Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.     

Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.     

Photodynamic inactivation and its repair in mycoplasmas

Photodynamic inactivation is the loss in viability observed when organic dye-treated cells are exposed to visible light and molecular oxygen. The photodynamic inactivation of mycoplasmas, the smallest free living cells, has been studied. Depending on the extent of inactivation in Acholeplasma laidlawii, photodynamic induced damage can be repaired if the irradiated cells are incubated in the dark in buffer. Analysis of the DNA of these cells shows that photodynamic inactivation induces single strand breaks which can be repaired during liquid holding. To examine possible damage to the cell membrane, glucose uptake was studied as a permeability measure. Neither acriflavine nor photodynamic inactivation had any measurable effect on membrane permeability.     

Ribonuclease from Alcaligenes eutrophus: Activation by heat treatment,partial purification and some properties

Summary Heat treatment (10 min at 65° C) of cells of Alcaligenes eutrophus, which resulted in the release of RNA degradadation products into the medium, was found to activate cellular ribonucleases. Two ribonucleases degrading yeast RNA were found, one localized in the periplasmic space and the other in the soluble fraction of the ribosomes. Compared to non-heated cells, in the heat-treated cells the former enzyme, the cell debris ribonuclease, was present at an eightfold increased specific activity, and the latter, the cytoplasmic ribonuclease, was present at a fourfold increased specific activity. This increase was due to the inactivation of a thermolabile inhibitor and to denaturation of part of the soluble protein during heat treatment. With respect to their properties the enzymes were similar; they had endonuclease activity and hydrolysed only RNA. They were heat-stable, resistent to trypsin, highly sensitive to a ribonuclease inhibitor isolated from the same bacterium and were partially inhibited by ATP and GTP. These properties provided a partial explanation for the mechanism of the release of RNA dagradation products from A. eutrophus cells after heat treatment.Abbreviations DNA Deoxyribonucleic acid - RNA Ribonucleic acid - tRNA Transfer RNA - DNase Deoxyribonuclease - RNase Ribonuclease - d-RNase debris RNase - c-RNase cytoplasmic RNase     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.     

The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex

Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin.     

Effects of Pressure-Induced Membrane Phase Transitions on Inactivation of HorA, an ATP-Dependent Multidrug Resistance Transporter, in Lactobacillus plantarum

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37°C and pressure treated at 15°C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15°C and pressure treated at 37°C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.     

Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease

The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates.     

Extracellular DNA and Type IV pili mediate surface attachment by <Emphasis Type="Italic">Acidovorax</Emphasis><Emphasis Type="Italic">temperans</Emphasis>

Extracellular DNA can play a structural role in the microbial environment. Here evidence is presented that an environmental isolate of Acidovorax temperans utilises extracellular DNA for intercellular and cell-surface attachment and that Type IV pili and electrostatic interactions play a role in this interaction. Preliminary attempts to isolate and purify extracellular polysaccharides from A. temperans strain CB2 yielded significant amounts of DNA raising the question of whether this molecule was present as a structural component in the extracellular matrix. The role of DNA in attachment was indicated by experiments in which the addition of DNase to liquid medium inhibited the attachment of Acidovorax to glass wool. A Tn5 insertional mutant, lacking Type IV pili, was unable to initiate attachment. Addition of DNase caused rapid detachment of bound cells, but no detachment occurred when proteinase, RNase or inactivated DNase were used. Addition of MgCl₂ also caused significant detachment, supporting the possible mechanistic role of electrostatic interactions in the attachment process. Although attachment was apparent in early to mid-log phase growth, surprisingly DNA was not detected in the culture supernatant until late stationary phase and coincided with an appreciable loss of cell viability. This suggests that during log-phase growth attachment is mediated by eDNA that is released in low quantities and/or is highly localised within the extracellular matrix and also that stationary phase DNA release through widespread cell lysis may be a separate and unrelated event.     

Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying

Summary The mechanism of inactivation of freeze-dried Lactobacillus bulgaricus during storage in maltodextrin under controlled humidity was investigated. Evidence is presented supporting the hypothesis that membrane damage occurs during storage. A study on the lipid composition of the cells by gas chromatography showed a decrease in the unsaturated and saturated fatty acid content of the cell. Further evidence indicating membrane damage includes a decrease in membrane bound proton-translocating ATPase activity.     

Biological Effect and Inactivation Mechanism of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Exposed to Pulsed Magnetic Field: Morphology,Membrane Permeability and Intracellular Contents

Biological effect and inactivation mechanism of Bacillus subtilis exposed to pulsed magnetic field (PMF) were investigated. Cell morphology, membrane permeability, release of intracellular proteins and damage of deoxyribonucleic acid (DNA) were monitored. The inactivation effect of PMF treatment on B. subtilis was enhanced with the increase in intensity and pulse number. Scanning electron microscopy (SEM) analysis showed that the PMF treated bacterial cells had rough surfaces and damaged cellular membranes. Flow cytometry measurements showed that the cell membrane permeability increased after the PMF treatment. Leakage of intracellular contents, measured as OD, significantly increased with the increase in PMF intensity and pulsed number. A maximum leakage of cytoplasmic contents was detected at intensity of 3.30 T and 30 pulses. Enterobacterial repetitive intergenic consensus - polymerase chain reaction (ERIC-PCR) fingerprint signaled that the DNA was fragmented after the PMF treatment at the intensity of 3.0 T with 30 pulses and 3.3 T with 10, 20 and 30 pulses. These findings may give clues to the mechanism of bacterial cell death due to the PMF treatment.     

