Archives: Methods

Reference:

Schindler and Teuber. Antimicrob. Ag. & Chemother. 8:95-104, 1975.

Method:

1. Dissolve 40mgs of Polymyxin B sulfate in 1.2mls of 0.1M NaHCO₃.
2. Dissolve 10mgs of Dansyl-chloride in 0.8mls of acetone.
3. Add the polymyxin to the Dansyl-chloride and place in the dark for 90 minutes at room temperature.
4. After incubation load the mixture onto a Sephadex G-50 column (50 x 2.5 cm) equilibrated with 10mM Na-phosphate buffer (pH 7.1) containing 0.145M NaCl.
5. Collect 5-6ml fractions.
6. The Dansyl-Polymyxin comes out in a fairly broad peak ahead of the unreacted Dansyl-chloride peak. Location of the Dansyl-Polymyxin in the collected fractions can be determined by holding a UV lamp over the fractions and looking for fluorescence. The fluorescence of the Dansyl-Polymyxin is yellowish, while the unreacted Dansyl-chloride is more blue-green. The fractions containing the Dansyl-polymyxin are extracted into about 1/2 volume of n-butanol. The butanol is then evaporated to dryness in a glass petri dish place inside a dessicator which is then evacuated and placed @ 37^oC. This takes about 24hrs.
7. The dried Dansyl-Polymyxin is dissolved in 3mls of buffer (5mM Hepes, pH 7.0) and stored in aliquots @ -20^oC. The concentration of the Dansyl-polymyxin is determined by dinitrophenylation assay.
8. NOTE: To make column buffer, make up 10mM Na₂HPO₃ and NaH₂PO₃ plus salt separately and add together to obtain proper pH (about a 1:5 ratio of NaH₂PO₃ to Na₂HPO₃).

Preparation of chambers:

• Immediately after use, rinse with distilled water a couple of times and then with 70% or 100% ethanol (can repeat this rinsing), then invert the cells and let them air-dried. This should work for getting rid of most porins.
• When used for small peptides which can get into the porous Telfon cell wall and are hard to clean, soak overnight in RBS (diluted) at room temperature. Then rinse under distilled water for half a day (detergent gets into cell wall too and may cause problems during experiment). Then rinse as above. Don’t heat the chambers (e.g. with hair-drier, that’s not good for the chamber)
• Oxidized cholesterol stored at –20^oC in chloroform CHCl₃ as a 2% solution in 100ul aliquots, may be partially dried down.
• Dry the aliquot down completely in a vacuum until can’t smell the CHCl₃, resuspend the white powder in 0.133 ul decane with 5 ul of n-butanol to yield 1.5% solution of cholesterol, vortex, can store this in fridge, sealed to prevent moisture getting in. Also, don’t open vial until warm (room temperature) or moisture will get into vial and hydrate the lipid.
• Coat the hole in chamber with about 3ul of oxidized cholesterol, let air-dry. For single channel, coat one side; for selectivity, coat both sides. Another lipid commonly used is diphytanoylphosphatidyl choline (DiphPC). It is prepared in the same method used for oxidized cholesterol (dried down a 2% solution, resuspend in decane and n-butanol to yield the desired %). There are small vials with 2mg/vial DiphPC in freezer. These were originally in CHCl₃ but probably all dried down after the CHCl₃ evaporated.

Doing the experiment:

Make sure the table is pumped up so the equipment will be less sensitive to motion around.

