ATCC medium: 2010 Chromatium medium (ATCC medium 37) with 3% NaCl
ATCC Medium 37 (see below) with 3% NaCl
ATCC Medium 37: Solution 1:
CaCl2.................................... 2.0 g
Distilled water.......................... 2.5 L
Distribute 2000 ml in 75-80 ml amounts into 127 ml (4 oz) screw-capped Boston round bottles and autoclave.
Solution 2:
Heavy Metal Solution (see below)........ 50.0 ml
Vitamin B12 Solution (see below)......... 3.0 ml
Vitamin Solution (see below)............ 15.0 ml
KH2PO4 ................................... 1.0 g
KCl...................................... 1.0 g
NH4Cl .................................... 0.8 g
MgCl2 . 6H2O ............................. 0.8 g
Sodium ascorbate*........................ 2.4 g
Distilled water......................... 32.0 ml
*May be omitted if large inocula are used.
Solution 3:
NaHCO3................................... 4.5 g
Distilled water........................ 900.0 ml
Bubble gaseous CO2 through for at least 30 minutes. After CO2 saturation of Solution 3, add Solution 2 and immediately filter-sterilize using CO2 pressure to push the liquid through (no suction). Add this sterile filtered solution to the 127 ml bottles containing 75-80 ml of Solution 1 so that it completely fills the bottle.
Solution 4:
Na2S . 9H2O.............................. 3.0 g
Distilled water........................ 200.0 ml
Autoclave with a magnetic-stirrer rod in the flask. Partially neutralize the sterilized Solution 4 by adding, on a magnetic stirrer, drop by drop, 2.0 ml sterile 2 M H2SO4. 1 Directions for completion of Medium: Remove 6 ml of medium in the Boston round bottles that were filled to full capacity as directed under Solutions 2 and 3. Replace this volume with an equal volume of neutralized Solution 4. Allow the bottles to stand at least overnight to develop a hazy, white precipitate before inoculating. At this time, the final pH will be 6.7 +/- 0.1. To inoculate, remove 6 ml of completed medium and replace it with an equal volume of inoculum. Grow cultures under tungsten light.
Heavy Metal Solution:
EDTA (should be dissolved first)........................... 1.5 g
Modified Hoagland Trace Element Solution (see below)....... 6.0 ml
FeSO4 . 7H2O ............................................... 0.2 g
ZnSO4 . 7H2O ............................................... 0.1 g
MnCl2 . 4H2O ............................................... 0.02 g
Distilled water............................................ 1.0 L
Vitamin B12 Solution:
Vitamin B12 (Cyanocobalamine)...... 2.0 mg
Distilled water.................. 100.0 ml
Vitamin Solution:
Biotin.............................0.2 mg
Nicotinic acid..................... 2.0 mg
Thiamine........................... 1.0 mg
p-Aminobenzoic acid................ 0.1 mg
Pantothenic acid................... 0.5 mg
Pyridoxamine 2HCl.................. 5.0 mg
Distilled water.................. 100.0 ml 2
Modified Hoagland Trace Element Solution:
AlCl3 .............................. 1.0 g
KI................................. 1.0 g
KBr................................ 0.5 g
LiCl............................... 0.5 g
MnCl2 . 4H2O ....................... 7.0 g
H3BO3 ............................. 11.0 g
ZnCl2 .............................. 1.0 g
CuCl2 .............................. 1.0 g
NiCl2 .............................. 1.0 g
CoCl2 .............................. 1.0 g
SnCl2 . 2H2O ....................... 0.5 g
BaCl2 .............................. 0.5 g
Na2MoO4 ............................ 0.5 g
NaVO3 . H2O ........................ 0.1 g
Na2SeO3 ............................ 0.5 g
Dissolve each salt in distilled water before mixing. Adjust the pH of each solution to below 7.0. Adjust the final pH to 3-4. The total final volume is 3.6 L. The flaky yellow precipitate which is formed after mixing transforms after a few days into a very fine white precipitate. Mix the solution thoroughly before use.