suggest that more than one mechanism is responsible for the observed changes. In general, however,

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp June, 1964 Copyright a 1964 by the American Society for Microbiology Printed in U.S.A. FACTORS WHICH MODIFY THE EFFECT OF SODIUM AND POTASSIUM ON BACTERIAL CELL MEMIBRANES1 DOROTHY H. HENNEMAN2 AND W. W. UMBREIT Department of Bacteriology, Rutgers, The State University, New Brunswick, New Jersey, and Department of Medicine, Seton Hall College of Medicine, Jersey City, New Jersey Received for publication 22 November 1963 ABSTRACT HENNEMAN, DOROTHY H. (Rutgers, The State University, New Brunswick, N.J.), AND W. W. UMBREIT. Factors which modify the effect of sodium and potassium on bacterial cell membranes. J. Bacteriol. 87: Suspensions of Escherichia coli B, when placed in 0.2 to 0.5 M solutions of NaCl, KCl, or LiCl, show an increased turbidity. With NaCl, this increased turbidity is stable with time; with KCl and LiCl, it is gradually lost. The stability to NaCl with time is due to substances removable from the cell by incubation in phosphate buffer; these materials exist in water washings from such phosphate-incubated cells. Considerable work has been done on the relation of changes produced by osmotic environmental modifications to bacterial structure and the manner in which structure determines permeability of a given substance. The evidence for the suggested mechanisms whereby simple diffusion, exchange diffusion, or specific permeation are related to cell structure was thoroughly reviewed by Mitchell and Moyle (1956) and Kleinzeller and Kotyk (1960). It has been shown repeatedly that increases in external osmotic pressure elicit corresponding changes in the turbidity of cell suspensions of both gram-negative and gram-positive bacteria, and that these turbidity responses vary not only for the substance producing the osmotic change but also for the particular species of bacteria studied (Mager et al., 1956; Avi-Dor et al., 1956; Kuczynski- Halmann, Avi-Dor, and Mager, 1958). Differences in the turbidity response of any single species to different osmotically active conditions I Presented in part at the Annual Meeting of the American Society for Microbiology, Cleveland, Ohio, 5-9 May Present address: E. R. Squibb and Sons, Instituite for Medical Research, New Brunswick, N.J. suggest that more than one mechanism is responsible for the observed changes. In general, however, changes in external osmotic pressure are associated with varying degrees and duration of plasmolysis. It is presumed that plasmolysis is accompanied by compression of the protoplasm lying below the cell wall, thereby causing the cell membrane to pull away from the cell wall. This separation of cell membrane and cell wall results in another surface of irregular length and shape which might be expected to increase the scattering of light. Changes in light scatter subsequent to the production of plasmolysis by varying osmotic conditions have been measured as a means of studying the rate of penetration of different substances. Light scattering can be measured by direct methods (Shibata, 1962; Amez, Duysens, and Brandt, 1961), by refractive index methods as described by Barer and Joseph (1958), or by phase microscopy (Orskow, 1946, 1947). A simple technique is to determine under standard conditions changes in optical density which occur when suspensions of bacteria are placed in salt solutions of varying concentrations. Using this technique, we designed the present study to ascertain the effect of monovalent cations on the optical density of bacterial suspensions and to determine whether changes in environmental conditions (such as ph, buffers, substrates) modified the response to these cations. It was anticipated that the effects of environmental modifications might disclose the manner in which certain cations permeated the cell membrane and, hence, provide some evidence as to the nature of the cell membrane itself. MATERIALS AND METHODS The cultures used were strains carried for several years in the departmental collection. Unless otherwise indicated, Escherichia coli B was the organism used. The organisms (except where specified) were grown on a fluid media 1266

