Handbook of Microbiological Media, Fourth Edition part 162 pdf

Spirochaeta litoralis Medium 1605
Glucose Salts Solution:
Composition
per liter:
NaCl 49.3g
MgSO
4
·7H
2
O 49.2g
CaCl
2
·2H
2
O 5.9g
Glucose solution 100.0mL
Sulfide solution 100.0mL
Preparation of Glucose Salts Solution: Add components, except
glucose solution and sulfide solution, to distilled/deionized water and
bring volume to 800.0mL. Mix thoroughly. Autoclave for 15 min at 15
psi pressure–121°C. Cool to 25°C. Aseptically add sterile glucose so-
lution and sulfide solution. Mix thoroughly.
Sulfide Solution:
Composition
per 100.0mL:
Na
2
S·9H
2
O 0.5g
Preparation of Sulfide Solution: Add Na

2
S·9H
2
O to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 25°C.
Glucose Solution:
Composition
per 100.0mL:
Glucose 5.0g
Preparation of Glucose Solution: Add glucose to distilled/deion-
ized water and bring volume to 100.0mL. Mix thoroughly. Filter ster-
ilize.
Yeast Extract Peptone Solution:
Composition
per 30.0mL:
Yeast extract 4.0g
Peptone 2.0g
Preparation of Yeast Extract Peptone Solution: Add compo-
nents to distilled/deionized water and bring volume to 30.0mL. Mix
thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
25°C.
Preparation of Medium: Aseptically combine 30.0mL of yeast ex-
tract peptone solution with 970.0mL of glucose salts solution. Mix
thoroughly. Aseptically distribute into sterile tubes or flasks.
Use: For the isolation and cultivation of Spirochaeta halophila.
Spirochaeta isovalerica Medium
Composition per liter:
Glucose 2.0g
Pancreatic digest of casein 1.0g

Yeast extract 0.5g
L-Cysteine·HCl 0.05g
Resazurin 0.001g
Seawater 750.0mL
Tris·HCl buffer
(0.2M solution, pH 7.5) 250.0mL
pH 7.5 ± 0.2 at 25°C
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Combine components. Mix thoroughly. Sparge with 100%
N
2
. Adjust pH to 7.5 while continuing to sparge with 100% N
2
. Anaer-
obically distribute into tubes or flasks. Autoclave for 15 min at 15 psi
pressure–121°C.
Use: For the cultivation and maintenance of Spirochaeta isovalerica.
Spirochaeta litoralis Medium
Composition per liter:
Pancreatic digest of casein 3.0g
NaCl 2.0g
Yeast extract 0.5g
Glucose solution 2.0mL
Potasssium phosphate
buffer (1M, pH 7.4) 2.0mL
Sulfide solution 0.5mL
Salts solution 0.2mL
pH 7.3 ± 0.2 at 25°C

Glucose Solution:
Composition per 100.0mL:
D-Glucose 25.0g
Preparation of Glucose Solution: Add D-glucose to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.
Sulfide Solution:
Composition
per 100.0mL:
Na
2
S·9H
2
O 10.0g
Preparation of Sulfide Solution: Add Na
2
S·9H
2
O to distilled/de-
ionized water and bring volume to 100.0mL. Autoclave for 15 min at
15 psi pressure–121°C. Cool to 25°C.
Salts Solution:
Composition
per 100.0mL:
MgSO
4
·7H
2
O 12.5g
CaCl

2
·2H
2
O 3.75g
EDTA 1.0g
FeSO
4
·7H
2
O 0.5g
Trace elements solution 25.0mL
Preparation of Salts Solution: Add components to distilled/deion-
ized water and bring volume to 100.0mL. Mix thoroughly.
Trace Elements Solution:
Composition
per 1800.0mL:
H
3
BO
3
5.5g
MnCl
2
·4H
2
O 3.5g
AlCl
3
·6H
2

O 0.5g
CoCl
2
·6H
2
O 0.5g
CuCl
2
·2H
2
O 0.5g
NiCl
2
·6H
2
O 0.5g
ZnCl
2
0.5g
KI 0.25g
LiCl 0.25g
Na
2
MoO
4
·2H
2
O 0.25g
BaCl
2

·2H
2
O 0.15g
SnCl
2
·2H
2
O 0.15g
NaVO
3
0.05g
Preparation of Trace Elements Solution: Add components to
distilled/deionized water and bring volume to 1800.0mL. Mix thor-
oughly. Adjust pH to 3–4 with HCl.
Preparation of Medium: Add components, except glucose solu-
tion and sulfide solution, to distilled/deionized water and bring volume
to 997.5mL. Mix thoroughly. Gently heat and bring to boiling. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Asepti-
cally add sterile glucose solution and sulfide solution. Mix thoroughly.
Aseptically distribute into sterile tubes or flasks.
Use: For the isolation of Spirochaeta litoralis from marine habitats.
© 2010 by Taylor and Francis Group, LLC
1606 Spirochaeta litoralis Medium
Spirochaeta litoralis Medium
Composition per liter:
Glucose 2.0g
Pancreatic digest of casein 1.0g
Yeast extract 1.0g
L-Cysteine 0.5g
Resazurin 1.0mg

