TSBD Trypticase Soy Broth Dialysate

Use for producing stable exoenzyme S.

1. 1 litre
2. 30 g TSB
3. 90 ml dH₂O
4. 10 g Chelex 100-400 (other Chelex is ok)
5. Stir 6 hours at 25°C.
6. Spin 16,000 g for 30 minutes.
7. Filter through 0.45 um filter.
8. Concentrate to almost dry over a PM-30 (PM-10 is ok) Amicon membrane.
9. Discard concentrate on the filter, the rest is 10X and filtered.
10. Add nitrilotriacetic acid to 10 mM.
11. Adjust to pH 7.0.
12. Autoclave in 2 litre flask. Cool.
13. Add 20 ml sterile 50% glycerol.
14. Add 50 ml sterile 2 M MSG (or glutamate, Na salt)
15. Add sterile MgSO₄ to 1 mM