2 VOL. 87, 1964 SALT EFFECTS ON BACTERIAL CELL MEMBRANES 1267 composed of 0.3% beef extract, 0.5% Tryptone, and 0.2 to 1.0% glucose at 37 C for 18 hr on a rotary shaker. The final ph of these cultures (designated as "G") was generally 4.5. Cells grown in the same media without added glucose, or with less than 0.2% glucose (designated as "N"), had a final culture ph of 7.0 to 8.5. After, 18 hr, the cultures were harvested by cold centrifugation at 13,500 X g for 15 min. The cells were then washed and recentrifuged twice with distilled water (100 ml per 100 ml of culture media) at 13,500 X g for 15 min. After washing, they were resuspended in distilled water. This final suspension is designated as SH,o, and samples of this were used throughout the study. SH,O was used immediately or stored in the cold for 0 to 72 hr; the duration of storage is indicated in all figures and tables. We recognize that such treatment of the bacterial cells has released much of the amino acid pool, and that perhaps other damage has taken place (see Britten and McClure, 1962). However, in spite of these effects, the cells still respond in a characteristic and reproducible manner, and are a suitable object for study of membrane properties. Varying concentrations of chemically pure sodium, potassium, and lithium chloride were added to samples of SH20 so that the final molarity of the salts ranged between 0.07 and 2.62 M. Initially, no attempt was made to control variations in ph; the salt solution was added directly to the sample of SH2O. Later studies controlled any ph change by the use of fixed amounts of tris(hydroxymethyl)aminomethane (tris) buffer (0.1 M, ph 7.2) or phosphate buffer (KH2PO4- K2HP04, 0.1 M, ph 7.2). When buffer was present, it will be so indicated. For measuring turbidity changes, portions (0.5 to 1.0 ml) of bacterial suspensions (SH20 ) were diluted to 3 ml with water so that on a Beckman DU spectrophotometer at 500 m,a the optical density was between and This served as the blank. Similar portions of SH20 were diluted with solutions of sodium, potassium, and lithium chloride so that the final molarity in a volume of 3 ml ranged between 0.07 and 2.62 M. All studies were run in this fashion unless otherwise indicated. When tris and phosphate buffers were added to the bacterial suspension (SH20 ) prior to the addition of salt (to maintain ph constant), the final volume was still 3 ml and the final molarity of the salt content was still in the range of 0.07 to 2.62 M. Generally, 0.5 ml of 0.1 M tris (ph 7.2) or 1.0 ml of 0.05 M tris and 0.3 ml of 0.1 M KH2PO4-HPO4 buffer (ph 7.2) were used. The change produced by the several factors studied in the turbidity of the bacterial suspension (SH20 ) at 500 m,u was expressed in the following manner (according to Kuczynski-Halmann et al., 1958): OE (optical effect) = % change in optical density (OD Sni,o + NaCl) - (OD Sn,o, + H20 X 100 (OD SH20 + H20) where OD = optical density at 500 m,u. For convenience, we have used the term "turbidity increase" (or decrease) to express this quantity. RESULTS When a water-washed suspension (SH2o ) Of optical density at 500 ma of approximately 0.5 (after threefold dilution with water) is placed in solutions of KCl, LiCl, and NaCl, an increase in turbidity results. The increase is more marked in basic- or neutral-grown cells ("N") than in acid-grown cells ("G"). In the case of NaCl, an increase of as much as 30 to 40% in optical density occurs (Fig. 1). A somewhat more rapid increase in optical density results with KCI, and a slower increase is observed with LiCl. Dense water suspensions (prior to the threefold dilution with water) of cells could be held in the cold for as long as 48 hr with only slight loss in ability to respond to salts in this fashion. After 72 hr of cold storage in water, the ability to respond was markedly decreased-particularly if the suspension was stored as dilute or acid-grown ("G" cells). The increase in turbidity was not