Basal Medium
PPLO Broth w/o Crystal Violet (BD 255420)…….………….3.5 g
Pancreatic Digest of Casein (Hardy C6530)……….……….10.0 g
Pancreatic Digest of Gelatin (Hardy C6540)………………..5.0 g
Agar (if desired)………………………….…………………….15 g
DI Water………………………..……………………………….500 ml
Adjust pH to 7.4 +/- 0.2. Autoclave at 121?C. If agar is used, cool to 50-55 ?C in a water bath.
Aseptically add the following additive solution:
Additive Solution
10X CMRL 1066 Medium (ATCC 20-2207).………………….50.0 ml
Yeast Extract Solution (see below)………….…………..…….35.0 ml
TC Yeastolate 10% solution (BD 255772)…………….………20.0 ml Fetal
bovine serum (see below)……….………………………170 ml
Phenol Red Solution0.1% Solution (see below)….…...…….20 ml
Urea (Sigma U5218)……………………………………………1.0 g
DI Water.……………………………..…………………………..205 ml
Combine ingredients and filter sterilize through a 0.22 μm filter.
Final pH at 25 ?C of complete medium should be 6.0 +/- 0.2. Dispense as required.
Yeast Extract Solution
Yeast Extract (Hardy C7340)…………………………………..15 g
DI Water…………………………………………………………..100 ml
Dissolve yeast extract with constant stirring. Filter once using a crepe fluted filter (VWR 28331-106 or equivalent). Add 35 ml to additive solution.
Fetal Bovine Solution (heat inactivated)
Thaw a bottle of Fetal Bovine Serum (ATCC 30-2020). Place into the water bath (55 ?C). Place a large beaker over the bottle to hold heat. Allow the bottle to remain in the water bath for 1 hour and 15 minutes. (15 minutes to come to 55?C and 1 hour to heat inactivate).
Phenol Red Solution
Add Phenol Red powder to 1/5th required volume of 0.1 N NaOH to dissolve. When dissolved, add remaining 4/5th of volume (water). Examine to confirm that phenol red has dissolved. Add additional 6 N NaOH drop by drop, if necessary to complete the dissolving of the phenol red.
Note: CMRL 1066 Medium is made as a 10X solution from custom CMRL Powder (20-2206) manufactured by Sigma. Custom formulation in use at ATCC, as of 1/2/03, contains L-glutamine
CMRL Solution and FBS, if known to be sterile, may be added directly to the basal medium and omitted from the Additive Solution. This makes filtersterilization easier.