FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein‐AM], 5‐chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2′,7′‐dichlorofluorescein diacetate [H₂DCFDA]; and two membrane probes: bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and SYTOX‐Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold‐water, 26 temperate, and four warm‐water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein‐AM and H₂DCFDA (P < 0.001). Of the two membrane probes, DIBAC₄(3) stained rhodophytes and euglenophytes much better than SYTOX‐Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC₄(3), and SYTOX‐Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.     

FLOW CYTOMETRIC ANALYSES OF VIRAL INFECTION IN TWO MARINE PHYTOPLANKTON SPECIES, MICROMONAS PUSILLA (PRASINOPHYCEAE) AND PHAEOCYSTIS POUCHETII (PRYMNESIOPHYCEAE)

Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry. Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P. pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures. DNA analyses were performed using the nucleic acid–specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis. The present study provides insight into basic virus–algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

Cell death in individual freshwater phytoplankton species: relationships with population dynamics and environmental factors

ABSTRACT

Understanding and predicting changes in phytoplankton populations requires knowledge of losses due not only to sedimentation and grazing, but also to intrinsic processes (here, collectively termed ‘cell death’). Cell death is poorly understood, especially in freshwater phytoplankton, but experiments in culture often suggest involvement of abiotic factors (e.g. temperature, light, nutrients). The occurrence of cell death was examined in a simple, natural environment: a small, well-mixed, temperate, urban pond during a period of phytoplankton growth, from mid-July to mid-November. Abundances of 18 phytoplankton taxa were measured weekly and fluorescence microscopy and staining was used to detect dead cells (using SYTOX which measures loss of membrane integrity) and cells undergoing cell death (using Annexin-V, which measures lipid inversions of membranes, an early signal of cell death). Dead and dying cells occurred in most phytoplankton taxa, but incidence and timing varied considerably, e.g. species like the chlorophyte Ankistrodesmus spiralis showed 20–30% of cells staining with SYTOX and Annexin in late autumn when the population was decreasing, while the dinoflagellate Peridinium sp. showed staining of up to 50% of cells with STYOX throughout the period, and the cyanobacterium Microcystis aeruginosa occasionally showed staining of 100% of cells with SYTOX. Overall, there was some association between cell death staining and growth phase with 10–15% of the total community showing SYTOX and Annexin staining in late autumn, when most populations were declining. Cell death could not be correlated with thresholds or rapid changes in abiotic conditions (e.g. temperature, irradiance) or with indicators of nutrient limitation (e.g. N:P ratios). While abiotic factors have been clearly implicated in cell death within unialgal culture experiments, in natural freshwater ecosystems interactions between biotic factors, such as pathogens or allelopathy, may play greater roles in losses related to cell death and be distinct for different taxa.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.     

Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide     

Genetic diversity of Cladophora oligocladoidea forming a bloom in the coastal area of Korea

Green algal blooms by Cladophora species are primarily reported in freshwater and coastal regions and cause severe ecological problems. A taxonomic report of the geographic distribution of Cladophora blooms is essential to explore the cause and impact of macroalgal blooms and for eco-physiological studies. The identification of blooming species is necessary for monitoring and controlling algal blooms. Genetic information of DNA sequences is useful for identifying species in the genus Cladophora. In the coastal area of Sangrok, Korea, a large-scale Cladophora bloom was reported for the first time in September 2015. In the present study, we identified the taxonomic entity of Cladophora oligocladoidea (Ulvophyceae, Cladophorales) in Korea. We report for the first time a green algal bloom by this species globally. This is the second report about genetic diversity of C. oligocladoidea since the species was established in Japan. Four ribotypes in the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA represent the genetic diversity of Korean C. oligocladoidea. The results from this taxonomic report and our analysis of the green algal bloom of Korean C. oligocladoidea can provide valuable data to understand the geographic distribution and the genetic diversity of the species.     

Green Autofluorescence in Dinoflagellates, Diatoms, and Other Microalgae and Its Implications for Vital Staining and Morphological Studies

Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.     

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5?μmol/l SYTOX Green for 40?min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6?h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617?mg/l 1-pentanol (95?% confidence limits 4,670-9,327) and 12,818?mg/l 1-pentanol (95?% confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.     

Direct cell penetration of the antifungal peptide,MMGP1, in Candida albicans

An antifungal peptide, MMGP1, was recently identified from marine metagenome. The mechanism of cellular internalization of this peptide in Candida albicans was studied using fluorescein 5–isothiocynate (Sigma, California, USA) labeling followed by fluorescence microscopy and flow cytometry analyses. The peptide could enter C. albicans cells even at 4 °C, where all energy‐dependent transport mechanisms are blocked. In addition, the peptide internalization was not affected by the endocytic inhibitor, sodium azide. The kinetic study has shown that the peptide was initially localized on cell membrane and subsequently internalized into cytosol. The MMGP1 treatment exhibited time‐dependent cytotoxicity in C. albicans as evidenced by SYTOX Green (Molecular Probes Inc., Eugene, Oreg) uptake. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.     

