An improved microelectrophoresis apparatus and technique for studying biological cell surfaces

,\NALYTI(:41,

HIOCII~:MISTRY

An Improved Technique

1,

475485

(1960)

Microelectrophoresis for Studying Biological G. J. GITTENS

From

the

AND

Apparatus and Cell Surfaces

A. M. JAMES

Department

of Chemistry, Chelsea College Technology, London, England

Received August

of Science

and

17, 1960

At the present time the biological cell surface is the subject of extensive investigation. Physical, rather than chemical, methods are preferred for characterizing the nature of components at such surfaces, since the manipulation of the cells and hence cellular disorganization is minimized. Microelectrophoresis is an important technique with diagnostic (1, 2) as well as research uses. Many different types of microelectrophoresis apparatus and techniques for using them have been described (3-5) ; all suffer from one or more of the following disadvantages: failure to conform to the theoretical requirements, inadequacy of temperature control, difficulty of construction and manipulation. In the apparatus described here based essentially on previous types of apparatus (6, 7)) these disadvant,ages, and in consequence errors arising from them, have been eliminated. The only drawback remaining, which is common to all fused rect.angular observation chambers, is that the apparatus requires calibration with particles of known electrophoretic mobility; this disadvantage is offset by ease of use. The apparatus has been extensively tested by statistically designed experiments, and a standard procedure has been established. Using these conditions, the results obtained day to day over a period of weeks are reproducible to -t370. METHODS Apparatus (a) Cell and Electrode Compartmen.ts. The cell (Fig. l), used in the horizontal position (8), is constructed by fusing two optically flat Hysil glass plates (40 X 25 x 0.5 mm thick) round the edgesusing spacers to give a cell depth of 0.5mm. Spherical joints sealed to the cell arms and the electrode compartments simplify assembly and mounting. The rlec475

MICROELECTROPHORESIS

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479

trode compartments are constructed by sealing side arms, carrying taps of vacuum quality, onto a lo-mm Pyrex tube containing the sintered-glass disk (No. 2) as close as possible to the disks. This prevents the accumulation of pockets of liquid which cannot be flushed out. (The glassware can be obtained from Linskey Bros., 72, Holloway Road, London N.7). (b) Electrodes. These are made of pure silver foil (8 X 3 X 0.015 cm) rolled into a cylinder and welded to 3 cm of 0.05 cm diameter platinum wire. The wire is sealed into a length of bent glass tubing, filled with

Ag.AgCI

electrode

strengthening struts

FIG.

1. The

microelectrophoresis

npparatus.

mercury for electrical connection, which carries a rubber bung to fit the electrode compartment (Fig. 1). The electrodes and platinum wire are cleaned with acid, silver plated, and then anodized at a current density of 1 ma/sq cm for 1 hr. The electrodes are always kept in potassium chloride solution (3.5 M). (c) Electric Circuit. The best one is that proposed by Lerche (9) (Fig. 2), supplied by two 90-v H. T. batteries. r1 and rZ are wire-wound potentiometers each of 50,000 ohms dimensioned for 10 w. The current passing through the microelectrophoresis cell is set with rs, and with r1 it can be kept constant during observation. The current is measured with a multirange milliameter (e.g., Sangamo-Weston). 8, is a shorting switch for the electrodes, & the battery switch, and S, a reversing switch. (d) Microscope cmd Stage. Any microscope in which the focusing is by movement of the objective is suitable. It should be fixed securely to a firm bench. A x 40 phase-contrast objective (N.A. 0.731 and an angled

480

tiITTENS

AND

JAMES

eyepiece of 1.5 magnification carrying a X 10 focusing eyepiece containing a cross-hatch graticule are suitable for observing bacteria. The phasecontrast condenser and object.ive lenses are waterproofed. A high-intensity microscope lamp provides t(hc illumination. The normal microscope stage is replaced by a thick brass plate (n, Figs. 3 and 4) permitting the subst,age condenser b to pass up into the thermostat tank c. Adjustable mounting clamps d, made from two brass plates separated by four adjustable bolts, are bolted to the stage for holding the cell arms. These are clamped to the top plates of each using a Perspex strip and foam rubber pads to protect the glass. The electrode compartments are held on to t.his assembly by clips e and suitably supported on the bench f, The condenser pushes up into the thermostat c through a tight-fitting hole in a rubber sheet fixed to the bottom of the tank.

FIG,

2. The

electric

circuit.

