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Inhibition of cell division in escherichia coli exposed to cis-platinum(II)diamminodichloride
Micron, 1978, VoI.: 9, pp. 45-46. :t~ Pergamon Press Ltd. Printed in Great Britain
0047-7206/78/0301-0045 $02.00/0
I N H I B I T I O N OF CELL DIVISION IN ESCHERICHIA COLI EXPOSED TO
CIS-PLATINUM(II)DIAMMINODICHLORIDE* DOROTHY A. GINGRICH and DORIS J. BECK Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio
The antitumor agent cis-platinum(lI)diamminodichloride (PDD) inhibits cell division in gram negative bacteria (Rosenberg et al., 1967). Filamentation of Escherichia coli in the presence of P D D and resumption of division by elongated cells after removal of P D D from medium were studied using scanning electron microscopy (SEM). A mutant lacking polymerase 1 and therefore deficient in D N A repair, E. coli P3478 (polA), and the wild-type isogenic parent strain, W3110 (pol÷), were utilized. Bacteria were grown for 3hr at 37°C with aeration in 10 pg P D D per ml tryptone-E broth (Beck and Brubaker, 1973). To remove P D D the cells were collected onto a millipore filter (pore size, 0.45 lam), washed with and suspended in fresh broth. The cultures were incubated at 37°C and 0.5 ml samples collected onto a nucleopore filter (0.6 lam pore size). Samples were prepared for SEM as described by Crang and Ward (1975). A sandwich was made of two nucleopore filters enclosing cells which was supported between two metal washers and fastened by a paper clip. The cells enclosed in nucleopore filters were washed * This research was supported by a grant to D.J.B. from the Faculty Research Committee of Bowling Green State University.
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in 5 ml cold 0.25M Sorensen's phosphate buffer (pH 7.2) for 15min, fixed in 3 ~ glutaraldehyde in phosphate buffer at 4°C for 2hr, and washed twice with cold buffer for 15rain. The bacteria on filters were dehydrated using a graded acetone series. Cells were dried by the critical point method of Anderson (1951), and filters attached to stubs. The specimens were coated with approximately 20nm of gold on a glowdischarge apparatus. Observations and photo micrographs were made with a Hitachi H H S - 2 R scanning electron microscope at 20kV. Examination of scanning electron micrographs of P D D treated bacteria confirmed that wild-type cells and those having a defective polymerase 1 formed filaments when grown in the presence of P D D which divided upon removal of the drug.
REFERENCES Anderson, T. F., 1951. Transactions" N. Y. Acad. Sci., 13" 130. Beck, D. J. and Brubaker, R. R., 1973. J. Bacteriol., 116: 1247. Crang, R. E. and Ward, J. A., 1975. In: Electron Microscopy: Principles and Practices, W. B. Saunders Co., PA. Rosenberg, B., Renshaw, E., Van Camp, L., Hartwick, J. and Drobnik, J., 1967. J. Bacteriol., 93: 716.
46
I). A. Gingrich and I). J. Beck
Fig. 1 4 . Scanning electron micrographs of Escherichia coli ceils, (1) strain W3110 wild-type bacteria and (2) strain P3478 polA mutant, after 3hr growth in the presence of 10t~g PDD per ml medium. The platinum drug was removed by filtration and the bacteria after growth in fresh broth are shown in (3) strain W3110, 2hr growth period and (4) strain p3478 pol,4, 4hr growth period. 1500.
0047-7206/78/0301-0045 $02.00/0
I N H I B I T I O N OF CELL DIVISION IN ESCHERICHIA COLI EXPOSED TO
CIS-PLATINUM(II)DIAMMINODICHLORIDE* DOROTHY A. GINGRICH and DORIS J. BECK Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio
The antitumor agent cis-platinum(lI)diamminodichloride (PDD) inhibits cell division in gram negative bacteria (Rosenberg et al., 1967). Filamentation of Escherichia coli in the presence of P D D and resumption of division by elongated cells after removal of P D D from medium were studied using scanning electron microscopy (SEM). A mutant lacking polymerase 1 and therefore deficient in D N A repair, E. coli P3478 (polA), and the wild-type isogenic parent strain, W3110 (pol÷), were utilized. Bacteria were grown for 3hr at 37°C with aeration in 10 pg P D D per ml tryptone-E broth (Beck and Brubaker, 1973). To remove P D D the cells were collected onto a millipore filter (pore size, 0.45 lam), washed with and suspended in fresh broth. The cultures were incubated at 37°C and 0.5 ml samples collected onto a nucleopore filter (0.6 lam pore size). Samples were prepared for SEM as described by Crang and Ward (1975). A sandwich was made of two nucleopore filters enclosing cells which was supported between two metal washers and fastened by a paper clip. The cells enclosed in nucleopore filters were washed * This research was supported by a grant to D.J.B. from the Faculty Research Committee of Bowling Green State University.
45
in 5 ml cold 0.25M Sorensen's phosphate buffer (pH 7.2) for 15min, fixed in 3 ~ glutaraldehyde in phosphate buffer at 4°C for 2hr, and washed twice with cold buffer for 15rain. The bacteria on filters were dehydrated using a graded acetone series. Cells were dried by the critical point method of Anderson (1951), and filters attached to stubs. The specimens were coated with approximately 20nm of gold on a glowdischarge apparatus. Observations and photo micrographs were made with a Hitachi H H S - 2 R scanning electron microscope at 20kV. Examination of scanning electron micrographs of P D D treated bacteria confirmed that wild-type cells and those having a defective polymerase 1 formed filaments when grown in the presence of P D D which divided upon removal of the drug.
REFERENCES Anderson, T. F., 1951. Transactions" N. Y. Acad. Sci., 13" 130. Beck, D. J. and Brubaker, R. R., 1973. J. Bacteriol., 116: 1247. Crang, R. E. and Ward, J. A., 1975. In: Electron Microscopy: Principles and Practices, W. B. Saunders Co., PA. Rosenberg, B., Renshaw, E., Van Camp, L., Hartwick, J. and Drobnik, J., 1967. J. Bacteriol., 93: 716.
46
I). A. Gingrich and I). J. Beck
Fig. 1 4 . Scanning electron micrographs of Escherichia coli ceils, (1) strain W3110 wild-type bacteria and (2) strain P3478 polA mutant, after 3hr growth in the presence of 10t~g PDD per ml medium. The platinum drug was removed by filtration and the bacteria after growth in fresh broth are shown in (3) strain W3110, 2hr growth period and (4) strain p3478 pol,4, 4hr growth period. 1500.