80/20).
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* Trace minerals
805
Yeast extract
Mannitol
K2HPO4
KH2PO4
MgSO4·7H2O
Distilled water
Agar (if needed)
807
Methanospirillum Medium
KH2PO4
K2HPO4
(NH4)2SO4
NaCl
MgSO2·7H2O
FeSO2·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Trypticase peptone (BBL)
Sodium acetate
Sodium formate
Trace minerals*
Trace vitamins**
NaHCO3
Na2S·9H2O
Cysteine-HCl
Resazurin
Distilled water
1
g
5
g
0.7
g
0.1
g
1
g
1
L
15
g
pH 7.0 - 7.2
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
Thioglycolate Medium*
Polypepton**
Yeast extract
Glucose
NaCl
L-Cystine
Sodium thioglycolate
Resazurin
Distilled water
Agar (if needed)
15
g
5
g
5
g
2.5
g
0.5
g
0.5
g
1
mg
1
L
15
g
pH 7.0 - 7.2
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
g
g
g
g
g
g
g
g
g
g
g
g
L
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
** Trace vitamins
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
* Thioglycolate Medium I for JP general test, Wako Pure
Chemicals Ind. Ltd., Osaka, Japan.
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
964
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
809
Glucose
Bacto Yeast Extract (Difco)
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
NaHCO3
Na2S·9H2O
Cysteine-HCl
Trace elements solution*
Vitamin solution**
Resazurin solution (1 mg/ml)
Distilled water
Mix ingredients except vitamin solution, cysteine-HCl and
Na2S·9H2O, and autoclave under a H2/CO2 (80/20)
atmosphere. Separately autoclave cysteine-HCl and
Na2S·9H2O as 5% solutions, respectively, under a N2
atmosphere. Prior to inoculation, aseptically and
anaerobically add filter-sterilized vitamin, cysteine-HCl
and Na2S·9H2O. Pressurize the inoculated bottles to 200
kPa with H2/CO2 (80/20).
2.2
g
2.3
g
0.15 g
0.5
g
0.2
g
0.15 g
2.5
g
0.3
g
0.3
g
1
ml
1
ml
1
ml
1
L
Adjust pH to 7.0 at 25℃
* Trace element solution
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
Prepare the medium anaerobically under a N2/CO2
(80/20) atmosphere. Neutralized substrates were added
to the vials from stock solutions prior to inoculation.
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Na2WO4·H2O
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
0.12
0.026
0.003
1
g
g
g
g
g
g
g
g
g
g
g
g
L
810
2
2
10
5
5
5
5
0.1
5
5
1
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
0.14
0.14
0.25
3.4
2.7
0.33
0.75
0.5
30
1
10
10
1
1
0.5
0.5
1
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
811
Yeast extract
Ethanol
Disodium succinate
Ammonium acetate
Ferric citrate solution* (0.1%)
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Vitamin B12 solution (0.01%)
Trace element solution SL-6**
Neutralized sulfide solution***
Resazurin
Distilled water
M e t h a n o c a l d o c o c c u s j an n a s c h i i M e d i u m
K2HPO4
CaCl2·2H2O
NH4Cl
MgSO4·7H2O
MgCl2·2H2O
KCl
NiCl2·6H2O
Na2SeO3·5H2O
NaCl
Fe(NH4)2(SO4)2·6H2O
Trace element solution*
Vitamin solution**
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
L
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
g
g
g
g
g
g
mg
mg
g
mg
ml
ml
mg
g
g
g
L
965
0.3
0.5
1
0.5
5
0.5
0.4
0.4
0.4
0.05
0.4
1
5
1
1
g
ml
g
g
ml
g
g
g
g
g
ml
ml
ml
mg
L
Mix ingredients except ethanol, vitamin B12 solution and
neutralized sulfide solution, and adjust pH to 6.8 with 6 N
H2SO4. Dispense the medium into suitable culture
vessels under a stream of nitrogen gas, seal with butyl
rubber stoppers, and autoclave at 121°C for 15 min.
Separately autoclave neutralized sulfide solution under a
N2 atmosphere. Sterilize ethanol and vitamin B12 by
filtration. Sterile syringes are used to inoculate and
remove samples. Prior to inoculation, aseptically and
anaerobically add ethanol, vitamin B12 solution and
neutralized sulfide solution.
Media
*** Neutralized sulfide solution
* Soluble in hot water.
Na2S·9H2O
Distilled water
** Trace element solution SL-6
ZnSO4·7H2O
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
0.1
0.03
0.3
0.2
0.01
0.02
0.03
1
g
g
g
g
g
g
g
L
1.5
100
g
ml
813
NaCl
KCl
MgSO4·7H2O
CaCl2·2H2O
NaBr
NaHCO3
Proteose-Peptone No.3 (Difco)
Bacto Yeast Extract (Difco)
Glucose
Distilled water
Agar (if needed)
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
812
R h o d o m i c r ob i u m M e d i u m
Yeast extract
Disodium succinate
Ammonium acetate
Ferric citrate solution* (0.1%)
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Vitamin B12 solution (0.01%)
Trace element solution SL-6**
Neutralized sulfide solution***
Distilled water
0.2
g
1
g
0.5
g
5
ml
0.5
g
0.4
g
0.4
g
0.4
g
0.05 g
0.4 ml
1
ml
5
ml
1
L
pH 5.7
814
Trypticase Soy Yeast Extract Medium
Trypticase Soy Broth (Difco)
Yeast extract
Agar (if needed)
Distilled water
815
816
g
g
g
g
g
g
g
L
966
3
1
15
g
L
g
Tetragenococcus halophilus Medium (To
MRS Medium add 6.5% NaCl)
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
NaCl
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
** Trace element solution SL-6
30
g
3
g
15
g
1
L
pH 7.0 - 7.2
1/10 Tryptic Soy Medium
Bacto Tryptic Soy Broth (Difco)
Distilled water
Agar (if needed)
* Soluble in hot water.
0.1
0.03
0.3
0.2
0.01
0.02
0.03
1
50
g
2
g
1
g
0.36 g
0.23 g
0.06 g
5
g
10
g
1
g
1
L
15
g
pH 7.2 - 7.4
Trace amount of FeCl3 is added to the medium.
Mix ingredients except vitamin B12 solution and
neutralized sulfide solution, and adjust pH to 5.7 with 6 N
H2SO4. Dispense the medium into suitable culture
vessels (e.g. 20 ml of the medium in 120 ml serum
bottles) under a stream of nitrogen gas, seal with butyl
rubber stoppers, and autoclave at 121°C for 15 min.
Separately autoclave neutralized sulfide solution under a
N2 atmosphere. Sterilize vitamin B12 by filtration. Sterile
syringes are used to inoculate and remove samples. Prior
to inoculation, aseptically and anaerobically add vitamin
B12 solution and neutralized sulfide solution.
ZnSO4·7H2O
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
g
ml
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
*** Neutralized sulfide solution
Na2S·9H2O
Distilled water
1.5
100
10
g
10
g
5
g
20
g
1
g
65
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 6.0 - 6.5
Media
817
T e t r a g e n o c o c c u s m ur i a t i c u s M e d i u m ( T o
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
NaCl
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
818
821
MRS Medium add 10% NaCl)
10
g
10
g
5
g
20
g
1
g
100
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 6.0 - 6.5
822
10
g
10
g
10
g
10
g
0.2
g
10
mg
10
mg
10
mg
1
L
pH 6.8
40
20
20
20
10
20
1
Bacto PPLO Broth (Difco)
Horse serum*
L-Arginine
Phenol red
Penicillin G** (20,000 U/ml)
Distilled water
g
g
g
g
ml
g
L
pH 6.8
820
4
0.2
0.2
0.2
100
21
g
100
ml
3
g
0.02 g
5
ml
900
ml
pH 6.8-7.0
** Sterilized by filtration.
Dissolve 21 g of PPLO broth, 3 g of L-arginine, and 0.02 g
of phenol red in 900 ml of water (adjusted pH to 6.8-7.0.
sterilized by autoclave at 121℃ for 15 min). Aseptically
add 100 ml of horse serum and 5 ml of Penicillin G
(200,000 U/ml, sterilized by filtration) to the cooled broth.
g
g
g
g
ml
824
Medium for Co-culture of Strain JT with
Methanospirillum hungatei NBRC 100397
Na-terephthalate
Yeast extract
KH2PO4
MgCl2·H2O
CaCl2·2H2O
NH4Cl
Vitamin solution*
Trace element solution**
Se/W solution***
Resazurin solution****
NaHCO3
Na2S·9H2O
Cysteine-HCl
Distilled water
L e u c o n o s t oc o e n o s M e d i u m
Casein peptone, tryptic digest
Yeast extract
Glucose
Fructose
MgSO4·7H2O
MnSO4·H2O
Diammonium hydrogencitrate
Tween 80
Tomato juice, filtered
Cysteine-HCl
Distilled water
0.5
g
0.5
g
0.5
g
0.5
g
0.5
g
0.1
g
0.1
g
1
L
pH 7.0
* Heat inactivated at 56℃ for 30 min.
* Solution B
MgSO4·7H2O
MnSO4·xH2O
FeSO4·7H2O
NaCl
Distilled water
g
g
g
g
g
g
mg
g
g
L
pH 7.2
823
819
Glucose
Yeast extract
Peptone
Sodium acetate
Solution B*
Agar (if needed)
Distilled water
10
5
5
5
2
2
50
1
20
1
M i c r o l u n a t us M e d i u m
Glucose
Peptone
Yeast extract
Sodium glutamate
KH2PO4
(NH4)2SO4
MgSO4·7H2O
Distilled water
GYP-Sodium Acetate-Mineral Salts Broth
Glucose
Bacto Yeast Extract (Difco)
Bacto Peptone (Difco)
Sodium acetate
MgSO4·7H2O
MnSO4·xH2O
FeSO4·7H2O
NaCl
Distilled water
R Agar
Bacto Peptone (Difco)
Bacto Yeast Extract (Difco)
Bacto Malt Extract (Difco)
Bacto Casamino Acids (Difco)
Bacto Beef Extract (Difco)
Glycerol
Tween 80
MgSO4·7H2O
Agar
Distilled water
10
g
5
g
10
g
5
g
0.2
g
0.05 g
3.5
g
1
ml
100
ml
0.5
g
900
ml
pH 4.8
0.42
0.2
0.14
0.2
0.15
0.54
3
1
1
1
2.5
0.3
0.3
1
This medium is for co-culture of strain JT with
Methanospirillum hungatei strain NBRC 100397.
967
g
g
g
g
g
g
ml
ml
ml
ml
g
g
g
L
Media
828
For vial cultures, dissolve all ingredients except for
Na-terephthalate, Na2S·9H2O and cysteine-HCl (up to
900ml), flush N2/CO2 (80/20), and autoclave. Add 0.5 ml
of each of filter-sterilized Na2S·9H2O solution (0.6g
Na2S·9H2O/50 ml) and cysteine-HCl solution (0.6g
cysteine-HCl/ 50ml) per each vial separately. Add 0.2 ml
of neutralized and filter-sterilized 0.2 M Na-terephthalate
solution. Yeast extract is also added at final concentration
about 0.02%. Inoculum size 10-20%. Growth was
observed after 3-4 weeks of incubation.
829
0.5
0.9
0.4
0.7
0.8
0.2
0.5
2.7
0.3
0.4
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
12.8
1.35
0.1
24
0.1
0.1
25
10
24
0.12
1
g
g
g
mg
g
g
mg
mg
mg
g
L
830
mg
mg
831
**** Resazurin solution
Resazurin
Distilled water
825
1
1
g
L
0.132 g
0.052 g
0.041 g
0.49 g
0.009 g
14.7
g
1
mg
2
mg
1
mg
0.5 mg
0.5 mg
1
mg
5
mg
1
L
Adjust pH to 1.3 with H2SO4.
Acidianus manzaensis Medium for
Heterotrophic Growth
Yeast extract
(NH4)2SO4·7H2O
KCl
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
MnSO4·5H2O
NaMoO4·2H2O
CoCl2·6H2O
NiCl2·6H2O
FeSO4·7H2O
Distilled water
Acidic MRS Medium (pH 4.8)
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
Autotrophic Growth
In aerobic condition, H2 is necessary for autotrophic
growth as an electron donor. In anaerobic condition, H2
and Fe3+ are necessary for autotrophic growth as an
electron donor and acceptor, respectively.
*** Se/W solution
4
4
2.5
g
0.5
g
11.92 g
0.5
g
100
mg
5
mg
75
mg
1
L
pH 7.8 - 8.0
Acidianus manzaensis Medium for
(NH4)2SO4·7H2O
KCl
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
Fe2(SO4)3·nH2O
ZnSO4·7H2O
CuSO4·5H2O
MnSO4·5H2O
NaMoO4·2H2O
CoCl2·6H2O
NiCl2·6H2O
FeSO4·7H2O
Distilled water
First dissolve NTA in 200 ml of water and adjust the pH to
6.5 with KOH.
Na2SeO3
Na2WO4
g
g
g
L
pH 7.2
* N-2-Hydroxyethyl piperazine-N'-2-ethane sulfonic acid
** Trace element solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
10
1
15
1
HEPES Medium
(NH4)2SO4
KH2PO4
HEPES*
NaHCO3
MgSO4·7H2O
CaCl2·2H2O
Fe-EDTA
Distilled water
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
YCA Medium
Dry yeast
CaCl2·2H2O
Agar (if needed)
Distilled water
10
g
10
g
5
g
20
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 4.8
968
1
g
0.132 g
0.052 g
0.041 g
0.49 g
0.009 g
1
mg
2
mg
1
mg
0.5 mg
0.5 mg
1
mg
5
mg
1
L
Adjust pH to 1.3 with H2SO4.
Media
832
Pyrodictium Medium
NaCl
MgSO4·7H2O
MgCl2·6H2O
KCl
NaBr
H3BO3
SrCl2·6H2O
KI
CaCl2
KH2PO4
(NH4)2Ni(SO4)2·6H2O
Trace minerals*
Bacto Yeast Extract (Difco)
Citric acid
Sulfur (powder)
Resazurin
Na2S·9H2O
Distilled water
833
13.85 g
3.5
g
2.75 g
0.325 g
0.05 g
15
mg
7.5 mg
0.05 mg
0.75 g
0.5
g
2
mg
10
ml
2
g
5
mg
30
g
1
mg
0.3
g
1
L
Mix ingredients except sulfur and Na2S·9H2O, adjust pH
to 5.5 with H2SO4, and autoclave under H2/CO2 (80/20)
conditions. Separately autoclave neutralize Na2S·9H2O
solution. Sulfur is sterilized separately by autoclaving for
60 min at 105℃ on each of 3 successive days.
Aseptically and anaerobically add sterile sulfur to the
medium under a H2/CO2 (80/20) atmosphere. Prior to
inoculation, add Na2S·9H2O solution, and readjust pH to
5.5 if necessary. Pressurize inoculated bottles to 200 kPa
with H2/CO2 (80/20).
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
13.85 g
3.5
g
2.75 g
0.325 g
0.05 g
15
mg
7.5 mg
0.05 mg
0.75 g
0.5
g
2
mg
10
ml
2
g
5
mg
2.5
g
30
g
1
mg
0.3
g
1
L
Mix ingredients except sulfur and Na2S·9H2O, adjust pH
to 5.5 with H2SO4, and autoclave under H2/CO2 (80/20)
conditions. Separately autoclave neutralize Na2S·9H2O
solution. Sulfur is sterilized separately by autoclaving for
60 min at 105℃ on each of 3 successive days.
Aseptically and anaerobically add sterile sulfur to the
medium under a H2/CO2 (80/20) atmosphere. Prior to
inoculation, add Na2S·9H2O solution, and readjust pH to
5.5 if necessary. Pressurize inoculated bottles to 200 kPa
with H2/CO2 (80/20).
* Trace minerals
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H20
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
Pyrodictium occultum Medium
NaCl
MgSO4·7H2O
MgCl2·6H2O
KCl
NaBr
H3BO3
SrCl2·6H2O
KI
CaCl2
KH2PO4
(NH4)2Ni(SO4)2·6H2O
Trace minerals*
Bacto Yeast Extract (Difco)
Citric acid
Na2S2O3·5H2O
Sulfur (powder)
Resazurin
Na2S·9H2O
Distilled water
* Trace minerals
g
g
g
g
g
g
g
g
g
g
g
g
L
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H20
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
g
g
g
g
g
g
g
g
g
g
g
g
L
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
969
Media
835
Thermoproteus tenax Medium
(NH4)2SO4
FeSO4·7H2O
MgSO4·7H2O
CaSO4·2H2O
KH2PO4
NaF
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO7·2H2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Soluble starch
Sulfur (powder)
Na2S·9H2O
Resazurin
Distilled water
837
0.264 g
0.556 g
0.492 g
0.344 g
0.014 g
0.84 mg
0.18 mg
0.45 mg
0.22 mg
0.05 mg
0.03 mg
0.01 mg
0.2
g
5
g
10
g
0.5
g
1
mg
1
L
1.3
0.28
0.25
0.07
0.02
1.8
4.5
0.22
0.05
0.03
0.03
0.01
0.85
0.2
25
8
0.5
1
g
g
g
g
g
g
g
g
ml
ml
g
g
g
mg
ml
* Wako Pure Chemicals Ind. Ltd., Osaka, Japan.
** Trace mineral solution
Nitrilotriacetic acid
FeCl2·4H2O
MnCl2·4H2O
CoCl2·6H2O
ZnCl2
CaCl2·2H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
T h e r m o p r o t e u s n e u t r o ph i l u s M e d i u m
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·xH2O
CoSO4·7H2O
NaHCO3
Bacto Yeast Extract (Difco)
Na2S2O4
Sulfur (powder)
Resazurin
Distilled water
0.75
0.75
0.36
1
0.1
0.1
2.5
8
9
10
0.8
0.5
0.5
1
981
Mix ingredients except trace vitamins, Na2S·9H2O and
cysteine-HCl, and autoclave under a N2/CO2 (80/20)
atmosphere. Filter-sterilize vitamin solution while gassing
with N2. Separately autoclave Na2S·9H2O and
cysteine-HCl as 5% solutions under a N2 atmosphere.
Aseptically and anaerobically add vitamin solution to the
medium under a N2/CO2 (80/20) atmosphere. Prior to
inoculation, add the sterile Na2S·9H2O and cysteine-HCl
solutions, and adjust pH to 6.8.
Mix ingredients except yeast extract, starch, sulfur and
Na2S·9H2O, and filter-sterilize while gassing with N2.
Autoclave yeast extract and starch as 10% solutions and
Na2S·9H2O as a 5% solution under a N2 atmosphere.
Sulfur is sterilized separately by autoclaving for 60 min at
105℃ on each of 3 successive days. Aseptically and
anaerobically add the yeast extract and starch solutions,
and sulfur to the medium. Prior to inoculation, add the
sterile Na2S·9H2O solution, and adjust pH to 5.5 with
sterile 1 N H2SO4.