modified by the addition of small quantities (0.003 M, final molarity) of metabolizable materials (lactose, glucose), and thus differs from the response described by Packer and Perry (1961) and Bovell, Packer and Helgerson (1963). If the turbidity of these suspensions was measured for 30 min, a distinct difference was observed between cells suspended in KCl and cells suspended in NaCl. With NaCl, the increased turbidity remained constant (less than a 5% decrease); with KCl, the increased turbidity was gradually lost, the loss being more rapid at the lower concentrations. Cells suspended in solutions of LiCl also showed this re-equilibration

3 1268 HENNEMAN AND UMBREIT J. BACTERIOL. O.E. (*/. increa 30-1 in O.D. [St Ax 8 xx 0 % NaC I to E to 10' I os Molar 0.4 OS /. NaCI O.E K - v xe O.Sm 1 NaCI A _,., 0.2.m to0 10 K CI YS%MEX-A xemm \ LiCI oftllw_ 0***Oftft 0. 5 m arn I I I I,U I,I I M inutes Minutes Molar FIG. 1. Per cent increase in optical density (OE) at 500 m,u in water suspensions of E. coli (G cells) placed in solutions of sodium, potassium, and lithium chloride. Each symbol represents a mean of five observations made on separate occasions; different symbols are used for separate studies. toward control. The "stability" to NaCl was a constant finding in neutral- or basic-grown cells; it was a less constant finding in acid-grown cells. Accordingly, cells were grown in varying amounts of glucose (0.01 to 1.0%) added to peptone broth so that the final culture ph at the time of harvesting ranged from 4.5 to 9.0. Cells whose ph reached 4.5 the fastest (i.e., cells grown with more than 0.2% glucose) were the most likely to show some re-equilibration of the turbidity increase with NaCl. At no time or ph, however, did the rate or degree of re-equilibration reach that observed with KCl. Suspensions of E. coli cells exposed to either KCl or NaCl showed marked plasmolysis when observed in a phase microscope. The degree of plasmolysis was more marked the higher the molarity of the salt solution. As with the turbidity increase, the plasmolytic effect of KCl was unstable in time (plasmolysis no longer being visible after 30 min), whereas that of NaCl was stable in time. When salts were added to SH2O, there was usually a slight drop in ph, presumably due to replacement of surface H+ by Na or K; the drop in ph was more pronounced with KCl than with NaCl. This ph change was readily controlled by tris buffer (0.03 M, ph 7.4) without altering the described response of the suspension to salt. The ph per se, however, (in the absence of NaCl or KCl) did have a profound effect upon the turbidity of the cell suspension. This was readily observed when phosphate buffers of different ph (4.9 to 9.0) were used as suspending media for samples of SH20. With either N or G cells, there was a stepwise increase in turbidity as the ph of the suspending phosphate buffer (0.67 M; Table 1A) was lowered from 9.0 to 4.5. This ph effect was observed with to 0.67 M phosphate buffers. The change in turbidity was not stable with time, the fall off being more rapid as the ph was increased (Table 1B). When NaCl was added to cells which were suspended in phosphate buffers of different ph, the further increase in

4 VOL. 87, 1964 SALT EFFECTS ON BACTERIAL CELL MEMBRANES 1269 TABLE 1. Effect of ph on turbidity changes produced by phosphate (0.67 M) and subsequent NaCi (0.36 M) Observation Acid-grown "G" Basic-grown Phos- (ph 4.5) "N" (ph 9.0) phate FreshStored (48 hr) Fresh FrShtored (48 h r) (A) Immediate per cent in crease in OD at 500 m,u upon addition of phosphate (B) Per cent re equilibration in 30 min of ph - induced OD increase (C) Per cent in crease in OD with addition of 0.36 M NaCl 30 min after phosphate buffer turbidity was directly proportional to the ph. That is, as the ph increased (with its lesser rise in turbidity and more rapid re-equilibration), so did the turbidity rise produced by NaCl (Table 1C). This effect of ph probably explains the greater increase in turbidity observed when N cells (final culture ph 9) were placed in NaCl plus water solutions as compared with that