Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

A rapid cell membrane permeability test using flourescent dyes and flow cytometry

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein—fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.Abbreviations DMA dimethyl sulfate - DMSO dimethyl sulfoxide - EB ethidium bromide - F fluorescein - FDA fluorescein diacetate - FS₂₅ concentration of test substance resulting in a F-peak left-shift of 25% from control - PBS phosphate buffered saline - SCT forward light scatter - SDS sodium dodecyl sulfate     

The viability assessment of ethanol-producing yeast by computer-aided fluorescence microscopy

Vital staining of the ethanol-producing yeast Saccharomyces cerevisiae with ethidium bromide and DAPI allows intact and damaged cells to be differentiated by fluorescence microscopy. A computer image analysis procedure has been developed for the automatic determination of the relative number of damaged cells using ImageJ software (National Institute of Health, United States; http://rsb.info.nih.gov./ij/). A good correlation has been found between the viability rates determined by the plate count method and the relative numbers of intact cells assessed by the developed procedure in the dry-yeast preparations rehydrated under various conditions.     

Nucleus of the boar spermatozoon: structure and modifications in frozen, frozen-thawed, or sodium dodecyl sulfate-treated cells

After cryosubstitution and Epon embedding, or after Nanoplast embedding and very thin sectioning, the chromatin of ejaculated or diluted boar spermatozoa appears to be formed of DNA fibers embedded in a quite homogeneous matrix. After sodium dodecyl sulfate (SDS) treatment, and to a lesser extent after freeze-thawing, the DNA fibers are present mostly between cords, probably proteinaceous in nature. The quantity of free sulfhydryl (SH) groups, as calculated from staining by DACM and flow fluorometry, is increased in thawed or SDS-treated cells. The quantity of NH2 groups, calculated from electron microscopy image analysis of alcoholic phosphotungstic acid-stained cells, is decreased in thawed nuclei. The DNA is more accessible to the fluorochrome ethidium bromide after freeze-thawing, and its sensitivity to HCl hydrolysis is modified, during the Feulgen-like staining procedure using acriflavine. The X-ray energy dispersive analysis of cryosections of nuclei indicates that the slight separation of DNA and nucleoproteins in freeze-thawed spermatozoa could result from a dramatic modification of the nuclear ionic environment during thawing.     

Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis and B. cereus: a flow cytometric study

For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA.     

The viability assessment of ethanol-producing yeast by computer-aided fluorescence microscopy

A vital staining of the ethanol-producing yeast Saccharomyces cerevisiae with ethidium bromide and DAPI allows intact and damaged cells to be differentiated by fluorescence microscopy. A computer image analysis procedure is developed for the automatic determination of the relative number of damaged cells using ImageJ software (National Institute of Health, United States; http://rsb.info.nih.gov./ij/). A good correlation has been found between the viability rates determined by the plate count method and the relative numbers of intact cells assessed by the developed procedure in the dry-yeast preparations rehydrated under various conditions.     

Permeability of the membrane of the Escherichia coli envelope for ethidium bromide

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.     

Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.     

Kinetic analysis of the action of cetyltrimethylammonium bromide on the membranes of the capsular variant of Escherichia coli CA189 recorded by ethidium bromide fluorescence

A mathematical model, describing the kinetics of ethidium bromide probe fluorescence in the suspension of E. coli CA 189 capsular antigen after introduction of cetyltrimethylammonium bromide (a detergent), has been developed. Experimental testing of this mathematical model has confirmed that it describes the kinetics of the probe fluorescence augmentation sufficiently accurately. The new model permits the quantitative characterization of the rate of surfactant diffusion through the bacterial capsule.     

Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures

A method was developed that enabled an analysis of the proportion of permeable cells in a culture of Lactococcus lactis . This used the fluorescence of propidium iodide (PI) when in contact with DNA and the impermeability of the intact cell membrane to this compound. A permeability index was suggested that expresses the PI-induced fluorescence of a cell suspension as a percentage of the value obtained from wholly permeabilized cells after treatment with cetyltrimethylammonium bromide. This method was applied to the determination of cell permeability in death phase cultures. A large proportion of unlysed cells was freely permeable to PI, a finding that may have some significance for the investigation of the role of cell lysis in cheese maturation. This method is suggested as a useful addition to the techniques available for the study of cell damage in a variety of fields, and for the screening of cheese starter bacteria.     

Cell membrane integrity and lysis in Lactococcus lactis: the detection of a population of permeable cells in post-logarithmic phase cultures

A method was developed that enabled an analysis of the proportion of permeable cells in a culture of Lactococcus lactis. This used the fluorescence of propidium iodide (PI) when in contact with DNA and the impermeability of the intact cell membrane to this compound. A permeability index was suggested that expresses the PI-induced fluorescence of a cell suspension as a percentage of the value obtained from wholly permeabilized cells after treatment with cetyltrimethylammonium bromide. This method was applied to the determination of cell permeability in death phase cultures. A large proportion of unlysed cells was freely permeable to PI, a finding that may have some significance for the investigation of the role of cell lysis in cheese maturation. This method is suggested as a useful addition to the techniques available for the study of cell damage in a variety of fields, and for the screening of cheese starter bacteria.     

Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes.

Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

How endo- is endo-? Surface sterilization of delicate samples: a <Emphasis Type="Italic">Bryopsis</Emphasis> (Bryopsidales,Chlorophyta) case study

In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant’s cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatic approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.     