836
Methanosarcina Medium
KH2PO4
K2HPO4
MgCl2·6H2O
NH4Cl
Bacto Yeast Extract (Difco)
Polypepton*
Sodium acetate
Trimethylamine-HCl
Trace mineral solution**
Trace vitamins***
NaHCO3
Na2S·9H2O
Cysteine-HCl
Resazurin
Distilled water
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
g
mg
g
mg
L
4.5
g
0.4
g
0.1
g
0.17 g
0.1
g
0.02 g
0.019 g
0.01 g
0.03 g
1
L
Adjust pH to 7.0 with 1 N KOH
*** Trace vitamins
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
Mix ingredients except yeast extract, NaHCO3, Na2S2O4
and sulfur, and adjust pH to 6.5. Autoclave, then cool
under a H2/CO2 (80/20) atmosphere. Separately
autoclave yeast extract as a 10% solution under a N2
atmosphere. Filter-sterilize NaHCO3 as a 8% solution
while gassing with N2. Sulfur is sterilized separately by
autoclaving for 60 min at 105℃ on each of 3 successive
days. Aseptically and anaerobically add the yeast extract
and NaHCO3 solutions, and sulfur to the medium under a
H2/CO2 (80/20) atmosphere. Readjust pH to 6.5 by further
gassing with H2/CO2 (80/20) gas mixture, if necessary.
Prior to inoculation, add filter-sterilized 2.5% Na2S2O4
solution. Pressurize inoculated bottles to 150 kPa with
H2/CO2 (80/20).
970
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
838
Thermoproteus tenax Medium 2
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·xH2O
CoSO4·7H2O
NaHCO3
Bacto Yeast Extract (Difco)
Soluble starch
Na2S·9H2O
Sulfur (powder)
Resazurin
Distilled water
1.3
0.28
0.25
0.07
0.02
1.8
4.5
0.22
0.05
0.03
0.03
0.01
0.85
0.2
5
0.5
8
0.5
1
Mix ingredients except KP buffer, vitamin solution,
Na2CO3 and Na2S·9H2O. Dispense the medium into
suitable culture vessels under a stream of H2/CO2 (80/20)
and seal with butyl rubber stoppers. Separately autoclave
KP buffer and Na2S·9H2O (5% solution) under a N2
atmosphere. Sterilize vitamin and 10% Na2CO3 solutions
by filtration. Prior to inoculation, aseptically and
anaerobically add KP buffer, vitamin, Na2CO3 and
Na2S·9H2O solutions. Pressurize the inoculated vessels
to 150 kPa with H2/CO2 (80/20).
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
g
g
g
g
mg
L
* KP buffer
KH2PO4
K2HPO4
Distilled water
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
840
1
g
1
g
1
g
5
g
2.5
g
0.5
g
0.25 g
15
g
1
L
pH 7.4
g
g
g
g
g
g
g
g
g
g
g
g
g
L
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
Archaeoglobus Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
NaCl
Na2S2O3
Sodium acetate
Trace elements solution**
Vitamin solution***
Coenzyme M
Resazurin
Na2CO3
Na2S·9H2O
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
SP Medium
Raffinose
Sucrose
Galactose
Soluble starch
Casitone
MgSO4·7H2O
K2HPO4
Agar (if needed)
Distilled water
g
g
L
Autoclaved under N2.
Mix ingredients except NaHCO3, yeast extract, soluble
starch, Na2S·9H2O and sulfur, and adjust pH to 5.5.
Autoclave, then cool under N2/CO2 (80/20) gas mixture.
Under a N2 atmosphere, separately autoclave yeast
extract, soluble starch and Na2S·9H2O as 2%, 10% and
5% solutions, respectively. Filter-sterilize NaHCO3 as a
8% solution while gassing with N2. Sulfur is sterilized
separately by autoclaving for 60 min at 105℃ on each of
3 successive days. Aseptically and anaerobically add the
yeast extract, soluble starch and NaHCO3 solutions, and
sulfur to the medium under a N2/CO2 (80/20) atmosphere.
Readjust pH to 5.5, if necessary. Prior to inoculation, add
Na2S·9H2O.
839
119
21
1
5
ml
0.75 g
0.15 g
0.54 g
30
g
3.2
g
1.6
g
2
ml
2
ml
0.14 g
1
mg
1
g
0.36 g
1
L
pH unadjusted
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
841
Polypepton*
Yeast extract
MgSO4·7H2O
Kanamycin sulfate solution (250 μg/ml)**
Distilled water
Agar (if needed)
10
2
1
100
900
15
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Sterilize separately by filtration.
971
g
g
g
ml
ml
g
pH 7.0
Media
842
Mycoplasma Medium with Sucrose
Bacto Heart Infusion Broth (Difco)
Horse serum*
15% Yeast Extract Solution (Gibco 0689)
Sucrose
Phenol red
Distilled water
845
17.5
200
150
40
40
650
g
ml
ml
g
mg
ml
L-Asparagine
Glycerol
K2HPO4
Distilled water
Trace salts solution*
Agar
* Heat-inactivated at 56℃ for 30 min.
Dissolve 17.5 g of heart infusion broth and 40 mg of
phenol red in 450 ml of water (sterilized by autoclaving at
121℃ for 15 min). Dissolve sucrose in 200 ml of water
(sterilized by filtration). Aseptically add the horse serum,
yeast extract solution, sterile sucrose, and 1 ml of
Penicillin G (200,000 U/ml, sterilized by filtration) to the
cooled heart infusion broth. Final pH 7.4 +/- 0.2.
843
844
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
846
10
5
5
1
15
1
(NH4)2SO4
KCl
KH2PO4
MgSO4·7H2O
Ca(NO3)2·4H2O
Yeast extract
Distilled water
Glucose
Agar (if needed)
Distilled water
847
3
2.5
1
g
g
L
ml
ml
2
g
0.1
g
0.5
g
0.5
g
0.001 g
0.1
g
500
ml
Adjust pH to 3.5 with 1 N H2SO4
972
1
20
500
g
g
ml
T h e r m o p r o t e u s a n d V u lc a n i s a e t a M e d i u m
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·nH2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Sulfur (powder)
Na2S·9H2O
Resazurin
Distilled water
** Solution 2
H3BO3
SrCl2·6H2O
Distilled water
500
500
** Solution B
* Solution 1
g
g
g
L
g
g
g
ml
* Solution A
13.5
g
4
g
10.8
g
1.5
g
1
g
5
g
1
mg
10
ml
1
ml
30
g
0.25 g
1
L
7
2
1
1
0.1
0.1
0.1
100
Agar solid medium of BYG contained 2% agar. In order to
prevent the hydrolysis of agar and glucose by autoclaving
at low pH condition, Solutions A and B are autoclaved
separately and combined before solidified (50-60℃).
Mix ingredients except solutions 1 and 2, sulfur, and
Na2S·9H2O, and autoclave under a N2 atmosphere. Sulfur
is sterilized separately by autoclaving for 60 min at 105℃
on each of 3 successive days. Neutralize Na2S·9H2O as a
5% solution, and autoclave under a N2 atmosphere.
Aseptically and anaerobically add sterile sulfur and
filter-sterile solutions 1 and 2 to the medium. Prior to
inoculation, add the sterile Na2S·9H2O solution.
KCl
NaHCO3
NaBr
Distilled water
g
g
g
L
ml
g
pH 5.0
B Y G M e d i u m ( f o r A c i d ip h i l i u m m u l t i v o r u m )
Solution A*
Solution B**
g
g
g
g
g
L
pH 7.0
Pyrococcus Medium
NaCl
Na2SO4
MgCl2·6H2O
CaCl2·2H2O
Yeast extract
Tryptone
Resazurin
Solution 1*
Solution 2**
Sulfur (powder)
Na2S·9H2O
Distilled water
1
10
1
1
1
20
* Trace salts solution
L Medium
Bacto Tryptone (Difco)
Bacto Yeast Extract (Difco)
NaCl
Glucose
Agar (if needed)
Distilled water
Acidic Glycerol-Asparagine Agar (ISP
Medium No.5)
1.3
0.28
0.25
0.07
0.02
1.8
4.5
0.22
0.05
0.03
0.03
0.01
1
5
0.5
1
1
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
g
g
mg
L
Media
* Trace element solution
Mix ingredients except yeast extract, sulfur and
Na2S·9H2O, and autoclave under a N2 atmosphere.
Separately autoclave yeast extract as a 10% solution and
Na2S·9H2O as a 5% solution under a N2 atmosphere.
Sulfur is autoclaved for 60 min at 105℃ on each of 3
successive days. Aseptically and anaerobically add yeast
extract and sulfur to the medium. Prior to inoculation, add
sterile Na2S·9H2O solution, and adjust pH to 4.5-5.0 with
sterile 1 N H2SO4.
848
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Distilled water
BL Agar
BL Agar*
Defibrinated horse blood
Distilled water
58
50
950
g
ml
ml
Autoclave except horse blood and cool to about 50℃.
Aseptically add 5 % (final) sterile defibrinated horse blood.
Mix and quickly dispense into sterile test tubes or petri
dishes.
* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.
849
Bacto Yeast Extract (Difco)
Bacto Proteose Peptone No. 3 (Difco)
Bacto Casamino Acids (Difco)
Glucose
Soluble starch
Sodium pyruvate
K2HPO4
MgSO4·7H2O
Agar (if needed)
Distilled water
850
T h e r m o t o g a m a r it i m a M e d i u m
Soluble starch
KH2PO4
Trace minerals*
NaCl
Bacto Yeast Extract (Difco)
Resazurin
Na2S·9H2O
Artificial sea water**
Distilled water
0.5
g
0.5
g
0.5
g
0.5
g
0.5
g
0.3
g
0.3
g
0.05 g
15
g
1
L
pH 7.2+/-0.2
5
0.5
10
20
0.5
1
0.5
250
750
g
g
ml
g
g
mg
g
ml
ml
Mix ingredients except soluble starch and Na2S·9H2O,
and adjust pH to 6.5 with NaOH. Sterilize by filtration
under a N2 atmosphere. Soluble starch and neutralize
Na2S·9H2O are prepared as 10 and 5% solutions,
respectively, and autoclaved under a N2 atmosphere.
Prior to inoculation, add the sterile soluble starch and
Na2S·9H2O solutions.
Modified MB Medium
NH4Cl
K2HPO4
KH2PO4
MgCl2·6H2O
NaCl
KCl
CaCl2·2H2O
Yeast extract
Bacto Tryptone (Difco)
Resazurin
Soluble starch
Na2S2O3·5H2O
Cysteine-HCl
Trace element solution*
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
851
R2A Medium
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
0.02 g
0.01 g
0.01 g
0.025 g
0.3 mg
1
L
1
g
0.3
g
0.3
g
0.5
g
2
g
0.2
g
0.05 g
1
g
2
g
0.5 mg
10
g
5
g
0.5
g
10
ml
1
L
Adjust pH to 7.5 with 2 M NaOH
* Trace minerals
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Na2WO4
KAl(SO4)2·12H2O
Distilled water
Dissolve ingredients except thiosulfate and cysteine-HCl,
boil medium for 3 min, then cool to room temperature
under a N2 atmosphere, and add cysteine-HCl. Adjust pH
to 7.5, dispense under same gas atmosphere in culture
vessels and autoclave. Before inoculation, add thiosulfate
from a sterile, anaerobic stock solutions prepared under
N2.
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
0.01 g
0.01 g
0.025 g
0.8 mg
0.8 mg
4
mg
1
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
** Artificial sea water
973
Media
NaCl
MgSO4·7H2O
MgCl2·6H2O
CaCl2·2H2O
KCl
NaBr
H3BO3
SrCl2·6H2O
Citric acid
KI
Distilled water
852
27.7
7
5.5
2.25
0.65
0.1
30
15
10
0.05
1
g
g
g
g
g
g
mg
mg
mg
mg
L
853
125
10
0.25
2.2
0.07
2
2
0.5
1
865
ml
ml
g
g
g
mg
mg
g
g
ml
854
855
g
g
L
45
ml
2.5 ml
2
ml
0.5 ml
pH 6.8
After aseptically inject solution II, sulfide solution and
acetate solution into vessel of solution I, the pH is
adjusted to 6.8 by adding of sterile 0.6 N H2SO4 with a
syringe without opening the vessel. If it is necessary, 0.5
ml thiosulfate solution***** is injected.
* Solution I
g
g
g
g
g
g
g
mg
mg
mg
mg
mg
g
L
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
KCl
NH4Cl
Ammonium acetate
Ferric citrate solution (0.1%)
Vitamin B12 solution (0.001%)
Trace element solution SL-6
Resazurin
Distilled water
0.5
g
0.5
g
0.05 g
0.025 g
0.5
g
0.5
g
5
ml
2
ml
2
ml
1
mg
950
ml
pH 5.0
Mix ingredients except vitamin solution, and distribute the
medium into culture vessels under a stream of nitrogen
gas and seal with butyl rubber stoppers. After autoclaving
(121°C, 15 min), aseptically inject vitamin solution.
** Trace elements
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
Na2WO4
KAl(SO4)2·12H2O
Distilled water
3
15
1
Green Sulfur Bacterium Medium
Solution I*
Solution II**
Neutralized sulfide solution***
Acetate solution****
* Marine medium/synthetic seawater mix solution
47
18
7
3
3.13
0.1
0.2
72
52
8.1
2.4
0.05
0.4
1
g
g
g
g
g
g
L
Shrimp Agar
Dried shrimp
Agar
Distilled water
After flushing with N2/CO2, adjust pH to 7.0 with 1 N
H2SO4. Sterilize by filtration. Dispense the medium into
vessels under a N2/CO2/O2 (79/20/1) atmosphere.
Pressurize the inoculated vessels to 200 kPa with
N2/CO2/O2.
NaCl
MgCl2·6H2O
MgSO4·7H2O
CaCl2·2H2O
Na2SO4
Na2CO3
NaBr
SrCl2·6H2O
H3BO3
Na2HPO4
NaF
KI
Sodium silicate
Distilled water
3
0.5
0.25
2
10
0.2
1
Mix ingredients except glucose, and adjust pH to 1.0 with
10 N H2SO4. After autoclaving, add filter-sterilized 40%
glucose solution.
Pyrobaculum aerophilum Medium
Marine medium/synthetic seawater mix solution*
Trace elements**
NH4Cl
NaHCO3
KH2PO4
Fe(NH4)2(SO4)2·6H2O
(NH4)2Ni(SO4)2·6H2O
Bacto Yeast Extract (Difco)
Na2S2O3·5H2O
Distilled water
Picrophilus torridus Medium
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Glucose
(NH4)2SO4
Distilled water
1.5
g
3
g
0.5
g
1
g
0.1
g
0.18 g
0.1
g
0.18 g
0.01 g
0.01 g
0.01 g
0.025 g
0.4 mg
0.4 mg
2
mg
1
L
** Solution II
NaHCO3
Distilled water
1.5
g
50
ml
pH 8.2, Filter sterile
*** Neutralized sulfide solution
Na2S·9H2O
Distilled water
1.5
100
g
ml
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add minerals. Final pH 7.0 (with KOH).
974
Media
** Trace elements solution
**** Acetate solution
Ammonium acetate
Magnesium acetate
Fructose
Distilled water
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
2.5
g
2.5
g
2.5
g
100
ml
pH 7.2
***** Thiosulfate solution
Na2S2O3·5H2O
Distilled water
5
100
g
ml
pH 7.1
Solution is prepared in a vessel sealed with a butyl rubber
stopper. Autoclave at 121℃ for 15 min under a N2
atmosphere.
856
Potato Dextrose Agar (PDA)
Potato
Glucose
Agar
Distilled water
200
10
15
1
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
Sulfate Reducer Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2S2O3
Na2SO4
Trace elements solution**
Vitamin solution***
Resazurin
Na2CO3
Na2S·9H2O
Distilled water
10
ml
0.75 g
0.15 g
0.54 g
1.6
g
1.4
g
2
ml
2
ml
1
mg
0.5
g
0.36 g
1
L
pH unadjusted
859
* KP buffer
119
21
1
g
g
L
Autoclaved under N2.
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Methanopyrus Medium
NH4Cl
K2HPO4
KH2PO4
NaCl
MgSO4·7H2O
MgCl2·6H2O
Na2SO4
CaCl2·2H2O
KCl
NaHCO3
Fe(NH4)2(SO4)2·6H2O
(NH4)2Ni(SO4)2·6H2O
NaBr
SrCl2·6H2O
H3BO3
KI
Sodium silicate
NaF
KNO3
Na2HPO4·12H2O
Bacto Yeast Extract (Difco)
Coenzyme M
Trace elements solution*
Vitamin solution**
Resazurin
Na2S·9H2O
Distilled water
Mix ingredients except KP buffer, vitamin solution,
Na2CO3 and Na2S·9H2O. Dispense the medium into
suitable culture vessels under a stream of H2/CO2 (80/20)
and seal with butyl rubber stoppers. Separately autoclave
KP buffer and Na2S·9H2O (5% solution) under a N2
atmosphere. Sterilize vitamin and 10% Na2CO3 solutions
by filtration. Prior to inoculation, aseptically and
anaerobically add KP buffer, vitamin, Na2CO3 and
Na2S·9H2O solutions. Pressurize the inoculated vessels
to 150 kPa with H2/CO2 (80/20).
KH2PO4
K2HPO4
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
g
g
g
L
Wash potatoes with tap water, peel and cut into 1 cm
cubes. Rinse with tap water quickly and boil 200 g of
potato cubes with 1 L of distilled water for 20 min. Mash
and squeeze through a muslin bag. Add agar and boil till
melted. Add glucose and stir till dissolved. Make up to 1 L.
Adjust pH to 5.4 - 5.6. Autoclave to sterilize.
857
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
0.25 g
0.05 g
0.09 g
11.8
g
1.75 g
4.5
g
0.81 g
0.78 g
0.3
g
1
g
2
mg
2
mg
0.04 g
0.018 g
0.013 g
0.0125mg
1
mg
0.6 mg
0.4 mg
4.6 mg
0.2
g
0.14 g
10
ml
10
ml
1
mg
0.5
g
1
L
Mix ingredients except Na2S·9H2O, and adjust pH to 6.0
with H2SO4. Sterilize by filtration while gassing H2/CO2
(80/20). Separately autoclave Na2S·9H2O (5% solution)
under a N2 atmosphere. Prior to inoculation, aseptically
and anaerobically add Na2S·9H2O solution. Pressurize
the inoculated vessels to 150 kPa with H2/CO2 (80/20).
975
Media
* Trace elements solution
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
860
** Vitamin solution
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
M e t h a n o s a r c i n a a c e t i v or a n s M e d i u m
NaCl
MgSO4·7H2O
Na2CO3
NH4Cl
KCl
CaCl2·2H2O
Na2HPO4
Bacto Yeast Extract (Difco)
Trace elements solution*
Vitamin solution**
Methanol
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
23.4
9.45
5
1
0.8
0.14
0.6
1
10
10
6.5
1
0.3
0.3
1
861
mg
g
g
g
g
g
g
g
ml
ml
ml
mg
g
g
L
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Sulfurisphaera Medium (pH 2.5)
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·nH2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Distilled water
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
1.8 mg
4.5 mg
0.22 mg
0.05 mg
0.03 mg
0.03 mg
0.01 mg
1
g
1
L
pH 2.5
Mix ingredients except Bacto Yeast Extract, adjust pH to
2.5 with H2SO4, and autoclave at 121°C for 15 min.
Separately autoclave Bacto Yeast Extract as a 10%
solution. Prior to inoculation, add Bacto Yeast Extract
solution.
Mix ingredients except vitamin solution, cysteine-HCl,
and Na2S·9H2O. Flush with N2/CO2, and adjust pH to 7.0
with HCl. Dispense the medium into suitable culture
vessels under a stream of N2/CO2 (80/20) and seal with
butyl rubber stoppers. Separately autoclave cysteine-HCl
and Na2S·9H2O (5% solution) under a N2 atmosphere.
Sterilize vitamin solution by filtration. Prior to inoculation,
aseptically and anaerobically add vitamin, cysteine-HCl
and Na2S·9H2O solutions.
