Turbidity Changes in Suspensions of Gram-Positive Bacteria in Relation to Osmotic Pressure

Abstract

The turbidity of suspensions of Gram-negative micro-organisms is known to be influenced by the tonicity of the suspending medium. In contrast to this, suspensions of Gram-positive micro-organisms have the same optical density in 0.2 M-NaC1 as in distilled water. At higher salt concentrations, however, occasionally some batches were found to display changes in turbidity. Correlation was shown between the concentration of the amino acid pool of the organisms and their response to changes in osmotic pressure of the suspending medium. Depletion of the amino acid pool resulted in an increase in the changes in turbidity, with the simultaneous suceptibility of Gram-positive bacteria to plasmolysis. A possible correlation between this optical effect and the separation of cytoplasmic membrane from the bacterial cell wall is discussed.It was reported in preceding communications (Mager, Kuczynski, Schatzberg & Avi-Dory 1956; , that variations in the osmotic pressure of the suspending medium elicited corresponding changes in turbidity of Gram-negative bacteria. The light-scattering ability of suspensions of Gram-positive micro-organisms was found, however, to be independent of the prevailing tonicity. The object of the present study was to investigate the cause of the different response of these two classes of micro-organisms to changes in the osmotic pressure. METHODSCulture media. Escherichia coli was grown on nutrient agar (Difco) at 37".Bacillus megaterium was cultivated in a 2 yo (w/v) solution of an enzymic casein hydrolysate (N.B.C.), fortified with 0-1 yo (w/v) Marmite and incubated at 30' with shaking. Staphylococcus aureus was grown under the same conditions as B. meguterium with the addition of 1% (w/v) glucose to the culture medium.Inoculum. A volume of 0.1 ml. of 24 hr. slope culture of bacteria suspended in 5 ml. saline was used for inoculation of 100 ml. medium.Duration of incubation: 18 hr. Chemicals. All chemicals used were of reagent grade. Determination of ninhydrin-reacting substances released by boiling. A 2 ml. Procedure for measuring the optical eflect. The optical effect (o.E.) was measured as described before (Mager et al. 1956). The magnitude of the effect (E') was expressed as a percentage where E , is the light extinction of the suspension in water, and E , the extinction in the solute. RESULTSIn a previous communication, Mager et al. (1956) reported an increase in the light extinction of Gram-negative bacterial suspensions in isotonic saline or glucose as compared with suspensions in water (optical effect: o.E.). Under these conditions of assay no appreciable change could be demonstrated with Gram-positive bacteria. In further studies, however, when an increased concentration of NaCl was used ( 0 . 5~) occasionally batches of Gram-positive The amount of ninhydrin-reacting substances released by boiling of the organism was determined as described in 'Methods' and is calculated as 'glutamate'. micro-organisms were encountered which showed the optical effect in varying degree. It was also observed...

PROPIONATE INDUCED LYSIS OF PROTOPLASTS OF BACILLUS MEGATERIUM

“…Conversely, protoplast contraction results in an increase in optical density. A similar dependence of optical density on volume has been found for intact bacteria (Mitchell and Moyle, 1956;Kuczynski-Halmann, Avi-Dor, and Mager, 1958), rat liver mitochondria (Tedeschi and Harris, 1958), and erythrocytes (Wilbur and Collier, 1943).…”

Mechanism of Optical Effects in Suspensions of a Marine Pseudomonad

“…Potassium, which is ordinarily present in these cells at a concentration of 0.2 M, is lost from the cells when they are washed with 0.05 M MgSO4 (V. S. Srivastava, P. T. S. Wong, and R. A. MacLeod, unpublished data). As suggested by Kuczynski et al (9) in connection with gram-positive bacteria which have lost their amino acid pool, perhaps cells which have lost small molecules have a lower internal osmotic pressure and, hence, are able to contract to a gater extent than cells with their intracellular solutes intact.…”

“…Gram-positive bacteria ordinarily do not show 1 From a thesis submitted by Tibor I. Matula in partial fulfillment of the requirements for the Ph.D. degree at McGill University, May 1967. A preliminary report of these findings was presented at such optical effects, although after incubation in phosphate buffer followed by washing in distilled water, the cells of a number of species of grampositive bacteria became susceptible to optical changes comparable to those of gram-negative organisms (6,9). Our interest in the mechanism of these optical effects was aroused when we observed that suspensions of a marine pseudomonad showed optical changes typical of those of other gram-negative bacteria upon the addition of NaCl to the suspending medium.…”

Biochemical Cytology of Microorganisms

“…arises from the presence within the pro toplast of a high osmotic concentration of certain solutes [Mitchell (45) ; (25,48,49,50); Gale (148, 149»). The internal pressure must be created by the so-called active transport systems, that is to say, by the coupling of certain chemical bond exchanges of metabolism to the uptake, through the osmotic link, of nutrients [e.g., amino acids (40,48,149,150) ; phosphate (45,151)) which are accumulated in the protoplast. .…”