ATCC Medium: 2479 4YACO Medium
NaCl…………………………………………………1.0 g
KCl………………………………….........................0.5 g
MgCl2 . 6H2O…………………………………………0.4 g
CaCl2 . 2H2O…………………………………………0.1 g
NH4Cl………………………………………………..0.3 g
KH2PO4………………………………………………0.2 g
NaSO4……………………………………………….0.15 g
0.1 M MOPS* (pH 7.2)…………………………….100.0 ml
Resazurin…………………………………………..0.1 mg
DI Water……………………………………………848 ml
*3-N-morpholino propanesulfonate Adjust pH to 7.2-7.4 then autoclave at 121oC. Cool under 80% N2-20% CO2.
Prior to inoculation, the medium is reduced by adding 100.0 mM dithiothreitol (separately sterilized under 100% N2. to a final concentration of 1.0 mM).
The headspace is exchanged with 80% H2-20% CO2 for dispensing into culture tubes.
The headspace should be at lease four times the liquid volume.
Filter-sterilize and aseptically add the following solutions:
1.0 M NaHCO3 (gas under CO2)…………………..60.0 ml
Trace Element Mixture w/EDTA (see below)…….1.0 ml
Six Vitamin Solution (see below)………………….1.0 ml
Vitamin B12 (see below)…………………………….1.0 ml
Co-Factor Mixture (see below)…………………….20.0 ml
Yeast Autolysate (see below)………………………40.0 ml
Trace Element Mixture w/ EDTA
Na EDTA………………………………………………5.2 g
FeSO4 . 7H2O…………………………………………..2100.0 mg
H3BO3…………………………………………………30.0 mg
MnCl2 . 4H2O…………………………………………..100.0 mg
CoCl2 . 6H2O…………………………………………..190.0 mg
NiCl2 . 6H2O……………………………………………24.0 mg
CuCl2 . 2H2O…………………………………………..2.0 mg
ZnSO4 . 7H2O………………………………………….144.0 mg
Na2MoO4 . 2H2O………………………………………1200.0 mg
DI Water………………………………………………999.0 ml
Adjust pH to 6.0 with NaOH. Deoxygenate; sterilize under 100% N2.
Six-Vitamin Solution
4-Aminobenzoic acid…………………………………………….4.0 mg
Nicotinic acid……………………………………………………...10.0 mg
Calcium-D-(+)-pantothenate…………………………………….5.0 mg
Pyridoxine HCl……………………………………………………15.0 mg
Thiamine HCl……………………………………………………..10.0 mg
Biotin……………………………………………………………….1.0 mg
DI Water……………………………………………………………100.0 ml
While being stirred, the mixture is neutralized drop wise by addition of 0.1 M NaOH which facilitates solubility of all vitamins.Deoxygenate; filter-sterilize under N2.
Vitamin B12
Vitamin B12 (cyanocobalamin)……………………………………..5.0 mg
DI Water………………………………………………………………100.0 ml
Deoxygenate; filter-sterilize and store under N2. Protect from light.
Co-Factor Mixture
Pyridoxal HCl…………………………………………………………..25.0 mg
Pyridoxal phosphate…………………………………………………..5.0 mg
Ca folinic acid………………………………………………………….25.0 mg
beta-NAD……………………………………………………………….5.0 mg
Coenzyme A……………………………………………………………5.0 mg
FAD……………………………………………………………………..5.0 mg
Nicotinamide…………………………………………………………..2.5 mg
Folic acid………………………………………………………………250.0 ug
Riboflavin……………………………………………………………...50.0 ug
Thiamine pyrophosphate……………………………………………250.0 mg
DI Water……………………………………………………………….100.0 ml
Deoxygenate; filter-sterilize and store under N2.
Yeast Autolysate
Allow 56.0 g of dry baker’s yeast( Red Star Yeast only) to autolyze in 200.0 ml distilled water for 24 hrs at 56oC.Remove cell debris by centrifugation.
Neutralize the supernatant liquid with 5.0 N NaOH while stirring. Deoxygenate with N2; filter-sterilize and store under N2