# 1012a: BDELLOVIBRIO BACTERIOVORUS MEDIUM


## Main sol. 1012a

0.8	g	Nutrient broth (BD Difco)	
0.5	g	Casamino acids (BD Bacto)	
0.1	g	Yeast extract (OXOID)	
12	g	Agar (BD Bacto)	
0.6	g	MgCl2 x 6 H2O	
0.3	g	CaCl2 x 2 H2O	
1000	ml	Distilled water	

1. Preparation of double-layered agar plates:<p>

<b>Bottom layer:</b>
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.<p>

<b>Top layer:</b>
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.<p>
2. Inoculate the top layer of the double-layer plates the same day when prepared.