399. GEMMATA PALUSTRIS MEDIUM

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

Peptone 5.0 g

Yeast extract 0.25 g

Distilled water 965.0 ml (less for plates)

Adjust pH to 6.8-7.5 with 5 M KOH

After autoclaving and cooling (please see below for plates) add to the medium

Vitamin solution 5.0 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 0.0127 g

FeSO₄ × 7 H₂O 0.099 g

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. Sterilize by filtration, store at +4°C.

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.

Sterilize by filtration, store at +4°C.

Vitamin solution:

Biotin 4.0 mg

Folic acid 4.0 mg

Pyridoxine-HCl 20.0 mg

Riboflavine 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

Sterilize by filtration, store in the dark and cold (+4°C)

For plates:

8.0 g/l Phytagel in 200.0 ml distilled water (mix before autoclaving), autoclave separately. Gelrite gets solid below 80°C after autoclaving and in general polymerization starts fast after mixing with media.

Updated 08.08.2021