-
| |
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.
Mycoplasma Broth
| |
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 |
|
