Bacillus Medium

Culture of Geobacillus thermodenitrificans

94A5 Geobacillus thermodenitrificans G11MC16  cannot be grown at 37°C. Growth at 50°C is acceptable, but it will grow faster up to about 63°C.

94A5 grows very poorly in LB medium. For growth on solid medium, use Difco TBAB (Tryptose Blood Agar Base) straight from the tube, with no blood or other supplements. The strain also grows well in mLB agar or MG agar, with recipes below. Growth on Trypticase Soy Agar is also acceptable. For growth in liquid medium, we have put together a liquid version of TBAB from its components, minus the agar. It would have, per liter of water, 10 g of tryptose, 3 g of beef broth, and 5 g of NaCl. The liquid version of mLB and MG work well. We haven’t tested Trypticase Soy Broth, but I suspect it could work, too.

Third, it is also important to realize that 9A5 does not sporulate well under normal laboratory conditions and so will eventually lyse in stationary phase. If you wish to grow it in liquid medium, you should harvest it in late exponential phase or early stationary phase. The growth rate of this bacterium is high, with a doubling time of about 15 minutes at 60°C. It is easy to let it go too long in stationary phase. For this reason, we usually streak 9A5 heavily on TBAB and incubate it overnight at 60°C. The next morning we scrape cells off the surface of the agar with a sterile loop.

mLB Medium (Modified L-Broth)—N. Welker, Northwestern University, personal communication.

Tryptone	10	g
Yeast Extract	5	g
NaCl	5	g
Agar (if desired)	15	g
Water	995	ml
NaOH (10% w/v)	1.25	ml

Autoclave.  Cool to 55˚C and add 1 ml each of the following sterile stocks:

Nitrilotriacetic acida	1.05	M
MgSO4.7H2O	0.59	M
CaCl2.2H2O	0.91	M
FeSO4.7H2O	0.04	M

 MG Medium

(Minimal Glucose Medium)—N. Welker, Northwestern University, personal communication.

Minimal salts (below)	20	ml
Agar, if desired	15	g
Water	955	ml
10% KOH (w/v)	0.68	ml

After autoclaving, add 1 ml each of the following sterile stocks:

Nitrilotriacetic acida	1.05	M
MgSO4.7H2O	0.59	M
CaCl2.2H2O	0.91	M
FeSO4.7H2O	0.04	M

Then add 25 ml of:

Glucose, filter-sterilized

20% (w/v)

Minimal salts (do not sterilize; store refrigerated):

K2HPO4	26.1	g
KH2PO4	11.3	g
NH4NO3	25.0	g
Water	1000	ml

a CAUTION: Possible carcinogen.  Wear gloves and face mask. Dissolve by adding NaOH pellets directly to NTA and water, then adjust the pH to 6.6-6.8 by adding 1 M NaOH.  Filter sterilize and store at room temperature.