Handbook of Microbiological Media, Fourth Edition part 179 pps

Thioglycolate Medium, USP 1775
Thioglycolate Medium without Indicator
Composition per liter:
Pancreatic digest of casein 17.0g
Glucose 6.0g
Papaic digest of soybean meal 3.0g
NaCl 2.5g
Agar 0.7g
Sodium thioglycolate 0.5g
L-Cystine 0.25g
Na
2
SO
3
0.1g
pH 7.0 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from Oxoid
Unipath.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Prepare
freshly or boil and cool the medium just before use.
Use: For the growth of aerobic and anaerobic microorganisms in diag-
nostic bacteriology.
Thioglycolate Medium without Indicator-135C
Composition per liter:
Pancreatic digest of casein 17.0g
Glucose 6.0g
Papaic digest of soybean meal 3.0g
NaCl 2.5g
Agar 0.7g

Sodium thioglycolate 0.5g
Na
2
SO
3
0.1g
L-Cystine 0.25g
pH 7.0 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems.
Preparation of Medium:
Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat while
stirring and bring to boiling. Distribute into tubes or flasks, filling them
half full. For maintenance of cultures, a small quantity of CaCO
3
may
be added to tubes before adding medium. Autoclave for 15 min at 13
psi pressure–118°C. Prepare freshly or boil and cool the medium just
before use. Store prepared medium at 2°–8°C in the dark.
Use: For the isolation and cultivation of a wide variety of microorgan-
isms, particularly obligate anaerobes, from clinical specimens and
other materials.
Thioglycolate Medium without Indicator with Hemin
(Thioglycolate Medium, Supplemented)
Composition per liter:
Pancreatic digest of casein 17.0g
CaCO
3
, chips or powder 10.0g

Glucose 6.0g
Papaic digest of soybean meal 3.0g
NaCl 2.5g
Agar 0.7g
Sodium thioglycolate 0.5g
L-Cystine 0.25g
Na
2
SO
3
0.1g
Hemin 5.0mg
Na
2
CO
3
solution 10.0mL
Vitamin K
1
solution 10.0mL
pH 7.2 ± 0.2 at 25°C
Na
2
CO
3
Solution:
Composition
per 10.0mL:
Na
2

CO
3
1.0g
Preparation of Na
2
CO
3
Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster-
ilize.
Vitamin K
1
Solution:
Composition
per 100.0mL:
Vitamin K
1
1.0g
Ethanol, absolute 99.0mL
Preparation of Vitamin K
1
Solution: Add vitamin K
1
to 99.0mL
of absolute ethanol. Mix thoroughly.
Preparation of Medium: Add components, except CaCO

3
, Na
2
CO
3
solution, and vitamin K
1
solution, to distilled/deionized water and
bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to
boiling. Add 0.1g of CaCO
3
chips or powder to each of 100 test tubes.
Distribute broth into the same tubes in 10.0mL volumes. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add
0.1mL of sterile Na
2
CO
3
solution and 0.1mL of sterile vitamin K
1
so-
lution to each tube. Mix thoroughly.
Use: For the cultivation of a wide variety of obligate anaerobes.
Thioglycolate Medium, Supplemented
See: Thioglycolate Medium without Indicator with Hemin
Thioglycolate Medium, USP
Composition per liter:
Pancreatic digest of casein 15.0g
Glucose 5.5g
Yeast extract 5.0g

NaCl 2.5g
Agar 0.5g
L-cystine 0.5g
Sodium thioglycolate 0.5g
Resazurin 1.0mg
pH 7.1 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from Oxoid
Unipath.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C.
Use: For the cultivation of both aerobic and anaerobic organisms in
the performance of sterility tests.
Thioglycolate Medium with Vitamin K
1
and Hemin
See: Thioglycolate Medium, Enriched
Thioglycolate Peptone Glucose Yeast Extract Medium
See: TPGY Medium
Thioglycolate Potato Liver Medium
See: TPL Medium
© 2010 by Taylor and Francis Group, LLC
1776 Thiol Broth
Thiol Broth
Composition per liter:
Proteose peptone, No. 3 10.0g
Thiol complex 8.0g
Yeast extract 5.0g
NaCl 5.0g

Glucose 1.0g
p-Aminobenzoic acid 0.05g
pH 7.1 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. For neutralization of penicil-
lin, distribute medium into tubes to a depth of 60.0mm. For neutraliza-
tion of streptomycin, distribute medium into tubes in shallow layers.
Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of bacteria from body fluids and other mate-
rials containing penicillin, streptomycin, or sulfonamides. Also used
for the cultivation and maintenance of Bifidobacterium species.
Thiohalophilus Medium
(DSMZ Medium 1058)
Composition per liter:
NaCl 120.0g
K
2
HPO
4
1.5g
NH
4
Cl 0.5g
Bicarbonate solution 10.0mL
Calcium chloride solution 10.0mL
Magnesium chloride solution 10.0mL
Seven vitamin solution 1.0mL

Trace elements solution SL-10 with EDTA 1.0mL
Carbonate solution variable
pH 7.6 ± 0.2 at 25°C
Thiosulfate Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3
·5H
2
O 5.0g
Preparation of Thiosulfate Solution: Add components to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Bicarbonate Solution:
Composition per 10.0mL:
NaHCO
3
5.0g
Preparation of Bicarbonate Solution: Add NaHCO
3
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N

