# 169b: TREPONEMA AZOTONUTRICIUM MEDIUM
## Main sol. 169b
952 ml **Solution A**
20 ml **Solution B**
20 ml **Solution C**
1 ml **Solution D**
10 ml **Solution E**
1. Dissolve ingredients of solution A (except cysteine) and sparge medium with 100% N2 gas for 30 - 45 minto make it anoxic. Add cysteine and adjust pH with KOH, then distribute under the same gas atmosphere in anoxic Hungate-type tubes or serum vials and autoclave. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere, solution C is autoclaved under 100% N2 gas atmosphere and solutions D and E are sterilized by filtration under 100% N2 gas atmosphere. To complete the medium appropriate amounts of solutions B, C, D and E are added to the sterile solution A in the sequence as indicated. Final pH of the complete medium should be 7.5.
## Solution A
0.04 g CaCl2 x 2 H2O
0.5 g MgSO4 x 7 H2O
4 g Yeast extract (OXOID)
4 g D-Glucose
1 ml **Trace element solution SL-10**
1 ml **Selenite-tungstate solution**
0.5 ml Sodium resazurin (0.1% w/v)
0.5 g L-Cysteine HCl x H2O
950 ml Distilled water
## Solution B
1 g NaHCO3
20 ml Distilled water
## Solution C
16.04 K2HPO4 (0.5 M)
3.96 KH2PO4 (0.5 M)
1. Combine 16.04 ml of 0.5 M K2HPO4 with 3.96 ml of 0.5 M KH2PO4 stock solution to reach a pH of around 7.4.
## Solution D
1 ml **Seven vitamins solution**
## Solution E
0.15 g DL-Dithiothreitol
10 ml Distilled water
## 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
## Seven vitamins solution
100 mg Vitamin B12
80 mg p-Aminobenzoic acid
20 mg D-(+)-biotin
200 mg Nicotinic acid
100 mg Calcium pantothenate
300 mg Pyridoxine hydrochloride
200 mg Thiamine-HCl x 2 H2O
1000 ml Distilled water