1022 .
CRYPTOANAEROBACTER MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.5 - 8.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
4-Hydroxybenzoic acid | 0.45 | g | ||
|
|
K2HPO4 | 0.40 | g | ||
|
|
NH4Cl | 0.40 | g | ||
|
|
Yeast extract
(BD Bacto) |
5.00 | g | ||
|
|
Casamino acids
(BD Bacto) |
1.00 | g | ||
|
|
Trace element solution SL-10 | 2.00 | ml | ||
|
|
Selenite-tungstate solution | 1.00 | ml | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.50 | ml | ||
|
|
NaHCO3 | 4.00 | g | ||
|
|
C. sporogenes supernatant | 350.00 | ml | ||
|
|
MgCl2 x 6 H2O | 0.08 | g | ||
|
|
CaCl2 x 2 H2O | 0.06 | g | ||
|
|
Wolin's vitamin solution (10x) | 1.00 | ml | ||
|
|
Distilled water | 650.00 | ml | ||
| 1 Dissolve ingredients (except bicarbonate, C. sporogenes supernatant, magnesium chloride, calcium chloride, and vitamins), adjust pH to 7.0 - 7.5 and boil medium for 1 min, then cool to room temperature under 80% N2 and 20% CO2 gas mixture. Dissolve solid bicarbonate, adjust pH to 7.8, dispense the solution under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving add the appropriate amount of sterile and anoxic supernatant of C. sporogenes and complete the medium by adding magnesium chloride, calcium chloride and vitamins (sterilized by filtration) from sterile anoxic stock solutions prepared under 100% N2 gas. The final pH of the medium should be 7.5 - 8.0. | |||||
| 2 It may be necessary to add 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 gas and filter-sterilized), if the medium is not completely reduced after inoculation. | |||||
| 3 Note: For transfers use 10% (v/v) inoculum. | |||||
Trace element solution SL-10 #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
HCl
(25%) |
10.0 | ml | ||
|
|
FeCl2 x 4 H2O | 1.5 | g | ||
|
|
ZnCl2 | 70.0 | mg | ||
|
|
MnCl2 x 4 H2O | 100.0 | mg | ||
|
|
H3BO3 | 6.0 | mg | ||
|
|
CoCl2 x 6 H2O | 190.0 | mg | ||
|
|
CuCl2 x 2 H2O | 2.0 | mg | ||
|
|
NiCl2 x 6 H2O | 24.0 | mg | ||
|
|
Na2MoO4 x 2 H2O | 36.0 | mg | ||
|
|
Distilled water | 990.0 | ml | ||
| 1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. | |||||
Selenite-tungstate solution #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
NaOH | 0.5 | g |
|
|
Na2SeO3 x 5 H2O | 3.0 | mg |
|
|
Na2WO4 x 2 H2O | 4.0 | mg |
|
|
Distilled water | 1000.0 | ml |
C. sporogenes supernatant #
1000
| 1 Cultivate Clostridium sp. DSM 754 for 5 to 8 days at 37°C in the medium 1022, but omit 4-hydroxybenzoic acid, replace the C. sporogenes supernatant with distilled water and add after autoclaving 0.30 g/l Na2S x 9 H2O from a sterile anoxic stock solution prepared under 100% N2 gas. Adjust pH of the complete medium to 7.0. Disrupt cells of the grown culture by autoclaving at 121°C for 20 min . Centrifuge autoclaved culture at 18000 x g for 20 min. Discard cell pellet and store the supernatant in screw capped bottles at -20°C . Before use sterilize the supernatant by autoclaving under 100% N2 gas atmosphere in vials suitable for anaerobic cultivation. |
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |