자원검색

자원검색

KCTC Media No. 1363 : Kam1 Medium

조성 용량 단위
Mineral stock solution    
조성 용량 단위
 NH4Cl 1.0 
 MgSO4· 7H2O 1.0 
 CaCl2·2H2O 0.2 
 D.W 0.98 
Iron stock solution(sterile-filtered)    
조성 용량 단위
 FeNaEDTA 4.5 
 D.W 1.0 
Trace element stock solution(stored in the fridge)    
조성 용량 단위
 NaEDTA 0.25 
 ZnSO4 0.4 
 CoCl2·6H2O 0.05 
 MnCl2·4H2O 0.02 
 NiCl2·6H2O 0.01 
 Na2MoO4·2H2O 0.04 
 CuSO4·5H2O 0.2 
 H3BO4 0.015 
 D.W 1.0 
Phosphate buffer stock solution    
조성 용량 단위
 KH2PO4 37.325 
 Na2HPO4·2H2O 48.950 
 D.W 1.0 
 Cerium(III) chloride - 0.3 mM autoclaved solution    

pH 3.5

For cultivation of strain Kam1 we have used a 10-fold dilution of a standard methanotrophic medium (AMS) adjusted to pH 3.5. It requires a low-salinity medium for growth.

Stock solutions: as below

Preparation of medium:
1. Add 1 ml of the trace element solution and 1 ml of the iron stock solution to the mineral stock solution and autoclave. Also autoclave the phosphate buffer stock solution.
2. After cooling of the autoclaved solutions, add 20 ml of the phosphate buffer to the mineral stock solution
3. Dilute the mineral medium (containing trace elements, iron and macromineral-elements including phosphate) 1:10 in autoclaved dH2O. Add 1 ml of 0.3 mM Cerium(III) chloride solution to 1 liter of the diluted medium to obtain a final concentration of 300 nM Cerium(III) chloride.
4. Adjust pH to 3.5 with 1M HCl to obtain the final mineral medium (termed 0.1 x AMS).
Cultivation:
We use 50 or 100 ml serum flasks containing 20 to 40 ml liquid medium and a headspace of air. We inoculate with 10% inoculum with a syringe through the rubber stopper. Methane and CO2 is added with a syringe to 10% and 1% final head space concentration.
An alternative to methane is to use 0.1% methanol (final concentration) as growth substrate, but this increases the risk of contamination with other methylotrophs.
Incubation is at 55oC and preferably with shaking (if with methane). It takes about one week for turbidity to develop.