observed when G cells (final culture ph 4.5) were placed in NaCl plus water solutions: e.g., OE or turbidity increase of N cells in 0.5 M NaCl was generally 35.0 to 40.0, whereas it was 25.0 to 30.0 with G cells (Fig. 1). Study of the response to salts of other gramnegative organisms showed that there were two types of response. One, typical of E. coli (Fig. 2A), showed a gradual increase in turbidity as the molarity of NaCl was increased, which was stable for at least 30 min (Fig. 2B). In the other response, typical of Pseudomonas (Fig. 2C) and related organisms, turbidity, especially at lower molarities of NaCl, was decreased with time (Fig. 2D). Both types of organisms showed a loss with time of the increase in turbidity produced by KCl. These results are in good agreement with recent studies on halophilic bacteria, which show the type of response characteristic of Pseudomonas but whose turbidity response requires a somewhat higher salt concentration than nonhalophiles (Abram and Gibbons, 1960, 1961; Boring, Kushner, and Gibbons, 1963; MacLeod and Matula, 1962; Mohr and Larsen, 1963). Phosphate incubation. Kuczynski-Halmann et al. (1958) reported that gram-positive organisms showed decreased rather than increased turbidity when placed in moderately concentrated NaCl solutions. They correlated the response of grampositive organisms to NaCl with the presence of an amino acid pool, and demonstrated that with depletion of this pool gram-positive organisms responded to salt in a fashion similar to that of gram-negative organisms. (Depletion of the amino acid pool in gram-positive organisms was accomplished by incubation for 2 to 24 hr in 0.01 to 0.05 M phosphate buffer, ph 7.3.) They suggested that the higher internal osmotic pressure (due to the higher amino acid pool) in gram- so , E0 (A E. COLI x olar 0.: O4 O NaCIl 30ix-x-x-x-x-x 0.5 M O. E M B 10 - PSEUDOMONAS SP / IC MaCr D 0.5 M -b 0.2M I. I I I l to Min. Wo Min. FIG. 2. Per cent increase in optical density (OE) at 500 m/a in water suspensions of Escherichia coli B (A and B) and Pseudomonas (C and D) placed in solutions of sodium chloride.

5 1270 HENNEMAN AND UMBREIT J. BACTERIOL. positive organisms might be responsible for the absence of plasmolysis when these organisms are placed in salt solutions. When we incubated water-washed suspensions of Bacillus megaterium, Streptococcus faecalis, and Lactobacillus arabinosus in 0.06 M phosphate buffer at ph 7.4 for 2 hr, washed the cells three times with water, and resuspended them in water, the organisms (still reacting positively to Gram stain) responded to NaCl and KCl in a manner comparable to that of Pseudomonas, i.e., an increased optical density of 20 to 30% with a gradual decline over a 30-min period (comparable to Fig. 2D). However, if the gram-positive phosphate-incubated cell suspension was not washed with water, the cells failed to show any increase in turbidity when placed in solutions of KCl and NaCl. Since under these latter circumstances the internal amino acid pool would presumably be equally depleted (not checked experimentally), it was evident that phosphate incubation was modifying the cells in a manner other than simply by depleting the internal amino acid pool. Hence, gram-negative organisms were similarly incubated with phosphate. Treatment of freshly harvested cells with phosphate buffer and subsequent water-washing was not associated with any discernible lysis (Fig. 3). When cells of E. coli B were incubated in 0.06 M phosphate buffer at ph 7.2 to 7.7 (Fig. 3A) but not washed with water, there was an increased turbidity due to O.E 20- CELLS NOT SUBSEQUENTLY WASHED ph --_^e A %A N6210 CELLS WASHED WITH WATER AFTER INCUBATION MIN MIN. FIG. 3. Effect of incutbation (at ph 6.2 to 7.7) of water suspensions of Escherichia coli B for 2 hr in 0.06 m phosphate buffer, with and without subsequent water washing, on turbidity response to 0.25,M sodium chloride. NaCl which was stable with time; at ph 6.8 there was some loss; and at ph 6.2 this loss was marked. If such incubated cells were washed three times (Fig. 3B), there was a loss of