# 858: ANAEROBIC CHOLESTEROL MEDIUM


## Main sol. 858

9	ml	**Solution A**	
0.5	ml	**Solution B**	
0.025	ml	**Solution C**	
0.05	ml	**Solution D**	
0.025	ml	**Solution E**	
0.5	ml	**Solution F**	

1. Prepare solution A, distribute approbiate amounts of cholesterol (e.g. 5 mg for 10 ml medium) and solution A in anaerobic tubes, and autoclave anaerobically under 80% N2 + 20% CO2. Solutions B and F are also autoclaved under these conditions. Solution C, D and E may be prepared aerobically. After cooling, add to 9 ml solution A: 500 µl sol. B, 500 µl sol. F, 25 µl (one drop) sol. C and E, each, and 50 µl sol. D under anaerobic conditions. Adjust pH to 7.0±0.2.


## Solution A

0.1	g	MgSO4 x 7 H2O	
0.1	g	NH4Cl	
0.02	g	CaCl2	
0.16	g	NaNO3	
200	ml	Distilled water	


## Solution B

1	g	KH2PO4	
100	ml	Distilled water	


## Solution C

1	ml	**Trace element solution SL-10**	

1. Trace elements SL-10 (see medium 320)


## Solution D

1	ml	**Vitamin solution**	


## Solution E

1	ml	**Selenite-tungstate solution**	


## Solution F

8.4	g	NaHCO3	
100	ml	Distilled water	


## Vitamin solution

50	mg	Vitamin B12	
50	mg	Pantothenic acid	
50	mg	Riboflavin	
10	mg	Pyridoxamine hydrochloride	
20	mg	Biotin	
20	mg	Folic acid	
25	mg	Nicotinic acid	
25	mg	Nicotine amide	
50	mg	alpha-lipoic acid	
50	mg	p-Aminobenzoic acid	
50	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water	

1. Stir for some hours, filter sterilize the solution.


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

1. First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water