976
Media
862
Sulfurisphaera Medium (pH 5.0)
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·nH2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH is 7.0.
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
1.8 mg
4.5 mg
0.22 mg
0.05 mg
0.03 mg
0.03 mg
0.01 mg
1
g
1
L
pH 5.0
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
864
Mix ingredients except Bacto Yeast Extract, adjust pH to
5.0 with H2SO4, and autoclave at 121°C for 15 min.
Separately autoclave Bacto Yeast Extract as a 10%
solution. Prior to inoculation, add Bacto Yeast Extract
solution.
863
500
500
ml
ml
* Solution A
Yeast extract
(NH4)2SO4
MgSO4·7H2O
CaCl2·2H2O
KH2PO4
Distilled water
20
g
0.14 g
1
g
1
g
3.4
g
4.2
g
0.33 g
0.1
g
0.2
g
0.2
g
0.2
g
10
ml
10
ml
0.1
g
1
mg
0.5
g
1
L
1
g
0.2
g
0.5
g
0.25 g
0.6
g
500
ml
pH 2.5 - 3.0
** Solution B
Glucose
Agar (if needed)
Distilled water
1
g
20
g
500
ml
pH 5.5 - 6.0
Solutions A and B are separately sterilized by autoclaving
and mixed.
865
Haroarcula Medium
Casamino Acids (Difco)
Bacto Yeast Extract (Difco)
Sodium glutamate
Trisodium citrate
MgSO4·7H2O
KCl
NaCl
FeCl2·4H2O
MnCl2·4H2O
Distilled water
Agar (if needed)
Mix ingredients except vitamin solution, NaHCO3 and
Na2S·9H2O, and adjust pH to 7.5 - 8.0 with HCl. Dispense
the medium into suitable culture vessels under a stream
of H2/CO2 (80/20) and seal with butyl rubber stoppers.
Separately autoclave Na2S·9H2O (5% solution) under a
N2 atmosphere. Sterilize vitamin and NaHCO3 solutions
by filtration. Prior to inoculation, aseptically and
anaerobically add vitamin, NaHCO3 and Na2S·9H2O
solutions, and pressurize the inoculated vessels to 150
kPa with H2/CO2 (80/20).
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
M e d i u m f o r A l i c y c l o b a c il l u s a c i d i p h i l u s
Solution A*
Solution B**
Modified MMJY Medium
NaCl
K2HPO4
CaCl2·2H2O
NaNO3
MgSO4·7H2O
MgCl2·6H2O
KCl
Bacto Yeast Extract (Difco)
Sodium pyruvate
Sodium lactate
Sodium acetate
Trace elements solution*
Vitamin solution**
NaHCO3
Resazurin
Na2S·9H2O
Distilled water
2
2
10
5
5
5
5
1
0.01
1
866
12.8
g
1.35 g
0.1
g
0.024 g
0.1
g
0.1
g
0.025 g
0.01 g
0.024 g
1
g
0.12 g
0.8 mg
0.8 mg
4
mg
1
L
Thermus Nutrient Medium
Polypeptone
Bacto Yeast Extract (Difco)
NaCl
CaCl2·2H2O
MgCl2·6H2O
Distilled water
867
8
g
4
g
2
g
0.05 g
0.08 g
1
L
Adjust pH to 7.5 with NaOH
Thermus Medium
Polypeptone
Bacto Yeast Extract (Difco)
NaCl
Distilled water
977
5
g
5
g
1
g
3
g
20
g
2
g
200
g
36
mg
0.26 mg
1
L
20
g
pH 7.0-7.2
8
g
4
g
2
g
1
L
Adjust pH to 7.2 with NaOH
Media
*** Vitamin mixture
For solid medium, equal volumes of separately
autoclaved 1.6% gelrite and double-strength liquid
medium containing 2 g/L MgSO4·7H2O are mixed.
868
4-Aminobenzoic acid
D(+)-Biotin
Nicotinic acid
Calcium D(+)-panthothenate
Pyridoxine dihydrochloride
Thiamine-HCl
Distilled water
1/5 Marine Broth + 0.5% Sodium Pyruvate
Bacto Marine Broth 2216 (Difco)
Sodium pyruvate
Agar (if needed)
Sea water*
Distilled water
7.5
g
5
g
15
g
800
ml
200
ml
pH 7.6
Cyanocobalamin
Distilled water
84
1
g
L
***** Bicarbonate solution (1.0 M)
0.2
0.25
20
3
0.5
0.15
1
1
1
1
30
20
1
g
g
g
g
g
g
ml
ml
ml
ml
ml
mg
L
NaHCO3
Distilled water
Sterilized by filtration.
870
ml
mg
mg
mg
mg
mg
mg
mg
mg
ml
0.4
6
8
1
g
mg
mg
L
4
0.2
0.25
20
3
0.5
0.15
1
1
1
1
30
24
1
g
g
g
g
g
g
g
ml
ml
ml
ml
ml
ml
L
Mix ingredients except vitamin mixture, vitamin B12
solution, bicarbonate solution and sulfide solution, and
adjust pH to about 7.5. Dispense the medium into suitable
culture vessels under a stream of nitrogen gas, seal with
butyl rubber stoppers, and autoclave at 121°C for 15 min.
Aseptically and anaerobically add vitamin mixture,
vitamin B12 solution, bicarbonate solution and sulfide
solution using sterile syringes, and the N2/CO2 (80/20)
mixed gas is enclosed with the vessels. Adjust pH to 7.27.8 using compact pH meter. An aqueous solution of
sodium lactate (final concentration, 20 mM) is added as
the electron donor and carbon source before inoculation.
Addition of yeast extract (0.5 - 1 g/L) greatly improves the
growth of the strain.
* Trace element mixture
12.5
2100
30
100
190
24
2
144
36
987
Medium for Strain C/L2
Na2SO4
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element mixture*
Selenite-tungstate solution**
Vitamin mixture***
Vitamin B12 solution****
Bicarbonate solution (1.0 M)*****
Sulfide solution******
Distilled water
Mix ingredients except vitamin mixture, vitamin B12
solution, bicarbonate solution and dithionite, and adjust
pH to about 7.5. Dispense the medium into suitable
culture vessels under a stream of nitrogen gas, seal with
butyl rubber stoppers, and autoclave at 121°C for 15 min.
Aseptically and anaerobically add vitamin mixture,
vitamin B12 solution, bicarbonate solution and dithionite
solution using sterile syringes, and the N2/CO2 (80/20)
mixed gas is enclosed with the vessels. Adjust pH to 7.27.8 using compact pH meter. An aqueous solution of
sodium glutamate (final concentration, 10 mM) and
sodium thiosulfate (final concentration, 20 mM) are added
before inoculation. Addition of yeast extract (0.5 - 1 g/L)
greatly improves the growth of the strain.
* The trace element mixture
HCl (25%)
FeSO4·7H2O
H3BO3
MnCl2·4H2O
CoCl2·6H2O
NiCl2·6H2O
CuCl2·2H2O
ZnSO4·7H2O
Na2MoO4·2H2O
Distilled water
Sterilized by autoclaving.
** Selenite-tungstate solution
NaOH
Na2SeO·5H2O
Na2WO4·2H2O
Distilled water
mg
ml
Sterilized by filtration.
Medium for Strain C/G2
HCl (25%)
FeSO4·7H2O
H3BO3
MnCl2·4H2O
CoCl2·6H2O
NiCl2·6H2O
CuCl2·2H2O
ZnSO4·7H2O
Na2MoO4·2H2O
Distilled water
5
100
**** Vitamin B12 solution
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element mixture*
Selenite-tungstate solution**
Vitamin mixture***
Vitamin B12 solution****
Bicarbonate solution (1.0 M)*****
Dithionite (ca.)
Distilled water
mg
mg
mg
mg
mg
mg
ml
Sterilized by filtration.
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**)
869
4
1
10
5
15
10
100
Sterilized by autoclaving.
Sterilized by autoclaving.
978
12.5
2100
30
100
190
24
2
144
36
987
ml
mg
mg
mg
mg
mg
mg
mg
mg
ml
Media
874
** The selenite-tungstate solution
NaOH
Na2SeO·5H2O
Na2WO4·2H2O
distilled water
0.4
6
8
1
g
mg
mg
L
(NH4)2SO4
K2HPO4
MgSO4·7H2O
KCl
Ca(NO3)2
NaCl
Sulfur (powder)*
Distilled water
Sterilized by autoclaving.
*** The vitamin mixture
4-Aminobenzoic acid
D(+)-Biotin
Nicotinic acid
Calcium D(+)-panthothenate
Pyridoxine dihydrochloride
Thiamine-HCl
Distilled water
4
1
10
5
15
10
100
mg
mg
mg
mg
mg
mg
ml
875
mg
ml
876
g
L
877
******Neutralized sulfide solution
1.5
100
g
ml
Tryptic Soy Broth
Bacto Tryptic Soy Broth (Difco)
Distilled water
872
30
g
1
L
pH unadjusted
40
g
1
L
pH unadjusted
873
Skim milk
Tomato juice*
Yeast extract
Distilled water, make up to
100
100
5
1
5
ml
0.75 g
0.15 g
0.54 g
30
g
2.4
g
2.4
g
0.8
g
1.1
g
0.5
g
2
mg
2
ml
2
ml
1
mg
1
g
0.36 g
1
L
pH unadjusted
Mix ingredients except KP buffer, vitamin solution,
Na2CO3 and Na2S·9H2O. Dispense the medium into
suitable culture vessels under a stream of H2/CO2 (80/20)
and seal with butyl rubber stoppers. Separately autoclave
KP buffer and Na2S·9H2O (5% solution) under a N2
atmosphere. Sterilize vitamin and 10% Na2CO3 solutions
by filtration. Prior to inoculation, aseptically and
anaerobically add KP buffer, vitamin, Na2CO3 and
Na2S·9H2O solutions. Pressurize the inoculated vessels
to 150 kPa with H2/CO2 (80/20).
Tryptic Soy Agar
Bacto Tryptic Soy Agar (Difco)
Distilled water
g
g
L
g
A r c h a e o g l o b u s p r o f u n du s M e d i u m
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
NaCl
Na2SO4
Na2S2O3·5H2O
Sodium acetate
Sodium pyruvate
Bacto Yeast Extract (Difco)
Fe(NH4)2(SO4)2·7H2O
Trace elements solution**
Vitamin solution***
Resazurin
Na2CO3
Na2S·9H2O
Distilled water
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
871
2
10
1
15
Adjust pH 5 after autoclaving.
Sterilized by filtration.
Na2S·9H2O
Distilled water
Yeast-Starch Agar (pH 5)
Yeast extract
Soluble starch
Distilled water
Agar
***** The bicarbonate solution (1.0 M)
84
1
37.25 g
950
ml
After sterilization by autoclaving at 121°C for 15 min,
aseptically add 50 ml of sterile lactose solution (10% w/v).
Sterilized by filtration.
NaHCO3
Distilled water
g
g
g
g
g
g
g
L
pH 4
M17 Broth
M17 Broth (OXOID)
Distilled water
**** The vitamin B12 solution
5
100
3
5
5
1
0.3
20
10
1
* Sterilize separately by autoclaving for 60 min at 105℃ on
each of 3 successive days and add after inoculation.
Sterilized by filtration.
Cyanocobalamin
Distilled water
Marine Acidophilic Sulfur-oxidizing
Bacteria Medium
g
ml
g
L
* KP buffer
KH2PO4
K2HPO4
Distilled water
Autoclave at 121℃ for 15 minutes.
* Filter canned tomatoes through paper. Leave overnight at
10℃. Adjust pH to 7.0.
Autoclaved under N2.
979
119
21
1
g
g
L
Media
** Vitamin solution
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
L
880
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
878
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
0.75 g
0.75 g
1
g
0.36 g
0.1
g
0.1
g
10
ml
10
ml
1
mg
4.8
g
0.5
g
0.5
g
1
L
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
Mix ingredients except for vitamin solution, cysteine-HCl
and Na2S·9H2O, and autoclave under H2/CO2 (80/20)
condition. Separately autoclave cysteine-HCl and
Na2S·9H2O as a 5% solution under a N2 atmosphere.
Aseptically and anaerobically add filter-sterile vitamin
solution, and cysteine-HCl and Na2S·9H2O solutions, and
pressurize inoculated bottles to 150 kPa with H2/CO2.
4.1
0.36
0.09
0.15
0.018
0.09
0.017
0.009
0.018
1
4.1
0.36
0.09
0.15
0.018
0.09
0.017
0.009
0.018
1
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
0.25 g
0.34 g
4
g
3.5
g
0.14 g
0.14 g
6
g
10
ml
10
ml
2
g
2
g
1
g
1
mg
5
g
0.5
g
0.5
g
1
L
Mix ingredients except for vitamin solution, NaHCO3,
cysteine-HCl and Na2S·9H2O, and autoclave under
H2/CO2 (80/20) condition. Separately autoclave
cysteine-HCl and Na2S·9H2O as a 5% solution under a N2
atmosphere. Aseptically and anaerobically add
filter-sterile vitamin and NaHCO3 solutions, and
cysteine-HCl and Na2S·9H2O solutions, and pressurize
inoculated bottles to 150 kPa with H2/CO2.
Methanobacterium Medium
KH2PO4
K2HPO4
NH4Cl
MgCl2·6H2O
Bacto Yeast Extract (Difco)
Polypeptone
Trace elements solution*
Vitamin solution
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Methanoculleus Medium
NH4Cl
KCl
MgCl2·6H2O
MgSO4·7H2O
CaCl2·2H2O
K2HPO4
NaCl
Trace elements solution*
Vitamin solution**
Bacto Yeast Extract (Difco)
Polypeptone
Sodium acetate
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
2
2
10
5
5
5
5
1
0.01
1
** Vitamin solution
g
g
g
g
g
g
g
g
g
L
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
980
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
881
GYP Medium (pH 6)
Glucose
Bacto Yeast Extract (Difco)
Polypepton*
Sodium acetate
Tween 80 solution**
Salts solution***
Distilled water
** Vitamin solution
10
10
5
2
10
5
1
g
g
g
g
ml
ml
L
pH 6.0
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
Add 5 g/L CaCO3 and 15 g/L agar after the pH is adjusted
for the solid medium.
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
883
Tween 80
Distilled water
50
1
mg
ml
40
2
2
2
1
mg
mg
mg
mg
ml
*** Salts solution
MgSO4·7H2O
MnSO4·4H2O
FeSO4·7H2O
NaCl
Distilled water
884
0.535 g
0.136 g
0.204 g
0.147 g
1
ml
1
ml
0.82 g
0.85 g
2.52 g
1
mg
0.5
g
1
L
20
0.5
15
1
g
g
g
L
* Sterilize separately by autoclaving and add to other
ingredients. The final pH is around 8.0.
885
Nutrient Broth + 0.5%(w/v) Na2CO3
Bacto Nutrient Broth (Difco)
Na2CO3*
Agar (if needed)
Distilled water
8
5
15
1
g
g
g
L
* Sterilize separately by autoclaving and add to other
ingredients. The final pH is around 10.5.
886
Allantoin Mineral Medium
K2HPO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeSO4·7H2O
MnSO4·H2O
Allantoin
Agar
Distilled water
* Trace elements solution
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
LB Broth + 0.05%(w/v) Na2CO3
Bacto LB Broth, Lennox (Difco)
Na2CO3*
Agar (if needed)
Distilled water
Mix ingredients except for vitamin solution and
Na2S·9H2O, and autoclave under H2/CO2 (80/20)
condition. Separately autoclave Na2S·9H2O as a 5%
solution under a N2 atmosphere. Aseptically and
anaerobically add filter-sterile vitamin solution and
Na2S·9H2O solution, and pressurize inoculated bottles to
150 kPa with H2/CO2.
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
g
g
g
L
** Sterilize separately by autoclaving and add to other
ingredients. The final pH is around 8.5.
D e f e r r i b a c t er M e d i u m
NH4Cl
KH2PO4
MgCl2·6H2O
CaCl2·2H2O
Trace elements solution*
Vitamin solution**
Sodium acetate
Sodium nitrate
NaHCO3
Resazurin
Na2S·9H2O
Distilled water
30
2
15
1
* Becton Dickinson and Company, Cockeysville, MD USA.
Add a drop of 12 N HCl to 500 ml of the salts solution.
882
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
TSB + 0.2%(w/v) Na2CO3
Bacto Tryptic Soy Broth*
Na2CO3**
Agar (if needed)
Distilled water
** Tween 80 solution
2
2
10
5
5
5
5
1
0.01
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
887
Guaiacol-CMC Agar
Difco Yeast Carbon Base
Ammonium tartrate
CM-Cellulose
Agar
Guaiacol
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
981
0.8
g
0.2
g
0.5
g
0.05 g
0.01 g
1
mg
20
g
15
g
1
L
pH 7.2
11.7
g
0.2
g
0.1
g
15
g
0.1
g
1
L
pH 4.5
Media
889
Tre-P-Y Agar
Trehalose
Peptone
Bacto Yeast Extract (Difco)
Distilled water
Agar
890
897
20
3
1
1
15
g
g
g
L
g
pH 6.7
10
5
10
100
900
15
g
g
g
ml
ml
g
pH 7.0
LB + Ampicillin Medium
Bacto Tyrptone (Difco)
Yeast extract
NaCl
Sodium ampicillin solution (750 μg/ml)*
Distilled water
Agar (if needed)
Soybean Casein Digest Agar
Casein peptone
Soybean peptone
NaCl
Agar
Distilled water
892
893
15
g
5
g
5
g
15
g
1
L
pH 7.1-7.5
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
KAl(SO4)2·12H2O
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3
Na2WO4
KAl(SO4)2·12H2O
Distilled water
Soybean Casein Digest Broth
Casein peptone
Soybean peptone
K2HPO4
Glucose
NaCl
Distilled water
17
g
3
g
2.5
g
2.5
g
5
g
1
L
pH 7.1-7.5
2
1
15
1
g
g
g
L
pH 7.0
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**).
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
895
Solution A*
Solution B**
500
500
ml
ml
898
* Solution A
Yeast extract
Malt extract
Glucose
Distilled water
1
g
2.5
g
1
g
500
ml
pH 4 (by H2SO4)
899
18
500
Solutions A and B are separately sterilized by autoclaving
and mixed.
982
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
4.6
14
1
g
g
L
GYP Medium
Glucose
Bacto Yeast Extract (Difco)
Polypepton*
Sodium acetate
Tween 80 solution**
Salts solution***
Distilled water
g
ml
2
2
10
5
5
5
5
1
0.01
1
1/5 Nutrient Agar
Bacto Nutrient Agar (Difco)
Agar
Distilled water
** Solution II
Agar
Distilled water
4.1
g
0.36 g
0.09 g
0.15 g
0.018 g
0.09 g
0.017 g
0.02 g
0.009 g
0.018 g
1.6 mg
1.6 mg
8
mg
1
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1/5 LBM Medium
Bacto Tryptone (Difco)
Bacto Yeast Extract (Difco)
Agar (if needed)
Seawater*
6.56 g
1
g
0.3
g
0.6
g
0.1
g
0.08 g
5
ml
10
ml
1
mg
3.5
g
0.5
g
0.5
g
1
L
Mix ingredients except for vitamin solution, cysteine-HCl
and Na2S·9H2O, and autoclave under N2/CO2 (80/20)
condition. Separately autoclave cysteine-HCl and
Na2S·9H2O as a 5% solution under a N2 atmosphere.
