ATCC Medium: 2611 Spiroplasma Medium – Special Modified Formulation
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 required)…………………………………………………...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. Combine the 500 ml of additive solution to the basal medium. Gently mix; if necessary, readjust pH to 7.4 +/- 0.2.
Dispense as required.
Additive Solution
10X CMRL 1066 Medium (ATCC 20-2207)…………………….50.0 ml
Yeast Extract Solution (see below)…..………………………….35.0 ml
TC Yeastolate (BD 255772) 10% solution…………..…………20.0 ml
Fetal bovine serum (see below)……….………………………..170.0 ml
Phenol Red Solution 0.1% Solution (see below)……..……….20.0 ml
Glucose……………………………………………………………5.0 g
DI Water…………………………………………………………...205 ml
Combine ingredients and filter sterilize through 0.22 microns.
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, 2611 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.