2
+ 20% CO
2
. Filter sterilize.
Carbonate Solution:
Composition per 10.0mL:
Na
2
CO
3
2.0g
Preparation of Carbonate Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Magnesium Chloride Solution:
Composition
per 10.0mL:
MgCl
2
·6H
2
O 0.4g

Preparation of Magnesium Chloride Solution: Add 0.4g of
MgCl
2
·6H
2
O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to room temperature. Sparge with 100% N
2
.
Calcium Chloride Solution:
Composition
per 10.0mL:
CaCl
2
·2H
2
O 0.05g
Preparation of Calcium Chloride Solution: Add components to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room tem-
perature. Sparge with 100% N
2
gas.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 300.0mg
Nicotinic acid 200.0mg
Thiamine·HCl 200.0mg

Calcium
DL-pantothenate 100.0mg
Cyanocobalamine 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize. Sparge with 100% N
2
.
Trace Elements Solution SL-10 with EDTA:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
Na
2
-EDTA 0.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2

O 100.0mg
ZnCl
2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10 with EDTA:
Add FeCl
2

·4H
2
O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min
at 15 psi pressure–121°C. Adjust pH to 7.0.
Preparation of Medium: Add components, except magnesium
chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/
deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
gas mixture for at least 30 min to remove dis-
solved oxygen and to saturate the solution with CO
2
. Dispense into an-
aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air
atmosphere. Close vials with butyl rubber septa to prevent free ex-
change of oxygen with the external atmosphere. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to room temperature. Aseptically add
magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions.
Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium
carbonate (5% w/v).
Use: For the cultivation of Thiohalomonas denitrificans, Thiohalo-
philus thiocyanatoxydans, and Thiomicrospira halophila.
Thiohalophilus Medium
(DSMZ Medium 1058)

Composition per liter:
NaCl 233.0g
K
2
HPO
4
1.5g
© 2010 by Taylor and Francis Group, LLC
Thiohalophilus Medium 1777
NH
4
Cl 0.5g
Bicarbonate solution 10.0mL
Calcium chloride solution 10.0mL
Magnesium chloride solution 10.0mL
Seven vitamins solution 1.0mL
Trace elements solution SL-10 with EDTA 1.0mL
Carbonate solution variable
pH 7.2 ± 0.2 at 25°C
Thiosulfate Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3
·5H
2

O 5.0g
Preparation of Thiosulfate Solution: Add Na
2
S
2
O
3
·5H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Bicarbonate Solution:
Composition per 10.0mL:
NaHCO
3
5.0g
Preparation of Bicarbonate Solution: Add NaHCO
3
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Carbonate Solution:
Composition per 10.0mL:

Na
2
CO
3
2.0g
Preparation of Carbonate Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Magnesium Chloride Solution:
Composition
per 10.0mL:
MgCl
2
·6H
2
O 0.4g
Preparation of Magnesium Chloride Solution: Add 0.4g of
MgCl
2
·6H
2
O to distilled/deionized water and bring volume to 10.0mL.

Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to room temperature. Sparge with 100% N
2
.
Calcium Chloride Solution:
Composition
per 10.0mL:
CaCl
2
·2H
2
O 0.05g
Preparation of Calcium Chloride Solution: Add CaCl
2
·2H
2
O
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room
temperature. Sparge with 100% N
2
.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 300.0mg
Nicotinic acid 200.0mg
Thiamine·HCl 200.0mg
Calcium
DL-pantothenate 100.0mg

Cyanocobalamine 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize. Sparge with 100% N
2
.
Trace Elements Solution SL-10 with EDTA:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
Na
2
-EDTA 0.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl

2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10 with EDTA:
Add FeCl
2
·4H
2

O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min
at 15 psi pressure–121°C. Adjust pH to 7.0.
Preparation of Medium: Add components, except magnesium
chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/
deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
gas mixture for at least 30 min to remove dis-
solved oxygen and to saturate the solution with CO
2
. Dispense into an-
aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air
atmosphere. Close vials with butyl rubber septa to prevent free ex-
change of oxygen with the external atmosphere. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to room temperature. Aseptically add
magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions.
Mix thoroughly. Adjust pH to 7.2 using a sterile solution of sodium car-
bonate (5% w/v).
Use: For the cultivation of Thiomicrospira halophila DSM 15071.
Thiohalophilus Medium
(DSMZ Medium 1058)
Composition per liter:
NaCl 180.0g
K

2
HPO
4
1.5g
KNO
3
1.0g
NH
4
Cl 0.5g
Bicarbonate solution 10.0mL
Calcium chloride solution 10.0mL
Magnesium chloride solution 10.0mL
Seven vitamin solution 1.0mL
Trace elements solution SL-10 with EDTA 1.0mL
Carbonate solution Variable
pH 7.6 ± 0.2 at 25°C
Thiosulfate Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3
·5H
2
O 5.0g
Preparation of Thiosulfate Solution: Add Na

2
S
2
O
3
·5H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Bicarbonate Solution:
Composition per 10.0mL:
NaHCO
3
5.0g
Preparation of Bicarbonate Solution: Add NaHCO
3
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Carbonate Solution:
Composition per 10.0mL:
Na
2

CO
3
2.0g
Preparation of Carbonate Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
© 2010 by Taylor and Francis Group, LLC
1778 Thiohalophilus Medium
Magnesium Chloride Solution:
Composition
per 10.0mL:
MgCl
2
·6H
2
O 0.4g
Preparation of Magnesium Chloride Solution: Add 0.4g of
MgCl
2
·6H
2
O to distilled/deionized water and bring volume to 10.0mL.

Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to room temperature. Sparge with 100% N
2
.
Calcium Chloride Solution:
Composition
per 10.0mL:
CaCl
2
·2H
2
O 0.05g
Preparation of Calcium Chloride Solution: Add CaCl
2
·2H
2
O
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room
temperature. Sparge with 100% N
2
.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 300.0mg
Nicotinic acid 200.0mg
Thiamine·HCl 200.0mg
Calcium
DL-pantothenate 100.0mg

Cyanocobalamine 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize. Sparge with 100% N
2
.
Trace Elements Solution SL-10 with EDTA:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
Na
2
-EDTA 0.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl

2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10 with EDTA:
Add FeCl
2
·4H
2

O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min
at 15 psi pressure–121°C. Adjust pH to 7.0.
Preparation of Medium: Add components, except magnesium
chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/
deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
gas mixture for at least 30 min. to remove dis-
solved oxygen and to saturate the solution with CO
2
. Dispense into an-
aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air
atmosphere. Close vials with butyl rubber septa to prevent free ex-
change of oxygen with the external atmosphere. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to room temperature. Aseptically add
magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions.
Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium
carbonate (5% w/v).
Use: For the cultivation of Thiohalomonas denitrificans.
Thiohalophilus Medium
(DSMZ Medium 1058)
Composition per liter:
NaCl 180.0g
K

2
HPO
4
1.5g
KNO
3
2.0g
NH
4
Cl 0.5g
Bicarbonate solution 10.0mL
Calcium chloride solution 10.0mL
Magnesium chloride solution 10.0mL
Seven vitamin solution 1.0mL
Trace elements solution SL-10 with EDTA 1.0mL
Carbonate solution Variable
pH 7.6 ± 0.2 at 25°C
Thiosulfate Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3
·5H
2
O 5.0g
Preparation of Thiosulfate Solution: Add Na

2
S
2
O
3
·5H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Bicarbonate Solution:
Composition per 10.0mL:
NaHCO
3
5.0g
Preparation of Bicarbonate Solution: Add NaHCO
3
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Carbonate Solution:
Composition per 10.0mL:
Na
2

CO
3
2.0g
Preparation of Carbonate Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Magnesium Chloride Solution:
Composition
per 10.0mL:
MgCl
2
·6H
2
O 0.4g
Preparation of Magnesium Chloride Solution: Add 0.4g of
MgCl
2
·6H
2
O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to room temperature. Sparge with 100% N

2
.
Calcium Chloride Solution:
Composition
per 10.0mL:
CaCl
2
·2H
2
O 0.05g
Preparation of Calcium Chloride Solution: Add CaCl
2
·2H
2
O
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room
temperature. Sparge with 100% N
2
.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 300.0mg
Nicotinic acid 200.0mg
Thiamine·HCl 200.0mg
Calcium
DL-pantothenate 100.0mg
Cyanocobalamine 100.0mg
p-Aminobenzoic acid 80.0mg

D(+)-Biotin 20.0mg
© 2010 by Taylor and Francis Group, LLC
Thiomicrospira denitrificans Agar 1779
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize. Sparge with 100% N
2
.
Trace Elements Solution SL-10 with EDTA:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
Na
2
-EDTA 0.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl

2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10 with EDTA:
Add FeCl
2
·4H
2

O to 10.0mL of HCl solution. Mix thoroughly. Add dis-
tilled/deionized water and bring volume to 1.0L. Add remaining com-
ponents. Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min
at 15 psi pressure–121°C. Adjust pH to 7.0.
Preparation of Medium: Add components, except magnesium
chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/
deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
gas mixture for at least 30 min. to remove dis-
solved oxygen and to saturate the solution with CO
2
. Dispense into an-
aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air
atmosphere. Close vials with butyl rubber septa to prevent free ex-
change of oxygen with the external atmosphere. Autoclave for 15 min
at 15 psi pressure–121°C. Cool to room temperature. Aseptically add
magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions.
Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium
carbonate (5% w/v).
Use: For the cultivation of Thiohalomonas nitratireducens.
Thiol HiVeg Broth
Composition per liter:
Plant peptone No. 3 10.0g
Thiol compound 8.0g
NaCl 5.0g