Aseptically and anaerobically add filter-sterile vitamin,
cysteine-HCl and Na2S·9H2O solutions.
* Sterilize separately by filtration.
891
M e t h a n o t h r ix M e d i u m
Sodium acetate
NH4Cl
KH2PO4
NaCl
MgCl2·6H2O
CaCl2·2H2O
Trace elements solution*
Vitamin solution**
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
10
10
5
2
10
5
1
g
g
g
g
ml
ml
L
pH 6.8
Media
** Trace element solution
Add 5 g/L CaCO3 and 15 g/L agar after the pH is adjusted
for the solid medium.
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO3·5H2O
Distilled water
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Tween 80 solution
Tween 80
Distilled Water
50
1
mg
ml
40
2
2
2
1
mg
mg
mg
mg
ml
*** Salts solution
MgSO4·7H2O
MnSO4·4H2O
FeSO4·7H2O
NaCl
Distilled Water
901
Add a drop of 12 N HCl to 500 ml of the salts solution.
900
0.78 g
0.75 g
0.04 g
0.01 g
0.25 g
0.03 g
0.2
g
0.5
g
5
g
0.8
g
0.5
g
0.5
g
10
ml
10
ml
0.5
g
1
L
10
ml
0.75 g
0.15 g
0.54 g
3.2
g
0.1
g
2
ml
2
ml
0.5
g
1
L
Mix ingredients except KP buffer and vitamin solution.
Dispense the medium into suitable culture vessels under
a stream of N2/CO2/O2 (75/20/5) and seal with butyl
rubber stoppers. Separately autoclave KP buffer under a
N2 atmosphere. Sterilize vitamin solution by filtration.
Prior to inoculation, aseptically add KP buffer and vitamin
solution. Pressurize the inoculated vessels to 150 kPa
with N2/CO2/O2.
Mix ingredients except vitamin solution and cysteine-HCl.
Dispense the medium into suitable culture vessels under
a stream of N2/CO2 (80/20), seal with butyl rubber
stoppers and autoclave. Separately autoclave 5%
cysteine-HCl solution under a N2 atmosphere. Sterilize
vitamin solution by filtration. Prior to inoculation,
aseptically add vitamin and cysteine-HCl solutions.
* KP buffer
KH2PO4
K2HPO4
Distilled water
119
21
1
g
g
L
Autoclaved under N2.
** Trace elements solution
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
L
Sulfur-oxidizing Bacteria Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2S2O3
Bacto Yeast Extract (Difco)
Trace element solution**
Vitamin solution***
Na2CO3
Distilled water
Thermanaeromonas Medium
K2HPO4
KH2PO4
3Na-EDTA
FeSO4·7H2O
MgSO4·7H2O
CaCl2·2H2O
NaCl
NH4Cl
NaHCO3
Na2S2O3
Glucose
Bacto Yeast Extract (Difco)
Vitamin solution*
Trace element solution**
Cysteine-HCl
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.026
1
2
2
10
5
5
5
5
0.1
5
5
1
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
983
Media
** Vitamin solution
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
902
2
2
10
5
5
5
5
1
0.01
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Reinforced Clostridial Medium
CM0149 Broth (OXOID)
Distilled water
Agar (if needed)
38
1
15
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
NaCl
Agar (if needed)
Distilled water
TcP Medium
NaCl
KCl
NH4Cl
MgCl2·6H2O
MgSO4·7H2O
FeSO4·7H2O
K2HPO4
NaBr
Trace minerals*
Vitamin solution**
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
PIPES
Sulfur (powder)
Resazurin
Na2S·9H2O
Distilled water
30
g
0.33 g
0.25 g
2.8
g
3.4
g
0.025 g
0.3
g
0.01 g
10
ml
10
ml
3
g
3
g
6
g
10
g
1
mg
0.5
g
1
L
905
M Medium (EBR45)
KH2PO4
K2HPO4·3H2O
NH4Cl
MgCl2·6H2O
Yeast extract
NaHCO3
Cysteine-HCl
Na2S·9H2O
Resazurin
Cellulose (or Cellobiose)
Vitamin solution*
Mineral solution**
Distilled water
0.4
g
0.4
g
1
g
0.1
g
0.2
g
6
g
0.5
g
0.25 g
1
mg
10
g
10
ml
10
ml
1
L
pH 7.5
Mix ingredients except NaHCO3, cysteine-HCl,
Na2S·9H2O and vitamin solution, distribute the medium
into culture vessels under a stream of nitrogen gas, and
seal with butyl rubber stoppers. After autoclaving (121°C,
15 min), aseptically inject other ingredients. The pH is
adjusted to 7.5 by adding of sterile 0.6 N H2SO4 with a
syringe without opening the vessel.
* Trace minerals
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
5
g
5
g
5
g
1
g
10
g
15
g
1
L
pH 7.0 - 7.5
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Mix ingredients except vitamin solution, sulfur and
Na2S·9H2O, and adjust pH to 7. Autoclave under a N2
atmosphere. Filter-sterilize vitamin solution. Sulfur is
sterilized separately by autoclaving for 60 min at 105℃
on each successive days. Neutralize Na2S·9H2O as 5%
solution and autoclave under a N2 atmosphere. Prior to
inoculation, aseptically and anaerobically add vitamin,
sulfur and Na2S·9H2O.
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
904
g
L
g
Autoclave at 121℃ for 15 minutes.
903
2
2
10
5
5
5
5
0.1
5
5
1
g
g
g
g
g
g
g
g
g
g
g
g
L
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH solution. Then proceed to add minerals. Adjust final
pH to 7.0.
984
2
2
10
5
5
5
5
1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
* Trace elements solution
** Mineral solution
Nitrilotriacetic acid (NTA)
FeCl2·4H2O
CoCl2·6H2O
AlK(SO4)2
NaCl
CaCl2
Na2MoO4·2H2O
MnCl2·4H2O
ZnCl2
H3BO3
CuSO4·5H2O
NiCl2·6H2O
Distilled water
906
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
909
Glucose
Peptone
Yeast extract
Monosodium glutamate
KH2PO4
(NH4)2SO4
MgSO4·7H2O
Distilled water
Agar
g
g
g
g
L
g
0.14 g
0.54 g
0.2
g
0.15 g
0.1
g
1
g
1
ml
2
ml
1
mg
2.5
g
0.3
g
0.3
g
1
L
910
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
0.5
g
0.5
g
0.5
g
0.5
g
0.44 g
0.1
g
0.1
g
1
L
15
g
Adjust pH to 7.0
GS Medium
KCl
KH2PO4
MgCl2·6H2O
MgSO4·7H2O
NH4Cl
CaCl2·2H2O
NaCl
Fe(NH4)2(SO4)2·6H2O
S medium trace mineral solution*
Trace vitamins**
NaHCO3
Bacto Yeast Extract (Difco)
Trypticase Peptone (BBL)
Glucose
Na2S-cysteine solution***
Resazurin
Distilled water
Mix ingredients except for vitamin solution, cysteine-HCl
and Na2S·9H2O, and autoclave under H2/CO2 (80/20)
condition. Separately autoclave cysteine-HCl and
Na2S·9H2O as a 6% solution under a N2 atmosphere.
Aseptically and anaerobically add filter-sterile vitamin
solution, and cysteine-HCl and Na2S·9H2O solutions, and
pressurize inoculated bottles to 150 kPa with H2/CO2.
2
2
10
5
5
5
5
1
0.01
1
M i c r o l u n a t us M e d i u m
Medium for Strain SANAE
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Sodium acetate
Trace elements solution*
Vitamin solution
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
g
g
g
g
g
g
g
g
g
g
g
L
** Vitamin solution
10
g
8
g
4
g
20
g
1
ml
2
g
5
g
2
g
0.2
g
0.05 g
80
g
1
L
15
g
pH 6.0 - 6.4
1
1
1
1
1
15
4.1
0.36
0.09
0.15
0.018
0.09
0.017
0.009
0.018
0.004
0.004
1
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
ChrysPYG
Chrysalis extract
Proteose peptone
Yeast extract
Glucose
Distilled water
Agar
908
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
L
MRS+8% NaCl
Peptone
Lab-Lemco powder
Yeast extract
Glucose
Sorbitan mono-oleate
K2HPO4
Sodium acetate
Triammonium citrate
MgSO4·7H2O
MnSO4·4H2O
NaCl
Distilled water
Agar (if needed)
907
4.5
0.4
0.12
0.01
1
0.02
0.01
0.1
0.1
0.01
0.01
0.02
1
0.355 g
0.14 g
4
g
3.45 g
0.25 g
0.14 g
6
g
2
mg
10
ml
10
ml
5
g
2
g
2
g
1.8
g
10
ml
1
mg
970
ml
pH 7.0
Mix ingredients except for trace vitamins and
Na2S-cysteine solution, and autoclave under N2/CO2
(80/20) condition. Separately autoclave Na2S-cysteine
solution under a N2 atmosphere. Aseptically and
anaerobically add filter-sterile trace vitamins and
Na2S-cysteine solution.
985
Media
Mix ingredients except KP buffer, vitamin solution,
Na2CO3, Na2S·9H2O and cysteine-HCl. Dispense the
medium into suitable culture vessels under a stream of
H2/CO2 (80/20) and seal with butyl rubber stoppers.
Separately autoclave KP buffer, Na2S·9H2O (5% solution)
and cysteine-HCl (5% solution) under a N2 atmosphere.
Sterilize vitamin and 10% Na2CO3 solutions by filtration.
Prior to inoculation, aseptically and anaerobically add KP
buffer, vitamin, Na2CO3 and Na2S·9H2O solutions.
Pressurize the inoculated vessels to 150 kPa with
H2/CO2.
* S medium trace mineral solution
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·2H2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
(NH4)2Ni(SO4)2·6H2O
Na2WO4·2H2O
Distilled water
1.5
g
3
g
0.5
g
1
g
0.1
g
0.1
g
0.1
g
0.1
g
0.01 g
0.01 g
0.01 g
0.01 g
0.025 g
0.3
g
2
g
10
mg
1
L
* KP buffer
KH2PO4
K2HPO4
Distilled water
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
** Trace vitamins
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
5
5
100
g
g
ml
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
Marine H2-using Methanogen Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2SO4
NaCl
Bacto Yeast Extract (Difco)
Sodium acetate
Trace elements solution**
Vitamin solution***
Resazurin
Na2CO3
Na2S·9H2O
Cysteine-HCl
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
*** Vitamin solution
The gas phase is replaced with N2.
911
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
*** Na2S-cysteine solution
Cysteine-HCl
Na2S·9H2O
Distilled water
g
g
L
Autoclaved under N2.
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH, then add other minerals. Final pH 7.0 (with KOH).
Sterilize by filtration.
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
53
106
1
5
ml
3.05 g
0.15 g
0.54 g
0.3
g
30
g
0.02 g
0.8
g
2
ml
2
ml
1
mg
1.5
g
0.5
g
0.5
g
1
L
pH unadjusted
986
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
912
Methanogenium Medium
KCl
MgCl2·6H2O
MgSO4·7H2O
NH4Cl
CaCl2·2H2O
KH2PO4
NaCl
Trace element solution*
Vitamin solution**
Fe(NH4)2(SO4)2·6H2O
NaHCO3
Sodium acetate
Bacto Yeast Extract (Difco)
Trypticase Peptone (BBL)
Na2S·9H2O
Cysteine-HCl
Distilled water
913
0.355 g
4
g
3.45 g
0.25 g
0.14 g
0.14 g
18
g
10
ml
10
ml
2
mg
5
g
1
g
2
g
2
g
0.5
g
0.5
g
1
L
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Distilled water
Agar (if needed)
g
g
g
g
g
mg
g
g
L
5
g
3
g
0.87 g
1
L
15
g
pH 7.0
915
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Rifampicin solution (500 μg/ml)*
Spectinomycin solution (500 μg/ml)*
Distilled water
Agar (if needed)
* Trace element solution
5
g
3
g
0.87 g
100
ml
100
ml
800
ml
15
g
pH 7.0
* Sterilize separately by filtration.
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
0.025
0.3
2
0.01
1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
916
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Spectinomycin solution (500 μg/ml)*
Distilled water
Agar (if needed)
917
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Kanamycin solution (500 μg/ml)*
Rifampicin solution (500 μg/ml)*
Distilled water
Agar (if needed)
** Vitamin solution
2
2
10
5
5
5
5
1
0.01
1
5
g
3
g
0.87 g
100
ml
900
ml
15
g
pH 7.0
* Sterilize separately by filtration.
Dissolve nitrilotriacetic acid and adjust pH to 6.5 with
KOH. Then add minerals. Adjust final pH to 7.0.
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
1
2
1
0.5
0.5
10
30
15
1
914
Mix ingredients except NaHCO3, Na2S·9H2O and
cysteine-HCl, and adjust pH to 6.5. Bring to a boil for 5-10
seconds, then cool under a stream of H2/CO2 (80/20) and
add NaHCO3 to the medium. After autoclaving under a
H2/CO2 atmosphere, stand the medium overnight.
Separately autoclave cysteine-HCl and Na2S·9H2O as 5%
solutions under a N2 atmosphere. Prior to inoculation,
aseptically and anaerobically add the cysteine-HCl and
Na2S·9H2O solutions. Pressurize the inoculated bottles to
200 kPa with H2/CO2.
Nitrilotriacetic acid
MgSO4·7H2O
MnSO4·xH2O
NaCl
FeSO4·7H2O
CoSO4·7H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4·5H2O
AlK(SO4)2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3·5H2O
(NH4)2Ni(SO4)2·6H2O
Na2WO4·2H2O
Distilled water
Casamino Acids-Peptone-Czapek’s Agar
Casamino Acids (Difco)
Bacto Peptone (Difco)
K2HPO4
KCl
MgSO4·7H2O
FeSO4·7H2O
Sucrose
Agar
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
5
g
3
g
0.87 g
100
ml
100
ml
800
ml
15
g
pH 7.0
* Sterilize separately by filtration.
918
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Kanamycin solution (500 μg/ml)*
Rifampicin solution (500 μg/ml)*
Spectinomycin solution (500 μg/ml)*
Distilled water
Agar (if needed)
* Sterilize separately by filtration.
987
5
g
3
g
0.87 g
100
ml
100
ml
100
ml
700
ml
15
g
pH 7.0
Media
919
** Vitamin solution
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Rifampicin solution (500 μg/ml)*
Distilled water
Agar (if needed)
5
g
3
g
0.87 g
100
ml
900
ml
15
g
pH 7.0
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
* Sterilize separately by filtration.
920
Bacto Tryptone (Difco)
Yeast extract
CaCl2·2H2O
Kanamycin solution (500 μg/ml)*
Distilled water
Agar (if needed)
5
g
3
g
0.87 g
100
ml
900
ml
15
g
pH 7.0
923
922
O c e a n i t h e r m u s p r of u n d u s M e d i u m
KH2PO4
K2HPO4
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
NaHCO3
Trace elements solution*
Vitamin solution**
Yeast extract
Sucrose
NaNO3
NaCl
Distilled water
0.75 g
0.78 g
0.36 g
0.1
g
0.54 g
5
g
10
ml
10
ml
2
g
6.8
g
1.7
g
20
g
1
L
pH unadjusted
10
ml
0.75 g
0.15 g
0.54 g
3.2
g
0.1
g
1
g
0.2
g
0.2
g
2
ml
2
ml
0.5
g
1
L
Mix ingredients except KP buffer and vitamin solution.
Dispense the medium into suitable culture vessels under
a stream of H2/CO2/O2 (75/20/5) and seal with butyl
rubber stoppers. Separately autoclave KP buffer under a
N2 atmosphere. Sterilize vitamin solution by filtration.
Prior to inoculation, aseptically add KP buffer and vitamin
solution. Pressurize the inoculated vessels to 150 kPa
with H2/CO2/O2.
* KP buffer
KH2PO4
K2HPO4
Distilled water
Mix ingredients except sucrose, NaHCO3 and vitamin
solution. Dispense the medium into suitable culture
vessels under a stream of N2 and seal with butyl rubber
stoppers. After autoclaving, add filter-sterile sucrose,
NaHCO3 and vitamin solutions.
119
21
1
g
g
L
Autoclaved under N2.
** Trace elements solution
* Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Hydrogenobacter Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2S2O3
Bacto Yeast Extract (Difco)
Peptone (BBL)
Glucose
Sodium succinate
Trace elements solution**
Vitamin solution***
Na2CO3
Distilled water
* Sterilize separately by filtration.
2
2
10
5
5
5
5
1
0.01
1
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.025
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
988
Media
** Vitamin solution
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
924
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
1/4 YpSs
Soluble starch
Yeast extract
K2HPO4
MgSO4·7H2O
Agar
Distilled water
925
2
2
10
5
5
5
5
1
0.01
1
928
3.75
1
0.25
0.125
15
1
g
g
g
g
g
L
930
1.7
g
1
g
0.3
g
0.6
g
0.1
g
0.08 g
10
ml
10
ml
0.5
g
0.5
g
1
mg
4.5
g
0.3
g
0.3
g
1
L
39
1
g
L
0.5
g
0.15 g
0.2
g
0.15 g
0.5
g
0.86 g
2.5
g
0.5
g
0.5
g
1
ml
1
ml
1
mg
1
L
Mix ingredients except NaHCO3, cysteine-HCl,
Na2S·9H2O and vitamin solution, and autoclave under a
N2 atmosphere. Separately autoclave cysteine-HCl and
Na2S·9H2O as 5% solutions under a N2 atmosphere. After
adding filter-sterile NaHCO3 solution to the medium,
adjust pH to 7.0. Prior to inoculation, aseptically and
anaerobically add filter-sterile vitamin, cysteine-HCl and
Na2S·9H2O solutions.
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3
Na2WO4
Distilled water
g
g
g
L
g
pH 5.0
MPA Medium
NH4Cl
KH2PO4
MgCl2·6H2O
CaCl2·2H2O
Yeast extract
Crotonic acid
NaHCO3
Cysteine-HCl
Na2S·9H2O
Trace element solution*
Vitamin solution**
Resazurin
Distilled water
Mix ingredients except for vitamin solution, cysteine-HCl
and Na2S·9H2O, and autoclave under N2/CO2 (80/20)
condition. Separately autoclave cysteine-HCl and
Na2S·9H2O as a 3% solution under a N2 atmosphere.
Aseptically and anaerobically add filter-sterile vitamin,
cysteine-HCl and Na2S·9H2O solutions. Final pH is
7.2-7.5.
4
10
4
1
20
Potato Dextrose Agar
Nissui Potato Dextrose Agar
Distilled water
931
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Yeast Extract-Malt Extract Agar (ISP
Medium No. 2, pH 5.0)
Yeast extract
Malt extract
Glucose
Distilled water
Agar
Methanosaeta harundinacea Medium
Sodium acetate
NH4Cl
KH2PO4
NaCl
MgCl2·6H2O
CaCl2·2H2O
Trace elements solution*
Vitamin solution**
Bacto Yeast Extract (Difco)
Polypepton
Resazurin
KHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
2
2
10
5
5
5
5
1
0.01
1
4.1
g
0.36 g
0.09 g
0.15 g
0.018 g
0.09 g
0.017 g
0.009 g
0.018 g
1.6 mg
1.6 mg
1
L
* Trace element solution
FeCl2
CoCl2·6H2O
MnCl2·4H2O
ZnCl2
H3BO3
NiCl2·6H2O
AlCl3
Na2MoO4·2H2O
Na2SeO3·5H2O
Na2WO4·H2O
CuCl2·2H2O
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add other minerals. Final pH 7.0.