turbidity with time at all ph levels. Incubation of E. coli B SH20 in buffers other than phosphate did not modify the response to sodium chloride, i.e., the cells still responded to NaCl with a turbidity increase which was stable to time. Phase microscopy of washed, phosphate-incubated E. coli cells showed that, in contrast to cells not phosphate-incubated, sodium chloride induced plasmolysis which was unstable with time. Other procedures were tested, and the following conditions abolished the stability with time to NaCl manifested by E. coli B: (i) incubation in P04 buffer (ph 6.8 to 7.7) followed by washing and resuspension in distilled water (as described above); (ii) incubation in P04 buffer at ph 6.2 (Fig. 3A); (iii) incubation of a water suspension of cells with an equal volume of 5% butanol in water at room temperature for 30 min. This last method is presumed to remove a phospholipid component of the cell wall which protects the lysozyme-sensitive substrate in gram-negative bacteria (Noller and Hartsell, 1961). Butanol may have other effects as well. Treatment of E. coli B with butanol produced the most marked loss of stability to NaCl. Complete abolishment of any response (i.e., turbidity was not increased when suspended in salt) to either NaCl or KCl was accomplished by the following procedures: (i) incubation for 30 min with ether (5 volumes of a water suspension of E. coli plus 1 volume of ether) wtith and without subsequent washing with water; (ii) vacuum drying, or lyophilization, of a water suspension of E. coli. (This procedure not only destroyed the ability of the cell to show an increase in turbidity but suspension in NaCl, KCl, or phosphate resulted in a 10 to 20% decrease in turbidity.) Existence of "stability" factors. The previous studies have demonstrated that E. coli B (G or N cells), when exposed to NaCl, shows an increased turbidity which is stable in time (Table 2, line 1). If incubated in phosphate buffers, washed, resuspended in water, and subsequently placed in a NaCl, there is a smaller initial increase in turbidity which is lost with time (Table 2, line 2). In addition, these phosphate-incubated and washed cells respond to higher concentra-

6 VOL. 87, 1964 SALT EFFECTS ON BACTERIAL CELL MEMBRANES 1271 tions of NaCl with a lesser increase in turbidity. [With normal cells, there is a progressive increase in turbidity with NaCl solutions up to 0.67 M (up to 0.5 M, shown in Fig. 1 and 3). Above this point, there is a lesser turbidity increase and at 1 M, or above, turbidity increase is less than that observed at 0.5 M. After phosphate incubation, the increase in turbidity is not only less for a given molarity of salt but the entire response curve has shifted to lower salt concentrations. As such, phosphate-treated cells show less of a turbidity increase at 0.5 M NaCl than they do at 0.2 M NaCl. This observation is consistent with those described by Bernheim (1963).] If phosphate-incubated cells are washed and then resuspended in the water-wash, they are stable in time to NaCl (Table 2, line 3); i.e., the waterwash contains "stability factors." These factors are also present to a lesser extent in the spent phosphate (Table 2, line 4) but are not present in fresh phosphate buffer per se (Table 2, line 5). Attempts were made to isolate and identify the stability factors in the water-wash. Vacuum drying at 60 C and reconstitution in water to provide 10- to 100-fold concentrations did not destroy activity. Ferric and magnesium ions (102 to 10-5 M) did not replace stability to phosphate-incubated, water-washed cells. Washings obtained from Pseudomonas cells (similarly phosphate-incubated) did not contain activity. Vacuum-dried concentrates were separated into an ether-soluble and water-soluble phase by acid extraction with ether. The water-soluble phase, neutralized and reconstituted with tris buffer to the original volume of the washings used for the extraction, contained activity (as tested on phosphate-incubated E. coli cells), whereas the ether-soluble phase did not. Hence, "activity" did not appear to be related to substances