J . gen. Microbiol. (19691, 59, 91-95
With I plate
Printed in Great Britain
The Lysis of Cholera and El Tor Vibrios
By P. C. A D H I K A R I , C. R A Y C H A U D H U R I A N D
S . N. C H A T T E R J E E
Department of Biophysics, School of Tropical Medicine, Calcutta-la, India
(Acceptedfor publication 6 August 1969)
SUMMARY
Vibrio cholerae and V. eltor underwent considerable lysis after treatment
with tris EDTA, tris + EDTA +lysozyme or sodium lauryl sulphate. Lysis
induced by tris + EDTA or tris + EDTA + lysozyme was completely inhibited
by 0.3 M-sucrose, 0.15 M-NaCl or 0.01 M-MgCl,. The extent of death after
treatment with tris+ EDTA depended on EDTA concentration: 10 min.
with 10pg. EDTA/ml. left 43 to 50 yo of V. eltor viable and 71 to 75 % of
V. cholerae.Labelled organisms exhibited maximum leakage of 32Pcompounds
when exposed to 71 % (v/v) ethanol for V. cholerae and 50 % (v/v) ethanol
for V. eltor; leakage in IOO % ethanol was about 77 yo of maximum for V.
cholerae and 66% for V. eltor. Both 32P labelled and unlabelled vibrios
released considerable amounts of nucleic acid, phospholipid, protein and
carbohydrate and practically all their acid soluble phosphates with EDTA.
+
INTRODUCTION
Studies on the sensitivity of different bacteria to agents such as ethylene
diaminetetra-acetic acid (EDTA), lysozyme, sodium lauryl sulphate, etc., are likely to
throw light on the physico-chemical structure of their surface layers. According to
Wilkinson (1967), sensitivity of an organism to EDTA depends on the wall structure
and may have some taxonomic value. Similarly the nature of release of the 32P
compound by bacteria in ethanol of different concentrations can be broadly correlated
with their response to Gram-staining and hence to wall structure (Salton, 1963).
This communication records our study of the sensitivity of cholera and el tor
vibrios to di-sodium EDTA, di-sodium EDTA + lysozyme and sodium lauryl sulphate
and also of the mode of release of 32Pcompounds by the vibrios in ethanol.
METHODS
Organisms. Vibrio cholerae, Ogawa 154; V. cholerae, Inaba 66/64; V. eltor, 0~127;
V. eltor, E 249/67; V. eltor, E 252167; and V. eltor, E 256/67 were obtained from Dr
K. N. Neogy, Department of Bacteriology of this institute.
Test procedure. Vibrios harvested after 18 hr at 37" on peptone agar or from the
logarithmic phase of growth in alkaline peptone water were washed twice in tris buffer
(IOO p ~pH
, 8.0)and finally obtained as a thick suspension in the buffer. I ml. portions
were added to 9 ml. each of five test solutions: (a) IOO pM-tris buffer, (b) EDTA
(10, 50, 150 or 266pg./ml.) in ~ o o p m t r i sbuffer (Repaske, 1958) with or without
0.15 M-NaCl, 0.3 M-sucrose or 0.01 M-Mgcl,, (c) lysozyme (50 pg./ml.) in 100 pM-triS
buffer, ( d ) EDTA (266 pg./ml.) and lysozyme (50 pg./ml.) in IOO pmtris buffer
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92
P.
c. A D H I K A R I , c. R A Y C H A U D H U R I A N D s. N.
CHATTERJEE
with or without 0.15 M-NaCl, 0.3 M-sucrose or 0.01 M-MgCl and (e)0.5 % (w/v) sodium
lauryl sulphate in 0-I 5 M-saline (in this case the washing fluid was 0.I 5 M-saline) ;pH value
in all tests was adjusted to 8.0, initial optical density to about 0.8. Changes in optical
density relative to the test solutions were then measured in a photoelectric colorimeter
at 650 mp at room temperature (22 to 25').
Viability determination. Bacteria were treated with EDTA in 100 pM-tris buffer for
10min. and their viability relative to a control without EDTA was determined after
serial dilution in 0.15 M-saline; 0.1ml. volumes of suitable dilutions were spread on
peptone agar plates and colonies were counted after incubation at 37" for 18 to 24 hr.
Electron microscopy. Vibrios treated with (a) EDTA (50 pg./ml.) in tris buffer
( IOO p ~ for
) 10 min. (b) EDTA (50 pg./ml.) + lysozyme (50pg./ml.) in tris buffer for
10min. and (c) sodium lauryl sulphate, 0.5 yo (w/v) in 0.15M-saline for 80 min. were
immediately fixed in formaldehyde (4 yo,w/v) for 2 hr, washed, resuspended in distilled
water and prepared for electron microscopy (Das & Chatterjee, 1966).