Seawater 750.0mL
Tris·HCl buffer (1.0M solution, pH 7.5) 50.0mL
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components to distilled/deionized water and bring vol-
ume to 1.0L. Mix thoroughly. Sparge with 100% N
2
. Adjust pH to 7.5
while continuing to sparge with 100% N
2
. Anaerobically distribute into
tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Final
pH of medium after autoclaving should be 7.2.
Use: For the cultivation and maintenance of Spirochaeta litoralis.
Spirochaeta smaragdinae Medium
(DSMZ Medium 819)
Composition per liter:
NaCl 50.0g
Yeast extract 5.0g
NH
4
Cl 1.0g
Cysteine-HCl·H
2
O 0.5g
K
2
HPO

4
0.3g
KH
2
PO
4
0.3g
MgCl
2
·6H
2
O 0.2g
KCl 0.2g
CaCl
2
·2H
2
O 0.1g
Resazurin 0.5mg
NaHCO
3
solution 80.0mL
Na
2
S·9H
2
O solution 10.0mL
Trace elements solution 10.0mL
pH 7.8 ± 0.2 at 25°C
Na

2
S·9H
2
O Solution:
Composition
per 10.0mL:
Na
2
S·9H
2
O 0.2g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Neutralize to pH 7.0 with sterile HCl.
NaHCO
3
Solution:
Composition
per 100.0mL:

NaHCO
3
5.0g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
. Filter sterilize.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO

4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2

O 0.02g
H
3
BO
3
0.01g
Na
2
MoO
4
·4H
2
O 0.01g
CuSO
4
·5H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under 80%

N
2
+ 20% CO
2
gas atmosphere. Add components, except NaHCO
3
solu-
tion and Na
2
S·9H
2
O solution, to distilled/deionized water and bring vol-
ume to 910.0mL. Mix thoroughly. Adjust pH to 7.8. Gently heat and
bring to boiling. Cool while sparging with 80% N
2
+ 20% CO
2
. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to room temperature.
Aseptically and anaerobically add 80.0mL NaHCO
3
solution and
10.0mL Na
2
S·9H
2
O solution. Aseptically and anaerobically distribute
into sterile tubes or bottles.
Use: For the cultivation of Spirochaeta smaragdinae.
Spirochaeta stenostrepta Medium

Composition per liter:
Glucose 5.0g
Peptone 2.0g
Yeast extract 0.3g
Vitamin B
12
0.01mg
Salts solution 100.0mL
Phosphate solution 15.0mL
Sulfide solution 10.0mL
pH 7.0 ± 0.2 at 25°C
Phosphate Solution:
Composition
per liter:
KH
2
PO
4
30.0g
K
2
HPO
4
70.0g
Preparation of Phosphate Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize.
Salts Solution:
Composition
per liter:

MgSO
4
·7H
2
O 2.0g
CaCl
2
·2H
2
O 0.75g
EDTA 0.2g
FeSO
4
·7H
2
O 0.1g
Trace elements solution 5.0mL
Preparation of Salts Solution: Add EDTA to approximately
800.0mL of distilled/deionized water. Gently heat until dissolved. Ad-
just pH to 7.0 with 2.5% KOH. Add the remaining components. Mix
thoroughly. Bring volume to 1.0L with distilled/deionized water.
Trace Elements Solution:
Composition
per 1800.0mL:
H
3
BO
3
5.5g
MnCl

2
·4H
2
O 3.5g
AlCl
3
·6H
2
O 0.5g
CoCl
2
·6H
2
O 0.5g
CuCl
2
·2H
2
O 0.5g
NiCl
2
·6H
2
O 0.5g
ZnCl
2
0.5g
© 2010 by Taylor and Francis Group, LLC
Spirochete Medium 1607
KI 0.25g

LiCl 0.25g
Na
2
MoO
4
·2H
2
O 0.25g
BaCl
2
·2H
2
O 0.15g
SnCl
2
·2H
2
O 0.15g
NaVO
3
0.05g
Preparation of Trace Elements Solution: Add components to
distilled/deionized water and bring volume to 1800.0mL. Mix thor-
oughly. Adjust pH to 3–4 with HCl.
Sulfide Solution:
Composition
per 100.0mL:
Na
2
S·9H

2
O 2.0g
Preparation of Sulfide Solution: Add Na
2
S·9H
2
O to distilled/de-
ionized water and bring volume to 100.0mL. Autoclave for 15 min at
15 psi pressure–121°C. Cool to 25°C. Prepare solution freshly.
Preparation of Medium: Add components, except sulfide solution,
to distilled/deionized water and bring volume to 990.0mL. Mix thor-
oughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile
sulfide solution. Mix thoroughly. Aseptically distribute into sterile
tubes or flasks.
Use: For the isolation of Spirochaeta stenostrepta.
Spirochaeta stenostrepta Medium
Composition per liter:
Glucose 5.0g
Peptone 2.0g
Yeast extract 2.0g
L-Cysteine 0.5g
Resazurin 1.0mg
pH 7.3–7.6 at 25°C
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components to distilled/deionized water and bring vol-
ume to 1.0L. Mix thoroughly. Sparge with 100% N
2