989
1.27 g
0.24 g
0.2
g
0.14 g
6.2 mg
0.024 g
0.013 g
0.024 g
2.6 mg
3
mg
1.7 mg
1
L
Media
* Vitamin solution
** Vitamin solution
Biotin
p-Aminobenzoic acid
Ca-pantothenate
Pyridoxine-HCl
Nicotinic acid
Thiamine-HCl
Lipoic acid
Folic acid
Vitamin B12
Riboflavin
Distilled water
932
5
5
5
5
5
5
5
2
0.1
5
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Nitrilotriacetic acid (NTA)
FeCl2·4H2O
CoCl2·6H2O
AlK(SO4)2
NaCl
CaCl2
Na2MoO4·2H2O
MnCl2·4H2O
ZnCl2
H3BO3
CuSO4·5H2O
NiCl2·6H2O
Distilled water
1.3
g
0.28 g
0.25 g
0.07 g
0.02 g
1.8 mg
4.5 mg
0.22 mg
0.05 mg
0.03 mg
0.03 mg
0.01 mg
1
g
1
g
1
L
pH 2.5
934
935
0.4
g
0.4
g
1
g
0.1
g
0.2
g
6
g
0.5
g
0.25 g
1
mg
10
g
10
ml
10
ml
1
L
pH 7.5
g
g
g
g
g
g
g
g
g
g
g
g
L
37.4
g
5
g
15
g
1
L
pH unadjusted
Alkaline Halophile Medium
KH2PO4
KCl
NH4Cl
MgSO4·7H2O
CaSO4·2H2O
NaCl
Sodium glutamate
Bacto Yeast Extract (Difco)
Casamino acids
Trace elements solution
Na2CO3
Agar (if necessary)
Distilled water
M Medium (EBR46, EBR599)
KH2PO4
K2HPO4·3H2O
NH4Cl
MgCl2·6H2O
Yeast extract
NaHCO3
Cysteine-HCl
Na2S·9H2O
Resazurin
Bacto Tryptone (Difco)
Vitamin solution*
Mineral solution**
Distilled water
4.5
0.4
0.12
0.01
1
0.02
0.01
0.1
0.1
0.01
0.01
0.02
1
Marine Broth 2216 + 0.5% Alginate
Bacto Marine Broth 2216 (Difco)
Sodium alginate
Agar (if needed)
Distilled water
Mix ingredients except Bacto Yeast Extract and sulfur,
adjust pH to 2.5 with H2SO4, and autoclave at 121°C for
15 min. Separately autoclave Bacto Yeast Extract as a
10% solution. Sulfur is sterilized separately by
autoclaving for 60 min at 105°C on each of 3 successive
days. Prior to inoculation, add Bacto Yeast Extract
solution and sulfur powder. Cultivate under air containing
1-10% CO2.
933
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Mineral solution
Sulfolobus metallicus Medium
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·nH2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Sulfur (powder)
Distilled water
2
2
10
5
5
5
5
1
5
5
1
1
g
1
g
1
g
0.24 g
0.17 g
200
g
1
g
5
g
5
g
1
ml
5
g
20
g
1
L
Mix ingredients except Na2CO3 and adjust pH to 6.5
before autoclaving. Add Na2CO3 as a filter-sterile 10%
solution after autoclaving. Check final pH to be 9.0-9.5. If
agar medium is prepared, dissolve agar by heating before
adding NaCl.
Mix ingredients except NaHCO3, cysteine-HCl,
Na2S·9H2O and vitamin solution, distribute the medium
into culture vessels under a stream of nitrogen gas, and
seal with butyl rubber stoppers. After autoclaving (121°C,
15 min), aseptically inject other ingredients. The pH is
adjusted to 7.5 by adding of sterile 0.6 N H2SO4 with a
syringe without opening the vessel.
990
Media
*** Vitamin solution
* Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.01
0.011
0.025
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
KH2PO4
K2HPO4
MgSO4·7H2O
CaSO4·2H2O
FeCl3·6H2O
Na2MoO4·2H2O
Yeast extract
Sucrose
Agar (if needed)
Distilled water
Hydrogen-oxidizing Bacteria Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2S2O3
Bacto Yeast Extract (Difco)
Trace elements solution**
Vitamin solution***
Na2CO3
Distilled water
10
ml
0.75 g
0.15 g
0.54 g
1.6
g
0.1
g
2
ml
2
ml
0.5
g
1
L
939
10
2
1
50
1
15
940
802 + 10% NaCl
Polypepton*
Yeast extract
MgSO4·7H2O
NaCl
Distilled water
Agar (if needed)
g
g
L
Autoclaved under N2.
10
2
1
100
1
15
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
g
g
g
g
L
g
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* KP buffer
119
21
1
0.2
g
0.8
g
0.2
g
0.1
g
10
mg
10
mg
0.5
g
20
g
15
g
1
L
pH 7.2
802 + 5% NaCl
Polypepton*
Yeast extract
MgSO4·7H2O
NaCl
Distilled water
Agar (if needed)
Mix ingredients except KP buffer and vitamin solution.
Dispense the medium into suitable culture vessels under
a stream of H2/CO2/O2 (75/20/5) and seal with butyl
rubber stoppers. Separately autoclave KP buffer under a
N2 atmosphere. Sterilize vitamin solution by filtration.
Prior to inoculation, aseptically add KP buffer and vitamin
solution. Pressurize the inoculated vessels to 150 kPa
with H2/CO2/O2.
KH2PO4
K2HPO4
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
937
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
936
2
2
10
5
5
5
5
1
0.01
1
g
g
g
g
L
g
pH 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
941
Methanolinea Medium
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Sodium acetate
Sodium formate
Trace elements solution*
Vitamin solution
Resazurin
NaHCO3
Cysteine-HCl
Na2S·9H2O
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
991
0.14 g
0.54 g
0.2
g
0.15 g
0.1
g
0.1
g
2.72 g
1
ml
2
ml
1
mg
2.5
g
0.3
g
0.3
g
1
L
Media
* Vitamin solution
Mix ingredients except for sodium formate, vitamin
solution, cysteine-HCl and Na2S·9H2O, and autoclave
under N2/CO2 (80/20) condition. Separately autoclave
cysteine-HCl and Na2S·9H2O as a 6% solution under a N2
atmosphere. Aseptically and anaerobically add
filter-sterile vitamin and neutralized 1.6 M formate
solutions, and cysteine-HCl and Na2S·9H2O solutions.
Biotin
Folic acid
Thiamine-HCl
Ca-pantothenate
Vitamin B12
Riboflavin
Distilled water
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
4.1
0.36
0.09
0.15
0.018
0.09
0.017
0.009
0.018
0.004
0.004
1
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
CaCl2·2H2O
(NH4)6Mo7O24·2H2O
FeSO4·7H2O
Metal salt solution 44***
Dissolve and neutralize the nitrilotriacetic acid with KOH
(7.3 g); add the other ingredients and adjust the pH to
6.6-6.8 before bringing the volume to 1.0 L with distilled
water.
*** Metal salt solution 44
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
2
2
10
5
5
5
5
1
0.01
1
Na-EDTA
ZnSO4·7H2O
FeSO4·7H2O
MnSO4·H2O
CuSO4·5H2O
Co(NO3)2·6H2O
Na2B4O7·10H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
g
g
g
g
ml
g
944
0.15 g
0.15 g
0.25 g
1
g
10
ml
20
ml
15
g
970
ml
pH unadjusted
945
10
2
1
1
900
15
250
1095
0.5
154
39.2
24.8
17.7
100
mg
mg
g
mg
mg
mg
mg
ml
Dissolve the EDTA and add a few drops of concentrated
H2SO4 to retard precipitation of the heavy metal ions.
Store in the dark and cold (5℃).
Modified 802 (pH 10.0)
Polypepton*
Yeast extract
MgSO4·7H2O
K2HPO4
Distilled water
Agar (if needed)
Peptone Yeast Extract Alkaline Medium
Bacto Peptone (Difco)
8
g
Yeast extract
3
g
K2HPO4
1
g
EDTA-2Na
3.5 mg
ZnSO4·7H2O
3
mg
FeSO4·7H2O
10
mg
MnSO4·nH2O
2
mg
CuSO4·5H2O
1
mg
Co(NO3)2·6H2O
2
mg
H3BO3
1
mg
Agar (if needed)
15
g
100 mM NaHCO3/100 mM Na2CO3 buffer (pH 10)
1
L
pH unadjusted
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
After autoclaving, aseptically add 100 ml of a 10%
Na2CO3 solution to the medium. Check final pH to be
about 10.0.
Modified Prosthecomicrobium and
A n c a l o m i c r ob i u m M e d i u m
Bacto Peptone (Difco)
Yeast extract
(NH4)2SO4
Glucose
Vitamin solution*
Modified Hutner's solution**
Agar (if needed)
Distilled water
10
g
29.7
g
3.34 g
9.25 mg
99
mg
50
ml
Adjust volume to 1 L with distilled water.
** Vitamin solution
943
mg
mg
mg
mg
mg
mg
L
** Modified Hutner's solution
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
942
2
2
5
5
0.1
5
1
Maltose-Yeast Extract Medium
Maltose
Yeast extract
KH2PO4
NaCl
Agar (if needed)
Distilled water
992
20
10
2
5
15
1
g
g
g
g
g
L
pH 6.0
Media
946
Tryptic Soy Medium with 10% Horse Serum
Bacto Tryptic Soy Broth (Difco)
Horse serum*
Agar (if needed)
Distilled water
954
30
g
100
ml
15
g
900
ml
pH unadjusted
955
* Heat inactivated at 56°C for 30 min.
947
37.4
g
5
ml
15
g
1
L
pH unadjusted
* Sterilize separately by filtration.
948
YSG Medium
Solution A*
Solution B**
500
500
ml
ml
* Solution A
Yeast extract
Distilled water
2
g
500
ml
Adjust pH to 3.7 with H2SO4
2
1
15
500
g
g
g
ml
Solutions A and B are separately sterilized by autoclaving
and mixed.
949
950
30
g
1
L
Adjust pH to 5.5
1/2MY
Malt Extract Agar (Nissui)
Yeast extract
Bacto Peptone (Difco)
Glucose
Agar
Distilled water
952
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
40
g
1
L
Adjust pH to 5.5
TSB (pH5.5)
Bacto Tryptic Soy Broth (Difco)
Distilled water
951
* Trace elements solution
TSA (pH 5.5)
Bacto Tryptic Soy Agar (Difco)
Distilled water
2.7
g
1.5
g
2.5
g
5
g
18.8
g
1
L
pH unadjusted
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
** Vitamin solution
LCA
Glucose
KH2PO4
MgSO4·7H2O
KCl
NaNO3
Yeast extract
Agar
Distilled water
0.355 g
4
g
3.45 g
0.25 g
0.14 g
0.14 g
2
mg
0.82 g
2
g
2
g
10
ml
10
ml
5
g
1
mg
0.5
g
0.5
g
1
L
Mix ingredients except vitamin solution, NaHCO3,
cysteine-HCl and Na2S·9H2O, and adjust pH to 6.5 with
NaOH. Dispense the medium into suitable culture vessels
under a stream of H2/CO2 (80/20) and seal with butyl
rubber stoppers. Separately autoclave cysteine-HCl and
Na2S·9H2O as 5% solutions under a N2 atmosphere.
Sterilize vitamin and 8% NaHCO3 solutions by filtration.
Prior to inoculation, aseptically and anaerobically add
vitamin, NaHCO3, cysteine-HCl and Na2S·9H2O solutions.
Pressurize the inoculated vessels to 150 kPa with H2/CO2.
Adjust pH to 6.8-7.0.
** Solution B
Soluble starch
Glucose
Agar (if needed)
Distilled water
13
g
1
L
15
g
pH unadjusted
Hydrogen-using Methanogen Medium
KCl
MgCl2·6H2O
MgSO4·7H2O
NH4Cl
CaCl2·2H2O
KH2PO4
Fe(NH4)2(SO4)2·6H2O
Sodium acetate
Bacto Yeast Extract (Difco)
Polypepton (Nihon seiyaku)
Trace elements solution*
Vitamin solution**
NaHCO3
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
Marine Broth with 0.5% Methanol
Bacto Marine Broth 2216 (Difco)
Methanol*
Agar (if needed)
Distilled water
Nutrient Agar/Broth
Nutrient Broth (OXOID)
Distilled water
Agar (if needed)
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
1
g
1
g
0.2
g
0.2
g
2
g
0.2
g
13
g
1
L
pH 6.5-7.0
993
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
957
Trypticase Soy Agar + 7% NaCl
Trypticase Soy (BBL)
NaCl
Distilled water
Agar
959
* P IV Metals solution
30
70
1
20
g
g
L
g
pH 7.3
FeCl3·6H2O
MnCl2·4H2O
ZnSO4·7H2O
CoCl2·6H2O
Na2MoO4·2H2O
Na2EDTA·2H2O
Distilled water
Medium-H
Glucose
Yeast extract
Monosodium glutamate
Agar (if needed)
Seawater
2
0.2
0.5
12
1
g
g
g
g
L
965
Agar
Distilled water
961
15
g
1
L
pH around 5.5
TSB (pH 9.0 with K2CO3)
Bacto Tryptic Soy Broth (Difco)
Distilled water
Agar (if needed)
30
900
15
H3BO3
MnCl2·4H2O
ZnSO4·7H2O
CuSO4·5H2O
MoO3
Distilled water
Matsuaria Medium
Chitosan
Yeast extract
Peptone
K2HPO4
KH2PO4
MgSO4·7H2O
Distilled water
963
10
g
0.25 g
0.25 g
0.25 g
0.7
g
0.3
g
1
L
pH 7.0
968
0.253 g
12
g
1
L
H3BO3
MnCl2·4H2O
ZnSO4·7H2O
CuSO4·5H2O
MoO3
Distilled water
AF6
NaNO3
NH4NO3
MgSO4·7H2O
KH2PO4
K2HPO4
CaCl2·2H2O
CaCO3
Fe-citrate
Citric acid
Biotin
Thiamine-HCl
Vitamin B6
Vitamin B12
P IV Metals solution*
MES
Agar (if needed)
Distilled water
140
22
30
10
5
10
10
2
2
2
10
1
1
5
400
12
1
1.32 g
0.27 g
0.25 g
0.11 g
0.02 g
0.03 g
1
ml
20
g
1
L
pH 2
* N1 Metals solution
* Wako Pure Chemical Industries, Ltd. (Product Code:
398-01333).
964
2.86 g
1.81 g
0.22 g
0.08 g
15
mg
1
L
KO2(NH4)
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·6H2O
FeSO4·7H2O
EDTA-2Na
N1 Metals solution*
Agar (if needed)
Distilled water
IMK (SW)
Daigo's IMK Medium*
Agar (if needed)
Seawater
1.7
g
0.27 g
0.25 g
0.11 g
0.02 g
0.03 g
1
ml
20
g
1
L
pH 2
* N1 Metals solution
g
ml
g
After autoclaving, aseptically add 100 ml of a 10% K2CO3
solution to the medium. Check final pH to be about 9.0.
962
mg
mg
mg
mg
mg
mg
L
KO2(NO3)
NaNO3
KH2PO4
MgSO4·7H2O
CaCl2·6H2O
FeSO4·7H2O
EDTA-2Na
N1 Metals solution*
Agar (if needed)
Distilled water
960
19.6
3.6
2.2
0.4
0.25
100
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
μg
μg
μg
ml
mg
g
L
pH 6.6
970
M Medium (EBR596)
KH2PO4
K2HPO4·3H2O
NH4Cl
MgCl2·6H2O
Yeast extract
NaHCO3
Cysteine-HCl
Na2S·9H2O
Resazurin
Cellobiose
NaCl
Vitamin solution*
Mineral solution**
Distilled water
994
2.86 g
1.81 g
0.22 g
0.08 g
15
mg
1
L
0.4
g
0.4
g
1
g
0.1
g
2
g
6
g
0.5
g
0.25 g
1
mg
5
g
10
g
10
ml
10
ml
1
L
pH 6.5
Media
NaHCO3
Na2CO3 (anhydrous)
Distilled water
Mix ingredients except NaHCO3, cysteine-HCl,
Na2S·9H2O and vitamin solution, distribute the medium
into culture vessels under a stream of nitrogen gas and
seal with butyl rubber stoppers. After autoclaving (121°C,
15 min), aseptically inject other ingredients. The pH is
adjusted to 6.5 by adding of sterile 0.6 N H2SO4 with a
syringe without opening the vessel.
2
2
10
5
5
5
5
1
5
5
1
974
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
971
4.5
0.4
0.12
0.01
1
0.02
0.01
0.1
0.1
0.01
0.01
0.02
1
g
g
g
g
g
g
g
g
g
g
g
g
L
* Trace element solution
4.4
g
4.8
g
1
g
0.5
g
10
ml
10
ml
980
ml
20
g
pH unadjusted
Nitrilotriacetic acid
MnCl2·4H2O
CoCl2·6H2O
ZnCl2
CuCl2·2H2O
AlCl3
H3BO3
Na2MoO4·2H2O
SrCl2·6H2O
NaBr
KI
FeCl2·4H2O
NiCl2·6H2O
Na2SeO3
Na2WO4·2H2O
Distilled water
Solution A and B are autoclaved separately and add to
other autoclaved ingredients after cooling.
* Solution A.
Ferric ammonium citrate
CaCl2
Distilled water
5
0.5
1
g
g
L
972
100
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
g
L
Nutrient Broth + 5% NaCl (pH 10.0)
Nutrient Broth (OXOID)
NaCl
Sodium-sesquicarbonate solution*
Distilled water
Agar (if needed)
0.15
0.53
0.42
90
7
6
10
1
10
10
10
0.1
25
0.2
0.3
1
g
g
g
mg
mg
mg
mg
mg
mg
mg
mg
g
mg
mg
mg
L
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Vitamin solution
** Solution B.
Sucrose
Distilled water
1
g
30
g
90
mg
70
mg
1.06 g
4.18 g
0.33 g
0.535 g
2
g
0.3
g
0.3
g
10
ml
10
ml
1
mg
1
L
pH 7
For vial cultures, dissolve all ingredients except for yeast
extract, vitamin solution, Na2S·9H2O and cysteine-HCl
(up to 900 ml), flush N2/CO2 (80/20) (pH 7), dispense 19
ml into 50 ml vials, seal with butyl rubber stoppers with
flushing N2/CO2 (80/20) and autoclave. Add 0.5 ml of
each of filter-sterilized Na2S·9H2O solution (0.3 g
Na2S·9H2O/50 ml) and cysteine-HCl solution (0.3 g
cysteine-HCl/50 ml) per each vial separately. Add 0.5 ml
of filter-sterilized 40 g/l yeast extract solution. Inoculum
size 5%. Growth was observed after 2 days of incubation.
P s e u d o m o n a s s a c c h a r op h i l a M e d i u m
KH2PO4
Na2HPO4
NH4Cl
MgSO4·7H2O
Solution A*
Solution B**
Distilled water
Agar (if needed)
Medium for Strain RASEN
Bacto Yeast Extract (Difco)
NaCl
K2HPO4
KH2PO4
CaCl2·2H2O
MgCl2·6H2O
KCl
NH4Cl
NaHCO3
Na2S·9H2O
Cysteine-HCl
Trace element solution*
Vitamin solution**
Resazurin
Distilled water
** Mineral solution
Nitrilotriacetic acid (NTA)
FeCl2·4H2O
CoCl2·6H2O
AlK(SO4)2
NaCl
CaCl2
Na2MoO4·2H2O
MnCl2·4H2O
ZnCl2
H3BO3
CuSO4·5H2O
NiCl2·6H2O
Distilled water
g
g
ml
Sterilize separately and add to other autoclaved
ingredients. Check final pH to be about 10.0.