(e) Thermostat Tank. The depth of the large tank g constructed from 1.5 cm thick Perspex, depends upon the level of t,he microscope stage. The tank, c, constructed from 0.75 cm thick Perspex, is joined at one corner to the tank g and connected by a Polythene tube to the circulating pump h of the temperature control unit (e.g., Shandon’s Circotherm II Constant Temperature Unit). Thus a constant flow of water throughout the apparatus maintains any required temperature within kO.02W. Both tanks are covered and the reading thermometer is inserted through the lid of c close to the cell. (f) Additional Apparatus. A conductance bridge (c.g., Wayne-Kerr Magic Eye bridge) and conductivity cell of known constant are required to measure the conductance of the suspension for the calculation of the field strength. For multitemperature work the conductivity cell must be calibrated at the working temperature. Results indicate that the cell constant may not vary linearly with temperature as widely believed. (g) Assembly and Preparation. The graduated fine-adjustment screw of the microscope is calibrated over its complete range with a coverslip of known thickness; the most constant region is selected for subsequent use. To aid cell depth determinations, bacteria are deposited on the upper and lower inside surfaces of the cell by introducing a clilutc alcoholic bacterial suspension and drying out.

MICROELECTROPHORESIS

FIG.

3. The

FIG.

complete

4. The

microele~tIophoreeis

microelectrophoresis

481

APPARATUS

assembly.

cell.

482

GITTENS

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JAMES

When all the joints have been greased, the apparatlls is assembled and mounted in the tank. The electrode compartments are washed and finally filled with pot,assium chloride solution, and the ccl1 with distilled water. The condenser is raised as close to the cell as possible and, with the objective focused into the cell, the phase contrast is adjusted. Operational

P,rocedwe

This procedure, established after consideration of results to be described, should be followed for each electrophoretic measurement on a bacterial suspension to ensure reproducibility of results. 1. Check the temperature of the thermostat and adjust the regulator if necessary. 2. Close S, for 1 min to eliminate electrode polarization. 3. Flush through each electrode compartment with 50 ml of 3.5 M potassium chloride solution. With taps 1, 3, 3’ open and taps l’, 2, 2’ closed, allow a small amount of potassium chloride solution to pass through t,he sintered disks into the cell compartment. Finally pass another 25 ml potassium chloride solution through the electrode compartments and close all taps. 4. Thoroughly wash the observation cell and connections with glassdistilled water until all electrolytes have been removed. 5. Flush the experimental suspension (previously thermostatted at the working temperature) through the apparatus, and close taps 1, l’, thereby trapping a sample. 6. Determine the cell depth in arbitary units (using the fine adjustment over the region selected) by focusing on bacteria adhering to the t.op and bottom inside surfaces of the observation chamber. Always come up to focus from below to avoid backlash in the fine adjustment screw. Hence focus on the upper stationary level (4). 7. Check that the bacteria do not move systematically due to badfitting joints or incomplete temperature equilibrium. Switch on the current and adjust it to give a suitable excursion time (e.g., 5 set) of the bacteria. Select a cell in focus and record the time across a fixed distance in the eyepiece graticule, using a stopwatch accurate to 0.02 sec. Reverse the polarity and time a bacterium in the opposite direction. Repeat for 30 cells. On no account should the current be left flowing in one direction for more than 30 sec. The value of the current in either direction and throughout a set of readings should be kept constant with rl. 8. Wash out the cell and leave filled with distilled water with tap 1 open when not in use. 9. Determine the conductivity of the cell suspension at the working temperature.

MICROELECTROPHORESIS

483

APPARATUS

Calculation

The electrophoretic mobility Ge (cm/sec/v/cmj i.e., velocity field strength, at temperature 0 can be shown (9) to be:

per unit

where t (see) is the time to cover 1 (cm), Q (sq cm) cross-sectional area of the cell, K, (mhos/cm) the conductivity of the suspension and I (amp) the current. It is difficult to determine the cross-sectional area of fused rectangular cells; thus I and q are combined as a constant A and determined using standard particles, such as human erythrocytes in M/15 phosphate buffer solution at pH 7.35 when V,, = 1.31 k 0.02 p/set/v/cm (IO). Thus

RESULTS

AND

DISCUSSIOK

All results were obtained on overnight culture of Aerobacter aerogenes grown in synthetic medium (11). Cells were harvested, washed three times, and resuspended in phosphate buffer solution, pH 7.0, I = 0.0123. Measurements were made at 25OC. Velocity

Depth

Curves

The velocity of bacteria was determined at different depths in the cell at constant field strength. The equation to the velocity depth curve through the experimental points was calculated by the method of least squares, and the true electrophoretic velocity of the bacteria was determined by integration of this equation. The position of the stationary levels was obtained by substituting the velocity into t,he original equation and solving. This method is applicable to cells in which the ratio of width to depth is greater than 20 when the theoretical positions of the stationary levels are at 0.21 and 0.79 of the total cell depth (12). The positions of the stationary levels determined over a period of 6 months, on one cell, are list,ed as fractional depths from the lower inside surface of the ccl1 (Table 1). TABLE

1

THE POSITION OF TH,E STATIONARY LEVEIA Lower: Upper:

0.240 0.790

0.220 0.790

0.246 0.792

0.280 0.787

0.254 0.790

0.214 0.789

0.234 0.790

0.241 0.791

0.247 0.792

. 0,252 0.790

0.227 0.791

0.255 0.792

484

GITTENS

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JAMES

If the cell was geometrically asymmetrical, it would be expected that the upper and lower stationary levels, while not necessarily agreeing with the theoretical values, would remain fixed. The position of the upper stationary level is constant and in excellent agreement with theory. The lower stationary level, however, shifts erratically even over such a short period of time as 30 min ; this is most probably due to a change in the nature of the lower cell surface and the concomitant change of the electroosmotic velocity (13, 14). This can be accounted for by bacteria and bacterial debris depositing and adhering to this surface preferentially, the nature of which will thus depend on the extent of washing. Cleaning the cell with acid results in a marked shift of the Iower stationary level, but such a method cannot be used routinely as the debris rapidly reaccumulates. All subsequent measurements were made at t,he upper st,ationary level only. Statistical

Analysis

of Variables

Statistically designed experiments were carried out to observe any differences between mobility values of a suspension when determined by two observers over different distances, in both directions at the two stationary levels for different applied field strengths. The following are the conclusions: 1. There is a significant difference in measurements made between observers; thus all measurements should be taken by one observer. 2. The mobility value was independent of the distance over which timings were made. 3. There was a significant difference in one experiment between results obtained in both directions. This was traced to electrode polarization; after the introduction and use of the shorting switch S, no significant difference was found. 4. There was a significant difference between results obtained at the two theoretical stationary levels in confirmation of the previous results. 5. No significant difference between values at different field strengths, i.e., currents, was observed. Reprod,ucibility

of Results

A typical analysis of a group of 30 timings gives a mean of 6.04 set, standard deviation of 0.430 WC and a standard error of 0.079 sec. Analysis of timings obtained on t,he same and different days showed that the confidence limit for a single mean (15) at p = 0.05 is +-3% SUMMARY

A modified form of microelect.rophoresis apparat,us and technique for studying biological cell surfaces is described. The design of apparatus has

MICROELECTROPHORESI8

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485

been simplified thereby increasing the ease of assembly and manipulation. This is the first description of a microelectrophoresis cell using a rectangular observation chamber, conforming to the theoretical requirements in which the temperature can be accurately controlled. It thus makes an advance in design for it permits experimental verification of the thcorctical mobility t,emperature relation. The apparatus has been extensively tested by statistically designed experiments. These revealed t.he main sources of errors, which have been erradicated by modifications and the est,ablishment of a standard experimental procedure. Using this procedure, results obtained day to day and over a period of weeks are reproducible to k376, ACKNOWLEDGMENTS

The authors are grateful to Dr. G. A. Maccacaro (Instituto di Microbiologica, Milan) for valuable assistance in the application of statist.ical methods. One of us (G. J. G.) gratefully acknowledges financial support by the Department of Scientific and Industrial Resenrrl

1.

DAVIES,

2. GORDON, 3. THOMPSON,

REFERENCES D. F., Clint. Sci. 17, 563 (1958). M. J., Genetics 43, 913 (1957). M. R., Trims.

Electrochem.

Sot. 81, 147 (1942).

A. M., Progress in Biophys. and Biophys. Chem. 8,96 (1957). C. C., ASD LAUFFER, M. A., in “Electrophoresis” (Bier, ed.), p. 42i. Academic Press, New York, 1959. 6. MOYER, L. S., J. Bactetiol. 31, 531 (1936). 7. J.4MES, 8. M., .~ND LovEDaY, D. E. E., Chem. Prods. 21, 237 (1958). 8. HARTMAN, R. S., BATEMAN, J. B., AND LAUFFER, M. A., ATCIL. Biochem. Biophys. 39, 56 (1952). 9. LERCHE, C., Actn. Pnthol. Mimobiol. Scnnd. Suppl 98, 727 (1953). 10. HEARD, D. H., ASD SE.~MAX, G. V. F., J. Gen. Physiol. 43,635 (1960). 11. LOWICK, J. H. B., AND JAMES, A. M., Biochim. et Biophys. Acta 17, 424 (1955). 12. ABRAMSON, H. A., MOYER, I,. S.. AND GORIS, M. H., “Electrophoresis of Proteins,” pp. 48-52. Rheinhold Publ. Corp., Xcw York, 1942. 13. WHITE, P., Phil. Msg. 23, 811 (1937). 14. L.~NE, T. B., WHITE, P., Phil. Msg. 23,824 (1937). 15. BROWKLEE, K. A., “Indust,rial Experimentation,” p. 33. Her Majesty’s Stationery Offiw, London, 1957. 4. JAILS, 5. BRINTOP;,