Quantitative measurements of zeta-potentials have been made. (Joffe, Hitchcock and Mudd, 1933). A most striking observation

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1 VARIATIONS IN THE ELECTROPHORETIC MOBILITIES OF THE BRUCELLA GROUPS DOROTHEA E. SMITH AND ELEANORE W. JOFFE Department of Bacteriology, School of Medicine, University of Pennsylvania, Philadelphia, Pa. Received for publication November 5, 1933 The fact that serological methods fail to furnish any sharp, decisive tests in the differentiation of Brucella strains and that such differentiation now depends on the lesions produced in guinea pigs, the dye and the H2S test, has made it desirable that another distinguishing character be sought. It was thought that a differentiation might be effected by measuring the zetapotential or electro-kinetic p.d. resident upon the bacterial surface. Hence the following work was carried out. Dr. Theobald Smith kindly supplied us with strains which he had classified as bovine, porcine and caprine. We have retained his designations. Quantitative measurements of zeta-potentials have been made on rough and smooth acid-fast bacilli (Kahn and Schwarzkopf, 1931), and rough and smooth intestinal non-flagellate bacteria (Joffe, Hitchcock and Mudd, 1933). A most striking observation was made in the latter group; certain smooth intestinal organisms possess very little if any zeta-potential whereas the rough variants have high zeta-potentials. It was thought, therefore, that some definite relationship between the virulence and cultural differences on the one hand, and the zeta-potential on the other, might exist. METHODS The Kunitz (1928) modifications of the Northrop-Kunitz microcataphoresis cell was used with a dark field condenser and Bausch and Lomb 8 mm., 0 n.a., 21 X objective. Eight readings were 127 JOURNAL OF I;ACrERIOI,OGY, VOL. 28, NO. 2

2 128 DOROTHEA E. SMITH AND ELEANORE W. JOFFE taken for each suspension at the two stationary levels, i.e and 9 of the inside depth of the cell. Radio B batteries were used. The applied potential of 125 volts gave a gradient through the cell of about 6.5 volts. Electrophoretic mobilities are given in u per second per volt per centimeter fall in potential along the cell. The electrokinetic p.d. may be calculated from the electrophoretic mobility by multiplying by 12.6, if one assumes the dielectric constant and the viscosity of the suspending medium to be the same as those of pure water at 20 C. Care was taken to measure only those particles which were sharply in focus. Every attempt was made to avoid personal or systematic error. The cell was tested at intervals for symmetry with a suspension of relatively constant electrophoretic mobility. Bacterial preparations The organisms were grown on infusion agar slants at ph = 6.8; the cotton plug was burned and a little paraffin was added to insure optimal microaerophilic growth conditions. Loopsful of the growth were rubbed up in 2 or 5 ml. of saline, depending on the amount required for dispersion. The condensation water was carefully avoided (Smith, 1931) to minimize clumping., The organisms were washed twice in NaCl, and once in Walpole's sodium acetate-acetic acid buffer, ph = 5.2, at fiftieth molar concentration. After centrifuging, this first buffer wash-wvaterwas removed, and the bacteria were resuspended in the acetate buffer. The electrophoretic mobility was measured in this same solution. Glassware was chemically cleaned with concentrated sulfuric acid potassium dichromate cleaning solution, tap water and distilled water. RESULTS AND DISCUSSION Results are given in table 1. Measurements of the electrophoretic mobility revealed that except in a few instances, individ- ' Spontaneous agglutination occurs in certain strains of the Brucella group under certain conditions. Human 2a, swine 6a and swine 14, clumped wheni placed in m/50 acetate buffer. Swine 14 gave the appearance of precipitated or flocculated protein material.