• Check to ensure input voltage is ok: disconnect the blue box from the amplifier on the top and connect it to the voltmeter ( while the two electrodes are in a cell with some salt solution), check to see voltage applied = what you see on voltmeter. Change battery (2 x 9V) if it’s off.
• Don’t touch calibrated knob on power box.
• Set rise time to 10ms (can give better resolution if pores are small and close together but this setting is noisier); set to 30ms if channels are fast and fuzzy.
• Set the gain and suppression, generally 10⁹ and 10^-9 respectively; can use 10¹⁰ for greater sensitivity.
• Set power pack at 10-50 mV at DC for single channel measurements and selectivity measurements. (005 = 50mV).
• Oscilloscope: set at 20-50 mV / division for small pores and 100 mV or more for large pores.
• Chart recorder: set at 50-200 mV full scale (span) for small pores and 1-2V for large ones.
• Note on chart recorder paper: sample using (and amount used), salt solution and pH, gain, mV applied, chart speed, date, chart full scale.
• Fill the chamber with salt solution (after lipid coated has been dried around the hole). The salt solution is usually 1M KCl made up with dH2O, make sure there is no growth in solution (can filter before use). Other solutions: 0.1M or 3M KCl, 1M LiCl, 1M Kacetate, etc. (pH 7)
• Turn power pack on and check for baseline (about 20 min) to ensure there is no contamination in chamber.
• Add protein of interest. Usually 5ul of a 1:1000 dilution of the original sample is a good place to start. Dilute the protein in 0.1% Triton X-100 (10ul in 10ml total). If using diphytanoylphosphatidyl choline, use a 1:100 or even 1:10 dilution (depend on your sample conc.) because this lipid is less sensitive than cholesterol and need more proteins (but it also gives less artifact than with cholesterol).
• Record events on chart recorder. Watch the oscilloscope to ensure that the events on the chart recorder are real events and are single events. Mark these events on the chart recorder. Generally need to collect at least 100 pore events to generate a meaningful histogram. Can also check if the pore is voltage-dependent.
• If membrane breaks, reform it but do not keep adding more lipid unless you cannot reform the membrane without it.

Calculations:

V=IR

Volts = amps x ohms

S(siemen) = 1/ohm = amp/volt

# volts observed / volts applied x gain = siemen

e.g. 20 mV size steps, 10 mV applied, 10^-9 gain

20 x 10^-9 mV / 10 mV = 2 x 10^-9 S = 2nS “sized” channel

1. Outer membrane samples were resuspended to a concentration of 0.5 mg/ml in 0.1 ml sample buffer (125mM Tris-HCl, pH 6.8, 0.5% SDS, 10% (v/v) glycerol, 0.0001% (w/v) bromphenol blue).
2. This was heated at 100ºC for 2 minutes and cooled to 37ºC for protease digestion.
3. Protease was added in 1 µl amounts at the concentrations required and the outer membrane samples were digested for 1 hour at 37ºC.
4. Each sample was then divided in two aliquots.
   • To one aliquot, SDS was added to a final concentration of 2%.
   • The other aliquot was made up to 2% SDS and 10% (v/v) 2-ME, to identify the 2-ME modifiable fragments of protein F.
5. The aliquots were heated at 88ºC and loaded onto a 14% PAGE gel.

The initial solubilization step in 0.5% SDS was found to greatly aid the digestion of the fairly protease-resistant outer membrane proteins.

Reference:

Poole and Hancock. (1984.) Eur. J. Biochem. 144:607-612.

Method:

1. Purchase ³²P, phosphoric acid in HCl 2.0 mCi/ml from NEN (NEX-054).
2. Set up 10-25 ml overnight cultures in low phosphate media (see recipe file for Pseudomonas aeruginosa and E. coli low phosphate Hepes buffered minimal media). Harvest cells by filtration with Nalgene 0.45 µm filter units. wash cells by filtration 3X with 10 ms of Hepes buffered minimal media WITHOUT phosphate.
3. Resuspend washed bacteria in the same phosphate-free media to a final OD₆₀₀ = 0.2-0.3. Store on ice until needed. Cells are good for about 3 hours.
4. Place ³²P (2.5 µM = 1.25 µl of source) in a 10 ml diposable tube.
5. Warm bacterial suspension approximately 10 minutes at 37ºC before using in assay. Take one ml sample of bacterial suspension and add to the ³²P. Immediately start timing, vortex to insure good mixing. Remove 200 µl samples at the desired time intervals and filter using 0.45 nitrocellulose filters. (The old manifold filtration apparatus with filter cups from Amicon is useful for this.) Immediately wash the filter with Hepes buffered minimal media WITH 1 mM Phosphate (from a squirt bottle for fast washing). For a good time course try 15, 30, 45, 60 seconds after mixing cells with ³²P.
6. Remove filters , dry and count in 5 ml of scintillation cocktail.
7. Duplicate experiments should be done on two different days to verify results.
8. Controls: 200 µl of Hepes buffered media alone to get background counts and 1.25 µl of ³²P in 1 ml buffer, no cells, to get maximum counts.

