Archives: Methods

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 m m 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 m M = 1.25 m l of source) in a 10 ml diposable tube.
5. Warm bacterial suspension approximately 10 minutes at 37^oC 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 m l 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 moinimal 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 mls of scintillation cocktail.
7. Duplicate experiments should be done on two different days to verify results.
8. Controls: 200 m l of Hepes buffered media alone to get background counts and 1.25 m l of ³²P in 1 ml buffer, no cells, to get maximum counts.

Stock Solution:

0.001 M Phosphate Solution

use 100, 200, 300, 400, 500 µl and make up each sample to 500 µl final volume.

Reagents:

1. 10% Magnesium Nitrate in ethanol.
2. 1 M HCl.
3. 10% Ascorbic Acid (1 gm/10 ml).
4. Ammonium Molybdate, .42% in 1 N Sulphuric Acid

(.42 gm + 2.66 ml c/H₂SO₄ made up into 100 ml)

Method:

1. Weigh 0.5 mg LPS into Pyrex tube.
2. Add 0.05 ml 10% Magnesium Nitrate in ethanol.
3. Evaporate to dryness on flame.
4. Cool. Add 0.3 ml 1 N HCl. Heat at 100^oC for 15 min. in a boiling water bath.
5. Add 0.7 ml Ascorbic/Molybdate (1 part to 6 parts freshly mixed).
6. Keep at 45^oC for 20 min.
7. Read absorbance at 820 nm against reagent blank. (absorbence of 0.240 for 0.01 µmol phosphate).

* Ascorbic Acid is stable at 2 – 4^oC for approx. 7 weeks.

Reference:

Anal. Chem. 28:(1956) 350-356.

Objective:

To detect the presence of sugars. (Widely used method to monitor column eluates for elution of sugar containing compounds such as LPS, 0-antigen, etc.)

Materials:

• Standards:glucose 1 mg/ml stock solution
• use 10, 20, 40, 60, 80 µl and make up each sample with dH₂O to a final volume of 100 µl.
• Blank 100 µl water
• Samples:50 µl (of 5 mg/ml LPS solution) and make up to 100 µl.

Method:

1. Add 50 ul of 80% Phenol solution (80% Phenol by weight)
2. Vortex. Add 2.0 ml concentrated Sulphuric Acid in a stream.
3. Stand 10 min. at room temperature.
4. Read absorbance an 490nm.

(Purpose to assay muramic acid, a component of peptidoglycan)

Stock Solutions:

• 1.5% (w/v) p-hydroxydiphenyl in 96% ETOH (PHD)
• 1.0 M NaOH
• concentrated H₂SO₄
• 0.5 M H₂SO₄
• 4% (w/v) CuSO₄-5H₂O in H₂O

Procedure:

1. Bring sample to 0.5 ml with 1.0 M NaOH.
2. Incubate at 38^oC for 30 min.
3. Add 0.5 ml of 0.5 M H₂SO₄ + 5 ml conc. H₂SO₄, stopper tube well and place in boiling H₂O bath for 5 min.
4. Cool and add 0.05 ml CuSO₄ + 0.1 ml PHD.
5. Stopper tube and incubate at 30^oC for 30 min.
6. Read absorbance at 560 nm.

Standard curve:

0 – 20 µg muramic acid.

1. Place sample (containing 0.5 – 10 µg GlcN) in a Pyrex screw capped tube.
2. Add HCl to a final concentration of 2N and a final volume of 0.6 ml. Flush with nitrogen.
3. Stopper tightly and hydrolyze 16h at 100^oC.
4. Prepare standards containing 0 – 20 µg GlcN from a stock solution of 1.5 mg/ml. (Use 0, 3, 5, 8, 10, 12 ml of stock solution and add water to make up to 300 µl. Then add 300 µl 4N HCl to a final volume of 0.6 ml and final concentration of 2N). Standards need not be heated.
5. Neutralize samples and standards with 0.4 ml 2M Na₂CO₃ (10.6 g in 50 ml). (pH – 7 with the slight excess of Na₂CO₃)
6. Shake gently and add 0.5 ml (freshly prepared) 2% acetyl acetone in 1.5M Na₂CO₃. (15.9 g Sodium Carbonate + 2 ml acetyl acetone made up to 100 ml).
7. Stopper tightly and heat in boiling water bath for 20 min.
8. Cool. Add 1 ml EtOH.
9. Add 0.5 ml Ehrlichs reagent. (1 g p-dimethylaminobenzaldehyde in 15 ml EtOH and 15 ml c/HCl)
10. Shake tubes vigorously to expel excess CO₂.
11. Maximum colour development is reached in 5 min. Chromophore stable for 1 to 2 hours.
12. Read absorbance at 530 nm.

NOTE: NaCl affects colour. Therefore standards and sample should contain the same amount of salt to avoid colour depression and erroneous results. Dialysis helps to remove NaCl.

All dehydrogenase assays are preformed in the same way, although the electron donor (eg., succinate), the electron acceptor(s) and the final chromagenic substrate (eg., DCIP) must be varied according to the energy state of the dehydrogenase. Dehydrogenase enzyme assays are used by us as markers of the inner membrane.

Method:

1. Add the following to sample to be tested and incubate at 25^oC for six minutes. You will probably need to test a range of sample concentrations, but just do one at a time.
   