Release of 32P.The distribution of 32Pamong different chemical fractions of Vibrio
cholerae and V. eltor was determined using the membrane filter technique of Britten,
Roberts & French (1955) as modified by Roodyn & Mandel(rg60). The membrane
filters were the BAC-T-FLEX type B-6 of Schleicher and Schull Co., Keene, New
Hampshire, U.S.A. Release of 32P from the labelled vibrios treated with EDTA
(50 pg./ml.) in tris buffer (100p ~ was
) studied similarly. Release of 32Pin ethanol was
studied by Salton's (1963) method. In all cases, vibrios labelled during the logarithmic
phase of growth were used.
32Pactivity on the membrane filters was measured by a thin window G.M. Counter
and corrected for background and 32Pdecay; activity released was expressed as %
intact organism activity.
EDTA (50 pg./ml.) extracts of the labelled vibrios were subjected to one dimensional
ascending chromatography on Whatman No. I paper in the following solvents:
ammonium sulphate (sat.), 160 ml. +sodium acetate (8.2 g,/Ioo ml.), 36 ml. +isopropanol, 4ml. (Smith, 1960). The paper was dried at room temperature, cut into
I cm. strips perpendicular to the direction of the run and their 32P activities were
measured.
Chemical tests. The O.D. values of filtrates of vibrios treated with EDTA in tris
buffer were measured with a Backman spectrophotometer DB in the range 200 to
300 mp. Protein in the filtrate was estimated by the method of Lowry, Rosebrough,
Farr & Randall (I 95 I) using crystalline serum albumin as a standard. Carbohydrate
in the filtrate was determined by the method of Dubois et al. (1956) using glucose as a
standard. For comparison, equal amounts of bacteria were heated at 100' for 20 min.,
filtered and the protein and carbohydrate in the filtrates were also determined.
RESULTS
Decrease in turbidity. Vibrio suspensions of all strains, incubated with 266 pg.
EDTA/ml. in IOO pM-tris buffer decreased 50 % in O.D. in 5 min. ;with 50 pg. lysozymel
ml. in addition 72% lysis resulted in the same time. In tris and lysozyme without
EDTA, lysis was only 10%. No significant decrease in O.D. was noted when vibrios
were incubated for 80 min. in (i) 0.15 M-NacI; (ii) 100pM-tris buffer; (iii) 0.05 Msodium phosphate buffer; (iv) phosphate buffer EDTA (266 pglml) ; (v) NaCl
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93
Lysis of vibrios
(0.1
5 M)
+EDTA (266 pg./ml.) or (vi) phosphate buffer (0.05M) +EDTA (266 pg./ml.)
+lysozyme (50pg./ml.). The lytic action of tris (100,UM)+EDTA (266 pg./ml.) or
)
(266 ,ug./ml.) +lysozyme (50pg./ml.) was completely inhibited
tris (100p ~+EDTA
when NaCl (0.15 M), sucrose (0.3 M) or MgCl, (0.01M) was added. Decrease in O.D.
of the vibrios without these protective additives depended on the EDTA concentration,
was maximum with 50 ,ug./ml. The O.D. in 0.15M-NaCl containing sodium lauryl
sulphate (0.5yo,w/v) decreased by 80 yo after 70 min. at 37".
Loss of viability. Plate counts of Vibrio cholerae and V. eltor showed differences in
inactivation at low EDTA concentration. After 10min. exposure to 10pg. EDTA/ml.
at room temperature, viability of V. cholerae was 71 to 75 yo a,"d V. eltor 43 to 50 %.
After exposure for 10 min. to 50,I 50 and 266 pg. EDTA/ml. both species had viabilities
of 8 to I I, 15 to 20 and 36 to 40 % respectively. Controls without EDTA remained
IOO % viable.
25
50
Ethanol conc.
(%; v/v)
75
100
Fig. I . Release of 3zPcompounds from vibrios suspended in ethanol solutions for 10 min.
at room temperature (Salton, 1963; also see 'Methods'). A,V. cholerae; 0,
V. eltor.
Electron microscopy. Vibrio cholerae and V. eltor organisms lost most of their
electron opacity after EDTA treatment, but retained their original shape (Pl. I , fig. I).
EDTA+lysozyme left only round structures of different sizes, but of much less
electron opacity (Pl. I , fig. 2). Electron microscopy of the vibrios heated to IOOO for
40 to 50 sec. and then treated with sodium lauryl sulphate revealed vibrio shaped
ghosts only (Pl. I, fig. 3).
32Prelease in ethanol. Labelled vibrios suspended in 90 % ethanol leaked 32Pcompounds rapidly, attaining the maximum value within 5 min. Figure I shows that
leakage differed according to species and ethanol concentration; IOO yo ethanol
released about 77 yoof the maximum from vibrio cholerae and about 66 yoof maximum
from V. eltor.