. Adjust pH to 7.3–
7.6 while continuing to sparge with 100% N
2
. Anaerobically distribute
into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation and maintenance of Spirochaeta stenostrepta.
Spirochaeta zuelzerae Medium
Composition per liter:
Solution 1 480.0mL
Solution 2 480.0mL
Solution 3 20.0mL
Solution 4 20.0mL
pH 7.2 ± 0.2 at 25°C
Solution 1:
Composition
per 480.0mL:
KH
2
PO
4
0.75g
L-Cysteine·HCl·H
2
O 0.5g
NaH
2
PO
4
·H
2

O 0.25g
Preparation of Solution 1: Add components to distilled/deionized
water and bring volume to 480.0mL. Mix thoroughly. Adjust pH to 7.2
with 5.0% KOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to 45°–50°C.
Solution 2:
Composition
per 480.0mL:
NH
4
Cl 1.0g
MgSO
4
·7H
2
O 0.5g
Yeast extract 0.2g
CaCl
2
0.02g
Resazurin 1.0mg
FeCl
3
·6H
2
O solution (0.25g/L) 10.0mL
Trace elements solution 2.0mL
Preparation of Solution 2: Add components to distilled/deionized
water and bring volume to 480.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C. Cool to 45°–50°C.

Trace Elements Solution:
Composition
per 100.0mL:
Na
2
MoO
4
·2H
2
O 0.075g
H
3
BO
3
0.056g
ZnSO
4
·7H
2
O 0.044g
CoCl
2
·6H
2
O 0.02g
CuSO
4
·5H
2
O 2.0mg

MnCl
2
2.0mg
Preparation of Trace Elements Solution: Add components to
distilled/deionized water and bring volume to 100.0mL. Mix thorough-
ly.
Solution 3:
Composition
per 20.0mL:
NaHCO
3
1.0g
Preparation of Solution 3: Add NaHCO
3
to distilled/deionized
water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize un-
der pressure.
Solution 4:
Composition
per 20.0mL:
Glucose 2.0g
Preparation of Solution 4: Add glucose to distilled/deionized wa-
ter and bring volume to 20.0mL. Mix thoroughly. Filter sterilize.
Preparation of Medium: Aseptically add 480.0 mL of sterile solu-
tion 1 to 480.0mL of sterile solution 2 under 80% N
2
+ 20% CO
2
.
While gassing, add 20.0mL of sterile solution 3 and 20.0mL of sterile

solution 4. Mix thoroughly. Adjust pH to 7.2. Aseptically and anaero-
bically distribute into tubes. Cap with rubber stoppers.
Use: For the cultivation and maintenance of Spirochaeta zuelzerae.
Spirochete Enrichment Medium
Composition per liter:
Agar 10.0g
Beef extract 1.0g
Peptone 1.0g
Yeast extract 1.0g
Seawater 500.0mL
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 isolation of spirochetes from muds. A well is cut into the
agar plate and filled with mud samples. Spirochetes migrate out of the
mud into the agar surrounding the well.
Spirochete Medium
(ATCC Medium 164)
Composition per liter:
Agar 15.0g
KH
2
PO
4
1.0g
© 2010 by Taylor and Francis Group, LLC
1608 Spirochete Medium
NH
4

Cl 1.0g
Yeast extract 1.0g
MgSO
4
0.5g
CaCl
2
0.04g
FeCl
3
·6H
2
O 1.25mg
NaHCO
3
solution 20.0mL
Glucose solution 10.0mL
Na
2
S·9H
2
O solution 5.0mL
Glucose Solution:
Composition
per 100.0mL:
Glucose 10.0g
Preparation of Glucose Solution: Add glucose to distilled/deion-
ized water and bring volume to 100.0mL. Mix thoroughly. Filter ster-
ilize.
NaHCO

3
Solution:
Composition
per 100.0mL:
NaHCO
3
5.0g
Preparation of NaHCO
3
Solution: Add the NaHCO
3
to distilled/
deionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.

Na
2
S·9H
2
O Solution:
Composition
per 100.0mL:
Na
2
S·9H
2
O 10.0g
Preparation of Na
2
S·9H

2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C.

Preparation of Medium:
Add components—except NaHCO
3
solu-
tion, glucose solution, and Na
2
S·9H
2
O solution—to distilled/deionized
water and bring volume to 965.0mL. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to 50°C. Aseptically add 20.0mL of sterile
NaHCO
3
solution, 10.0mL of sterile glucose solution, and 5.0mL of
sterile Na
2
S·9H
2
O solution. Mix thoroughly. Pour into sterile Petri
dishes or distribute into sterile tubes.
Use: For the cultivation of spirochetes.