   2 ml 0.1M Tris-HCl, pH 8.0

   0.1 ml 0.2M KCN (13 mg/ml), freshly prepared

   0.1 ml 0.6M sodium succinate (162 mg/ml)

   10 µl enzyme or membrane preparation

   0.64 ml distilled water

2. Add 0.1 ml of freshly prepared 12.5mM phenazine methosulphonate (3.83mg/ml) and 0.05ml freshly prepared 2.5mM dichloroindole phenol (0.73mg/ml, DCIP) and mix briefly.
3. Measure the change in OD₆₀₀ using the spectrophotometer hooked up to the chart recorder. The blank contains no enzyme or membrane preparation.
4. Calculate the amount of enzyme using the appropriate molar extinction coefficient of the final elector acceptor (DCIP) reduction. For DCIP, this equals 22,000 l/mole.
5. Express the enzyme activity as umoles DCIP reduced/minutes/milligram of protein.

Reference:

Wright and Rebers, Anal. Biochem. 49: 307-319, 1972.

Objective:

To determine the relative amounts of LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars.

Methods:

Hexose Assay:

(sugars in the outer core and O-Ag)

1. Cool 200 µl of sample in an ice bath.
2. Add 900 µl reagent A (4 ml dH₂0 + 24 ml conc H₂SO₄). Shake carefully. Warm to room temperature (23ºC).
3. Boil in water bath for exactly 20 min.
4. Cool to room temperature under cold water tap.
5. Add 20 µl reagent B ( 0.3 g cysteine HCl in 10 mls dH₂O (fresh)). Shake and then let stand 30 min. in the dark at room temperature.
6. Assay the difference between 415 and 380 nm.
7. To zero the spec use all above ingredients except the bacterial sample. Standards are made of 1,2,3,4, and 5 µl of desired hexose sugar (eg. glucose) at 2 µg/ml.
8. To assay all three kinds of sugars do a wave length scan from 350 – 600 nm.

6-Deoxyhexose Assay: (outer core only)

Examples are fucose and rhamnose. The assay is the same for the hexoses but it is read at 396 and 427 nm. To make this assay more specific for 6-deoxyhexoses use the following modifications:

1. In step 3 above, boil for 5 min.
2. In step 6 above, let stand for 3 hours in the dark (this produces a green-yellow colour rather than pink)
3. Standards are the same as above using rhamnose.

Heptose Assay: (inner core only)

Same as above but uses the difference between 500 and 545 nm.

1. Grow bacteria overnight in Pi sufficient and Pi deficient media.
2. Read OD₆₀₀ of overnight culture. One OD₆₀₀ unit = 0.3 mg protein.
3. Pellet 1 ml of culture in microfuge (5 min). Save supernatant.
4. Add 500 µl of resulting supernatant to 500 µl of alkaline phosphatase substrate or phospholipase C substrate.
   

   Alakaline phosphatase substrate:

   • 2 mg/ml of p-nitrophenyl phosphate
   • One Sigma 104 tablet (5mg) per 2.5 ml of 0.1 M Tris-HCl, pH 8.5

   Phospholipase C substrate:

   • 6 mg/ml nitro-phenylphosphorylcholine
   • 0.25 M Tris-HCl pH 7.3
   • 60% (v/v) glycerol
5. Follow the A₄₁₀ “absorbance change” using the Perkin-Elmer spectrophotometer and attached chart recorder. 1-2 min is enough.
6. Let one sample hydrolyse the substrate to completion (i.e. no more change in absorbance) then read the A₄₁₀. This is the A₄₁₀ FINAL which is used to calculate the extinction coefficient.

Calculations:

One OD₆₀₀ culture = 0.3 mg/ml proteinMW Phospholipase C substrate = 304.2; 3 mg/ml final concentration = 9.9 umoles/ml

MW Alkaline phosphate substrate = 263.1;
1 mg/ml final concentration = 3.8 umoles/ml

Extinction coefficient = umoles substrate hydrolyzed/A₄₁₀ FINAL
(e.g. for APase: Ext. coef. = 3.8 umoles/A₄₁₀ FINAL)

Enzyme Activity = A₄₁₀ “absorbance change”/min x Extinction coefficient/mg protein = umoles substrate hydrolyzed/min./mg protein.

Purpose:

Can be used to test for the induction of the pho-regulon by monitoring the time of appearance of alkaline phosphatase in the culture medium. Although this assay could theoretically be used as a measure of outer membrane permeability, linearity of the initial rate of hydrolysis can not be obtained.

Materials:

The substate is 1 para-nitrophenyl phosphate (PNPP) tablet in 5ml of 0.1M Tris-HCl, pH 8.0 as a stock solution.

Methods:

Put 500µl of the substrate solution plus 500µl of the cells to be tested in a cuvette, mix, and record the hydrolysis at OD₄₀₅ on a strip chart recorder. The rate of hydrolysis is proportional to the amount of enzyme which has been synthesized and released from the cells.

• Position press plunger to the desired volumn marker.
• Invert the press onto the stand.
• Add the cells. Make sure the level is just below that of the outlet valve, otherwise move the plunger.
• Ensure the side valve is open.
• Insert the cap all the way.
• Close the side valve until finger tight.
• Place the cell plunger side-up into the press and lock into place.
• Speed: fast. Direction: up. Pump: on.
• When the pressure indicator moves, reduce the speed quickly to ensure a pressure of 15,000 psi with the large cell and 200 psi with the small one.
• Open the side valve gently until the cells leak out at 11,500 to 13,5000psi. NB: Hold the outlet tube against the wall of the sample container to prevent splashing. (A convenient sample container to use is a 50/12 ml sterile Falcon tube with a slash in the cap for insertion of the outlet tube.)
• When the run is completed open the side valve quickly to avoid spurting.
• Lower the press and repeat the breakage.
• Clean the press with distilled water and ethanol, then with ethanol and distilled water.