which were lipid in nature. Further attempts of identification by use of paper chromatography revealed that "activity" moved with a solvent system of normal butyl alcohol-acetic acid-water (4:0.5:5). This "activity" was ninhydrin-negative and did not fluoresce. It was acid- and heat-labile, but has not been further identified. DISCUSSION When cells of E. coli B are placed in NaCl (0.2 to 0.5 M), there is an increased turbidity compared with the same suspension of cells placed in water. This increased turbidity persists TABLE 2. Occurrence of stability factors in washings from phosphate-incubated Escherichia coli OE (% increase in turbidity due to NaCI) Line Condition 0.2 m( NaCl 0.5 mi NaCl 0 min 30 min 0 min 30 min 1 E. coli not incu bated in phosphate Incubated in phosphate (0. 06 M, ph 7.2),* washed in water, and cells suspended in: 2 Water Washings (w) "Spent" phos phate (s) 5 Fresh phosphate * The system employed was the following: 10 ml of cells of E. coli B (OD, approximately 2.0 at 500 m,u) were incubated with 20 ml of 0.1 M phosphate buffer at ph 7.2 for 2 hr on a shaking machine at room temperature. The cells were then centrifuged, and the supernatant "spent" buffer solution (30 ml) was removed. (It was labeled fraction s.) The residue of packed cells was suspended in 30 ml of distilled water and recentrifuged, and the washings were decanted. This process was repeated three times, and the washings were combined (90 ml, final volume) and designated fraction w. The cells were finally resuspended in about 5 ml of water and designated SH,O-PO4. Samples of SH,o-Po4 were then suspended in water, washings (w), spent phosphate (s), or fresh phosphate such that their OD in a 3-ml final volume was approximately 0.5 at 500 m/a. NaCl was added to equal portions of SH20-PO4 (suspended in water, washings, spent phosphate, or fresh phosphate) such that the final molarity was 0.2 or 0.5 M in a 3-ml total volume. for 30 min or longer. Stability with time to sodium is partially lost if the cells are grown in media with added glucose (more than 0.2%), and if the final culture media reaches ph 4.5 sooner than 6 hr before harvesting and 18- to 20-hr culture. With KCl, essentially the same turbidity increase occurs, but within 15 to 30 min the turbidity returns toward the control. Associated with this increase in turbidity is a

7 1272 HENNEMAN AND UMBREIT J. BACTERIOL. marked shrinking and twisting of the cell protoplasm so that it looks fragmented in a phase microscope. This protoplasmic compression presumably causes the cell membrane to pull away from the more rigid cell wall. Both effects tend to increase light scatter. The change in light scatter, whether observed in a phase microscope, a nephlometer, or measured spectrophotometrically, was strictly reversible (by dilution of the salt to concentrations which were not active) and did not involve lysis of the cells. Mitchell and Moyle (1956), using their technique for determining the phosphate-impermeable cell volume, demonstrated that E. coli B possesses an osmotic barrier which is relatively permeable to potassium chloride. Other gramnegative organisms, Pseudomonas sp. in type, appear to be about equally permeable to sodium and potassium chloride. This exclusion of sodium, or "stability" in time to sodium, of E. coli B is not modified by availability of external energy supply, since lactose or glucose did not modify the turbidity response to sodium chloride. However, increased acidity or prolonged exposure to acid in the culture media did modify the response with some loss of stability to NaCl. These acid-treated cells, however, placed in tris buffer, became stable to sodium while remaining unstable to potassium. Incubation of E. coli B in phosphate buffer also modified the turbidity response to sodium without producing any discernible lysis of the cells as a consequence of either the incubation or the subsequent washings. Bernheim (1963) demonstrated that phosphate incubation is associated with an increase in internal potassium concentration. He concluded that this explains the decreased turbidity response