258. NOCARDIOIDES AQUATICUS MEDIUM

Base medium (see below) 600.0 ml

Artificial sea water (see below) 400.0 ml

pH 7.5

Base medium:

Peptone Bacto 0.25 g

Yeast extract Bacto 0.25 g

Glucose solution (2.5%) 10.0 ml

Staley’s vitamin solution, double concentration (see below) 5.0 ml

Hutner’s basal salts medium (see below) 20.0 ml

Agar Bacto 15.0 g

Distilled water 965.0 ml

Artificial sea water (AWS):

NaCl 23.477 g

Na₂SO₄ 3.917 g

MgCl₂ × 6 H₂O 4.981 g

CaCl₂ × 2 H₂O 1.1 g

NaHCO₃ 192.0 mg

KCl 664.0 mg

KBr 6.0 mg

H₃BO₃ 26.0 mg

SrCl₂ 24.0 g

NaF 3.0 mg

Distilled water 1000.0 ml

Hutner’s basal salts medium:

Nitrilotriacetic acid (NTA) 10.0 g

MgSO₄ × 7 H₂O 29.7 g

CaCl₂ × 2 H₂O 3.34 g

Na₂MoO₄ × 2 H₂O 12.67 mg

FeSO₄ × 7 H₂O 99.0 mg

Metal salt solution “44” (see below) 50.0 ml

Dissolve NTA first by neutralizing with KOH, then add other salts.

pH 7.2 (adjust with KOH or H₂SO₄).

Adjust volume to 1000.0 ml with distilled water.

Metal solution “44”:

Na-EDTA 250.0 mg

ZnSO₄ × 7 H₂O 1095.0 mg

FeSO₄ × 7 H₂O 500.0 mg

MnSO₄ × 7 H₂O 154.0 mg

CuSO₄ × 5 H₂O 39.0 mg

Co(NO₃)₂ × 6 H₂O 24.8 mg

Na₂B₄O₇ × 10 H₂O 17.7 mg

Distilled water 1000.0 ml

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

Staley´s vitamin solution, double concentration:

Biotin 4.0 mg

Folic acid 4.0 mg

Pyridoxine-HCl 20.0 mg

Riboflavin 10.0 mg

Thiamine-HCl × 2 H₂O 10.0 mg

Nicotinamide 10.0 mg

D-Ca-pantothenate 10.0 mg

Vitamin B₁₂ 0.2 mg

p-Aminobenzoic acid 10.0 mg

Distilled water 1000.0 ml

Autoclave Base medium (except glucose solution and vitamin solution) and Artificial sea water separately at 121°C 15 min. After cooling to 60°C aseptically add to the Base medium glucose solution (sterilize by filtration) and vitamin solution (sterilize by filtration). Store vitamin solution in the dark and cold (5°C). Add 600.0 ml Base medium to 400 ml Artificial sea water and mix thoroughly.

 

Updated 1.02.2024