Discrimination of marine algal taxonomic groups using delayed fluorescence spectroscopy

We present a method for in situ monitoring of phytoplankton composition changes in a marine environment. The method is based on delayed fluorescence excitation spectra analyzed with CHEMTAX software, which is generally used for determination of phytoplankton communities with HPLC pigment data. Delayed fluorescence (DF) is a photosynthetic parameter that can only be measured in living cells. Algal DF excitation spectra are group-specific, based on their composition of photosynthetic pigments.DF excitation spectra of 14 marine algal species from different families were measured with a delayed fluorescence spectrometer. Mixtures were prepared from northern Adriatic algal species representing six taxonomic groups: dinoflagellates (Prorocentrum minimum), diatoms (Skeletonema costatum), cyanobacteria (Synechococcus sp.), prasinophytes (Micromonas sp.), cryptophytes (Teleaulax sp.), and prymnesiophytes (Isochrysis galbana). The DF excitation spectra (DFS) and HPLC pigment compositions of the mixtures were analyzed with CHEMTAX software. The prediction power of DFS–CHEMTAX method was comparable to HPLC–CHEMTAX.     

Spectroscopic and equilibrium dialysis studies of the binding of acridine and 10-methylacridine to DNA in the pH range 4–10

For a better understanding of the interactions between DNA and various acridine dyes, the binding of acridine (Acr) and 10-methylacridine (MeAcr) to native and heat-denatured calf-thymus DNA was studied in the pH range between 4 and 10 by the equilibrium dialysis and spectroscopic methods. The binding between DNA and the dyes was predominantly electrostatic. The amount of bound Acr varied with pH, mixing ratio (P/D), and the DNA conformation, and reached a maximum at pH = 5.2. The amount of bound MeAcr was constant in the pH range 5–9. The apparent binding constants of these dyes were obtained at some pH, and they were found to vary with P/D for native DNA-dye complexes. The pure spectra of bound Acr and MeAcr could be unmasked. The bound Spectra were bathochromic and hypochromic relative to the spectra of free days. Acridine bound to native DNA was shown to undergo structural changes from an acridiniumlike to a neutral acridinelike form as the pH of solutions was varied. The pK value for the transition between the bound forms was evaluated to be 7.3. The extrinsic Cotton effects of the bound dyes were observed in the DNA-Acr and-MeAcr complexes and varied with pH and the conformation of DNA.     

Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain

We have developed a simple, sensitive, fluorescence microplate-based assay for tumor necrosis factor (TNF) biological activity. The assay employs SYTOX Green nucleic acid stain to detect TNF-induced cell necrosis in actinomycin D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymmetrical cyanine dye that is excluded from live cells but can readily penetrate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is monitored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25-500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, using mouse fibroblast-derived WEHI 164, WEHI 13var, and L929 cell lines. The procedure can also be used to detect agents that modulate TNF activity. We demonstrated complete inhibition of rhTNF-alpha using monoclonal anti-human TNF-alpha antibody and determined that approximately 20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-alpha in these cell lines. Reagents are added in a single step, followed by a 6- to 8-h incubation period, during which the cytokine exhibits its effects. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures.     

Green autofluorescence in dinoflagellates, diatoms, and other microalgae and its implications for vital staining and morphological studies

Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP. USING TYRAMIDE SIGNAL AMPLIFICATION–FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY1

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA‐FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA‐FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.     

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp

Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus more costly and time‐consuming, or using only one fluorescent dye, thus less accurate. Methods and Results: A red primary fluorescence (autofluorescence) of the hyphal cell walls and septa of Rhizoctonia spp. with green excitation is evidenced in Rhizoctonia spp. This property is exploited and combined for the first time with a conventional DAPI fluorescence to accurately determine the nuclear condition of Rhizoctonia. This bi‐fluorescence imaging strategy depicted the nuclear condition in Rhizoctonia spp. more accurately than the conventional DAPI fluorescence used alone and was validated against isolates previously genotyped by DNA sequencing. Conclusions: We demonstrated that the bi‐fluorescence imaging strategy was safe, accurate and simple to perform and interpret. Significance and Impact of the Study: The developed bi‐fluorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia strains. Its simplicity is a key advantage when there are numerous cultures to be examined.     

Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.