Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10⁶ cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

The Use of Fluorescein Diacetate and Ethidium Bromide as a Viability Stain for Isolated Islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATF content A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol

A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC₍₄₎3, was used to quantify the total cell population and further divide this population into: (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads.     

Assessment of Cell Viability in Intact Glandular Tissue in <Emphasis Type="Italic">Chironomus ramosus</Emphasis> using Dye-exclusion and Colorimetric Assays

Conventionally, dye-exclusion test for determining cell viability has been restricted only for cells in suspension in tissue culture. In this paper, salivary gland of Chironomus has been proposed as a simple tissue model system where dye-exclusion test can be reliably employed for the intact gland. We have compared suitability of commonly used vital dyes and nigrosin was found suitable for the salivary gland cells. Biochemical tests using tetrazolium salts are also commonly used for determining quantitative indices of cell viability in metabolically active cells. Ours is the first attempt to extend the same technique for the whole tissue. We standardized the conditions and prepared a protocol for MTT-based colorimetric assay suitable for the salivary gland of Chironomus. A strong correlation (r² = 0.9893) was obtained where increasing O.D. correlated linearly with the number of live glands. We concluded that nigrosin dye-exclusion and MTT metabolic inclusion assays are suitable methods for the viability test of metabolically active intact salivary gland of Chironomus which can serve as a potential model for the assessment of cytotoxicity in future.     

The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.     

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     

A Novel Staining Protocol for Multiparameter Assessment of Cell Heterogeneity in Phormidium Populations (Cyanobacteria) Employing Fluorescent Dyes

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, ‘dead cell’ nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead.     

Short-term physiologic effects of mechanical flow sorting and the Becton-Dickinson cell concentrator in cultures of the marine phytoflagellata Emiliania huxleyi and Micromonas pusilla.

BACKGROUND: In contrast to large, high-efficiency cytometers, mechanically sorting benchtop instruments provide a feasible alternative for shipboard cell sorting of oceanic microbial communities. However, sorting efficiency of these instruments is constrained by their maximum sorting rate of approximately 300 cells/s and by constant dilution of sorted samples by sheath flow. These factors often render too low sorted cell concentrations for postsorting experiments of oceanic phytoplankton populations of low natural abundance. A Cell Concentrator module has been marketed to overcome these dilution effects. Postsorting experiments also have to consider potential physiologic effects of cell sorting. Short-term physiologic effects on phytoplankton photosynthetic rates and esterase activities by mechanical flow sorting and cell concentration and on the efficiency of the Cell Concentrator module are evaluated. METHODS: Increasing numbers of the oceanic phytoflagellates Micromonas pusilla and Emiliania huxleyi were sorted and concentrated, and recovery in the concentrated samples was compared with the sorted-only samples (concentration rate) and the total number of sorted cells (recovery rate). Photosynthetic rates and metabolic activities of sorted and sorted/concentrated cells were compared with unsorted cells. Photosynthetic rates were estimated from 14CO2 uptake experiments and metabolic activity quantified cytometrically after cleavage of fluorescein diacetate. RESULTS: Irrespective of the total number of sorted cells, concentration rates between concentrated and sorted cells remained mostly below 10-fold and did not increase with the number of concentrated cells. Recovery rates in the concentrated samples amounted to fewer than 10% of total sorted cells, except for forceful resuspension attempts in the Concentrator insert (25-44%), which might be unsuitable for delicate species. Cell sorting resulted in a 24-49% decrease in photosynthetic rates. Metabolic activity within metabolically active cells was not affected by cell sorting, but the share of metabolically active cells decreased by 32-37%. Cell concentration did not affect metabolic activity or the fraction of active cells but did increase photosynthetic rate several-fold compared with unsorted cells. CONCLUSION: Low recovery of concentrated cells, probably due to cell adhesion to the filer bottom of the Concentrator insert, render the Cell Concentrator of limited use to overcome dilution problems of mechanical flow sorting, particularly when results are extrapolated to natural, low-abundance populations. Severe changes in photosynthetic rates also render concentrated cells suspicious for subsequent physiologic experiments. Mechanical sorting alone also exhibited significant physiologic effects on sorted cells, some of which might not be temporary. Comparable effects between mechanical sorting and droplet sorting as previously reported confirm that physiologic effects might be caused predominantly by shear stress and laser exposure during cytometric analysis rather than the sorting process. Sufficient recovery time must be allowed before postsorting experiments, but potential changes in cell physiology from the natural conditions during postsorting recovery must be considered.     