Yeast extract 5.0g
Glucose 1.0g
p-Aminobenzoic acid (PABA) 0.05g
pH 7.1 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from Hi-
Media.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. For neutralization of penicil-
lin, distribute medium into tubes to a depth of 60.0mm. For neutraliza-
tion of streptomycin, distribute medium into tubes in shallow layers.
Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of bacteria from body fluids and other mate-
rials containing penicillin, streptomycin, or sulfonamides. Also used
for the cultivation and maintenance of Bifidobacterium species.
Thiol HiVeg Medium
Composition per liter:
Plant peptone No. 3 10.0g
Thiol compound 8.0g
Yeast extract 5.0g
NaCl 5.0g
Agar 1.0g
Glucose 1.0g
p-Aminobenzoic acid (PABA) 0.05g
pH 7.1 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from Hi-
Media.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. For neutralization of penicil-

lin, distribute medium into tubes to a depth of 60 mm. For neutraliza-
tion of streptomycin, distribute medium into tubes in shallow layers.
Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of bacteria from body fluids and other mate-
rials containing penicillin, streptomycin, or sulfonamides. Also used
for the cultivation and maintenance of Bifidobacterium species.
Thiol Medium
Composition per liter:
Proteose peptone No. 3 10.0g
Thiol complex 8.0g
Yeast extract 5.0g
NaCl 5.0g
Glucose 1.0g
Agar 1.0g
p-Aminobenzoic acid 0.05g
pH 7.1 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. For neutralization of penicil-
lin, distribute medium into tubes to a depth of 60mm. For neutraliza-
tion of streptomycin, distribute medium into tubes in shallow layers.
Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of bacteria from body fluids and other mate-
rials containing penicillin, streptomycin, or sulfonamides. Also used
for the cultivation and maintenance of Bifidobacterium species.
Thiomicrospira denitrificans Agar
Composition per 1001.0mL:
Solution A 940.0mL

Solution B 40.0mL
Solution C 20.0mL
Solution D 1.0mL
pH 7.0 ± 0.2 at 25°C
Solution A:
Composition
per 940.0mL:
Agar 15.0g
KH
2
PO
4
2.0g
KNO
3
2.0g
NH
4
Cl 1.0g
MgSO
4
·7H
2
O 0.8g
Trace elements solution SL-4 2.0mL
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 940.0mL. Mix thoroughly. Gently heat and
© 2010 by Taylor and Francis Group, LLC
1780 Thiomicrospira denitrificans Broth
bring to boiling. Adjust pH to 7.0 with NaOH. Autoclave for 15 min at

15 psi pressure–121°C. Cool to 45°–50°C.
Trace Elements Solution SL-4:
Composition per liter:
EDTA 0.5g
FeSO
4
·7H
2
O 0.2g
Trace elements solution SL-6 100.0
Preparation of Trace Elements Solution SL-4: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Trace Elements Solution SL-6:
Composition
per liter:
H
3
BO
3
0.3g
CoCl
2
·6H
2
O 0.2g
ZnSO
4
·7H
2
O 0.1g

MnCl
2
·4H
2
O 0.03g
Na
2
MoO
4
·H
2
O 0.03g
NiCl
2
·6H
2
O 0.02g
CuCl
2·
·2H
2
O 0.01g
Preparation of Trace Elements Solution SL-6: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Adjust pH to 3.4.
Solution B:
Composition per 40.0mL:
Na
2
S

2
O
3
·5H
2
O 5.0g
Preparation of Solution B: Add Na
2
S
2
O
3
·5H
2
O to distilled/deion-
ized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Solution C:
Composition per 20.0mL:
NaHCO
3
1.0g
Preparation of Solution C: Add NaHCO
3
to distilled/deionized
water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.
Solution D:
Composition per liter:
FeSO
4

·7H
2
O 2.0mg
H
2
SO
4
(0.1N solution) 1.0mL
Preparation of Solution D: Add FeSO
4
·7H
2
O to 1.0mL of 0.1N
H
2
SO
4
solution. Mix thoroughly. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 45°–50°C.
Preparation of Medium: Aseptically add 40.0mL of sterile solu-
tion B, 20.0mL of sterile solution C, and 1.0mL of sterile solution D to
940.0mL of sterile solution A. Mix thoroughly. Aseptically and anaer-
obically distribute into sterile tubes under 100% N
2
.
Use: For the cultivation and maintenance of Thiomicrospira denitrifi-
cans.
Thiomicrospira denitrificans Broth
Composition per 1001.0mL:
Solution A 940.0mL

Solution B 40.0mL
Solution C 20.0mL
Solution D 1.0mL
pH 7.0 ± 0.2 at 25°C
Solution A:
Composition
per 940.0mL:
KH
2
PO
4
2.0g
KNO
3
2.0g
NH
4
Cl 1.0g
MgSO
4
·7H
2
O 0.8g
Trace elements solution SL-4 2.0mL
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 940.0mL. Mix thoroughly. Adjust pH to 7.0
with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
45°–50°C.
Solution B:
Composition per 40.0mL:

Na
2
S
2
O
3
·5H
2
O 5.0g
Preparation of Solution B: Add Na
2
S
2
O
3
·5H
2
O to distilled/deion-
ized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Solution C:
Composition per 20.0mL:
NaHCO
3
1.0g
Preparation of Solution C: Add NaHCO
3
to distilled/deionized
water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.
Solution D:

Composition per liter:
FeSO
4
·7H
2
O 2.0mg
H
2
SO
4
(0.1N solution) 1.0mL
Preparation of Solution D: Add FeSO
4
·7H
2
O to 1.0mL of 0.1N
H
2
SO
4
solution. Mix thoroughly. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 45°–50°C.
Trace Elements Solution SL-4:
Composition per liter:
EDTA 0.5g
FeSO
4
·7H
2
O 0.2g

