IRPCM:Common culture media

SP-4 medium

This medium was originally developed for use with fastidious Spiroplasma species, but has found widespread use as an excellent medium for most Mollicutes.

Instructions: 1.) Prepare medium base and autoclave for at least 15 minutes 2.) While the base is sterilizing, mix the supplements. Remove large particles by repeated straining through coffee filters or filter paper 3.) Pass the supplements through a 0.2 μM vacuum filter. Multiple filters may be required. Passing through 0.45 μM filters prior to the 0.2 μM filter may alleviate this. 4.) Add filtered supplements aseptically to the sterile broth base. 5.) Adjust the final pH to 7.6-7.8 (glucose supplemented) or 7.0-7.2 (arginine supplemented) with filtered, 2N NaOH or HCl

Medium Base (amounts to prepare 1 liter): 1.) Mycoplasma Broth Base (w/o crystal violet) 3.3 g 2.) Tryptone 10 g 3.) Peptone 5.3 g 4.) Phenol red 20 mg 5.) 2N NaOH 2 mL 6.) Distilled Water 640 mL

• • Options**

1.) D-glucose (dextrose) 10g* 2.) Agar 15g** Supplements: 1.) Fetal bovine serum (heat inactivated) 167 mL 2.) CMRL 1066 (1x) 50 mL 3.) L-glutamine 50 mg 4.) Yeast Extract (Gibco) 35 mL 5.) 2% Yeastolate 100 mL

• • Options**

1.) L-arginine 2.1g* 2.) Nicotinamide adenine dinucleotide 100mg*** 3.) L-cysteine 100mg*** 4.) Antibiotics****

• The carbon source provided is determined by the species of interest. Glucose and arginine can both be added to the same stock of medium if desired.
  • Add only when making solid medium (i.e., plates or slants)
    • These supplements are essential for M. synoviae, but no other species
      • Adding β-lactams and/or polymixins (final concentration 250 mg/mL) can minimize contamination but is not necessary