Discrete changes of the fluorescence yield from cells vitally stained with ethidium bromide (EB), as determined by flow cytometry

Cultured L cells were scraped from the glass surface and directly suspended in solutions of ethidium bromide (EB) in Tris buffer. After different staining times the fluorescence distributions were analysed with a flow cytometer. At 5 min after staining the cells yielded a normal DNA-specific histogram, but then a second peak at about 30–50% beyond the normal G1 fluorescence appeared and grew with increasing staining time. This effect of a discrete transition into a state of higher fluorescence was not dependent on the proliferative state of the cultures but was influenced by the temperature of the suspension and by serum and RNase. The effect proved not to be correlated with a G0–G1 transition and shows the possibility of artefacts in the determination of G0/G1 ratios by vital staining.     

S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells

A modification, using S1-nuclease, of a simple and sensitive fluorometric assay with ethidium bromide was developed for the measurement of cellular DNA interstrand crosslinking induced by bifunctional alkylators. Cells are lysed and treated with proteinase K and sodium dodecyl sulfate followed by extensive dialysis to yield intact high-molecular-weight DNA, free of contaminating proteins, on which the crosslink assay is then performed. The assay depends on the differential binding of ethidium bromide to single- and double-stranded DNA. Because of the higher ethidium bromide binding capacity of double-stranded DNA, the fluorescence retained after a heating/cooling cycle is directly proportional to the drug-induced cellular DNA interstrand crosslinking. We demonstrate that the sensitivity of this assay can be increased up to fourfold by including an S1-nuclease digestion step. This modified technique is simple and suited to the quantitation of low levels of DNA-interstrand crosslinking in cells.     

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10(2) to 10(7) cfu/ml. Cytometric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.     

Freezing injury in the yeast respiratory system

The effect of freeze-thawing on the yeast respiratory system was studied at rapid rates of cooling. Freezing of whole cells with liquid nitrogen induced decrease of respiratory activity to under 20% of that of original cells. Mitochondria harvested from freeze-thawed cells have markedly decreased succinate oxidizing activity. Activity of succinate cytochrome c reductase was reduced significantly after freeze-thawing of whole cells while activities of succinate dehydrogenase and cytochrome c oxidase were reduced slightly. By spectrophotometric analysis it was found that about one-half the amount of cytochrome c + c1 was eluted from mitochondria to cytosol after freeze-thawing of cells. The activities of succinate oxidation in mitochondria from freeze-thawed cells were restored to normal levels by the addition of cytochrome c. Freeze-thawing of isolated mitochondria did not induce deactivation of succinate oxidizing activities and succinate cytochrome c reductase, and no elution of cytochrome c was observed. It was concluded that the decreased respiratory activities of yeast cells by freezing of cells with liquid nitrogen can be attributed primarily to the elution of cytochrome c from mitochondria.     

Neomycin inhibits mastoparan-induced lactate dehydrogenase release,ethidium bromide accumulation,and intracellular fluorescein depletion in MDCK cells

Mechanisms of cell death induced by toxins probably involve one or more processes such as inhibition of protein synthesis and impairment of plasma membrane integrity leading to an increase in membrane permeability. Since one of the possible actions of mastoparan, a cationic tetradecapeptide from wasp venom, is to perturb membrane phospholipids resulting in an increase in membrane permeability, we studied the effect of chemically synthesized mastoparan on lactate dehydrogenase release (LDH), ethidium bromide and fluorescein accumulation in Madin-Darby Canine Kidney (MDCK) cultured cells. Our results demonstrated that mastoparan induced cytosolic LDH release, ethidium bromide accumulation and intracellular fluorescein depletion in MDCK cells. Neomycin, a polycationic aminoglycoside, interacts with anionic polyphosphoinositides at the plasma membrane. Since both mastoparan and neomycin are cationic peptides and react with the negatively charged membrane phospholipids, we studied the interaction of these two peptides on membrane permeability. Our results demonstrated that neomycin inhibited mastoparan-induced LDH release, ethidium bromide accumulation and intracellular fluorescein depletion.Abbreviations LDH Lactate Dehydrogenase - MDCK Madin Darby Canine Kidney     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. II. Stabilization of the membranes by polyamines

The effects of polyamines, spermine, spermidine and putrescine on the stabilization of the membrane organization of Escherichia coli cells were studied using measurements of fluorescence polarization change of extrinsic fluorescence probes in membrane specimens as a function of temperature. The effects of the polyamines on the restoration of the cell viability after freeze-thawing were also investigated. In logarithmic-phase membrane specimens, polyamines depressed the polarization ratio increase below the transition temperatures in a dose-dependent manner. The physiologically relevant concentration of polyamines repressed the ratios to the same levels as are obtained with the stationary-phase specimens. In the stationary-phase specimens, no effect of polyamines on repression of the polarization increase was observed. A preliminary exposure of logarithmic-phase cells to polyamines protected the cells from the reduction of viability in freeze-thawing. However, a considerably high concentration and a certain length of preincubation time were required in order to an effect to be exerted. These results indicate that the intracellular polyamines could stabilize the membrane organization of logarithmic-phase cells to the same extent as in the stationary-phase cell membranes. It is conjectured that the membrane stability which is mediated by the polyamines results in cellular resistance to freeze-thawing, as it is attained by increasing the growth phase of the cells.