【 KCOM medium 1 】  

* Broth medium

Tryptic Soy (or Brain Heart Infusion) broth 30.0 (38) gm

Yeast extract 5.0 gm

0.025% Resazurin 4.0 ml

Cysteine HCl-H2O 0.5 gm (final conc. 0.05%)

Hemin solution (5 mg/ml) 1.0 ml (final conc. 5 μg/ml)

Vitamin K1 solution (10 mg/ml) 0.2 ml (final conc. 2 μg/ml)

DW upto 1000 ml

…………………………………………………………………………………………………………………… 

pH 7.2±0.2

-> Autoclave

* Agar medium

Tryptic Soy (or Brain Heart Infusion) broth 30.0 (38) gm

Yeast extract 5.0 gm (0.5%)

Bacto Agar 20.0 gm (2%)

0.025% Resazurin 4.0 ml

Cysteine HCl-H2O 0.5 gm (0.05%)

Hemin solution (5 mg/ml) 1.0 ml (5 μg/ml)

Vitamin K1 solution (10 mg/ml) 0.2 ml (2 μg/ml)

DW upto 1000.0 ml

…………………………………………………………………………………………………………………… 

pH 7.2±0.2

-> Autoclave

-> After autoclave, maintain at 45°C

-> Add 50 ml of defibrinated sheep blood (final conc. 5%)

Vitamin K1 can be replaced of Menadione (Vitamin K3, final conc. 1 μg/ml in both) but should be added after autoclaving.

* Hemin stock solution (5 mg/ml):

1. Dissolve 0.5 g of Hemin in 5 ml of 1 N NaOH.

2. Add up to 100 ml of deionized water and Autoclave 

3. Wrap bottle in foil and store at 4°C

* Vitamin K1 (Sigma V-3501) stock solution, 5 mg/ml

1. Mix 0.15 ml of Vit. K1 with 30 ml of 95% ethanol

2. Wrap bottle in foil and store at 4°C

    (※ Discard after one month)

* Vitamin K3 (Menadione) stock solution, 1 mg/ml

1. Dissolve 0.1 g of Vit. K3 in 100ml of deionized water

2. Wrap bottle in foil and store at 4°C

 (※ Cannot be autoclaved)