Trace elements solution SL-6 100.0mL
Preparation of Trace Elements Solution SL-4: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Trace Elements Solution SL-6:
Composition
per liter:
H
3
BO
3
0.3g
CoCl
2
·6H
2
O 0.2g
ZnSO
4
·7H
2
O 0.1g
MnCl
2
·4H
2
O 0.03g
Na
2
MoO
4

·H
2
O 0.03g
NiCl
2
·6H
2
O 0.02g
CuCl
2·
2H
2
O 0.01g
Preparation of Trace Elements Solution SL-6: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 3.4.
Preparation of Medium: Aseptically add 40.0mL of sterile solution
B, 20.0mL of sterile solution C, and 1.0mL of sterile solution D to
940.0mL of sterile solution A. Mix thoroughly. Aseptically and anaero-
bically distribute into sterile tubes under 100% N
2
.
Use: For the cultivation and maintenance of Thiomicrospira denitrifi-
cans.
© 2010 by Taylor and Francis Group, LLC
Thiomicrospira Medium 1781
Thiomicrospira denitrificans Medium
Composition per liter:
Part I 500.0mL
Part II 500.0mL

pH 7.0–8.0 at 25°C
Part I:
Composition
per liter:
NaCl 20.0g
KNO
3
4.0g
(NH
4
)
2
SO
4
2.0g
MgSO
4
·7H
2
O 1.5g
K
2
HPO
4
0.6g
KH
2
PO
4
0.4g

FeSO
4
solution 2.0mL
Trace metals solution 2.0mL
HCl, concentrated 1.0mL
Preparation of Part I: Add components to distilled/deionized wa-
ter and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at
15 psi pressure–121°C. Cool to 25°C.
FeSO
4
Solution:
Composition
per 100.0mL:
FeSO
4
·7H
2
O 0.5g
HCl (1N solution) 100.0mL
Preparation of FeSO
4
Solution: Combine the FeSO
4
·7H
2
O and
100.0mL of HCl solution. Mix thoroughly.
Trace Metals Solution:
Composition
per liter:

Disodium EDTA 50.0g
NaOH 11.0g
CaCl
2
·2H
2
O 7.34g
MnCl
2
·2H
2
O 2.5g
ZnSO
4
·7H
2
O 2.2g
CoCl
2
·6H
2
O 0.5g
(NH
4
)
6
Mo
7
O
24

·4H
2
O 0.5g
CuSO
4
·5H
2
O 0.2g
Preparation of Trace Metals Solution: Add EDTA to distilled/
deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust
pH to 6.0 with NaOH. Add remaining components, one by one. Main-
tain the pH at 6.0. After dissolution of all the salts, adjust the pH to 4.0
with HCl. Store at 4°C.
Part II:
Composition
per liter:
Na
2
S
2
O
3
·5H
2
O 10.0g
NaHCO
3
3.0g
NaOH 0.05g
Preparation of Part II: Add components to distilled/deionized wa-

ter and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at
15 psi pressure–121°C. Cool to 25°C.
Preparation of Medium: Aseptically combine 500.0mL of sterile
part I and 500.0mL of sterile part II. Mix thoroughly. Aseptically dis-
tribute into sterile tubes or flasks.
Use: For the isolation and cultivation of Thiomicrospira denitrificans.
Thiomicrospira Medium
(ATCC Medium 1036)
Composition per liter:
NaCl 25.0g
Na
2
S
2
O
3
·5H
2
O 8.0g
MgSO
4
·7H
2
O 1.5g
(NH
4
)
2
SO
4

1.0g
K
2
HPO
4
0.5g
CaCl
2
0.3g
Vitamin B
12
15.0μg
Vishniac and Santer trace metals 0.2mL
Bromcresol Purple (0.05% solution) 0.1mL
pH 7.2 ± 0.2 at 25°C
Vishniac and Santer Trace Metals:
Composition
per liter:
Ethylenediamine tetraacetic acid (EDTA) 50.0g
ZnSO
4
·7H
2
O 22.0g
CaCl
2
5.54g
MnCl
2
·4H

2
O 5.06g
FeSO
4
·7H
2
O 4.99g
CoCl
2
·6H
2
O 1.61g
CuSO
4
·5H
2
O 1.57g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 1.1g
Preparation of Vishniac and Santer Trace Metals: Add com-
ponents to distilled/deionized water and bring volume to 1.0L. Adjust

pH to 6.0 with KOH. Mix thoroughly.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Fil-
ter sterilize. Aseptically distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Thiomicrospira species.
Thiomicrospira Medium
(ATCC Medium 1422)
Composition per liter:
NaCl 25.1g
Tris·HCl 3.07g
Na
2
S
2
O
3
·5H
2
O 2.48g
MgSO
4
·7H
2
O 1.5g
(NH
4
)
2
SO
4