when such phosphate-incubated cells are exposed to sodium or potassium chloride. This increase in potassium does not, however, explain the effect of phosphate incubation on the "stability" to sodium chloride of E. coli cells or on the turbidity response of grampositive organisms to either potassium or sodium chloride. Similarily, depletion of the internal amino acid pool, as advocated by Avi-Dor et al. (1956), does not explain the loss of "stability" to sodium chloride since nonwashed phosphateincubated E. coli cells retain stability. The relation of the observed turbidity changes to plasmolysis is not a simple one. However, the patterns of turbidity change produced in different bacterial species are constant, reproducible, and can be quantitated. Identification of the factor responsible for stability to sodium chloride and present in the washings of phosphate-incubated E. coli cells should provide information not only on the mechanisms responsible for sodium exclusion but also on the nature of the cell-membrane itself. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grants A-3337 from the National Institute of Arthritis and Metabolic Diseases and 2G-505 from the Division of General Medical Sciences. LITERATURE CITED ABRAM, D., AND N. E. GIBBONS Turbidity of suspensions and morphology of red halophilic bacteria as influenced by the sodium chloride concentration. Can. J. Microbiol. 6: ABRAM, D., AND N. E. GIBBONS The effect of chlorides of monovalent cations, urea, detergents and heat on morphology and the turbidity of suspensions of red halophilic bacteria. Can. J. Microbiol. 7: AMESZ, J. L., N. M. DUYSENS, AND D. C. BRANDT Methods for measuring and correcting the absorption spectrum of scattering suspensions. J. Theoret. Biol. 1: AvI-DoR, Y., M. KUCZYNSKI, G. SCHATZBERG, AND J. MAGER Turbidity changes in bacterial suspensions; kinetics and relation to metabolic state. J. Gen. Microbiol. 14: BARER, R., AND S. JOSEPH Concentration and mass measurements in microbiology. J. Appl. Bacteriol. 21: BERNHEIM, F Factors which affect the size of the organisms and the optical density of suspensions of Pseudomonas aeruginosa and Escherichia coli. J. Gen. Microbiol. 30: BORING, J., D. J. KUSHNER, AND N. E. GIBBONS Specificity of the salt requirement of Halobacteriumn cutirubrum. Can. J. Microbiol. 9: BOVELL, C. R., L. PACKER, AND R. HELGERSON Permeability of Escherichia coli to organic compounds and inorganic salts as measured by light-scattering. Biochem. Biophys. Acta 75: BRITTEN, R. J., AND F. T. MCCLURE The amino acid pool in Escherichia coli. Bacteriol. Rev. 26: KLEINZELLER, A. G., AND A. KOTYK [ed.] 1960.

8 VOL. 87, 1964 SALT EFFECTS ON BACTERIAL CELL MEMBRANES 1273 Membrane transport and metabolism. Academic Press, Inc., New York. KUCZYNSKI-HALMANN, M., Y. AvI-DoR, AND J. MAGER Turbidity changes in suspensions of gram-positive bacteria in relation to osmotic pressure. J. Gen. Microbiol. 18: AMAcLEOD, R. A., AND T. I. MATULA Nutrition and metabolism of marine bacteria. XI. Some characteristics of the lytic phenomenon. Can. J. Microbiol. 8: MAGER, J., M. KuczYNSKI, G. SCHATZBERG, AND Y. AvI-DoR Turbidity changes in bacterial suspensions in relation to osmotic pressure. J. Gen. Microbiol. 14: MITCHELL, P., AND J. MOYLE Osmotic function and structure in bacteria. Symp. Soc. Gen. Microbiol. 6: MOHR, V., AND H. LARSEN On the structural transformations and lysis of Halobacterium salinarium in hypotonic and isotonic solutions J. Gen. Microbiol. 31: NOLLER, E. C., AND S. E. HARTSELL Bacteriolysis of enterobacteriaceae. II. Pre- and co-lytic treatments potentiating the action of lysozyme. J. Bacteriol. 81: ORSKOV, S. L Investigations on the permeability of yeast cells. Acta Pathol. Microbiol. Scand. 22: ORSKOV, S. L A method for microscopic examination of suspensions. Acta Pathol. Microbiol. Scand. 24: PACKER, L., AND M. PERRY Energy-linked light-scattering changes in Escherichia coli. Arch. Biochem. Biophys. 95: SHIBATA, K Spectrophotometry of opaque biological materials-reflection methods. Methods Biochem. Analy. 9: Downloaded from on June 8, 2018 by guest