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
4.2
5.3
100
13
50
100
900
15
g
g
ml
ml
g
* Sodium-sesquicarbonate solution
995
Media
975
Thermus Medium
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
CaSO4·2H2O
MgSO4·7H2O
NaCl
KNO3
NaNO3
Na2HPO4
Trace elements solution*
Agar (if needed)
Distilled water
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
1
g
1
g
0.06 g
0.1
g
0.008 g
0.1
g
0.69 g
0.11 g
10
ml
20
g
1
L
Adjust pH to 7.5 with NaOH
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
978
Sulfur-oxidizer Medium
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Na2S2O3
Trace elements solution**
Vitamin solution***
Na2CO3
Distilled water
10
ml
0.75 g
0.15 g
0.54 g
0.8
g
2
ml
2
ml
0.5
g
1
L
pH unadjusted
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Alkalibacterium
10
5
5
5
25
10
5
15
1
g
g
g
g
g
g
ml
g
L
pH 8.5
Sterilize by filtration.
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
** Salt solution
MgSO4·7H2O
MnSO4·4H2O
FeSO4·7H2O
Distilled water
* KP buffer
119
21
1
2
2
10
5
5
5
5
1
0.01
1
2.5% NaCl GYPFK Agar/Broth for
Glucose
Polypepton*
Yeast extract
Extract Bonito*
NaCl
K2HPO4
Salt solution**
Agar (if needed)
Distilled water
Mix ingredients except KP buffer and vitamin solution.
Dispense the medium into suitable culture vessels under
a stream of N2/CO2/O2 (60/20/20) and seal with butyl
rubber stoppers. Separately autoclave KP buffer under a
N2 atmosphere. Sterilize vitamin solution by filtration.
Prior to inoculation, aseptically and anaerobically add KP
buffer and vitamin solution. Pressurize the inoculated
vessels to 150 kPa with N2/CO2/O2 (60/20/20).
KH2PO4
K2HPO4
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
976
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
0.02
1
g
g
L
Autoclaved under N2.
** Trace elements solution
996
40
2
2
1
g
g
g
L
Media
979
C l o s t r i d i u m t h e r m o c e l l um M e d i u m
(NH4)2SO4
MgCl2·6H2O
KH2PO4
K2HPO4·3H2O
CaCl2·2H2O
Sodium glycerophosphate
FeSO4·7H2O
Glutathione
Yeast extract
Resazurin
Cellulose (Avicel or MN 300)
or Cellobiose
Distilled water
*** Artificial sea water
1.3
g
2.6
g
1.43 g
7.2
g
0.13 g
6
g
1.1 mg
0.25 g
4.5
g
1
mg
10
g
5
g
1
L
pH 7.0-7.2
NaCl
Na2SO4
MgCl2·6H2O
CaCl2
NaHCO3
KCl
KBr
H3BO3
SrCl2
NaF
Distilled water
**** Metal salt solution 44
Na-EDTA
ZnSO4·7H2O
FeSO4·7H2O
MnSO4·H2O
CuSO4·5H2O
Co(NO3)2·6H2O
Na2B4O7·10H2O
Distilled water
Mix ingredients except for cellobiose and autoclave under
H2/CO2 (95/5) condition. Aseptically and anaerobically
add filter-sterile 10% cellobiose solution before
inoculation.
980
S6 Medium
KH2PO4
Na2HPO4
(NH4)2SO4
MgSO4·7H2O
FeCl3·6H2O
MnSO4·xH2O
CaCl2·2H2O
Na2S2O3
Distilled water
1.8
g
1.2
g
0.1
g
0.1
g
30
mg
30
mg
40
mg
10
g
1
L
Adjust pH to 7
988
0.25
0.25
0.25
10
20
50
250
670
g
g
g
ml
ml
ml
ml
ml
0.1
2
5
5
2
5
5
1
mg
mg
mg
mg
mg
mg
mg
L
10
29.7
3.34
9.25
99
50
950
g
g
g
mg
mg
ml
ml
* Vitamin solution
* K solution
KH2PO4
NaCl
(NH4)2SO4
Distilled water
90
90
90
1
g
g
g
L
4
4
1
g
g
L
10.6
1
g
L
** M solution
MnCl2·4H2O
CoCl2·6H2O
Distilled water
** Hutner's basal salts
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
CaCl2·2H2O
(NH4)6MoO7O24·4H2O
FeSO4·7H2O
Metal salt solution 44****
Distilled water
2
g
2
g
5
g
0.2
g
10
ml
2.5 ml
2.5 ml
1
mg
20
ml
0.75 g
1
L
pH 7
Mix ingredients except for Na2CO3 and cysteine-HCl,
dispense the medium into suitable culture vessels under
a stream of N2/CO2 (80/20), seal with butyl rubber
stoppers, and autoclave at 115℃ for 25 min. Aseptically
and anaerobically add filter-sterile Na2CO3 and
cysteine-HCl solutions, and adjust pH to 7.0 with 3 N HCl.
Autoclave at 121°C for 15 min.
Vitamin B12
Biotin
Thiamine-HCl
Ca-pantothenate
Folic acid
Riboflavin
Nicotinamide
Distilled water
mg
mg
mg
mg
mg
mg
mg
L
TYG Medium
Trypticase Peptone (BBL)
Bacto Yeast Extract (Difco)
Glucose
Sodium n-hexanoate
K solution*
M solution**
C solution***
Resazurin
15% Na2CO3 solution
Cysteine-HCl
Distilled water
Verrucomicrobium Medium
Peptone
Yeast extract
Glucose
Vitamin solution*
Hutner's basal salts**
0.1 M Tris-HCl (pH 7.5)
Artificial sea water***
Distilled water
250
1095
500
154
39.2
24.8
17.7
1
Dissolve the EDTA and add a few drops of concentrated
H2SO4 to retard precipitation of the heavy metal ions.
Store in the dark and cold (5℃).
Separately sterilize 10% Na2S2O3 solution by filtration.
Aseptically add sterile Na2S2O3 solution to the medium.
981
23.477 g
3.917 g
4.981 g
1.102 g
192
mg
664
mg
6
mg
26
mg
24
mg
3
mg
1
L
*** C solution
CaCl2·2H2O
Distilled water
997
Media
989
TYL Medium
Trypticase Peptone (BBL)
Bacto Yeast Extract (Difco)
50% Sodium lactate
Biotin solution*
K solution**
M solution***
C solution****
Resazurin
15% Na2CO3 solution
Cysteine-HCl
Distilled water
992
2
g
2
g
25
ml
2
ml
10
ml
2.5 ml
2.5 ml
1
mg
20
ml
0.75 g
1
L
pH 7
mg
ml
993
90
90
90
1
g
g
g
L
4
4
1
g
g
L
10.6
1
g
L
MMJS Medium
NaCl
K2HPO4
CaCl2·2H2O
NH4Cl
NaNO3
MgSO4·7H2O
MgCl2·6H2O
KCl
Fe(NH4)2(SO4)2·6H2O
Bacto Yeast Extract (Difco)
Trace elements solution*
Vitamin solution**
NaHCO3
Sulfur (powder)
Resazurin
Na2S·9H2O
Distilled water
** K solution
KH2PO4
NaCl
(NH4)2SO4
Distilled water
g
g
g
g
L
** Wako Pure Chemical Ind., Ltd., Osaka, Japan.
* Biotin solution
5
100
10
4
2
18
1
* Filtered, aged seawater or artificial seawater (Daigo's
Artificial Seawater SP**).
Mix ingredients except for Na2CO3 and cysteine-HCl,
dispense the medium into suitable culture vessels under
a stream of N2/CO2 (80/20), seal with butyl rubber
stoppers, and autoclave at 115℃ for 25 min. Aseptically
and anaerobically add filter-sterile Na2CO3 and
cysteine-HCl solutions, and adjust pH to 7.0 with 3 N HCl.
Biotin
Ethanol
M1 Medium
Starch
Yeast extract
Peptone
Agar (if needed)
Seawater*
30
g
0.14 g
0.14 g
0.125 g
0.125 g
3.4
g
4.2
g
0.33 g
0.01 g
1
g
10
ml
2
ml
0.1
g
30
g
1
mg
0.5
g
1
L
*** M solution
MnCl2·4H2O
CoCl2·6H2O
Distilled water
Mix ingredients except for vitamin solution, sulfur,
NaHCO3, and Na2S·9H2O. Dispense the medium into
suitable culture vessels under a stream of H2/CO2 (80/20),
seal with butyl rubber stoppers, and autoclave at 121°C
for 15 min. Separately autoclave Na2S·9H2O (5%
solution) under N2 atmosphere. Sterilize vitamin and
NaHCO3 solutions by filtration. Sulfur is sterilized
separately by autoclaving for 60 min at 105°C on each of
3 successive days. Prior to inoculation, aseptically and
anaerobically add vitamin, sulfur, NaHCO3, and
Na2S·9H2O, and pressurize the inoculated vessels to 150
kPa with H2/CO2 (80/20). Final pH is 5.5-6.0.
**** C solution
CaCl2·2H2O
Distilled water
990
1/10 Marine Broth (Agar)
Bacto Marine Broth 2216 (Difco)
Agar (if needed)
Distilled water
991
3.7
g
15
g
1
L
pH unadjusted
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
KAl(SO4)2·12H2O
Distilled water
1/2 R2A-75%ASW
R2A Broth (DAIGO)
Agar (if needed)
Artificial sea water*
Distilled water
1.6
15
750
250
g
g
ml
ml
* Artificial sea water
NaCl
MgCl2
Na2SO4
CaCl2·2H2O
KCl
NaHCO3
KBr
H3BO3
SrCl2
NaF
Distilled water
23.5
4.9
3.9
1.1
0.66
0.19
96
26
24
3
1
g
g
g
g
g
g
mg
mg
mg
mg
L
12.8
g
1.35 g
0.1
g
0.024 g
0.1
g
0.1
g
0.025 g
0.01 g
0.024 g
1
g
0.12 g
0.8 mg
0.8 mg
4
mg
1
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH is 7.0.
998
Media
** Vitamin solution
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
994
2
2
10
5
5
5
5
1
0.01
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
*** Rumen fluid:
Methanosaeta concilii Medium
Sodium acetate
(NH4)2SO4
K2HPO4
KH2PO4
NaCl
MgSO4·7H2O
CaCl2·2H2O
Resazurin
Trace element solution*
Vitamin solution**
Rumen fluid***
FeSO4·7H2O****
Na2CO3
Cysteine-HCl
Na2S·9H2O
Distilled water
2
2
10
5
5
5
5
1
0.01
1
4.1
0.45
2.19
1.5
0.5
0.12
0.06
1
10
10
10
0.01
3
0.25
0.25
1
Remove solid particles from a fresh rumen fluid by
filtration and centrifugation, and then autoclave under a
N2 atmosphere. Stock at 4°C.
g
g
g
g
g
g
g
mg
ml
ml
ml
g
g
g
g
L
**** FeSO4·7H2O:
FeSO4·7H2O is dissolved in 0.1 N H2SO4 as a 0.2%
solution and then autoclaved under a N2 atmosphere.
996
YSG Medium (pH 4.5)
Solution A*
Solution B**
500
500
ml
ml
* Solution A
Yeast extract
Distilled water
Mix ingredients except for vitamin solution, rumen fluid,
FeSO4, Na2CO3, cysteine-HCl and Na2S·9H2O, and
autoclave under N2/CO2 (80/20) condition. A white
precipitate that appears in the medium after autoclaving
should dissolve again in about 2 days. Separately
autoclave cysteine-HCl and Na2S·9H2O as a 5% solution
under a N2 atmosphere. Vitamin and Na2CO3 (as a 10%
solution) are sterilized by filtration. Aseptically and
anaerobically add vitamin, rumen fluid, FeSO4, Na2CO3,
cysteine-HCl and Na2S·9H2O. Adjust the pH of the
medium to 7.3-7.5. Use 20% culture as an inoculum.
2
g
500
ml
Adjust pH to 4.5 with H2SO4
** Solution B
Soluble starch
Glucose
Agar (if needed)
Distilled water
2
1
15
500
g
g
g
ml
Solutions A and B are separately sterilized by autoclaving
and mixed.
Check the final pH around 4.5.
* Trace element solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3
Na2WO4
Distilled water
999
4.1
g
0.36 g
0.09 g
0.15 g
0.018 g
0.09 g
0.017 g
0.009 g
0.018 g
1.6 mg
1.6 mg
1
L
PmTG Agar
Peptonized Milk (Oxoid)
Tryptone
Glucose
Agar
Distilled water
1
1
5
10
1
g
g
g
g
L
1000 Xy Agar
Xylan
NH4NO3
K2HPO4
MgSO4·7H2O
Ca(NO3)2·4H2O
KCl
Thiamine
Agar
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
999
5
g
0.16 g
0.5
g
0.25 g
0.06 g
0.06 g
2
mg
10
g
1
L
Media
1 0 0 2 A c i d i m i c r o b iu m M e d i u m
MgSO4·7H2O
(NH4)2SO4
K2HPO4
KCl
Distilled water
* Trace element solution SL-10:
0.5
g
0.4
g
0.2
g
0.1
g
1
L
Adjust pH to 2.0 with H2SO4.
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
Heterotrophic growth on yeast extract:
Add 10 mg/l of FeSO4·7H2O to the medium. After
autoclaving, add yeast extract from sterile stock solution
to final concentration of 0.25 g/l.
Na2SO4
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element solution SL-10*
NaHCO3
Na-L-lactate
Selenite solution**
Sulfide solution***
Resazurin
Distilled water
Add 13.9 g/l of FeSO4·7H2O to the medium, and adjust
medium pH to 1.7 with H2SO4 prior to autoclaving.
1003
10
g
5
g
5
g
10
g
1
g
0.2
g
10
g
18
g
1
L
pH 10
1004 Leeming & Notman Agar Modified (MLNA)
10
10
2
8
10
0.5
5
20
15
965
g
g
g
g
ml
g
ml
ml
g
ml
* Trace element solution SL-10:
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
1008 Pelobacter carbinolicus Medium
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element solution SL-10*
Resazurin
NaHCO3
2,3-Butanediol
Na2S·9H2O
Distilled water
3
g
0.2
g
0.3
g
21
g
3
g
0.5
g
0.15 g
1
ml
2.5
g
2.3
g
1
ml
10
ml
1
mg
988
ml
pH 7
Dissolve ingredients except bicarbonate, lactate, selenite
and sulfide, dispense under a N2/CO2 atmosphere (80/20)
in culture vessels, and autoclave. Add bicarbonate,
lactate, vitamin and sulfide from sterile anaerobic stock
solutions prepared under N2 or N2/CO2 (bicarbonate). The
bicarbonate and vitamin solutions should be sterilized by
filtration. Adjust the final pH of the medium to 7.0.
Addition of 10 - 20 mg sodium dithionite per liter (e.g.
from 5% (w/v) solution, freshly prepared under N2 and
filter-sterilized) may stimulate growth at the beginning.
For transfers use 5 - 10% inoculum.
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
Bacteriological peptone
Glucose
Yeast extract
Ox bile, desiccated
Glycerol
Glycerol monostearate
Tween 60
Olive oil
Agar
Distilled water
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
1010
Autotrophic growth on ferrous sulfate:
Glucose
Polypepton*
Yeast extract
NaCl
K2HPO4
MgSO4·7H2O
Na2CO3
Agar (if needed)
Distilled water
10
1.5
70
100
6
190
2
24
36
990
0.2
g
0.25 g
20
g
3
g
0.5
g
0.15 g
1
ml
1
mg
2.5
g
0.9
g
0.36 g
1
L
pH to 7.2
10
1.5
70
100
6
190
2
24
36
990
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
0.4
3
1
g
mg
L
0.4
10
g
ml
** Selenite solution
NaOH
Na2SeO3·5H2O
Distilled water
Sterilized by autoclaving.
*** Sulfide solution
Gas atmosphere: N2/CO2 (80/20). Solutions of sodium
bicarbonate, trace elements, and 2,3-butanediol are
filter-sterilized separately. Sodium sulfide (3.6% w/v in
water) is autoclaved under nitrogen atmosphere.
Na2S·9H2O
Distilled water
1000
Media
1011 Modified Pfennig’s Medium I (Purple Sulfur
Bacteria)
CaCl2·2H2O
KH2PO4
NH4Cl
KCl
MgSO4
Vitamin B12 (0.002%) solution
Trace element solution SL-12B*
NaHCO3
Na2S·9H2O
Resazurin
Distilled water
Dissolve ingredients except bicarbonate, Vitamin B12 and
sulfide, dispense under a N2/CO2 atmosphere (80/20) in
culture vessels, and autoclave. Add bicarbonate, vitamin
and sulfide** from sterile anaerobic stock solutions
prepared under N2 or N2/CO2 (bicarbonate). The
bicarbonate and vitamin solutions should be sterilized by
filtration. Adjust the pH of the medium with sterile HCl or
Na2CO3 solutions (2 mol/L each) to pH 7.3.
0.25 g
0.34 g
0.34 g
0.34 g
0.5
g
1
ml
1
ml
1.5
g
0.4
g
0.5 mg
998
ml
pH 7.3
* Trace element solution SL-12B
Na2-EDTA
FeSO4·7H2O
CoCl2·6H2O
MnCl2·2H2O
ZnCl2
NiCl2·6H2O
Na2MoO4·2H2O
H3BO3
CuCl2·2H2O
Distilled water
Dissolve ingredients except bicarbonate, Vitamin B12 and
sulfide, dispense under a N2/CO2 atmosphere (80/20) in
culture vessels, and autoclave. Add bicarbonate, vitamin
and sulfide** from sterile anaerobic stock solutions
prepared under N2 or N2/CO2 (bicarbonate). The
bicarbonate and vitamin solutions should be sterilized by
filtration. Adjust the pH of the medium with sterile HCl or
Na2CO3 solutions (2 mol/L each) to pH 7.3.
3
1.1
190
50
42
24
18
300
2
1
Na2S·9H2O
Distilled water
g
g
mg
mg
mg
mg
mg
mg
mg
L
pH 6
1.5
100
g
ml
1013 Modified Pfennig’s Medium II (Green
Sulfur Bacteria)
g
ml
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
1 0 1 2 M e d i u m f o r E c t o t h i o r h od o s p i r a
CaCl2·2H2O
KH2PO4
NH4Cl
KCl
MgSO4
NaCl
Vitamin B12 (0.002%) solution
Trace element solution SL-12B*
NaHCO3
Na2S·9H2O
Resazurin
Distilled water
1.5
100
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
** Neutralized sulfide solution
Na2S·9H2O
Distilled water
g
g
mg
mg
mg
mg
mg
mg
mg
L
pH 6
** Neutralized sulfide solution
* Trace element solution SL-12B
Na2-EDTA
FeSO4·7H2O
CoCl2·6H2O
MnCl2·2H2O
ZnCl2
NiCl2·6H2O
Na2MoO4·2H2O
H3BO3
CuCl2·2H2O
Distilled water
3
1.1
190
50
42
24
18
300
2
1
CaCl2·2H2O
KH2PO4
NH4Cl
KCl
MgSO4
Vitamin B12 (0.002%) solution
Trace element solution SL-10B*
NaHCO3
Na2S·9H2O
Resazurin
Distilled water
0.25 g
0.34 g
0.34 g
0.34 g
0.5
g
1
ml
1
ml
1.5
g
0.6
g
0.5 mg
998
ml
pH 6.8
Dissolve ingredients except bicarbonate, Vitamin B12 and
sulfide, dispense under a N2/CO2 atmosphere (80/20) in
culture vessels, and autoclave. Add bicarbonate, vitamin
and sulfide** from sterile anaerobic stock solutions
prepared under N2 or N2/CO2 (bicarbonate). The
bicarbonate and vitamin solutions should be sterilized by
filtration. Adjust the pH of the medium with sterile HCl or
Na2CO3 solutions (2 mol/L each) to pH 6.8.
