Stereological analysis of plasmolysis in logarithmic-phase Bacillus licheniformis

Abstract

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.

Bacterial plasmolysis as a physical indicator of viability

“…The second scenario involves the cell internally accumulating a different solute (distinct from the external compound causing hyperosmotic conditions) which balances the intracellular water activity required for enzyme function. Numerous reports support the hypothesis that many bacteria accomplish osmotic regulation through accumulation and loss of amino acids (11,12,16,36).…”

“…In addition, bacteria isolated from marine or saline environments and bacteria isolated from nonsaline systems may respond differently. Gram-positive bacteria have also been shown to plasmolyze when they are subjected to hypertonic media (27,36), but their thick peptidoglycan layer can make the response more difficult to quantify than the response of gram-negative bacteria. It is thus essential to conduct control experiments, such as those described above, for different media or different bacteria, in order to assure that the salt concentrations are sufficient to induce a detectable but nontoxic plasmolytic response.…”

“…Hypertonic solutions have since been used to demonstrate gross morphological changes in a wide range of bacteria, with the cytoplasm shrinking and the plasma membrane contracting from the cell wall as a result of internal water loss (17,27,28,37). Research has subsequently been conducted with sucrose and various salts in an attempt to understand membrane transport phenomena, stress resistance, activity, and cell surface physiology (12,27,36,38).…”

“…While plasmolysis commonly occurs in gram-negative species, plasmolysis, as well as changes in cell area or volume, have also been documented in gram-positive bacteria (27,36). The ability to undergo plasmolysis indicates that a semipermeable membrane is present and functioning to maintain protoplast integrity.…”

Soil Electrical Conductivity and Water Content Affect Nitrous Oxide and Carbon Dioxide Emissions in Intensively Managed Soils

“…To survive under stress, soil microbial communities change their microbial functions and/or activity in response to environmental disturbances. Several reports suggest that bacteria involved in nitrification regulate intracellular solute concentrations through accumulation and loss of amino acids (Schall et al, 1981;Killham and Firestone, 1984). More generally, the ability to do this may vary among microbial communities.…”