Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation

Cultures of the copiotrophic bacterium Micrococcus luteus were stored in spent growth medium for an extended period of time following batch culture. After an initial decrease, the total cell counts remained constant at approximately 60 to 70% of the counts at the beginning of storage. The level of viability, as judged by plate counts, decreased to less than 0.05%, while respiration and the ability to accumulate the lipophilic cation rhodamine 123 decreased to undetectable levels. However, using penicillin pretreatment (to remove viable cells) and flow cytometry and by monitoring both the total and viable counts, we found that at least 50% of the cells in populations of 75-day-old cultures were not dead but were dormant. Resuscitation in liquid medium was accompanied by the appearance of a population of larger cells, which could accumulate rhodamine 123 and reduce the dye 5-cyano-2,3-ditolyl tetrazolium chloride to a fluorescent formazan, while a similar fraction of the population was converted to colony-forming, viable cells. We surmise that dormancy may be far more common than death in starving microbial cultures.     

Rapid assessment of bacterial viability by flow cytometry

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper     

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Analysis of the Physiological Heterogeneity within Starved Cultures of Micrococcus luteus by Flow Cytometry and Cell Sorting

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). We used flow cytometry and cell sorting to study populations of bacteria that had been starved for 5 months. These cells could be stained by the fluorescent lipophilic cation rhodamine 123, but such staining was almost independent of metabolically generated energy in that it was not affected by uncouplers. Two populations could be distinguished, one with a lower degree of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accumulating rhodamine 123 (and fluorescing in region B) and of larger cells exhibiting a yet-greater degree of fluorescence (region C). These more fluorescent cells accounted for as much as 50% of the total population, under conditions in which the viable and total counts were constant. Thus, metabolic resuscitation of at least one-half of the cells takes place under conditions in which cryptic growth cannot play any role. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C and that the extent of rhodamine staining under conditions of starvation therefore reflects the physiological state of the cells. Physical separation of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken from growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant (resuscitable) cells are concentrated in regions B and C.     

Membrane hyperpolarisation by valinomycin and its limitations for bacterial viability assessment using rhodamine 123 and flow cytometry

Abstract The ionophore, valinomycin, was investigated as a possible means of bacterial viability assessment using the fluorescent membrane potential dye rhodamine 123. Membrane hyperpolarisation in Escherichia coli , Pseudomonas fluorescent , Enterobacter aerogenes and Arthrobacter globiformis was examined during exponential growth and during stress by brief starvation in a high sodium, low potassium buffer using flow cytometric analysis of rhodamine 123 uptake. Dye uptake was variable both between species and amongst cells from the same culture. Exponential phase cells showed no increase in dye uptake due to valinomycin treatment. Stressed P. fluorescens cells responded to valinomycin treatment by increased dye uptake, while stressed E. coli and A. globiformis cells showed no response. Approximately 50% of stressed Eb. aerogenes cells responded to valinomycin. The results demonstrate the limitations of rhodamine dye for viability analysing the viability of diverse bacterial communities and underline the degree of cell heterogeneity in batch cultures.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Active cell membrane mechanisms involved in the exclusion of RH 123 allow distinction between normal and tumoral cells

Human cell lines derived from three epithelial carcinomas (CaSki, HeLa, SiHa), one B lymphoma (BL60), one promyelocytic (HL60), one monocytic (U937) leukemia, one chronic myelogenous leukemia (sensitive K562S; multichemoresistant K562R) and normal human skin fibroblasts were compared for their capacity of staining with rhodamine 123 (Rh 123) and their kinetics of dye exclusion. Cells were exposed for 30 min to 10 g/ml of Rh 123 in culture medium; fluorescence intensity was measured by flow cytometry immediately or 1, 2, 3 and 4 h after staining. The highest fluorescence intensity was observed in carcinoma cell lines; there was no incorporation in multichemoresistant K562R cells. Exclusion of Rh 123 was evaluated from 0 to 4 h, both by flow cytometry and by fluorimetry. Fluorescence intensity measured by flow cytometry decreased slightly in carcinoma and leukemia cells and rapidly in fibroblasts. In all cell lines Rh 123 exclusion was inhibited by 40 mol/L verapamil and 5 mmol/L probenecid. Thus, incorporation and exclusion of Rh 123 allows distinction between normal and tumoral cells; moreover, inhibition of exclusion by verapamil and probenecid favors the involvement of active cell membrane mechanisms in the exclusion process.Abbreviations PBS phosphate-buffered saline - Rh 123 rhodamine 123     

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.     

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Bac light bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation between the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcvs and Pseudomonas tested.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.     

Proliferation of mitochondria during the cell cycle of the human cell line (HL-60)

Using rhodamine 123 to stain mitochondria of the human cell line HL-60, we have followed their increase over the cell cycle by flow cytometry. A near-linear synthesis of mitochondrial mass was shown to occur over the cell cycle. A comparison with the cell's DNA synthesis pattern obtained by the same technique established a common time-base. The mitochondrial synthesis curve changes with culture age. As a control, thd dye was tested for its binding specificity and for its use to resolve mitochondria microscopically. Its stoichiometric range was established and, above 0.25 microgram/ml, it was shown to reduce growth rate and cell viability in culture.     

Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry

A triple staining procedure was developed to evaluate bull spermatozoa using flow cytometry. Flow cytometric estimates of cell viability, measured by propidium iodide (PI) exclusion, and acrosomal integrity, measured by Pisum sativum agglutinin (PSA) binding acrosomal contents, were equivalent to estimates made by using standard laboratory assays. Mitochondrial function, measured by rhodamine 123 (R123) fluorescence, was depressed by the mitochondrial inhibitors rotenone (64%) or monensin (52%), establishing that mitochondrial damage can be detected. Dilauroylphosphatidylcholine (PC12) or lysophosphatidylcholine (LPC) was used to destabilize sperm membranes. When challenged with 15-30 microM PC12, selective exposure of PSA binding sites occurred without induction of PI uptake or loss of R123 staining. However, PC12 concentrations greater than 60 microM resulted in a loss of R123 fluorescence intensity. In contrast, greater than 1200 microM LPC was required to expose PSA binding sites, which also resulted in PI uptake. By using flow cytometry, these three stains in combination can be used to correlate three different features simultaneously on individual spermatozoa and assay thousands of cells per sample without extensive preparation.     

Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol.

The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

Viability of starved Pasteurella piscicida in seawater monitored by flow cytometry and the effect of antibiotics on its resuscitation

The viability of starved Pasteurella piscicida cells in seawater was analysed by flow cytometry using two fluorescent dyes that stain only living cells. The fluorescent histograms using rhodamine 123 showed that, during the total experimental period, a considerable proportion of cells were able to incorporate this fluorochrome. In addition, the assays employing propidium iodide as dye, demonstrated that the Past. piscicida cells remained viable, maintaining their cellular integrity. Chloramphenicol or ampicillin totally inhibited the resuscitation of non-culturable cells when they were added 8 and 24 h after the incorporation of fresh medium to the microcosms, but had practically no effect after 48 h when cells were fully resuscitated, indicating that active protein and peptidoglycan synthesis were ongoing during all stages of resuscitation.     

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.     

Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion.     

Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation

The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low-power helium-neon laser source for flow cytometry of DNA content in ethanol-fixed or detergent-permeabilized cells. Cells in near-isotonic medium are stained with 10-30 microM dye, and fluorescence excited at 633 nm is measured at wavelengths above 665 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 microM Hoechst 33342 is added to cells simultaneously with a red-excited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.