Release of intracellular material. Chemical fractionation revealed that, after EDTA
treatment, 40% of the lipid, 50% of the nucleic acid and practically the whole of
acid-soluble phosphate fraction had been released from the vibrios. 5'-mono- and tri-
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94
P. C. A D H I K A R I , C. R A Y C H A U D H U R I A N D S. N. CHATTERJEE
phosphates of adenosine, guanosine, thymidine, cytidine, and uridine were identified
by chromatography. The ultraviolet absorption spectrum had the expected maximum
at 260 mp, ratio A 280:A 260 being 0.71. Carbohydrate in the EDTA extract was
almost double that released by vibrios heated at 100' for 2omin. while the protein
was almost the same. No significant chemical differences between the extracts of two
vibrio biotypes were observed.
DISCUSSION
The initial effect of tris+EDTA is probably at the cell surface (Goldschmidt &
Wyss, 1967; Gray & 9Wilkinson, 1965). Sensitive organisms possess surface layers
which are easily altered by EDTA, which allow the chelating agent to enter the cell
readily and to damage sensitive intracellular targets so as to produce inactivation of
the cell (Kaufman & McDonald, 1957; Martell & Calvin, 1953). The present study
reveals a significant difference in the sensitivity of cholera and el tor vibrios to EDTA
at concentrations round about 10,ug./ml. Thus there may be a small difference in the
physicochemical structures of the walls of the two vibrio biotypes. The release of 32P
compounds in ethanol of different concentrations also indicates some difference in
the physicochemical structures of their walls and recalls the two species of Pseudornonas
which exhibited different degrees of release of 32Pcompounds in ethanol (Salton, 1963).
Electron micrographs of ultrathin sections of Vibrio cholerae and V. eltor have
revealed no differentiatingfeature in wall structure (unpublished observation). Fimbriae
were detected on V. eltor (Barua & Chatterjee, 1964; Tweedy, Park & Hodgkiss, 1968)
and on one strain of V. cholerae (Tweedy et al. 1968). But only the el tor strains examined
by ourselves possessed fimbriae (unpublished observation). These fimbriae may have
some relation with the greater susceptibility of the V. eEtor strains to EDTA toxicity.
However, there may exist other differentiating features in the physico-chemical structures of their surface layers, such as presence of metallic cations, the nature of their
binding with the cell wall components, chemical composition of the cell wall, etc.
contributing to the difference in their sensitivities to EDTA and ethanol; a difference
in the sensitivity of the cell wall of cholera and el tor vibrios to polymyxin has recently
been reported (Takeya, Koike & Iida, 1967).
The authors are indebted to Dr J. B. Chatterjea, M.D., F.N.I., Director, School
of Tropical Medicine, for his kind interest in this work. This investigation was supported by research grants from the Indian Council of Medical Research to P. C. A. and
from the Department of Atomic Energy, Government of India to C. R.
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E. F. (1955).Amino acid absorption and protein synthesis
in Escherichia coli. Proc. natn. Acad. Sci. U.S.A.41,863.
DAS,J. & CHATTERJEE,
S. N.(1966).Electron microscopic studies on some ultrastructural aspects of
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M., GILLES,
K. A., HAMILTON,
J. K., REBERS,
P. A. & SMITH,
F. (1956).Colorimetric method
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M. C, & WYSS,0. (1967). The role of tris in EDTA toxicity and lysozyme lysis.
GOLDSCHMIDT,
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Journal of General Microbiology, YO/.59, NO. I
P. C. ADHIKARI, C. RAYCHAUDHURI
AND
S. N. CHATTERJEE
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Plate
I
Lysis of vibrios
95
GRAY,G. W. & WILKINSON,
S. G. (1965). The effect of ethylenediaminetetraaceticacid on the cell
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N. J., FARR,
A. L. & RANDALL,
R. J. (1951). Protein measurement with
the Folin phenol reagent. J. biol. Chem. 193,265.
MARTELL,
A. E. & CALVIN,
M. (1953). Chemistry of the Metal Chelate Compounds, p. 499. New York:
Prentice Hall, Inc.
REPASKE,
R. (1958). Lysis of Gram-negative organisms and the role of versene. Biochim. biophys.
Acta 30, 225.
ROODYN,
D. B. & MANDEL,
H. G. (1960). A simple membrane fractionation method for determining
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SALTON,
M. R. J. (1963). The relationship between the nature of the cell wall and the Gram-stain.
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EXPLANATION OF PLATE
Fig. I. Electron micrograph of Vibrio cholerae treated for 10 min. with tris (roo ~ M ) + E D T A
(50 pg./ml.) x 6000.
Fig. 2. Vibrio cholerae treated for 10 min. with tris (100p ~ EDTA
)
(50 pg./ml.) lysozyme
(50 ,ug./ml.)x I 1,400.
Fig. 3. Vibrio cholerae treated for 80 min. with sodium lauryl sulphate (0.5 %, w/v) in 0.15 M-NaCl.
The vibrios were preheated at 100' for 40 to 50 sec. x 9000.
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