ATCC Medium: 2734 VL55 Medium
For Broth
MES…………….………………………………1.95 g
20 mM MgSO4………………………………….10 ml
30 mM CaCl2……………………………………10 ml
20 mM (NH4)2HPO4……………………………10 ml
Se/W………………………………………………1 ml
SL-10.........................................................…...1 ml
DI Water………………………………………….960 ml
pH w/ NaOH/KOH to 5.5 (about 6 ml)
After autoclaving, add:
Vitamin 10 mix……………………………………3 ml
0.2M glucose………………………………..….10 ml
For Agar
MES…………….………………………………..3.9 g
20 mM MgSO4………………………………….20 ml
30 mM CaCl2……………………………………20 ml
20 mM (NH4)2HPO4……………………………20 ml
Se/W………………………………………………2 ml
SL-10 (pH w/ NaOH/KOH)…….………………..2 ml
DI Water………………………………………….920 ml
pH w/ NaOH/KOH to 5.5 (about 12 ml)
Autoclave in 250 ml lots, with a magnetic stirring bar in each bottle.
After autoclaving, keep at about 60°C, then add to each 250 ml lot:
Vitamin 10 mix……………………………..……1.5 ml
0.2M glucose………………………………..….10 ml
3% washed agar………………………………..250 ml (keep at about 60°C)
Stir and pour plate
NOTE: If the agar or gellan is added to the MES-containing medium, fewer
bubbles will be formed.
Stocks for VL55
20mM MgSO4 MgSO4x7H2O………………..0.49 g
DI Water………………………100 ml
30mM CaCl2 CaCl2x2H2O………………….0.44 g
DI Water……………………….100 ml
20 mM (NH4)2HPO4 (NH4)2HPO4……………..0.26 g
DI Water……………………100 ml
NaOH/KOH NaOH………………………..0.80 g (200mM)
KOH…………………………0.56 g (100mM)
DI Water…………………….100 ml
Washed Agar
Suspend about 10% more agar than finally required in a large volume of water
(i.e., 30g in 2000ml of glass-distilled H2O), and stir with magnetic stirrer for about
5 min. Turn off stirrer, and allow agar to settle (about 30 min). Decant off
supernatant and repeat this washing step about 5 times, to remove sugars etc
from the agar. The supernatant will at first be yellowish, but later clearer.
Resuspend agar in required final volume of water, and autoclave.
Trace element solution SL10
The components of the trace element solutions SL10 (Widdel et al. 1983) are
added and dissolved in the order listed.
25% HCl……………………………………………….10 ml
FeCl2x4H2O………………………………………….1.5 g
CoCl2x6H2O………………………………………….190 mg
MnCl2x4H2O………………………………………….100 mg
ZnCl2…………………………………………………..70 mg
H3BO3………………………………………………….6 mg
Na2MoO4x2H2O……………………………………..36 mg
NiCl2x6H2O…………………………………………..24 mg
CuCl2x2H2O………………………………………….2 mg
DI Water……………………………………………….1000 ml
The trace element solution is autoclaved under air, in 25 ml aliquots in 50 ml
screw-capped bottles. Large stocks do not need to be sterilized for storage.
Selenite/tungstate solution
NaOH…………………………………………………….0.5 g
Na2SeO3x5H2O………………………………………..3 mg
Na2WO4x2H2O………………………………………..4 mg
DI Water…………………………………………………1000 ml
The selenite/tungstate solution (Tschech and Pfennig 1984) is autoclaved under
air in 50 ml aliquots in 100 ml screw-capped bottles.
Vitamin 10 stock
4-aminobenzoate………………………………………13 mg
d-(+)-biotin………………………………………………3 mg
Nicotinic acid……………………………………………33 mg
Hemicalcium D-(+)-pantothenate……………………..17 mg
Pyridoxamine hydrochloride……………………………50 mg
Thiamine hydrochloride………………………………….33 mg
Cyanocobalamin…………………………………………17 mg
DL-6,8-thioctic acid……………………………………….10 mg
Riboflavin…………………………………………………..10 mg
Folic acid…………………………………………………...4 mg
Vitamin 10 solution is sterilized by filtration into sterile bottles though sterile
0.2uM pore-size cellulose acetate filters. The bottles are wrapped in aluminum
foil to protect against light, and stored at 4°C.