Main sol. 1012a

Equipment needed: Autoclave Petri dishes

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Nutrient broth (BD Difco)	0.8	g	0.8	-
Casamino acids (BD Bacto)	0.5	g	0.5	-
Yeast extract (OXOID)	0.1	g	0.1	-
Agar (BD Bacto)	12.0	g	12	-
MgCl₂ x 6 H₂O	0.6	g	0.6	2.951
CaCl₂ x 2 H₂O	0.3	g	0.3	2.041
Distilled water	1000.0	ml	-	-
1 Preparation of double-layered agar plates: Bottom layer: Dissolve ingredients and adjust the pH of the medium to 7.2 with NaOH. Then bring the medium to a boil to dissolve the agar and autoclave (121°C for 15 min). Dry plates at room temperature for 2 - 3 days. Top layer: Grow prey bacterium on agar slants using DSMZ medium 54 or medium 1. Prepare 5 ml aliquots of 1 % Bacto agar in 25 mM HEPES buffer (6.0 g/l HEPES, 0.6 g/l MgCl₂ x 6 H₂O, 0.3 g/l CaCl₂ x 2 H₂O, pH 7.2 ) and autoclave in test tubes. After cooling to 45°C in a water bath add 0.5 ml cell suspension of the host organism, mix, and pour the top layer. For the top layer of one plate use one 24 h old agar slant culture and wash off grown cells in 0.5 ml sterile water.
2 Inoculate the top layer of the double-layer plates the same day when prepared.

BDELLOVIBRIO BACTERIOVORUS MEDIUM