Mycoplasma Broth Recipes－PPLO Broth
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200 ml 1000 ml 4000 ml Mycoplasma broth base (Gibco) 4.5 22.5 90.0 DNA (Sigma, Degraded free acid, type IV) 0.05 0.20 0.80 Glass Distilled Water 142 ml 710 ml 2840 ml Phenol Red (1% aqueous) 0.4 ml 2.0 ml 8.0 ml For Agar Media using Noble Agar (Difco) 2.0 10.0 40.0 Using Seaplaque Agarose (FMC corp. Marine Colloids Div.) 1.6 8.0 32.0
Adjust pH to 7.8 - 8.0 with 1N NaOH
Autoclave at 121°C for 15 min. at 15 lbs pressure, slow exhaust.
Allow to cool for liquid medium, and bring to 50°C in a water bath for agar
Add aseptically:
| 200 ml | 1000ml | 4000 ml | |
| Agamma horse serum | 40 ml | 200 ml | 800 ml |
| 25% Yeast Extract | 10ml | 50 ml | 200 ml |
| Penicillin G (100,000 units/ml) | 1 ml | 5 ml | 20 ml |
| Cefobid 2.5 mg/ml (50:50 EtOH:50 water) | 2 ml | 10 ml | 40 ml |
| 50 % Dextrose (filter sterilized) | 2 ml | 10 ml | 40 ml |
pH should be around 7.8-8.0
Store at 4°C for one month or freeze for 3 months
Aseptically dispense agar into petri dishes with a minimum depth of 4mm, store in sealed plastic bags at 4°C for 3 weeks.
<center><strong><font>Mycoplasma Broth</font></strong></center>
| 4 L | 1 L | 0.5 L | |
| Dist. Water | 2640 ml | 660 ml | 330 ml |
| PPLO Broth Base | 84 g | 21 g | 10.5 g |
| Glucose | 20 g | 5 g | 2.5 g |
| 1% Phenol Red | 8 ml | 2 ml | 1 ml |
Adjust pH to 7.8-8.2 & Autoclave
| 4 L | 1 L | 0.5 L | |
| Yeast Extract | 400 ml | 100 ml | 50 ml |
| Penicillin (120,000 u/ml) | 20 ml | 5 ml | 2.5 ml |
| Thallium Acetate (1:50) | 100 ml | 25 ml | 12.5 ml |
| Horse Serum | 800 ml | 200 ml | 100 ml |
readjust pH after addition of above!
Mycoplasma Agar
Substitute Agar-PPLO for PPLO broth base
| 4 L | 1 L | 0.5 L |
| 60 g | 15 g | 7.5 g |