0.25 g
0.34 g
0.34 g
0.34 g
0.5
g
30
g
1
ml
1
ml
1.5
g
0.4
g
0.5 mg
998
ml
pH 7.3
1001
Media
1017 Pelobacter venetianus Medium
* Trace element solution SL-10B
HCl (25%)
FeSO4·7H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
7.7
1.5
70
100
300
190
2
24
36
1
ml
g
mg
mg
mg
mg
mg
mg
mg
L
1.5
100
g
ml
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element solution SL-10*
Resazurin
NaHCO3
Polyethylene glycol (Mw 106-20000)
Na2S·9H2O
Distilled Water
** Neutralized sulfide solution
Na2S·9H2O
Distilled water
Gas atmosphere: N2/CO2 (80/20). Solutions of sodium
bicarbonate, trace elements and polyethylene glycol are
filter-sterilized separately. Sodium sulfide (3.6% w/v in
water) is autoclaved under nitrogen atmosphere.
The sulfide solution is prepared in a vessel sealed with a
butyl rubber stopper. Autoclave at 121℃ for 15 min
under a N2 atmosphere. After cooling to room
temperature, the pH is adjusted to about 7.3 by adding of
sterile 2 N H2SO4 drop-wise with a syringe without
opening the bottle. Appearance of yellow colour indicates
the pH is about 8. The vessel should be shaken
continuously to avoid precipitation of elemental sulfur.
The final solution should be transparent yellow.
* Trace element solution SL-10:
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
1016 Pelobacter acetylenicus Medium
KH2PO4
NH4Cl
NaCl
MgCl2·6H2O
KCl
CaCl2·2H2O
Trace element solution SL-10*
Resazurin
NaHCO3
Acetoin
Na2S·9H2O
Distilled Water
0.2
g
0.25 g
1
g
0.4
g
0.5
g
0.15 g
1
ml
1
mg
2.5
g
1
g
0.36 g
1
L
pH to 7.2
KP buffer*
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
NaCl
Na2SO4
Sodium lactate
Bacto Yeast Extract (Difco)
Fe(NH4)2(SO4)2·7H2O
Trace element solution**
Vitamin solution***
Resazurin
Na2CO3
Na2S·9H2O
Distilled water
* Trace element solution SL-10:
10
1.5
70
100
6
190
2
24
36
990
10
1.5
70
100
6
190
2
24
36
990
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
1019 Archaeoglobus fulgidus Medium
Gas atmosphere: N2/CO2 (80/20). Solutions of sodium
bicarbonate, trace elements and acetoin are
filter-sterilized separately. Sodium sulfide (3.6% w/v in
water) is autoclaved under nitrogen atmosphere.
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
0.2
g
0.25 g
20
g
3
g
0.5
g
0.15 g
1
ml
1
mg
2.5
g
1
g
0.36 g
1
L
pH to 7.2
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
5
ml
0.75 g
0.15 g
0.54 g
30
g
2.4
g
2.2
g
0.5
g
2
mg
2
ml
2
ml
1
mg
1
g
0.36 g
1
L
pH unadjusted
Mix ingredients except KP buffer, vitamin solution,
Na2CO3 and Na2S·9H2O. Dispense the medium into
suitable culture vessels under a stream of N2/CO2 (80/20)
and seal with butyl rubber stoppers. Separately autoclave
KP buffer and Na2S·9H2O (5% solution) under a N2
atmosphere. Sterilize vitamin and 10% Na2CO3 solutions
by filtration. Prior to inoculation, aseptically and
anaerobically add KP buffer, vitamin, Na2CO3 and
Na2S·9H2O solutions.
* KP buffer
KH2PO4
K2HPO4
Distilled water
Autoclaved under N2.
1002
119
21
1
g
g
L
Media
* Vitamin solution
** Trace element solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
L
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Distilled water
*** Vitamin solution
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
0.46
0.1
0.14
0.2
0.15
0.54
0.82
2
2
1
1
2.5
0.3
0.3
1
12.8
1.35
0.1
24
0.1
0.1
25
10
24
0.12
1
g
g
g
mg
g
g
mg
mg
mg
g
L
First dissolve NTA in 200 ml of water and adjust the pH to
6.5 with KOH.
*** Se/W solution
Na2SeO3
Na2WO4
Distilled water
1 0 2 0 M e t h a n o f o l l i s e t h a n o l i c us M e d i u m
Ethanol
Yeast extract
KH2PO4
MgCl2·6H2O
CaCl2·2H2O
NH4Cl
Sodium acetate
Vitamin solution*
Trace element solution**
Se/W solution***
Resazurin solution****
NaHCO3
Na2S·9H2O
Cysteine-HCl
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace element solution
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
0.5
0.9
0.4
0.7
0.8
0.2
0.5
2.7
0.3
0.4
1
ml
g
g
g
g
g
g
ml
ml
ml
ml
g
g
g
L
4
4
1
mg
mg
L
1
1
g
L
**** Resazurin solution
Resazurin
Distilled water
1 0 2 1 D e s u l f o v i b r io M e d i u m
For vial cultures, dissolve all ingredients except for
ethanol, Na2S·9H2O and cysteine-HCl, flush N2/CO2
(80/20) and autoclave. Add each of filter-sterilized
Na2S·9H2O solution (0.6 g Na2S·9H2O/50 ml) and
cysteine-HCl solution (0.6 g cysteine-HCl/50 ml) per each
vial separately. Add filter-sterilized 0.8 M ethanol solution.
Inoculum size 10-20%.
K2HPO4
NH4Cl
Na2SO4
CaCl2·2H2O
MgSO4·7H2O
Sodium lactate
Yeast extract
FeSO4·7H2O
Resazurin
Thioglycolate solution*
Distilled water
Dissolve ingredients except thioglycolate solution,
dispense under a N2 atmosphere in culture vessels, and
autoclave. Adjust the final pH of the medium to 7.8.
* Thioglycolate solution
Sodium thioglycolate
Ascorbic acid
Distilled water
Sterilize by filtration.
1003
0.5
g
1
g
1
g
0.1
g
2
g
2
g
1
g
0.5
g
1
mg
10
ml
990
ml
pH 7.8
0.1
0.1
10
g
g
ml
Media
1022
1028 Modified WS Medium
(NH4)2SO4
K2HPO4
MgSO4·7H2O
KCl
Ca(NO3)2
NaCl
Sodium tetrathionate
Distilled water
3
5
5
1
0.3
20
2
1
g
g
g
g
g
g
g
L
pH 4
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
NaCl
Methanol
NaHCO3
Vitamin solution*
Trace element solution**
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
1024 Alkali A Medium
Solution A*
Solution B**
500
500
ml
ml
Mix ingredients except for methanol, vitamin solution,
cysteine-HCl and Na2S·9H2O, and autoclave under a
N2/CO2 atmosphere (80/20). Separately autoclave
cysteine-HCl and Na2S·9H2O solutions as 3% solutions
under a N2 atmosphere. Sterilize methanol and vitamin
solution by filtration. Prior to inoculation, add methanol,
vitamin solution, cysteine-HCl and Na2S·9H2O.
* Solution A
Glucose
Peptone
Yeast extract
K2HPO4
MgSO4·7H2O
Agar
Distilled water
10
5
5
1
0.2
12
500
g
g
g
g
g
g
ml
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
** Solution B
Na2CO3
Distilled water
10
500
g
ml
Solutions A and B are autoclaved separately and
combined before solidified. Final pH 10.3.
1026 Medium for Strains JA121 and JA322
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Sodium pyruvate
Yeast extract
Ferric citrate solution* (0.1%)
Vitamin B12 (0.002% (w/v))
Trace element solution SL7**
Na2S·9H2O
Distilled water
0.5
g
2
g
20
g
0.6
g
0.15 g
3
g
0.5
g
5
ml
1
ml
1
ml
0.48 g
1
L
pH 7
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
Mix ingredients except vitamin B12 and Na2S·9H2O, and
autoclave under a N2 atmosphere. Separately autoclave
Na2S·9H2O solution under a N2 atmosphere. Sterilize
vitamin B12 solution by filtration. Prior to inoculation,
aseptically and anaerobically add them.
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
* Soluble in hot water.
1029 Alkaline Yeast Extract-Malt Extract Agar
** Trace element solution SL7
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
0.136 g
0.54 g
3.05 g
0.147 g
20
g
0.8 ml
2.52 g
2
ml
1
ml
1
mg
0.3
g
0.3
g
1
L
10
1.5
70
100
60
200
20
20
40
990
Bacto Yeast Extract (Difco)
Bacto Malt Extract (Difco)
Glucose
Agar
Artificial seawater
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
4
10
4
15
1
After autoclaving, adjust pH to 10.0 with sterilized 10%
Na2CO3 solution.
Initially, dissolve FeCl2·4H2O in 25% HCl, then add
distilled water followed by the other components.
1004
g
g
g
g
L
Media
1030 Yeast Extract-Malt Extract Agar (ISP-2)
with Artificial Seawater
Bacto Yeast Extract (Difco)
Bacto Malt Extract (Difco)
Glucose
Agar
Artificial seawater
Mix ingredients except for Na2CO3, vitamin solution,
cysteine-HCl and Na2S·9H2O, and autoclave under a
N2/CO2 atmosphere (80/20). Separately autoclave
cysteine-HCl and Na2S·9H2O solutions as 3% solutions
under a N2 atmosphere. Prior to inoculation, add the
filter-sterile 5% Na2CO3 and vitamin solutions,
cysteine-HCl, and Na2S·9H2O.
4
g
10
g
4
g
15
g
1
L
Adjust pH to 7.3
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
1031
Sodium pyruvate
KH2PO4
(NH4)2SO4
Na2S2O3·5H2O
Yeast extract
MgCl2·6H2O
CaCl2
NaCl
Vitamin B12
Nicotinic acid
p-Aminobenzoic acid
Trace element solution*
Distilled water
2
g
1
g
1
g
0.5
g
0.5
g
0.2
g
45
mg
0.2
g
20
μg
10
μg
3
μg
1
ml
1
L
pH 6.8
5
1
0.3
0.11
50
30
30
30
10
1
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
g
g
g
g
mg
mg
mg
mg
mg
L
2
5
2
100
1
18
g
g
g
g
L
g
pH 7.3
0.05 g
0.09 g
0.25 g
0.25 g
0.15 g
5
g
2.5
g
0.25 g
2
ml
1
ml
1
mg
0.3
g
0.3
g
1
L
g
g
g
g
g
g
g
g
g
g
g
g
g
L
1040 1/2 ISP-2 + 15% NaCl
Yeast extract
Malt extract
Glucose
NaCl
Distilled water
Agar
1039 Thermoheterotroph Medium
K2HPO4
KH2PO4
NH4Cl
MgSO4·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Na2S2O3·5H2O
Na2CO3
Vitamin solution*
Trace element solution**
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1038 1/2 ISP-2 + 10% NaCl
Yeast extract
Malt extract
Glucose
NaCl
Distilled water
Agar
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace elements solution
* Trace element solution
2Na-EDTA
FeCl2·4H2O
H3BO3
CoCl2·6H2O
ZnCl2
MnCl2·4H2O
Na2MoO4·2H2O
NiSO4·6H2O
CuCl2·2H2O
Distilled water
2
2
10
5
5
5
5
1
0.01
1
2
5
2
150
1
18
g
g
g
g
L
g
pH 7.3
1041 Medium for Strain JA143
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Sodium pyruvate
Yeast extract
Na2S2O3·5H2O
Ferric citrate solution* (0.1%)
Trace element solution SL7**
Na2S·9H2O
Distilled water
0.5
g
0.2
g
0.4
g
0.6
g
0.05 g
3
g
0.5
g
5
g
5
ml
1
ml
0.48 g
1
L
pH 7
Mix ingredients except for Na2S·9H2O and autoclave
under a N2 atmosphere. Separately autoclave Na2S·9H2O
solution under a N2 atmosphere. Prior to inoculation,
aseptically and anaerobically add it.
1005
Media
1049 AS Medium
* Soluble in hot water.
NaCl
Bacto Yeast Extract (Difco)
MgCl2·6H2O
MgSO4·7H2O
(NH4)2SO4
CaCl2·2H2O
KH2PO4
KCl
NaBr
H3BO3
SrCl2·6H2O
Fe(III) ammonium citrate
Bis Tris Propane
Trace elements solution
Vitamin solution
Resazurin
Sulfur (powder)
Na2S·9H2O
Distilled water
** Trace element solution SL7
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
10
1.5
70
100
60
200
20
20
40
990
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
Initially, dissolve FeCl2·4H2O in 25% HCl, then add
distilled water followed by the other components.
1044 Hagem Medium
KH2PO4
MgSO4·7H2O
NH4Cl
Fe-citrate 1% solution
Malt extract
Glucose
Agar
Distilled water
0.5
g
0.5
g
0.5
g
0.5 ml
5
g
5
g
15
g
1
L
pH 5.6
Mix ingredients except for vitamin, sulfur and Na2S·9H2O,
adjust pH to 7 with H2SO4, and autoclave under a N2
atmosphere. Sulfur is separately sterilized by autoclaving
for 60 min at 105°C on each of 3 successive days.
Separately autoclave Na2S·9H2O as 5% solution under a
N2 atmosphere. Anaerobically add filter-sterile vitamin,
sulfur and Na2S·9H2O.
* Trace elements solution
1045 Trypticase Soy Agar + 10% NaCl
Trypticase Soy (BBL)
NaCl
Distilled water
Agar
30
100
1
20
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3
Na2WO4
AlK(SO4)2
Distilled water
g
g
L
g
pH 7.3
1046 Synthetic Low-Nutrient Agar (SNA)
Glucose
Sucrose
KH2PO4
KNO3
MgSO4·7H2O
KCl
1 N NaOH solution
Agar
Distilled water
0.2
g
0.2
g
1.0
g
1.0
g
0.5
g
0.5
g
0.6 ml
23
g
1
L
pH unadjusted
4.1
g
0.36 g
0.09 g
0.15 g
0.018 g
0.09 g
0.017 g
0.009 g
0.018 g
1.6 mg
1.6 mg
5
mg
1
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add other minerals. Final pH 7.0.
** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
1047 802 + 5% Glucose
Polypepton*
Yeast extract
Glucose
MgSO4·7H2O
Distilled water
Agar (if needed)
20
g
2
g
3
g
6
g
1
g
0.3
g
0.2
g
0.5
g
0.05 g
0.025 g
0.02 g
0.01 g
2.25 g
10
ml
10
ml
1
mg
10
g
0.3
g
1
L
5
g
5
g
50
g
1
g
1
L
15
g
pH 6.6 - 7.0
* Wako Pure Chemical Ind., Ltd., Osaka, Japan.
1006
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
1050 A Medium
NaCl
Bacto Yeast Extract (Difco)
MgCl2·6H2O
MgSO4·7H2O
(NH4)2SO4
CaCl2·2H2O
KH2PO4
KCl
NaBr
H3BO3
SrCl2·6H2O
Fe(III) ammonium citrate
Bis Tris Propane
Trace elements solution
Vitamin solution
Resazurin
Na2S·9H2O
Distilled water
1052 Oatmeal Agar (ISP Medium No.3, pH 5.5)
20
g
2
g
3
g
6
g
1
g
0.3
g
0.2
g
0.5
g
0.05 g
0.025 g
0.02 g
0.01 g
2.25 g
10
ml
10
ml
1
mg
0.3
g
1
L
Oatmeal
Trace salts solution*
Distilled water
Agar
FeSO4·7H2O
MnCl2·4H2O
ZnSO4·7H2O
Distilled water
Yeast extract
Soluble starch
Distilled water
Agar
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add other minerals. Final pH 7.0.
** Vitamin solution
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
4
10
4
1
20
2
10
1
15
g
g
L
g
pH 5.5
KH2PO4
NH4Cl
Cysteine-HCl
Ferric quinate solution*
Wolfe's vitamin solution**
Wolfe's mineral solution***
Sodium fumarate
Sodium pyruvate
Distilled water
0.2
g
0.06 g
0.05 g
2
ml
4
ml
2
ml
0.58 g
0.44 g
1
L
pH 7
Mix ingredients except for vitamin solution and
cysteine-HCl. Dispense the medium into vials suitable for
anaerobic cultures, sparge with argon gas to establish
anoxic condition (at least 30 min), seal with butyl rubber
stoppers, and autoclave at 121°C for 15 min. Separately
autoclave cysteine-HCl (0.5% solution) under argon
atmosphere. Sterilize vitamin solution by filtration. Prior to
inoculation, aseptically and anaerobically add the vitamin
and cysteine-HCl solutions to the medium, and sparge
the vials with sterile argon gas for 5 min. The
cysteine-HCl more than the above-mentioned amount
(0.05 g/L) is unsuitable for growth of strain RS-1. And the
strain produces only a small amount of magnetite in the
presence of sulfate.
* Ferric quinate solution
FeCl3
Quinate
Distilled water
0.27 g
0.19 g
100
ml
** Wolfe's vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
1051 Yeast Extract-Malt Extract Agar (ISP
Medium No. 2, pH 5.5)
Yeast extract
Malt extract
Glucose
Distilled water
Agar
g
g
g
ml
1 0 5 6 D e s u l f o v i b r io m a g n e t i c us M e d i u m f o r
Magnetosome Formation
4.1
g
0.36 g
0.09 g
0.15 g
0.018 g
0.09 g
0.017 g
0.009 g
0.018 g
1.6 mg
1.6 mg
5
mg
1
L
2
2
10
5
5
5
5
1
0.01
1
0.1
0.1
0.1
100
1053 Yeast Extract-Starch Agar (pH 5.5)
* Trace elements solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
g
ml
L
g
pH 5.5
* Trace salts solution
Mix ingredients except for vitamin and Na2S·9H2O, adjust
pH to 7 with H2SO4, and autoclave under a N2
atmosphere. Separately autoclave Na2S·9H2O as 5%
solution under a N2 atmosphere. Anaerobically add
filter-sterile vitamin and Na2S·9H2O.
