Beta-LACTAMASE INDUCTION (CRUDE PREPARATION)

REFERENCE: Gootz & Sanders. A.A.C.
 

METHOD:

1. Inoculate regular growth medium with overnight culture of bacteria. (1/20 - 1/50)
2. Grow to OD₆₀₀ = 0.3 under usual conditions (37^oC, shaking).
3. Add sterile antibiotics so the final concentration is 1/2 the MIC of the antibiotic for the bacterium tested. Include a control of no antibiotic for each strain.
4. Grow for another 3 hours. Check final OD₆₀₀. (Cells should still grow.)
5. Add 1mM sodium azide (final concentration) to stop cell metabolism.
6. Centrifuge cells in Sorvall 7,000 rpm, 15 min. in G3 or 10,000 rpm, 5 min. in SS34.
7. Wash 2X in 5mM sodium Hepes buffer pH 7.2.
8. Resuspend each pellet in small volume of buffer (approx. 2 mls) (or 1/100 of original growth volume).
9. Place each sample in small plastic test tube. Keeping samples on ice, sonicate each one 3X for 1 minute bursts, using small probe at 35%. Alternatively: add polymyxin B to a final concentration of 1mg/ml or French Press in the small cell.
10. Spin down in ultracentrifuge 70.1 Ti, 23K, 30', 5^oC.
11. Carefully pipet off supernatant. This is the crude B-lactamase extract. Store: short term: 4^oC (loses activity with time) long term: -20^oC (loses activity with freeze-thawing)
12. By doing protein assays and nitrocefin assays on each sample, you can then determine the specific activity. See "permeability Calculations: 10 easy steps" for calculations of specific activity.