3 TABLE 1 Electrophoretic mobility in.aper second per volt per centimeter SOURCE Human... Goat... Swine... Cow...1. liorse...{..1 NUMBER f. 1.I I 12 2a* 6 30a a 14t 6at b 515a Spain (2) RACE IDENTIFIED AS (Danish) (Danish) (Danish) 3? 3? AVERAGE MAXIMUM > 1.3 * Spontaneous agglutination. Shaken to homogenous suspension. t Clumped macroscopically. + Clumped macroscopically, giving loose floccular appearance. MINIMUM 1.7 () Give no H2S reaction, unlike our porcine strains. The figures given are the electrophoretic mobilities in micra per second per volt per centimeter. See text for further explanation. 1

4 130 I)OROTHEA E. SMIThI AND ELEANORE W. JOFFE ual bacteria did not have the same or nearly the same mobility, but varied over a wide range. Consequently a report of the mean electrophoretic mobility of a number of readings, in this instance, has little biological significance. It represents a statistical mean of a sample which may or may not be a true random sample. For this reason the inaximum and the minimum mobility are reported for each culture, and the variability may be judged from these figures. Determinations from several subcultures of the same strain occasionally differed. In order to obtain more constant results, strains Br. abortzts 1798, 1534 and 1465 were passed through guinea pigs, recovered from the spleen and plated. These presumably smooth strains varied as much after, as before, animal passage. Other thain this, there was no attempt to eliminate dissociated forms if present. Such variability of individuals within a single culture has not been found by one of us in numerous observations within the colon-typhoid group, using the same apparatus and equal care in washing. The values for the electrophoretic mobility obtained may eventually be correlated with the recent work of Wilson and Miles (1932). They conclude that the antigens present in abortus and melitensis strains are similar, but quantitatively different. There is more of the "a" antigen in the abortus than the "m" antigen, and more of the "m" than "a" in the melitensis strains. Such antigenic differences might possibly account for differences in the electrokinetic properties of the bacterial surfaces of closely related strains. At present it is impossible to say that there is a constant relation between electrokinetic p.d. and virulence or other characteristics in the Brucella group. Since it has been shown in this laboratory that the electrokinetic p.d. of certain rough strains in the intestinal group (colon-typhoid-dysentery) is very much greater than that of the smooth, one might suspect that the cultures were mixed rough and smooth. According to Wilson and Miles, the melitensis strains are more often rough than the abortus strains.2 There were no very recently isolated melitensis 2 Dr. Smith tells us that roughness as exhibited by spontaneous clumping is relatively rare in the genuine bovine types.

5 VARIATIONS IN ELECTROPHORETIC MOBILITIES strains available for examination. This problem awaits further work on antigenic structure and spontaneous dissociation in this group. It may be that a study of the electrophoretic mobility of strains dissociated into definite and stable variants, or of strains derived from single cells would yield results of theoretical and practical significance. Since the purpose of the authors was to characterize further and more definitely the strains as found ordinarily in the laboratory this has not been done. SUMMARY AND CONCLUSIONS The electrophoretic mobilities of certain strains of the Brucella group have been measured. The great variability within a single culture and the overlap of the groups prevents the establishment of any constant relationship between virulence or cultural characteristics and zeta-potentials. On account of the overlap of these groups it is impossible to refer an unknown strain definitely to one group. In general, the strains of the bovine group exhibit the lowest electrophoretic mobility, and some of the caprine strains from human sources the highest. The porcine strains show an intermediate mobility. Whether there is any difference in the electrophoretic mobility of dissociated strains, as is the case in the colontyphoid group, we hope to determine shortly. REFERENCES BROWN, H. C., AND BROOM, J. C Brit. Jour. Exper. Path., 13, 334. JOFFE, E. W., HITCHCOCK, C. H., AND MUDD, STUART Jour. Bacteriol., 25, 24. KAHN, M. E., AND SCHWARZKOPF, H Amer. Rev. Tuberc., 23, 45. KUNITZ, M Coll. Symp., p. 134, footnote. LISBONNE AND SEIGNEURIN 1933 Compt. Rend. Sea. Ac. Sci., 196, MUDD, S., NUGENT, R. L., BULLOCK, L. T Jour. Phys. Chem., 36, 2. SMITH, T Science, 74, 21. WILSON, G. S., AND MILES, A. A Brit. Jour. Exper. Path., 13,