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO3
Na2WO4
AlK(SO4)2
Distilled water
20
1
1
18
g
g
g
L
g
pH 5.5
1007
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
Media
* Wolfe's modified mineral solution
*** Wolfe's mineral solution
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
MnSO4·H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
Distilled water
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
NaCl
MnSO4·H2O
FeSO4·7H2O
Co(NO3)2·6H2O
CaCl2
ZnSO4·7H2O
CuSO4·5H2O
Boric acid
Na2MoO4·2H2O
Na2SeO3
g
g
g
g
g
g
g
g
g
g
g
g
L
** VFA
1061 Alkalic MRS Medium (pH 8.0)
Acetic acid
Propionic acid
Butyric acid
iso-Butyric acid
n-Valeric acid
iso-Valeric acid
DL-α-Methylbutyric acid
10
g
10
g
5
g
20
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 8.0
17
6
4
1
1
1
1
ml
ml
ml
ml
ml
ml
ml
0.2
40
0.2
200
40
20
40
40
20
100
2
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
μg
L
*** Vitamin mixture
Biotin
Ca-pantothenate
Folic acid
myo-Inositol
Niacin
p-Aminobenzoic acid
Pyridoxine-HCl
Riboflavin
Thiamine-HCl
Choline chloride
Cyanocobalamin
Distilled water
1066
Na2HPO4·12H2O
KH2PO4
Na2CO3
MgCl2·6H2O
NH4Cl
Yeast extract
Bacto Tryptone (Difco)
Na2WO4·2H2O
Wolfe's modified mineral solution*
VFA**
myo-Inositol
Vitamin mixture***
Cellobiose
Cysteine-HCl
Na2S·9H2O
Resazurin
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
Nitrilotriacetic acid is suspended in 500 ml water, and
dissolved by titrating with 2 N KOH until its pH stabilize at
6.5. The rest of the ingredients are added and dissolved.
Finally total volume is adjusted to 1 L.
Add nitrilotriacetic acid to approximately 500 ml of water
and adjust to pH 6.5 with KOH to dissolve the compound.
Bring volume to 1 L with remaining water and add
remaining compounds one at a time.
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
1.5
3
1
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.001
4.2
g
1.5
g
4
g
0.4
g
0.6
g
1
g
1
g
0.4 mg
50
ml
1
ml
1
mg
10
ml
5
g
0.5
g
0.5
g
1
mg
939
ml
pH 6.5
1067 Methanobacterium Medium
Dissolve ingredients except for vitamin, cysteine-HCl and
sulfide, dispense under a N2 atmosphere in culture
vessels and autoclave. Add vitamin, cysteine-HCl and
sulfide from sterile anaerobic stock solutions prepared
under a N2 atmosphere. The vitamin and cysteine-HCl
solutions should be sterilized by filtration. Adjust the pH of
the medium with sterile 10 N KOH solution to pH 6.5.
1008
KH2PO4
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
NaHCO3
Bacto Yeast Extract (Difco)
Sodium acetate
Vitamin solution*
Trace element solution**
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
0.136 g
0.54 g
0.2
g
0.147 g
2.5
g
0.2
g
0.8
g
10
ml
10
ml
1
mg
0.5
g
0.5
g
1
L
Mix ingredients except for vitamin solution, cysteine-HCl
and Na2S·9H2O, and autoclave under a H2/CO2
atmosphere (80/20). Concentrated cysteine-HCl and
sodium sulfide solutions are sterilized separately under a
N2 atmosphere in tightly closed vessels. Prior to
inoculation, add the filter-sterile vitamin solution,
cysteine-HCl and Na2S·9H2O. Pressurize the inoculated
vessels to 150 kPa with H2/CO2 (80/20).
Media
** Trace element solution SL7
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
2
2
10
5
5
5
5
1
0.01
1
HCl (25%; 7.7 M)
FeCl2·4H2O
ZnCl2
MnCl2·4H2O
H3BO3
CoCl2·6H2O
CuCl2·2H2O
NiCl2·6H2O
Na2MoO4·2H2O
Distilled water
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.025
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
0.5
g
0.2
g
0.4
g
0.6
g
0.05 g
3
g
0.5
g
5
ml
1
ml
1
ml
1
mg
1
L
pH 7
ml
g
mg
mg
mg
mg
mg
mg
mg
ml
Initially, dissolve FeCl2·4H2O in 25% HCl, then add
distilled water followed by the other components.
1071 PYGSW Agar
Peptone
Yeast extract
Glucose
Seawater (2% salinity)
Agar
1
1
0.5
1
15
g
g
g
L
g
1072 1% Tryptone Agar
Bacto Tryptone (Difco)
Bacto Agar
Distilled water
10
15
1
g
g
L
pH 7.0
1074 Alkaline Agar
Glucose
Bacto Peptone (Difco)
Bacto Yeast Extract (Difco)
KH2PO4
MgSO4·7H2O
20% NaCl solution
10% Na2CO3 solution
Agar
Distilled water
1068 Medium for Strains JA145, JA193, JA310,
JA334, JA415, JA430 and JA447
KH2PO4
MgSO4·7H2O
NaCl
NH4Cl
CaCl2·2H2O
Sodium pyruvate
Yeast extract
Ferric citrate solution* (0.1%)
Vitamin B12 (0.002% (w/v))
Trace element solution SL7**
Resazurin
Distilled water
10
1.5
70
100
60
200
20
20
40
990
10
5
5
1
0.2
200
100
15
700
g
g
g
g
g
ml
ml
g
ml
Autoclave NaCl and Na2CO3 solutions separately and
add the solutions to the medium aseptically.
1 0 7 8 D e s u l f o v i b r io m a g n e t i c us M e d i u m f o r
Sulfate-reducing Growth Mode
KH2PO4
NH4Cl
Cysteine-HCl
Ferric quinate solution*
Wolfe's vitamin solution**
Wolfe's mineral solution***
Na2SO4
Sodium lactate
Distilled water
Mix ingredients except for vitamin B12 and autoclave
under a N2 atmosphere. Prior to inoculation, aseptically
and anaerobically add vitamin B12 solution sterilized by
filtration.
For strain JA310, add 2 mM Na2S2O3 (separately
sterilized).
0.2
g
0.06 g
0.05 g
2
ml
4
ml
2
ml
0.3
g
0.45 g
1
L
pH 7
Mix ingredients except for vitamin solution and
cysteine-HCl. Dispense the medium into vials suitable for
anaerobic cultures, sparge with argon gas to establish
anoxic condition (at least 30 min), seal with butyl rubber
stoppers, and autoclave at 121°C for 15 min. Separately
autoclave cysteine-HCl (0.5% solution) under an argon
atmosphere. Sterilize vitamin solution by filtration. Prior to
inoculation, aseptically and anaerobically add the vitamin
and cysteine-HCl solutions to the medium, and sparge
the vials with sterile argon gas for 5 min. The
cysteine-HCl more than the above-mentioned amount
(0.05 g/L) is unsuitable for growth of strain RS-1.
* Soluble in hot water.
1009
Media
* Trace elements solution
* Ferric quinate solution
FeCl3
Quinate
Distilled water
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
NaSeO3·5H2O
0.27 g
0.19 g
100
ml
** Wolfe's vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
2
2
10
5
5
5
5
0.1
5
5
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
1.5
3
0.5
1
0.1
0.1
0.1
0.1
0.01
0.01
0.01
0.01
1
1082 LB Medium with 50 ppm Naphthalene
g
g
g
g
g
g
g
g
g
g
g
g
L
Peptone
Yeast extract
NaCl
Naphthalene solution (10,000 ppm)*
Distilled water
Agar (if needed)
10
5
5
5
1
15
g
g
g
ml
L
g
pH 7.0
50
5
mg
ml
* Naphthalene solution (10,000 ppm)
Naphthalene
Ethanol
Sterilize separately by filtration.
Add nitrilotriacetic acid to approximately 500 ml of water
and adjust to pH 6.5 with KOH to dissolve the compound.
Bring volume to 1 L with remaining water and add
remaining compounds one at a time.
1 0 8 3 M e t h a n o b a c t e r i u m a l c a l ip h i l u m M e d i u m
1 0 7 9 M a r i n e M i c r o a e r o b i c T he r m u s M e d i u m
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
NaCl
K2HPO4
CaCl2·2H2O
NH4Cl
MgSO4·7H2O
MgCl2·6H2O
KCl
Fe(NH4)2(SO4)2·6H2O
Trace elements solution*
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
*** Wolfe's mineral solution
Nitrilotriacetic acid (NTA)
MgSO4·7H2O
MnSO4·H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2
ZnSO4·7H2O
CuSO4·5H2O
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
Distilled water
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.05
0.05
1
g
1
g
30
g
0.14 g
0.14 g
0.25 g
3.4
g
4.18 g
0.33 g
0.01 g
10
ml
1
L
NH4Cl
MgCl2·6H2O
CaCl2·2H2O
K2HPO4
Bacto Yeast Extract (Difco)
Polypeptone
Resazurin
Carbonate mixture*
Tris-HCl buffer**
Vitamin solution***
Trace elements solution****
Cysteine-HCl
Na2S·9H2O
Distilled water
1
0.1
0.1
0.4
2
2
1
210
10
10
10
0.3
0.3
760
g
g
g
g
g
g
mg
ml
ml
ml
ml
g
g
ml
Mix ingredients except for carbonate mixture, Tris-HCl
buffer, vitamin solution, cysteine-HCl and Na2S·9H2O,
and autoclave under a H2 atmosphere. Carbonate
mixture, Tris-HCl buffer, cysteine-HCl (3% solution) and
Na2S·9H2O (3% solution) are separately autoclaved
under a N2 atmosphere. Prior to inoculation, add
filter-sterile vitamin solution, carbon mixture, Tris-HCl
buffer, cysteine-HCl and Na2S·9H2O. Pressurize the
inoculated vessels to 150 kPa with H2.
Mix ingredients, adjust pH to 7.0 and autoclave under a
N2/O2 atmosphere (95/5). Pressurize the vessels to 150
kPa with N2/O2.
* Carbonate mixture
NaHCO3
Na2CO3
Distilled water
10
0.5
210
g
g
ml
Mix ingredients and autoclave under a N2 atmosphere.
1010
Media
1091 MRS + Fructose
** Tris-HCl buffer
Peptone
Meat extract
Yeast extract
Glucose
Fructose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
Distilled water
Agar (if needed)
Dissolve 2 M Tris, adjust pH to 8.4 with HCl, and
autoclave under a N2 atmosphere.
*** Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
1095 LMG 157 Medium
**** Trace elements solution
2Na-EDTA
MgSO4·7H2O
MnSO4·4H2O
NaCl
FeSO4·7H2O
CoCl2·6H2O
CaCl2·2H2O
ZnSO4·7H2O
CuSO4
KAl(SO4)2·12H2O
H3BO3
Na2MoO4·2H2O
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
10
g
10
g
5
g
20
g
10
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
1
L
15
g
pH 6.0 - 6.5
0.64
6.2
0.55
1
0.1
0.17
0.13
0.18
0.05
0.018
0.01
0.011
0.025
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
L
Beef extract
Yeast extract
Tryptone
Sodium acetate
Glucose*
Distilled water
Agar (if needed)
0.5
g
0.5
g
2
g
0.2
g
0.25 mg
1
L
15
g
pH 7.2- 7.4
* Sterilize separately by autoclaving and add to other
ingredients. The final concentration is 0.025%.
1104 Thermus Medium
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
Sodium glutamate monohydrate
CaSO4·2H2O
MgSO4·7H2O
NaCl
KNO3
NaNO3
Na2HPO4
Trace elements solution*
Agar (if needed)
Distilled water
Dissolve the 2Na-EDTA in 500 ml of distilled water, and
after adding the salts, make up to 1 L.
1084 Actinotalea fermentas Medium
K2HPO4
2.21 g
KH2PO4
1.5
g
(NH4)2SO4
1.3
g
MgCl2·6H2O
0.1
g
CaCl2·2H2O
0.02 g
FeSO4·7H2O
1.25 mg
Yeast extract
5
g
Cellobiose or Cellulose (MN 300)
5
g
NaHCO3
0.8
g
Resazurin
1
mg
Cysteine-HC
0.5
g
Distilled water
1
L
Adjust pH to 7.4 with 8 M NaOH
1
g
1
g
1
g
0.06 g
0.1
g
0.008 g
0.1
g
0.69 g
0.11 g
10
ml
20
g
1
L
Adjust pH to 7.5 with NaOH
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
Prepare the medium anaerobically under 100% nitrogen.
Cellobiose, NaHCO3, and cysteine-HCl are sterilized
separately by autoclaving under N2. For aerobic growth,
omit resazurin and cysteine-HCl.
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1011
Media
1109 Modified Norkans' C (MNC)
Solution B
KH2PO4
MgSO4
ZnSO4 0.2 % solution
NH4-tartrate
Fe-citrate 1.0 % solution
Thiamine-HCl
Casein hydrolysate
Yeast extract
Glucose
Agar
Distilled water
1
g
0.5
g
0.5 ml
0.5
g
0.5 ml
50
μg
0.23 g
0.5
g
10
g
15
g
1
L
Ca(NO3)2·4H2O
Distilled water
Solution C
MnCl2·4H2O
Distilled water
500
100
400
K2HPO4
KH2PO4
NH4Cl
MgSO4·7H2O
CaCl2·2H2O
Bacto Yeast Extract (Difco)
Glucose
Na2CO3
Vitamin solution*
Trace element solution**
Resazurin
Cysteine-HCl
Na2S·9H2O
Distilled water
ml
ml
ml
Separately autoclave Solutions A, B and C. Mix
aseptically. Final pH around 8.5.
* Solution A
Peptone
Meat extract
Yeast extract
Glucose
Tween 80
K2HPO4
Sodium acetate
Diammonium hydrogencitrate
MgSO4·7H2O
MnSO4·nH2O
NaCl
Distilled water
10
g
10
g
5
g
20
g
1
g
2
g
5
g
2
g
0.2
g
0.05 g
30
g
500
ml
pH unadjusted
* Vitamin solution
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl
Riboflavin
Nicotinic acid
Ca-pantothenate
p-Aminobenzoic acid
Vitamin B12
Distilled water
5
g
100
ml
pH unadjusted
*** Solution C
Agar
Distilled water
15
g
400
ml
pH unadjusted
In the case of liquid medium, prepare Solution A with 900
ml of distilled water and mix only with Solution B.
1112 Anaero Columbia Agar with Rabbit Blood
Anaero Columbia agar with rabbit blood (BD-BBL)
1113 Sporulation Medium
Dissolve each regent in distilled water and sterilize them
individually. Cool them down to about 60℃, and mix all the
solution together.
Solution A
Nutrient broth
FeSO4·7H2O
MgSO4·7H2O
KCl
Agar (if needed)
Distilled water
0.05 g
0.09 g
0.25 g
0.25 g
0.15 g
1
g
1.8
g
0.25 g
2
ml
1
ml
1
mg
0.3
g
0.3
g
1
L
Mix ingredients except for Na2CO3, vitamin solution,
cysteine-HCl and Na2S·9H2O, and autoclave under a
N2/CO2 atmosphere (80/20). Separately autoclave
cysteine-HCl and Na2S·9H2O solutions as 3% solutions
under a N2 atmosphere. Prior to inoculation, add the
filter-sterile 5% Na2CO3 and vitamin solutions,
cysteine-HCl, and Na2S·9H2O.
** Solution B
Na2CO3
Distilled water
1.979 mg
100
ml
pH unadjusted
1118 Thermotoga Medium
1110 Alkalic MRS (pH 8.5) + 3% NaCl
Solution A*
Solution B**
Solution C***
236.15 mg
100
ml
pH unadjusted
8
g
0.278 mg
0.25 g
1
g
15
g
800
ml
pH unadjusted
2
2
10
5
5
5
5
1
0.01
1
mg
mg
mg
mg
mg
mg
mg
mg
mg
L
** Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1012
Media
1121 Thermus Medium
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
Sodium glutamate monohydrate
CaSO4·2H2O
MgSO4·7H2O
NaCl
KNO3
NaNO3
Na2HPO4
Trace elements solution*
Gelrite (if needed)
Distilled water
* Trace elements solution
1
g
1
g
1
g
0.06 g
0.1
g
0.008 g
0.1
g
0.69 g
0.11 g
10
ml
8
g
1
L
Adjust pH to 7.5 with NaOH
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
For solid medium, equal volumes of separately
autoclaved 1.6% gelrite and double-strength liquid
medium containing 2 g/L MgSO4·7H2O are mixed.
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
1128 Acidianus Medium
g
g
g
g
g
g
g
g
g
g
g
g
g
L
(NH4)2SO4
KH2PO4
MgSO4·7H2O
CaCl2·2H2O
FeCl3·6H2O
MnCl2·4H2O
Na2B4O7·10H2O
ZnSO4·7H2O
CuCl2·2H2O
Na2MoO4·2H2O
VOSO4·xH2O
CoSO4·7H2O
Bacto Yeast Extract (Difco)
Sulfur (powder)
Resazurin
Na2S·9H2O
Distilled water
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1124 Thermus Medium
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
Sodium glutamate monohydrate
CaSO4·2H2O
MgSO4·7H2O
NaCl
KNO3
NaNO3
Na2HPO4
Na2S2O3·5H2O
Trace elements solution*
Gelrite (if needed)
Distilled water
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
1
g
1
g
1
g
0.06 g
0.1
g
0.008 g
0.1
g
0.69 g
0.11 g
1.24 g
10
ml
8
g
1
L
Adjust pH to 7.5 with NaOH
1.3
0.28
0.25
0.07
0.02
1.8
4.5
0.22
0.05
0.03
0.03
0.01
0.5
1
1
0.5
1
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
g
mg
g
L
Mix ingredients except for Bacto Yeast Extract, sulfur and
Na2S·9H2O, and adjust pH to 2.0 with H2SO4. Dispense
the medium into suitable culture vessels under a stream
of H2/CO2 (80/20) and seal with butyl rubber stoppers.
Separately autoclave Bacto Yeast Extract and
Na2S·9H2O as 5% solutions under a N2 atmosphere.
Sulfur is sterilized separately by autoclaving for 60 min at
105°C on each of successive days. Prior to inoculation,
add Bacto Yeast Extract, sulfur and Na2S·9H2O, and
pressurize the inoculated vessels to 150 kPa with H2/CO2.
1129 Thermus Medium
Bacto Yeast Extract (Difco)
Bacto Tryptone (Difco)
Sodium glutamate monohydrate
CaSO4·2H2O
MgSO4·7H2O
NaCl
KNO3
NaNO3
Na2HPO4
Na2S2O3·5H2O
Trace elements solution*
Agar (if needed)
Distilled water
For solid medium, equal volumes of separately
autoclaved 1.6% gelrite and double-strength liquid
medium containing 2 g/L MgSO4·7H2O are mixed.
1013
1
g
1
g
1
g
0.06 g
0.1
g
0.008 g
0.1
g
0.69 g
0.11 g
0.1
g
10
ml
20
g
1
L
Adjust pH to 7.5 with NaOH
Media
* Trace elements solution
Nitrilotriacetic acid (NTA)
FeCl3·6H2O
MnCl2·4H2O
CoCl2·6H2O
CaCl2·2H2O
ZnCl2
CuCl2·2H2O
H3BO3
Na2MoO4·2H2O
NaCl
NiCl2·6H2O
Na2SeO4
Na2WO4
Distilled water
12.8
1.35
0.1
0.024
0.1
0.1
0.025
0.01
0.024
1
0.12
0.004
0.004
1
g
g
g
g
g
g
g
g
g
g
g
g
g
L
First dissolve nitrilotriacetic acid and adjust pH to 6.5 with
NaOH, and then add minerals. Final pH 7.0.
1130 Sazae YGS
Turban shell (sazae) meat cut
Yeast extract
Glucose
Agar
Seawater (3 % salinity)
75
10
30
12
1
g
g
g
g
L
20
1
1
15
g
g
L
g
1132 Malt Extract Ebios Agar (MEBA)
Malt extract
Ebios (tablets)*
Distilled water
Agar
* Manufactured by Asahi Breweries, Ltd., Tokyo, Japan.
Distributed by Tanabe Seiyaku Co. Ltd., Osaka, Japan.
1136 CHROMagerTM Malassezia/Candida*
* Kanto Chemical Co., Inc., Tokyo, Japan.
1014