Spirochete Medium
(ATCC Medium 1712)
Composition per liter:
Tris(hydroxymethyl)aminomethane buffer 7.52g
Pancreatic digest of casein 1.0g
Yeast extract 1.0g
L-Cysteine·HCl·2H
2
O 0.5g
Resazurin 1.0mg
Seawater 750.0mL
Glucose solution 20.0mL
pH 7.2 ± 0.2 at 25°C
Glucose Solution:
Composition
per 20.0mL:
Glucose 2.0g
Preparation of Glucose Solution: Add glucose to distilled/deion-
ized water and bring volume to 20.0mL. Mix thoroughly. Filter steril-
ize.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components, except glucose solution, to distilled/deion-
ized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH
to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.
Aseptically add sterile glucose solution. Mix thoroughly. Aseptically
distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Spirochaeta litoralis.
Spirochete Thermophile Medium

(DSMZ Medium 509)
Composition per 1012.0mL:
Solution A 920.0mL
Solution D 50.0mL
Solution E 20.0mL
Solution F 10.0mL
Solution G 10.0mL
Solution B 1.0mL
Solution C 1.0mL
pH 7.0 ± 0.2 at 25°C
Solution A:
Composition
per 920.0mL:
NaCl 4.0g
MgCl
2
·6H
2
O 0.8g
KCl 0.5g
NH
4
Cl 0.3g
KH
2
PO
4
0.2g
CaCl
2

·2H
2
O 0.03g
Resazurin 1.0mg
Preparation of Solution A: Add components to 920.0mL distilled/
deionized water. Mix thoroughly. Bring to boiling for a few minutes.
Cool to room temperature while gassing with 80% N
2
+ 20% CO
2
gas.
Adjust pH to 6.0. Immediately distribute under N
2
into anaerobic tubes.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.
Solution B:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
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 Solution B: Add FeCl

2
·4H
2
O to 10.0mL of HCl so-
lution. Mix thoroughly. Add distilled/deionized water and bring vol-
ume to 1.0L. Add remaining components. Mix thoroughly. Sparge with
80% N
2
+ 20% CO
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to 25°C.
Solution C:
Composition
per liter:
NaOH 0.5g
Na
2
WO
4
·2H
2
O 4.0mg
Na
2
SeO
3
·5H
2
O 3.0mg

Preparation of Solution C: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N
2
.
Filter sterilize.
Solution D:
Composition
per 100.0mL:
NaHCO
3
5.0g
Preparation of Solution D: Add NaHCO
3
to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Sparge with
© 2010 by Taylor and Francis Group, LLC
Spiroplasma Agar MID 1609
100% N
2
gas mixture. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to 25°C.
Solution E:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg
Riboflavin 5.0mg
Nicotinic acid 5.0mg

D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Solution E: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N
2
+ 20% CO
2
. Filter sterilize.
Solution F:
Composition
per 10.0mL:
Starch 1.0g
Preparation of Solution F: Add starch to distilled/deionized water
and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N
2
gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
25°C.
Solution G:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.3g

Preparation of Solution G: Add Na
2
S·9H
2
O to distilled/deionized
water and bring volume to 10.0mL. Mix thoroughly. Autoclave under
100% N
2
for 15 min at 15 psi pressure–121°C. Cool to 25°C.
Preparation of Medium: Solution A is distributed into anaerobic
tubes with rubber stoppers prior to autoclaving. Using aseptic and anaer-
obic conditions and syringes, appropriate volumes of sterile solutions B–
G are injected into each tube to yield the specified concentrations.
Use: For the cultivation of Spirochaeta thermophila.
Spirolate Broth
Composition per liter:
Pancreatic digest of casein 15.0g
Glucose 5.0g
Yeast extract 5.0g
NaCl 2.5g
L-Cysteine·HCl·H
2
O 1.0g
Sodium thioglycolate 0.5g
Palmitic acid 0.05g
Stearic acid 0.05g
Oleic acid 0.05g
Linoleic acid 0.05g
Serum 100.0mL
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, except serum, to dis-
tilled/deionized water and bring volume to 900.0mL. Mix thoroughly.
Distribute into screw-capped tubes in 20.0mL volumes. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 2.0mL
of serum to each tube. Heat-inactivated sheep, rabbit, or bovine serum
may be used. Tighten caps. Mix thoroughly.
Use: For the cultivation of Treponema phagedenis and other spiro-
chetes.
Spirolate HiVeg Broth, OMATA with Serum
Composition per liter:
Plant hydrolysate 15.0g
Glucose 5.0g
Yeast extract 5.0g
NaCl 2.5g
L-Cysteine·HCl 1.0g
Na-thioglycolate 0.5g
Sheep or rabbit blood serum, inactivated 10% 100.0mL
pH 7.1 ± 0.2 at 25°C
Source: This medium, without serum, is available as a premixed pow-
der from HiMedia.
Preparation of Medium: Add components, except serum, to dis-
tilled/deionized water and bring volume to 900.0mL. Mix thoroughly.
Distribute into screw-capped tubes in 20.0mL volumes. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 2.0mL
of serum to each tube. Heat-inactivated sheep, rabbit, or bovine serum
may be used. Tighten caps. Mix thoroughly.
Use: For the cultivation of Treponema phagedenis and other spiro-
chetes.

