Nitrocefin Assay on Whole Cells for Outer Membrane Permeability Assessment

Reference:

Angus et al. (1982). AAC 19:299

Materials:

• Double beam P.E. spectrophotometer and chart recorder
• Small French pressure cell

Cells:

1. Grow overnight in 1 ml LB normal salt (plus marker eventually)
2. Inoculate 0.4 ml into 20 ml LB normal salt (no marker anymore, since antibiotic may interfere with the cell surface.)
3. Grow to OD₆₀₀ = 0.5 -0.8 (mid-log)
4. Remove a small amount, if needed, to check cell phenotype
5. Centrifuge and resuspend so that all strains to be tested are at the same final OD (1.0) in: 10 mM Na-Hepes pH 7.0
6. 5 mM MgCl₂ (to maintain OM integrity)
7. Record OD₆₀₀ of resuspended cells for calculations
8. Break 3 ml of each cell sample 1 x in small French press, record OD₆₀₀ and keep on ice until needed.

Nitrocefin (NF):

1. Make stock solution of 1.0 mg/ml NF by dissolving 10 mg of NF in about 200 ul DMSO. Make sure all NF has been dissolved in the DMSO before adding Hepes buffer (same as above) to make up to 10 ml. Solution should be dark yellow-coloured and clear. A cloudy solution means that the NF has come out of solution. If this occurs, you can try to save the NF by pHing it in (almost never works) or by diluting your stock solution 10-fold to make a working solution of 0.1 mg/ml. Check the pH of your Hepes solution before making another attempt.
2. Place 1 ml of stock solution in each of 10 Falcon 10 ml tubes, cover with aluminium foil and freeze at -20 C. Dilute in the tube with 9 ml of buffer immediately prior to use. You will need about 5-10 ml at 0.1 mg/ml per strain for one day’s experiments.
3. The diluted stock solution can be refrozen and used later if you don’t use it all. Check colour.

NOTE: NF is a suspected carcinogen, so use gloves and a mask when weighing it out and don’t spill overmuch. NF 1 x solution should be yellow in colour , not orange. If it isn’t, either the compound has been destroyed by light or your concentration is wrong. You will need to repeat broken cell assays with several different concentrations of NF.

Assay:

Whole Cells:

• 0.65 ml 0.1 mg/ml NF (for P. aeruginosa)
• 0.1 ml whole cells or broken cells
• the concentration of NF to be used will depend on the strain and the amount of B-lactamase produced by the bacteria.
• mix briefly by upending with parafilm to seal the cuvette opening and read on strip chart recorder at OD₄₉₅.
• reference: 0.65 ml NF plus 0.1 ml cell supernatent (pellet 200 ul same cell suspension in Eppendorf for 1 min and use the supernatent to control for leakage of beta-lactamase out of the cell).

Settings:

• Ordinate limits = 0-0.2 for whole cells without plasmid beta-lactamase; 0-0.5 for whole cells with plasmid beta-lactamase
• Chart speed = 15 mm/min

Broken cells:

• 0.65 ml nitrocefin
• 0.1 ml broken cells
• same procedure as for whole cell

Settings:

• Ordinate limits = 0-2.0 for determination of rate of cleavage; 0-3.0 for determination of extinction coefficient (max. OD₄₉₅ after cleavage of all 0.1 mg NF in cuvette).