For assessing hydrophobicity of the cell surface.

1. Grow cells to midlog phase, centrifuge and concentrate 10 fold.
2. Prepare phases by adding volumes of 20% (w/w) polyethylene glycol 6000 Sigma) and 20% (w/w) Dextran T500 (Pharmacia) to give final concentrations of 4.4% and 6.2%, respectively, in 30mM Tris-HCl buffer, pH 7.0. Equilibriate at 4C. for several hours to allow separation into the 2 phases.
3. Add 1 ml of each of the top and bottom phases. Add 0.1 ml of Triton X-100 and 0.1 ml of the concentrated cells.
4. Invert tubes 20x and leave to equilibrate for 45 min. at room temperature.

Take a sample from both the top and the bottom phases. The OD of each of these samples is measured in a spectrophotometer at 600 nm. Results may be expressed as the ratio of the top/bottom phases.

1. Grow cells to mid log phase in Luria Broth.
2. Centrifuge and resuspend in 5mM Na-azide, 5 mM Na-Hepes pH 7.0, to about 0.5 OD₆₀₀. Note: KCN can be substituted for Na-azide
3. Measure OD₆₀₀ of cells only 10 seconds.
4. Add lysozyme to give a concentration of 50 µg/ml. (measure OD 10 seconds)
5. Add permeabilising compound. (EDTA, gentamicin, or polymyxin B) (measure OD for at least 1 minute).
6. Lysis is measured as the decrease in OD₆₀₀.
7. Measure OD of controls:

1. cells + antibiotic (no lysozyme)
2. buffer + lysozyme + antibiotic (no cells)
3. cells + lysozyme (no antibiotic)

• If compound does show permeabilising activity, measure the effect of adding Mg²⁺.
• Measure OD₆₀₀ of cells.
• Add lysozyme and measure OD₆₀₀.
• Add Mg²⁺ to final concentrationd of 3mM, 1mM, and 0.1mM; measure OD₆₀₀.
• Add antibiotic and measure OD₆₀₀.

1. Grow 2 litres of cells OD₆₀₀ = 0.8-1.0, pellet by centrifugation in the GS3 rotor for 10 minutes at 7000rpm and resuspend in 20ml cold 20% sucrose in 10 mM Tris pH 8.0, 50 µg/ml DNase I. Incubate at least 15 minutes at room temperature before french pressing.NOTE: The cells may be frozen in 20% sucrose to enhance breakage. The 20% sucrose plasmolyses cells, resulting in better separation of the outer and inner membranes.
2. Break twice at 15,000 psi in the large French Pressure cell and put broken cells on ice immediately to stop protease action.
3. Remove the cell debris by centrifugation at 3,000 rpm for 10 minutes at 4ºC.
4. A one-step sucrose gradient is used. The sucrose concentrations in the steps off the gradient may also be varied depending on the degree of separation desired. Use Beckman polyallomer tubes for the gradients.
5. For crude separation of outer and inner membranes, use a 2-step gradient:
   

   For the SW 41 rotor	For the Sw 27/8 rotor
   4 ml 70% sucrose	14 ml 70% sucrose
   4 ml 60% sucrose	14 ml 60% sucrose
   4 ml sample (in 20% sucrose)	9 ml sample (in 20% sucrose)
   Adjust the volume of the 70% cushion to fill the tube.	To increased yield of outer membranes use 50% sucrose in instead of 60% sucrose