Spiroplasma Agar MID
Composition per 291.2mL:
Schneider’s Drosophila medium 160.0mL
Solution 1 80.0mL
Fetal calf serum 50.0mL
Phenol Red (0.5% solution) 1.2mL
pH 7.4 ± 0.2 at 25°C
Schneider’s Drosophila Medium:
Composition
per liter:
MgSO
4
·7H
2
O 3.7g
NaCl 2.1g
Yeast extract 2.0g
Trehalose 2.0g
D-Glucose 2.0g
L-Glutamine 1.8g
L-Lysine·HCl 1.7g
L-Proline 1.7g
KCl 1.6g
Na
2
HPO
4
·7H
2
O 1.3g

L-Glutamic acid 0.8g
L-Methionine 0.8g
CaCl
2
, anhydrous 0.6g
KH
2
PO
4
0.5g
β-Alanine 0.5g
L-Tyrosine 0.5g
L-Arginine 0.4g
L-Aspartic acid 0.4g
L-Histidine 0.4g
L-Threonine 0.4g
NaHCO
3
0.4g
Glycine 0.3g
L-Serine 0.3g
L-Valine 0.3g
© 2010 by Taylor and Francis Group, LLC
1610 Spiroplasma Broth MID
L-Isoleucine 0.2g
L-Leucine 0.2g
L-Phenylalanine 0.2g
α-Ketoglutaric acid 0.2g
Fumaric acid 0.1g
Malic acid 0.1g

Succinic acid 0.1g
L-Cystine 0.1g
L-Tryptophan 0.1g
L-Cysteine 0.06g
Preparation of Schneider’s Drosophila Medium: Add compo-
nents to 1.0L of distilled/deionized water. Mix thoroughly. Filter ster-
ilize.
Solution 1:
Composition
per 80.0mL:
Sorbitol 7.0g
Noble agar 5.0g
Beef heart, solids from infusion 5.0g
Peptone 1.8g
Sucrose 1.0g
Pancreatic digest of casein 1.0g
NaCl 0.5g
D-Fructose 0.1g
D-Glucose 0.1g
Preparation of Solution 1: Add components to distilled/deionized
water and bring volume to 80.0mL. Mix thoroughly. Adjust pH to 7.8
with 1N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to 50°C.
Preparation of Medium: Bring fetal calf serum and Phenol Red so-
lution to 56°C. Rapidly bring Schneider’s Drosophila medium to 37°C.
Rapidly combine the components. Mix thoroughly. Pour into sterile
Petri dishes or distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Spiroplasma kunkelii and
Spiroplasma species.
Spiroplasma Broth MID

Composition per 291.2mL:
Schneider’s Drosophila medium 160.0mL
Solution 1 80.0mL
Fetal calf serum 50.0mL
Phenol Red (0.5% solution) 1.2mL
pH 7.4 ± 0.2 at 25°C
Schneider’s Drosophila Medium:
Composition
per liter:
MgSO
4
·7H
2
O 3.7g
NaCl 2.1g
Yeast extract 2.0g
Trehalose 2.0g
D-Glucose 2.0g
L-Glutamine 1.8g
L-Lysine·HCl 1.7g
L-Proline 1.7g
KCl 1.6g
Na
2
HPO
4
·7H
2
O 1.3g
L-Glutamic acid 0.8g

L-Methionine 0.8g
CaCl
2
, anhydrous 0.6g
KH
2
PO
4
0.5g
β-Alanine 0.5g
L-Tyrosine 0.5g
L-Arginine 0.4g
L-Aspartic acid 0.4g
L-Histidine 0.4g
L-Threonine 0.4g
NaHCO
3
0.4g
Glycine 0.3g
L-Serine 0.3g
L-Valine 0.3g
L-Isoleucine 0.2g
L-Leucine 0.2g
L-Phenylalanine 0.2g
α-Ketoglutaric acid 0.2g
Fumaric acid 0.1g
Malic acid 0.1g
Succinic acid 0.1g
L-Cystine 0.1g
L-Tryptophan 0.1g

L-Cysteine 0.06g
Preparation of Schneider’s Drosophila Medium: Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Filter sterilize.
Solution 1:
Composition
per 80.0mL:
Sorbitol 7.0g
Beef heart, solids from infusion 5.0g
Peptone 1.8g
Sucrose 1.0g
Pancreatic digest of casein 1.0g
NaCl 0.5g
D-Fructose 0.1g
D-Glucose 0.1g
Preparation of Solution 1: Add components to distilled/deionized
water and bring volume to 80.0mL. Mix thoroughly. Adjust pH to 7.8
with 1N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to 25°C.
Preparation of Medium: Bring fetal calf serum and Phenol Red so-
lution to 56°C. Rapidly bring Schneider’s Drosophila medium to 37°C.
Rapidly combine the components. Mix thoroughly. Aseptically distrib-
ute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Spiroplasma kunkelii and
Spiroplasma species.
Spiroplasma Medium
Composition per liter:
Sucrose 80.0g
Beef heart, solids from infusion 34.7g
Peptone 6.9g

