275. TELMATOCOLA MEDIUM

Mineral salts solution:

KH₂PO₄ 0.1 g

(NH₄)₂SO₄ 0.1 g

MgSO₄ × 7 H₂O 0.1 g

CaCl₂ × 2 H₂O 0.02 g

Trace elements solution "44" (see below) 1.0 ml

Staley vitamin solution (see below) 1.0 ml

Yeast extract 0.1 g

Glucose (1% v/v) 10.0 ml

Distilled Water 1000.0 ml

Trace elements solution "44":

Hutner's salts:

Nitrilotriacetic acid (NTA) 10.0 g

MgSO₄ × 7 H₂O 29.7 g

CaCl₂ × 2 H₂O 3.335 g

(NH₄)₆MoO₇O₂₄ × 4 H₂O 9.25 mg

FeSO₄ × 7 H₂2O 99.0 mg

"Metals 44" (see below) 50.0 ml

Distilled water 950.0 ml

Dissolve the nitrilotriacetic acid, adjust the pH to 7.0 with KOH (about 7.3 g). Dissolve other salts separately, combine and adjust the pH to 6.8.

"Metals 44":

Na-EDTA 250.0 mg

ZnSO₄ × 7 H₂O 1095.0 mg

FeSO₄ × 7 H₂O 500.0 mg

MnSO₄ × H₂O 154.0 mg

CuSO₄ × 5 H₂O 39.2 mg

Co(NO₃)₂ × 6 H₂O 24.8 mg

Na₂B₄O₇ × 10 H₂O 17.7 mg

Distilled water 1000.0 ml

Dissolve the EDTA and add a few drops of concentrated H2SO4 to retard precipitation of the heavy metal ions.

Staley’s vitamin solution:

Vitamin B₁₂ 0.2 mg

Biotin 4.0 mg

Thiamine-HCl × 2 H₂O 10.0 mg

Ca-pantothenate 10.0 mg

Folic acid 1.0 mg

Riboflavin 10.0 mg

Nicotinamide 10.0

p-Aminobenzoic acid 10.0 mg

Pyridoxine hydrochloride 10.0 mg

Distilled water 1000.0 ml

Prepare the liquid medium. Adjust to pH 4.8 - 5.5. Dispense the medium into serum bottles under gas-sing with CO₂ (5% v/v).

Sterilize at 121°C for 15 min. Before inoculation add from filter-sterilized Staley vitamin solution and autoclaved at 121°C for 15 min glucose.