6. Centrifuge for 4 hours or overnight at 39,000 rpm in the SW 41 rotor at 4^oC. Centrifuge overnight at 23,000 rpm in the SW 27/8 rotor at 4^oC.
7. There should be two bands for the 2-step gradient. The upper band should be reddish and the lower one should be white. The lower band is the outer membrane. Outer membranes will band at the junction between the 60% and 70% steps. Inner membranes will band at the junction between the 20% and 60% steps. Collect the membranes by poking a hole in the bottom of the tube with a hot needle, and let the sucrose drip out, collecting only the membrane band.
8. Collect the bands separately using the bottom-drip method.
9. Place each band in a 60Ti tube and add at least two volumes of distilled water to dilute the sucrose below 20%.
10. Centrifuge at 47,000 rpm for 1 hour.
11. Using a syringe, resuspend the pellets in approximately 1ml.

Reference:

Poole and Hancock. l984. Eur.J.Biochem.144:607-612.

Method:

For P. aeruginosa:

1. Grow bacteria overnight in 100 ml of the appropriate medium.
2. Harvest the bacteria by spinning at 10,000 X g (7,000 rpm in SS34 Sorvall rotor), 4ºC for 10 minutes.
3. Resuspend the pellet with 8 ml of 0.05 M Tris-HCl pH 7.3, 0.2 M MgCl. Incubate for 10 minutes at 30ºC.
4. Chill in water-ice bath for 15 minutes.
5. Warm at 30ºC. for 10 minutes.
6. Repeat step 4.
7. Pellet shocked cells as in step 2. The supernatant should contain the periplasmic contents. (save both the pellet and the supernatant to run on gels)
8. Concentrate the supernatant 10 fold by using either a 10,000 Mol wt cutoff Centricon filter or an Amicon filtration apparatus with a PM10 filter.
9. Dialyse concentrated supernatant versus 0.05 M Tris-HCl pH 7.3 to remove any residual MgCl.
10. Do Nitrocefin assay to see if shock fluids contain the known standard periplasmic enzyme ß-lactamase.

For E.coli:
Neu and Heppel. 1965. J.Biol.Chem. 240:3685; Benz et al. 1978 J. Bact. 135:1080

1. Steps 1 and 2 as above.
2. Resuspend pellet in 8 ml of 20% w/v sucrose, 0.03 M Tris-HCl pH 8.0. Add 2 ml of 5 mM sodium EDTA (pH 8.0). Mix on rotating shaker at 180 rpm for 10 minutes at room temperature.
3. Pellet cells at 4C for 10 minutes at 10,500 rpm in an SS34 Sorvall rotor.
4. Resuspend pellet in 10 ml of ice-cold water and mix as before in ice bath for 10 minutes.
5. repeat step 4.
6. See steps 8-10 above.

References:

• Hancock & Wong. AAC. Vol.26, No.1:48 – 52. July, 1984.
• Loh et. al. AAC. Vol. 26, No. 4:546 – 551. October, 1984.
• Hancock et. al. AAC. Vol. 35, No. 7:1309 – 1314. July, 1991.

Materials:

Buffer:

• 5 mM sodium Hepes pH7.2
• 5 uM CCCP (better) or 5mM KCN
• 5 mM glucose

Note: Filter sterilize buffer if desired but DO NOT AUTOCLAVE any of its components except dH₂O. If using CCCP (which is unstable and light sensitive) make only enough working solution for that day. Solubilize stock solution in EtOH and store at -20^oC in the dark.

1-N-phenylnapthylamine (NPN ):

• Make 5 mM stock solution = 10.95 mg in 10 ml acetone. Dilute 1/10 to make a 0.5 mM working solution. Store both stock and working solutions at -20^oC between experiments.