Independent functions of viral protein and nucleic acid in growth of bacteriophage

1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.     

Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays

The aim of this study was to investigate the mechanism(s) of X-ray-mediated cell damage in comparison to mechanism(s) of organic hydroperoxide cytotoxicity and to find the main targets for the two different kinds of cell inactivation. Damage of Chinese hamster fibroblasts induced by tert-butyl hydroperoxide (t-BHP) or X-irradiation was measured by the colony-formation assay and the average single colony volume. DNA double-strand breaks (dsb) were determined by constant-field gel electrophoresis. The contents of peroxides, of SH-groups and the size of inactivated cells were tested for oxidative modifications.Oxidative damage of fibroblasts induced by t-BHP or by X-rays inhibits cell proliferation. Simultaneously, irradiation causes an increase of DNA dsb with the dose, while incubation with t-BHP yields only a very few DNA dsb. Neither chemically induced oxidation nor irradiation significantly changed the amount of membrane lipid peroxides. Oxidation with t-BHP but not irradiation leads to a loss of the membrane SH-groups and to an increase of cell diameter.The similar decrease of cell proliferation can be caused by DNA dsb without detectable membrane damage (X-radiation) as by membrane damage with nearly no DNA dsb (chemically induced oxidative stress).     

Resisting Arrest: Recovery from Checkpoint Arrest Through Dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signalling, and speculate on models for checkpoint maintenance and release.     

Resisting arrest: recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1

The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release.     

Tert-butyl-2(4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2-pyrrolidine-1-carboxylic ester displays novel cytotoxicity through reactive oxygen species-mediated oxidative damage in MCF-7 and MDA-MB-231 cells

The cytotoxicity of a new nitroxyl nitroxide radical, tert-butyl-2 (4,5-dihydrogen-4,4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2-pyrrolidine-1-carboxylic ester (L-NNP) was examined in MCF-7 and MDA-MB-231 cells. L-NNP treatment resulted in a significant growth inhibition in MCF-7 and MDA-MB-231 cells. Compared with control, 10, 30, and 50 μg/ml L-NNP treatments for 48 h induced significant cell and nuclei swelling, and organelle distension. The marked cell death was seen in a concentration- and time-dependant manner in L-NNP treated groups. The L-NNP treated group displayed a concentration-dependant increase in DNA double strand damage compared to the control and the 1 Gy γ-rays exposure groups. These results suggest that L-NNP could result in more lethal genotoxicity than 1 Gy γ-radiation. Based on mitochondrial alteration (membrane potential loss and SDH activity descend), DNA damage, an increase in MDA production, and GSH-PX inactivation, we predicate that L-NNP induces lipid oxidation and oxidative damage in MCF-7 and MDA-MB-231 cells. Since L-NNP initiated a significant increase in reactive oxygen species, which could largely be inhibited by NAC pretreatment, the overall data strongly suggest that the mechanism of cytotoxicity of L-NNP was its ability to act as a strong free radical, and significantly increase intracellular reactive oxygen species production. This led to intracellular oxidative damage, and antioxidant enzyme inactivation, resulting in cell death. We hypothesize that the greater cytotoxicity of L-NNP in MDA-MB-231 cells than in MCF-7 cells might be due to more ROS production in MDA-MB-231 cells, leading to more oxidative damage.     

Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.     

Assessment of cellular damage during cooling to −196 °C using radiochemical markers

The release of ten radiochemical markers from MRC-5 and CHO cells after cooling at various rates and thawing from temperatures in the range of 0 to ?196 °C was measured. Many of these radiochemicals had specific sites of attachment on or within the cell and the aim was to determine the effect of freeze-thaw stresses on various parts of the cell. Cell death during cooling and thawing was, in most instances, accompanied by osmotic damage and loss of cytoplasmic constituents. Significant damage to the cell membrane occurred only after the cell was already dead and was related to the disruption of cells killed at higher temperatures and to osmotic stress during rewarming. The release of cations and other cytoplasmic markers was correlated to cell shrinkage and dehydration. The data were used to assess the relative effects of some of the proposed damaging factors in freeze-thaw injury (thermal shock, ice damage, dilution shock, etc.). CHO cells showed a much higher survival rate and release of cations after fast cooling than MRC-5 cells. This, and additional circumstantial information, indicated that CHO cells survived freeze-thaw cycles better than MRC-5 cells because they are able to dehydrate more readily, even at fast cooling rates.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.