# 1022: CRYPTOANAEROBACTER MEDIUM
## Main sol. 1022
0.45 g 4-Hydroxybenzoic acid
0.4 g K2HPO4
0.4 g NH4Cl
5 g Yeast extract (BD Bacto)
1 g Casamino acids (BD Bacto)
2 ml **Trace element solution SL-10**
1 ml **Selenite-tungstate solution**
0.5 ml Sodium resazurin (0.1% w/v)
4 g NaHCO3
350 ml **C. sporogenes supernatant**
0.08 g MgCl2 x 6 H2O
0.06 g CaCl2 x 2 H2O
1 ml **Wolin's vitamin solution (10x)**
650 ml Distilled water
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
10 ml HCl (25%)
1.5 g FeCl2 x 4 H2O
70 mg ZnCl2
100 mg MnCl2 x 4 H2O
6 mg H3BO3
190 mg CoCl2 x 6 H2O
2 mg CuCl2 x 2 H2O
24 mg NiCl2 x 6 H2O
36 mg Na2MoO4 x 2 H2O
990 ml Distilled water
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
0.5 g NaOH
3 mg Na2SeO3 x 5 H2O
4 mg Na2WO4 x 2 H2O
1000 ml Distilled water
## C. sporogenes supernatant
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)
20 mg Biotin
20 mg Folic acid
100 mg Pyridoxine hydrochloride
50 mg Thiamine HCl
50 mg Riboflavin
50 mg Nicotinic acid
50 mg Calcium D-(+)-pantothenate
1 mg Vitamin B12
50 mg p-Aminobenzoic acid
50 mg (DL)-alpha-Lipoic acid
1000 ml Distilled water