1.0g
KH
2
PO
4
0.42g
CaCl
2
·2H
2
O 0.29g
NaHCO
3
0.2g
Phenol Red (0.5% solution) 1.0mL
Vishniac and Santer trace metals 0.2mL
pH 7.5 ± 0.2 at 25°C
Vishniac and Santer Trace Metals:
Composition
per liter:
Ethylenediamine tetraacetic acid (EDTA) 50.0g
ZnSO
4
·7H
2
O 22.0g
CaCl
2
5.54g
MnCl

2
·4H
2
O 5.06g
FeSO
4
·7H
2
O 4.99g
CoCl
2
·6H
2
O 1.61g
CuSO
4
·5H
2
O 1.57g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 1.1g

Preparation of Vishniac and Santer Trace Metals: Add com-
ponents to distilled/deionized water and bring volume to 1.0L. Adjust
pH to 6.0 with KOH. Mix thoroughly.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Fil-
ter sterilize. Aseptically distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Thiomicrospira species.
© 2010 by Taylor and Francis Group, LLC
1782 Thiomicrospira pelophila Medium
Thiomicrospira pelophila Medium
Composition per liter:
NaCl 25.0g
Agar 10.0g
Na
2
S
2
O
3
·5H
2
O 5.0–8.0g
MgSO
4
·7H
2
O 1.5g
(NH
4
)

2
SO
4
1.0g
K
2
HPO
4
0.5g
CaCl
2
0.3g
Vitamin B
12
0.15mg
Trace metals solution 0.2mL
Trace Metals Solution:
Composition
per liter:
Disodium EDTA 50.0g
NaOH 11.0g
CaCl
2
·2H
2
O 7.34g
FeSO
4
·7H
2

O 5.0g
MnCl
2
·2H
2
O 2.5g
ZnSO
4
·7H
2
O 2.2g
CoCl
2
·6H
2
O 0.5g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 0.5g
CuSO
4
·5H

2
O 0.2g
Preparation of Trace Metals Solution: Add EDTA to distilled/
deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust
pH to 6.0 with NaOH. Add remaining components, one by one. Main-
tain the pH at 6.0. After dissolution of all the salts, adjust the pH to 4.0
with HCl. Store at 4°C.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation of Thiomicrospira pelophila.
Thiomicrospira pelophila Medium
Composition per 1000.2mL:
NaCl 25.0g
MgSO
4
·7H
2
O 1.5g
(NH
4
)
2
SO
4
1.0g
CaCl
2
·2H

2
O 0.42g
Bromthymol Blue 4.0mg
K
2
HPO
4
solution 100.0mL
Na
2
S
2
O
3
·5H
2
O solution 100.0mL
Vitamin B
12
solution 10.0mL
Trace elements solution 0.2mL
Na
2
CO
3
solution variable
pH 7.2 ± 0.2 at 25°C
K
2
HPO

4
Solution:
Composition
per 100.0mL:
K
2
HPO
4
0.5g
Preparation of K
2
HPO
4
Solution: Add K
2
HPO
4
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C.
Na
2
S
2
O
3
Solution:
Composition
per 10.0mL:
Na

2
S
2
O
3
·5H
2
O 5.0g
Preparation of Na
2
S
2
O
3
Solution: Add Na
2
S
2
O
3
·5H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C.
Vitamin B
12
Solution:
Composition
per 10.0mL:

Vitamin B
12
15.0mg
Preparation of Vitamin B
12
Solution: Add vitamin B
12
to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Filter sterilize.
Trace Elements Solution:
Composition
per liter:
Disodium EDTA 50.0g
ZnSO
4
·7H
2
O 22.0g
CaCl
2
·2H
2
O 5.54g
MnCl
2
·4H
2
O 5.06g
FeSO

4
·7H
2
O 5.0g
CoCl
2
·6H
2
O 1.61g
CuSO
4
·5H
2
O 1.57g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 1.1g
Preparation of Trace Elements Solution: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Na
2
CO

3
Solution:
Composition
per 100.0mL:
Na
2
CO
3
0.4g
Preparation of Na
2
CO
3
Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Add components, except K
2
HPO
4
solu-
tion, Na
2
S
2
O

3
·5H
2
O solution, vitamin B
12
solution, and Na
2
CO
3
solu-
tion, to distilled/deionized water and bring volume to 790.0mL. Mix
thoroughly. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure–
121°C. Aseptically add 100.0mL of sterile K
2
HPO
4
solution, 100.0mL
of sterile Na
2
S
2
O
3
·5H
2
O solution, and 10.0mL of sterile vitamin B
12
solution. Mix thoroughly. Aseptically adjust pH to 7.2 with the appro-
priate volume of sterile Na
2

CO
3
solution. Aseptically distribute into
sterile tubes or flasks.
Use: For the cultivation and maintenance of Thiomicrospira pelo-
phila.
Thiomonas delicata Medium
(DSMZ Medium 1037)
Composition per liter:
Na
2
S
2
O
3
·5H
2
O 5.0g
Na
2
HPO
4
4.5g
K
2
HPO
4
1.5g
Yeast extract 1.0g
Na-aspartate 1.0g

NH
4
Cl 0.3g
MgSO
4
·7H
2
O 0.1g
pH 6.0 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0.
Dispense into culture vessels. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For the cultivation of Thiomonas delicata.
Thiomonas delicata Medium
(DSMZ Medium 1037)
Composition per liter:
Na
2
S
2
O
3
·5H
2
O 5.0g
Na
2
HPO
4

4.5g
© 2010 by Taylor and Francis Group, LLC
Thiorhodococcus Medium 1783
K
2
HPO
4
1.5g
Yeast extract 1.0g
NH
4
Cl 0.3g
MgSO
4
·7H
2
O 0.1g
pH 6.0 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0.
Dispense into culture vessels. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For the cultivation of Thiomonas perometabolis.
Thiophaeococcus mangrovi Medium
(DSMZ Medium 1162)
Composition per liter:
NaCl 20.0g
Sodium pyruvate 3.0g
MgSO
4

·7H
2
O 2.0g
NH
4
Cl 0.64g
K
2
HPO
4
0.5g
Yeast extract 0.4g
CaCl
2
·2H
2
O 0.15g
Bicarbonate solution 10.0mL
Sulfide solution 10.0mL
Thiosulfate solution 10.0mL
Trace element solution SL-8 1.0mL
Vitamin solution 1.0mL
pH 7.5 ± 0.2 at 25°C
Sulfide Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 1.0g

Preparation of Sulfide Solution: Add Na
2
S·9H
2
O to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N
2
. Filter sterilize.
Thiosulfate Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3
·5H
2
O 1.5g
Preparation of Thiosulfate Solution: Add Na
2
S
2
O
3
·5H
2
O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Bicarbonate Solution:
Composition per 10.0mL:
NaHCO
3
1.0g
Preparation of Bicarbonate Solution: Add NaHCO
3
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with a gas mixture of 80% N
2
+ 20% CO
2
. Filter sterilize.
Vitamin Solution:
Composition
per 10.0mL:
Vitamin B
12
2.0mg
Preparation of Vitamin Solution: Add Vitamin B
12
to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Filter
sterilize.
Trace Elements Solution SL-8:

Composition per liter:
Disodium EDTA 5.2g
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 0.19g
MnCl
2
·4H
2
O 0.1g
ZnCl
2
0.07g
H
3
BO
3
0.06g
NaMoO
4
·2H
2
O 0.04g

CuCl
2
·2H
2
O 0.02g
NiCl
2
·6H
2
0 0.02g
Preparation of Trace Elements Solution SL-8: Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly.
Preparation of Medium: Add components, except sulfide, bicar-
bonate, thiosulfate, and vitamin solutions, to distilled/deionized water
and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N
2
+
20% CO
2
gas mixture. Dispense into culture vessels. Autoclave for 15
min at 15 psi pressure–121°C. Cool to room temperature. Aseptically
add sulfide, thiosulfate, bicarbonate, and vitamin solutions. Mix thor-
oughly. Adjust pH to 7.5.
Use: For the cultivation of Thiophaeococcus mangrovi.
Thiophene-2-Carboxylic Acid Hydrazide
See: TCH Medium
Thiorhodococcus Medium
(DSMZ Medium 28a)
Composition per 5.0L:

Solution A 4.0L
Solution B 860.0mL
Solution E 100.0mL
Solution F 20.0mL
Solution C 5.0mL
Solution D 5.0mL
pH 7.3 at 25°C
Solution A:
Composition
per 4.0L:
NaCl 100.0g
MgCl
2
·6H
2
O 5.0g
MgSO
4
2.5g
Na
2
S
2
O
3
·5H
2
O 2.5g
KH
2

PO
4
1.7g
NH
4
Cl 1.7g
KCl 1.7g
CaCl
2
·2H
2
O 1.25g
Preparation of Solution A: Add components to 4.0L distilled wa-
ter. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C in
a 5-liter special bottle or flask with four openings at the top, together
with a teflon-coated magnetic bar. In this 5-liter bottle, two openings
for tubes are in the central, silicon rubber stopper; one is a short, gas-
inlet tube with a sterile cotton filter, and the other is an outlet tube for
medium, which reaches the bottom of the vessel at one end and has, at
the other end, a silicon rubber tube with a pinch cock and a bell for
aseptic dispensing of the medium into bottles. The other two openings
have gas-tight screw caps; one of these openings is for the addition of
sterile solutions and the other serves as a gas outlet. After autoclaving,
cool to room temperature under a 100% N
2
atmosphere with a positive
pressure of 0.05– 0.1 atm (a manometer for low pressure will be re-
quired). Saturate the cold medium with CO
2
by magnetic stirring for 30

min under a CO
2
atmosphere of 0.05–0.1 atm.
Solution B:
Distilled water 860.0mL
© 2010 by Taylor and Francis Group, LLC
1784 Thiosphaera Agar
Preparation of Solution B: Autoclave distilled water for 15 min at
15 psi pressure–121°C in a cotton-stoppered Erlenmeyer flask. Cool to
room temperature under an atmosphere of N
2
in an anaerobic jar.
Solution C:
Composition
per 100.0mL:
Vitamin B
12
2.0mg
Preparation of Solution C: Add vitamin B
12
to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Sparge under
100% N
2
gas for 3 min. Filter sterilize Store under N
2
gas.
Solution D:
Composition
per liter:

Disodium ethylendiamine tetraacetate (Disodium EDTA) 3.0g
FeSO
4
·7H
2
O 1.1g
H
3
BO
3
0.3g
CoCl
2
·6H
2
O 0.19g
MnCl
2
·2H
2
O 50.0mg
ZnCl
2
42.0mg
NiCl
2
·6H
2
O 24.0mg
Na

2
MoO
4
·2H
2
O 18.0mg
CuCl
2
·2H
2
O 2.0mg
Preparation of Solution D: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min
at 15 psi pressure–121°C.
Solution E:
Composition
per 100.0mL:
NaHCO
3
7.5g
Preparation of Solution E: Add NaHCO
3
to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Sparge with
100% CO
2
until saturated. Filter sterilize under 100% CO
2
into a ster-
ile, gas-tight 100.0mL screw-capped bottle.