NaCl & MgCl₂ (may not need):

• Make serial dilutions over the range you wish to test. MgCl₂: make 300 mM, 100 mM, 30 mM & 10 mM (use 1/100). NaCl: make 3 M, 1.5 M, .75 M (use 1/10)

Antibiotics:

• Make up solutions in buffer or distilled water. Make serial dilutions 100X the desired final concentration so you are always adding a constant volume to the cuvette,
• i.e. Polymyxin B;
• adding 10 µl to a 1 ml cell solution
• make 0.64 mg/ml –> final concentration 6.4 µg/ml
• dilute 1/2: 0.32 mg/ml –> final concentration 3.2 µg/ml
• dilute 1/2: 0.16 mg/ml –> final concentration 1.6 µg/ml
• Each antibiotic is used over a different range

Preparing Cells:

A. Use 1 ml of overnight culture to innoculate 50 mls of media. Incubate 37^oC, shaking. Grow to OD₆₀₀ = 0.4 – 0.6.B. Spin down cells: Sorvall SS34, Room Temp., 10,000 rpm, 5 min. or Silencer, Room Temp., 3500 rpm, 10 min.

C. Wash 1X in buffer. (Turn on fluorescent spec. now)

D. Resuspend in buffer to OD₆₀₀ = 0.5. Record OD₆₀₀.

Note: Don’t grow up large batches of cells; they are not stable enough to assay all day. It is better to grow another batch while you are assaying the first ones if you need a lot of cells. This avoids getting leaky cells which results in high background fluorescence of NPN only

Note: For Pseudomonas: keep at room temp. DO NOT SPIN AT 4ºC. Use the silencer in room 235A if the Sorvall is too cold. (This also helps prevent leaky cells.)

Using the Fluorescent Spectrophotometer (Perkin-Elmer 650 – 105):

1. ON POWER SUPPLY: Switch power to on. Count to 10. Press Lamp button. Check if lamp is on by holding white piece of paper in front of the beam inside the spec. Make sure the side ‘door’ is pushed in (therefore open).
2. Set excitation wave length to 350. Set emission wave length to 420. Set both slit widths to 5. sett the range to 1. Turn the spec. on, the chart recorder on and switch the multimeter to “DCV2”.

Check the other settings which should be:

To “Standardize Assay”:

1. Add 1.0 ml cell solution to cuvette. Place inside spec.. Open the side door (i.e. push in). Using the zero,adjust and zero suppression on spec. (adjust the knobs till the multimeter reads 0.0) Now adjust with the ‘zero’ knob on the chart recorder till the pen is at the baseline (or + 5 – 10% if you prefer)
2. Add 20 ul of 0.5 mM NPN (final conc. = 10 uM). Top cuvette with parafilm and shake. Place in spec.; measure for 2 – 5 seconds.
3. Remove cuvette and add 10 µl of 0.64 mg/ml PxB. Shake; place in spec. Using the range and sensitivity knobs, adjust until the pen is at 90% full deflection.
4. Repeat steps 1 – 3 until you have PxB 6.4 µg/ml giving 90% deflection and you have zeroed properly after changing the range &/or sensitivity.

Now you are ready to start! Try adding solutions and returning the cuvette quickly.

The Assay:

1. Add 1 ml of cells (OD₆₀₀ = 0.5) to cuvette. Measure 2 – 5 seconds.
2. Add 20 ul NPN 0.5 mM (shake to mix). Measure 2 – 5 seconds.
3. Add 10 ul antibiotic 100X desired final concentration (shake to mix). Measure till max. level reached (1 – 5 min.). If testing effect of NaCl or MgCl₂, add each in between NPN and antibiotic.
4. Don’t assay more than 2 hours – the cells don’t work as well as time goes on.

Be sure to assay controls:

1. cells only
2. cells + NPN only (no antibiotic)
3. cells + NPN + antibiotic solvent
4. NPN in buffer only (no cells, no antibiotic)
5. NPN in buffer only + antibiotic (no cells)

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).