NaCl 3.5g
Horse serum, heat inactivated 100.0mL
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components, except horse serum, to
distilled/deionized water and bring volume to 900.0mL. Mix thorough-
ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Asep-
tically add horse serum. Mix thoroughly. Aseptically distribute into
sterile tubes or flasks.
Use: For the cultivation and maintenance of Spiroplasma species.
© 2010 by Taylor and Francis Group, LLC
Sporobacter Medium 1611
Spiroplasma Medium
Composition per liter:
Sorbitol 70.0g
Pancreatic digest of casein 7.0g
Yeast extract 5.0g
NaCl 5.0g
Beef extract 3.0g
Yeast extract 3.0g
Beef heart, solids from infusion 2.0g
Fructose 1.0g
Glucose 1.0g
Phenol Red 20.0mg
Horse serum 100.0mL
pH 7.8 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 900.0mL. Mix thoroughly. Gently heat and
bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool
to 50°–55°C. Aseptically add 100.0mL of sterile horse serum. Mix
thoroughly.

Use: For the cultivation of Spiroplasma citri.
Spiroplasma Medium
with 25 mg/L of Phenol Red
Composition per liter:
Sucrose 80.0g
Beef heart, solids from infusion 34.7g
Peptone 6.9g
NaCl 3.5g
Phenol Red 25.0mg
Horse serum, heat inactivated 100.0mL
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components, except horse serum, to
distilled/deionized water and bring volume to 900.0mL. 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 add heat-inactivated
horse serum. Mix thoroughly. Aseptically distribute into sterile tubes
or flasks.
Use: For the cultivation and maintenance of Spiroplasma floricola.
Spizizen Potato Agar
Composition per liter:
Potatoes 200.0g
Agar 15.0g
MnSO
4
5.0mg
pH 6.8 ± 0.2 at 25°C
Preparation of Medium: Peel and dice potatoes. Add potatoes to
1.0L of tap water. Gently heat and bring to boiling. Continue boiling
for 30 min. Filter through cheesecloth. Add MnSO
4

to filtrate and bring
volume to 1.0L with tap water. Mix thoroughly. Adjust pH to 6.8. Add
agar. 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 and maintenance of Bacillus amyloliquefa-
ciens.
SPMA
See: Mineral Salt Peptonized Milk Agar
Spore Strip Broth
Composition per liter:
Spore strip broth 9.0g
Preparation of Medium: Add 9.0g of spore strip broth powder (a
mixture of glucose, buffer salts, growth factors, and Bromthymol Blue)
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.
Use: For the recovery of spores of Bacillus stearothermophilus on
spore strips used to determine the sterilization efficiency of autoclaves.
Sporobacter Medium
(DSMZ Medium 711)
Composition per liter:
NH
4
Cl 1.0g
NaCl 0.6g
Na-acetate·3H
2
O 0.5g
Cysteine-HCl·H

2
O 0.5g
K
2
HPO
4
0.3g
KH
2
PO
4
0.3g
Yeast extract 0.2g
MgCl
2
·6H
2
O 0.2g
CaCl
2
·2H
2
O 0.1g
KCl 0.1g
Resazurin 0.5mg
NaHCO
3
solution 40.0mL
Trimethoxycinnamate solution 10.0mL
Na

2
S·9H
2
O solution 10.0mL
Trace elements solution SL-10 1.5mL
pH 7.1 ± 0.2 at 25°C
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
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: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 80% N
2
+ 20% CO

2
. Autoclave for 15 min at 15
psi pressure–121°C.
Na
2
S·9H
2
O Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
NaHCO
3

Solution:
Composition
per 100.0mL:
NaHCO
3
10.0g
© 2010 by Taylor and Francis Group, LLC
1612 Sporocytophaga Medium
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
. Filter sterilize.
Trimethoxycinnamate Solution:
Composition
per 10.0mL:
Trans-3,4,5-trimethoxycinnamate 1.2g
Preparation of Trimethoxycinnamate Solution: Add trans-
3,4,5-trimethoxycinnamate to distilled/deionized water and bring vol-
ume to 10.0mL. Mix thoroughly. Neutralize with NaOH. Sparge with
100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
room temperature.

Preparation of Medium: Prepare and dispense medium under 80%
N
2
+ 20% CO
2
gas atmosphere. Add components, except NaHCO
3
solu-
tion, Na
2
S·9H
2
O solution, trimethoxycinnamate solution, and trace ele-
ments solution SL-10, to distilled/deionized water and bring volume to
938.5mL. Mix thoroughly. Adjust pH to 7.1. Sparge with 80% N
2
+ 20%
CO
2
. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and an-
aerobically add 40.0mL NaHCO
3
solution, 10.0mL Na
2
S·9H
2
O solution,
10.0mL trimethoxycinnamate solution, and 1.5mL trace elements solution
SL-10. Mix thoroughly. Aseptically and anaerobically distribute into ster-
ile tubes or bottles.

Use: For the cultivation of Sporobacter termitidis.
Sporocytophaga Medium
Composition per liter:
NaNO
3
2.0g
K
2
HPO
4
1.2g
MgSO
4
·7H
2
O 1.0g
KCl 0.5g
KH
2
PO
4
0.14g
Yeast extract 0.02g
FeSO
4
·7H
2
O 6.0mg
Filter paper strips variable
pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper
strips, to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically add a sterile filter paper strip to
each tube so that 1.0–2.0cm of the strip extends above the medium.
Use: For the cultivation and maintenance of Sporocytophaga myxo-
coccoides.
Sporohalobacter lortetii Agar
Composition per liter:
NaCl 105.0g
L-Glutamic acid 4.0g
Agar 20.0g
CaCO
2
5.0g
Soluble starch 2.0g
Casamino acids 2.0g
Nutrient broth 2.0g
Yeast extract 2.0g
KCl 0.75g
L-Cysteine 0.5g
FeSO
4
·7H
2
O 0.002g
Resazurin 1.0mg
MgCl
2
·6H

2
O solution 40.0mL
CaCl
2
·2H
2
O solution 10.0mL
Trace elements solution 10.0mL
Vitamin solution 10.0mL
pH 6.5 ± 0.2 at 25°C
MgCl
2
·6H
2
O Solution:
Composition
per 40.0mL:
MgCl
2
·6H
2
O 0.01g
Preparation of MgCl
2
·6H
2
O Solution: Add MgCl
2
·6H
2

O to dis-
tilled/deionized water and bring volume to 40.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
CaCl
2
·2H
2
O Solution:
Composition
per 40.0mL:
CaCl
2
·2H
2
O 0.01g
Preparation of CaCl
2
·2H
2
O Solution: Add CaCl
2
·2H
2
O to dis-
tilled/deionized water and bring volume to 40.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.

Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g

FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
CuSO
4
·5H
2
O 0.01g
H
3
BO
3
0.01g
Na
2
MoO

4
·2H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L.
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium
DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg
p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12

0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components, except MgCl
2
·6H
2
O and CaCl
2
·2H
2
O so-
lutions, to distilled/deionized water and bring volume to 950.0mL. Mix
thoroughly. Gently heat and bring to boiling. Sparge with 100% N
2
.
Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add
40.0mL of sterile MgCl
2
·6H
2
O solution and 10.0mL of sterile
CaCl
2
·2H
2
O solution. Mix thoroughly. Aseptically and anaerobically

pour into sterile Petri dishes or distribute into sterile tubes.
Use: For the cultivation and maintenance of Sporohalobacter lortetii.
© 2010 by Taylor and Francis Group, LLC
Sporomusa Medium 1613
Sporohalobacter lortetii Broth
Composition per liter:
NaCl 105.0g
L-Glutamic acid 4.0g
Casamino acids 2.0g
Nutrient broth 2.0g
Yeast extract 2.0g
KCl 0.75g
L-Cysteine 0.5g
FeSO
4
·7H
2
O 0.002g
Resazurin 1.0mg
MgCl
2
·6H
2
O solution 40.0mL
CaCl
2
·2H
2
O solution 10.0mL
Trace elements solution 10.0mL

Vitamin solution 10.0mL
pH 6.5 ± 0.2 at 25°C
MgCl
2
·6H
2
O Solution:
Composition
per 40.0mL:
MgCl
2
·6H
2
O 0.01g
Preparation of MgCl
2
·6H
2
O Solution: Add MgCl
2
·6H
2
O to dis-
tilled/deionized water and bring volume to 40.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
CaCl
2
·2H

2
O Solution:
Composition
per 40.0mL:
CaCl
2
·2H
2
O 0.01g
Preparation of CaCl
2
·2H
2
O Solution: Add CaCl
2
·2H
2
O to dis-
tilled/deionized water and bring volume to 40.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g

Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H

2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
CuSO
4
·5H
2
O 0.01g
H
3
BO
3
0.01g
Na
2
MoO
4
·2H
2
O 0.01g
Na
2
SeO
3

·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L.
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium
DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg
p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components, except MgCl
2

·6H
2
O and CaCl
2
·2H
2
O so-
lutions, to distilled/deionized water and bring volume to 950.0mL. Mix
thoroughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pres-
sure–121°C. Aseptically add 40.0mL of sterile MgCl
2
·6H
2
O solution
and 10.0mL of sterile CaCl
2
·2H
2
O solution. Mix thoroughly. Asepti-
cally and anaerobically distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Sporohalobacter lortetii.
Sporomusa Medium
Composition per 877.0mL:
NaCl 2.25g
Pancreatic digest of casein 2.0g
Yeast extract 2.0g
MgSO
4

·7H
2
O .0.5g
NH
4
Cl 0.5g
K
2
HPO
4
0.35g
KH
2
PO
4
0.23g
CaCl
2
·2H
2
O 0.025g
FeSO
4
·7H
2
O 2.0mg
Resazurin 1.0mg
NaHSeO
3
15.0μg