Solution F:
Composition
per 100.0mL:
Na
2
S·9H
2
O 10.0g
Preparation of Solution F: Add Na
2
S·9H
2
O to distilled/deionized
water in a 250.0mL screw-capped bottle fitted with a butyl rubber sep-
tum and bring volume to 100.0mL. Mix thoroughly. Sparge under
100% N
2
gas for 3 min. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to room temperature.
Neutralized Sulfide Solution:
Composition
per 100.0mL:
Na
2
S·9H
2
O 1.5g
Preparation of Neutralized Sulfide Solution: Add Na
2
S·9H

2
O
to distilled/deionized water in a 250.0mL screw-capped bottle fitted
with a butyl rubber septum and bring volume to 100.0mL. Add a mag-
netic stir bar. Mix thoroughly. Sparge under 100% N
2
gas for 3 min.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temper-
ature. Adjust pH to about 7.3 with sterile 2M H
2
SO
4
. Do not open the
bottle to add H
2
SO
4
; use a sterile syringe. Stir the solution continuously
to avoid precipitation of elemental sulfur. The final solution should be
clear and yellow in color.
Preparation of Medium: Add solutions B, C, D, E, and F to solu-
tion A through one of the screw-cap openings against a stream of either
N
2
gas or, better, a mixture of 95% N
2
and 5% CO
2
while the medium
is magnetically stirred. Adjust the pH of the medium with sterile HCl

or Na
2
CO
3
solution (2M solutions) to pH 7.3. Distribute the medium
aseptically through the medium outlet tube into sterile, 100mL bottles
(with metal caps and autoclavable rubber seals) using the positive gas
pressure (0.05–0.1 atm) of the N
2
/CO
2
gas mixture. Leave a small air
bubble in each bottle to meet possible pressure changes. The tightly
sealed, screw-cap bottles can be stored for several weeks or months in
the dark. During the first 24 hr, the iron of the medium precipitates in
the form of black flocs. No other sediment should arise in the otherwise
clear medium. Incubate in the light at 500–1,000 lux intensity. Feed pe-
riodically with neutralized solution of sodium sulfide to replenish sul-
fide and with other supplement solutions.
Use: For the cultivation of Thiorhodococcus minor.
Thiosphaera Agar
Composition per liter:
Agar 15.0g
Na
2
HPO
4
4.2g
KH
2

PO
4
1.5g
NH
4
Cl 0.3g
MgSO
4
·7H
2
O 0.1g
KNO
3
0.1g
Vishniac and Santer trace metals 2.0mL
pH 8.0–8.2 at 25°C
Vishniac and Santer Trace Metals:
Composition
per liter:
Ethylenediamine tetraacetic acid (EDTA) 50.0g
ZnSO
4
·7H
2
O 22.0g
CaCl
2
5.54g
MnCl
2

·4H
2
O 5.06g
FeSO
4
·7H
2
O 4.99g
CoCl
2
·6H
2
O 1.61g
CuSO
4
·5H
2
O 1.57g
(NH
4
)
6
Mo
7
O
24
·4H
2
O 1.1g
Preparation of Vishniac and Santer Trace Metals: Add com-

ponents to distilled/deionized water and bring volume to 1.0L. Adjust
pH to 6.0 with KOH. Mix thoroughly.
Preparation of Medium: Add components, except agar, to dis-
tilled/deionized water and bring volume to 500.0mL. Mix thoroughly.
Adjust pH to 8.0–8.2. Filter sterilize. Warm to 45°–50°C. Add agar to
distilled/deionized water and bring volume to 500.0mL. Mix thorough-
ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to 45°–50°C. Aseptically combine the two ster-
ile solutions. Mix thoroughly. Pour into sterile Petri dishes or distribute
into sterile tubes.
Use: For the cultivation and maintenance of Thiosphaera pantotro-
pha.
Thiosphaera Broth
Composition per liter:
Na
2
HPO
4
4.2g
KH
2
PO
4
1.5g
NH
4
Cl 0.3g
MgSO
4
·7H

2
O 0.1g
KNO
3
0.1g
Vishniac and Santer trace metals 2.0mL
pH 8.0–8.2 at 25°C
Vishniac and Santer Trace Metals:
Composition
per liter:
Ethylenediamine tetraacetic acid (EDTA) 50.0g
ZnSO
4
·7H
2
O 22.0g
CaCl
2
5.54g
MnCl
2
·4H
2
O 5.06g
© 2010 by Taylor and Francis Group, LLC