Mesoplama florum:Culture media

ATCC 1161 Medium

Mix a base broth in a 1 liter 45mm cap bottle the following chemicals:

Heart Infusion Broth (Difco 0038)	17.5 g
Sucrose	40 g
Agar Noble (Difco 0142) if necessary	12 g
DI Water	710 ml

Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.

Centrifuge 2 x 45 ml of Gibco 18180-059 liquid yeast extract at high speed to remove sediment. Pour only 45 ml into centrifuge bottles, eliminating as much as possible of the sediment in the bottom of the Gibco stock.

Thaw Sigma H-1138 heat inactivated horse serum. For agar, bring both horse serum and centrifuged yeast extract to 37C. Horse serum at 55C will sediment protein.

Assemble 500 ml bottle top 0.22μ filter on the 1 liter bottle. For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling. Add by vacuum filtration:

Horse serum (Sigma H1138)	200 ml
Yeast extract	90 ml
Penicillin G 500,000 U/ml	400 μl
2% phenol red or 1% cresol red	2 ml (5 ml for cresol red)

Adjust pH to 7.4 with 1 M HCl or 1 M NaOH. Measured pH of as-made broth is 7.46, probably close enough. For cresol red containing solution, adjust pH to 7.8

Phenol red 2% solution must be neutralized with sufficient NaOH to convert it to the sodium salt. The phenol or cresol red amount can be adjusted to control the depth of color to taste.

SP-4 (ATCC 988) Medium

Mix a base broth containing:

Mycoplasma broth base (BBL 111458)	3.5 g
Tryptone	10 g
Peptone	5.3 g
Glucose	5.0 g
DI water	615 ml

Adjust pH to 7.5 and autoclave at 121C for 20 minutes.

Centrifuge 2 x 17.5 ml of liquid yeast extract (Gibco 18180-059) at high speed to remove sediment.

Mix 2 g of Yeastolate (Difco 5577) in 100 ml DI water.

Sterile filter into the cooled medium:

penicillin G 500,000 U/ml	400 μl
2% phenol red	2 ml
2% Yeastolate (Difco 5577)	100 ml
Newborn calf serum	170 ml
liquid yeast extract	35 ml
10x CMRL-1066 solution	50 ml