NaHCO
3
solution 50.0mL
Glycine betaine solution 50.0mL
Wolfe’s vitamin solution 10.0mL
L-Cysteine·HCl·H
2
O solution 10.0mL
Trace elements solution SL-6 3.0mL
pH 7.0–7.2 at 25°C
NaHCO
3
Solution:
Composition
per 50.0mL:
NaHCO
3
4.0g
Preparation of NaHCO
3
Solution: Add the NaHCO
3
to distilled/
deionized water and bring volume to 50.0mL. Mix thoroughly. Gas un-
der 80% N
2
+ 20% CO
2
for 20 min.
Glycine Betaine Solution:

Composition
per 50.0mL:
Glycine betaine 5.0g
Preparation of Glycine Betaine Solution: Add the glycine be-
taine to distilled/deionized water and bring volume to 50.0mL. Mix
thoroughly. Filter sterilize. Aseptically gas under 80% N
2
+ 20% CO
2
.
Wolfe’s Vitamin Solution:
Composition per liter:
Pyridoxine·HCl 0.01g
Thiamine·HCl 5.0mg
Riboflavin 5.0mg
Nicotinic acid 5.0mg
Calcium pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Thioctic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Cyanocobalamin 0.1mg
Preparation of Wolfe’s Vitamin Solution: Add components to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
© 2010 by Taylor and Francis Group, LLC
1614 Sporomusa Medium
L-Cysteine·HCl·H
2
O Solution:
Composition

per 10.0mL:
L-Cysteine·HCl·H
2
O 0.3g
Preparation of L-Cysteine·HCl·H
2
O Solution: Add L-cyste-
ine·HCl·H
2
O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Filter sterilize. Aseptically gas under 100% N
2
.
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.
Preparation of Medium: Add components—except NaHCO
3
solu-
tion, glycine betaine solution, and

L-cysteine·HCl·H
2
O solution—to
distilled/deionized water and bring volume to 890.0mL. Mix thorough-
ly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool
rapidly to 25°C under 80% N
2
+ 20% CO
2
. Add 50.0mL of NaHCO
3
solution. Mix thoroughly. Autoclave anaerobically for 15 min at 15 psi
pressure–121°C. Cool to 25°C. Aseptically add sterile glycine betaine
solution. Mix thoroughly. Immediately prior to inoculation, aseptically
and anaerobically add
L-cysteine·HCl·H
2
O solution.
Use: For the cultivation and maintenance of Sporomusa ovata and
Sporomusa sphaeroides.
Sporomusa Medium
Composition per 1010.0mL:
Betaine·H
2
O 6.7g
NaHCO
3
4.0g
NaCl 2.25g
Pancreatic digest of casein 2.0g

Yeast extract 2.0g
MgSO
4
·7H
2
O 0.5g
NH
4
Cl 0.5g
K
2
HPO
4
0.348g
CaCl
2
·2H
2
O 0.25g
KH
2
PO
4
0.227g
FeSO
4
·7H
2
O 2.0mg
Resazurin 1.0mg

NaHSeO
3
26.3μg
Vitamin solution 10.0mL
Reducing agent solution 10.0mL
Trace elements solution SL-10 1.0mL
pH 7.0 ± 0.2 at 25°C
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium
DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg
p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Reducing Agent Solution:
Composition
per 10.0mL:
L-Cysteine·HCl·H
2

O 0.3g
Na
2
S·9H
2
O 0.3g
Preparation of Reducing Agent Solution: Add 10.0mL of dis-
tilled/deionized water to a flask. Gently heat and bring to boiling. Con-
tinue to boil for 1 min while sparging with 100% N
2
. Cool to room
temperature. Add L-cysteine·HCl·H
2
O. Mix thoroughly. Adjust pH to
9 with 5N NaOH. Add Na
2
S·9H
2
O. Mix thoroughly. Autoclave for 10
min at 15 psi pressure–121°C.
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
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: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly.
Preparation of Medium: Prepare and dispense medium under 80%
N
2
+ 20% CO
2
. Add components, except reducing agent solution, to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 80% N
2
+ 20% CO
2
. Anaerobically distribute into tubes or
flasks. Autoclave for 15 min at 15 psi pressure–121°C. Using a sy-
ringe, aseptically and anaerobically add sterile reducing agent to each
tube (10.0mL per liter of medium).
Use: For the cultivation and maintenance of Sporomusa species.
Sporomusa Medium, Modified
Composition per liter:

NaHCO
3
4.0g
NaCl 2.25g
Pancreatic digest of casein 2.0g
Yeast extract 2.0g
MgSO
4
·7H
2
O 0.5g
NH
4
Cl 0.5g
K
2
HPO
4
0.348g
CaCl
2
·2H
2
O 0.25g
KH
2
PO
4
0.227g
FeSO

4
·7H
2
O 2.0mg
Resazurin 1.0mg
NaHSeO
3
26.3μg
Fructose solution 50.0mL
Reducing agent solution 10.0mL
Vitamin solution 10.0mL
Trace elements solution SL-10 1.0mL
pH 7.0 ± 0.2 at 25°C
© 2010 by Taylor and Francis Group, LLC