Inducible gene expression by nonculturable bacteria in milk after pasteurization

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.     

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE⁺ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.     

Plant derived coumestrol phytochemical targets human skin carcinoma cells by inducing mitochondrial-mediated apoptosis,cell cycle arrest,inhibition of cell migration and invasion and modulation of m-TOR/PI3K/AKT signalling pathway

The current study was undertaken to investigate anticancer activity of coumestrol phytoestrogen against human skin cancer. MTT assay was performed for cell viability assessment and clonogenic assay for cell colony formation assessment. Apoptosis was analysed by Annexin V/FITC staining, AO/EB staining and western blotting assays. Effects on the m-TOR/PI3K/AKT signalling pathway were investigated by western blotting. Results indicated that coumestrol induced significant toxicity in human skin cancer cells in contrast to mouse skin cancer cells. The proliferation rate in normal skin cells remained almost intact. Annexin V-FITC and AO/EB staining assays indicated coumestrol induced cytotoxicity in skin cancer cells is mediated through apoptosis stimulation. The apoptosis in skin cancer cells was mediated through caspase-activation. Cell migration and invasion was inhibited by coumestrol in human skin cancer cells via inhibition of MMP-2 and MMP-9 expressions. Moreover, m-TOR/PI3K/AKT signalling pathway in SKEM-5 cells was blocked by coumestrol.     

Cell-specific basolateral membrane sorting of the human liver Na(+)-dependent bile acid cotransporter

The human Na(+)-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 microM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells (P < 0.05). Brefeldin A (BFA; 1-2 microM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 microM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20 degrees C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.     

Basolateral Invasion and Trafficking of Campylobacter jejuni in Polarized Epithelial Cells

Campylobacter jejuni is a major cause of bacterial diarrheal disease. Most enteropathogenic bacteria including C. jejuni can invade cultured eukaryotic cells via an actin- and/or microtubule-dependent and an energy-consuming uptake process. Recently, we identified a novel highly efficient C. jejuni invasion pathway that involves bacterial migration into the subcellular space of non-polarized epithelial cells (termed subvasion) followed by invasion from the cell basis. Here we report cellular requirements of this entry mechanism and the subsequent intracellular trafficking route of C. jejuni in polarized islands of Caco-2 intestinal epithelial cells. Advanced microscopy on infected cells revealed that C. jejuni invades the polarized intestinal cells via the subcellular invasion pathway. Remarkably, invasion was not blocked by the inhibitors of microtubule dynamics colchicine or paclitaxel, and was even enhanced after disruption of host cell actin filaments by cytochalasin D. Invasion also continued after dinitrophenol-induced cellular depletion of ATP, whereas this compound effectively inhibited the uptake of invasive Escherichia coli. Confocal microscopy demonstrated that intracellular C. jejuni resided in membrane-bound CD63-positive cellular compartments for up to 24 h. Establishment of a novel luciferase reporter-based bacterial viability assay, developed to overcome the limitations of the classical bacterial recovery assay, demonstrated that a subset of C. jejuni survived intracellularly for up to 48 h. Taken together, our results indicate that C. jejuni is able to actively invade polarized intestinal epithelial cells via a novel actin- and microtubule-independent mechanism and remains metabolically active in the intracellular niche for up to 48 hours.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.     

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.     

Slashing the timelines: Opting to generate high‐titer clonal lines faster via viability‐based single cell sorting

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 ? 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry‐based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:198–207, 2016     

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.