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Exploration of SYTO® 9 Variabilities of Labeling Live Bacterial Cells and Analysis of MycoLight™ Fluorophore Alternatives for Live Bacterial Labeling and Viability Assessment

Abstract


The proprietary cell-permeable nucleic acid probe SYTO® 9 is commonly used in microbiology as either a standalone dye or in combination with propidium iodide (PI) in bacterial viability assays. The usefulness of SYTO® 9 in general overcomes the negative aspects such as high cytotoxicity, but cell-permeable fluorophores such as MycoLight™ Green JJ98 and MycoLight™ Red JJ94 provide alternatives with equivalent sensitivity and fewer downsides. MycoLight™ Green JJ98 in particular shows near-ideal results in combination with PI in bacterial viability assays, with highly similar spectra to SYTO® 9 and slightly improved performance with common instrumentation settings of both flow cytometers and fluorescence microscopes.

Keywords: Bacterial Viability, Nucleic Acid labeling, Cytotoxicity, Flow Cytometry, Fluorescent Imaging

 

Introduction


The small size of bacterial cells (typically 1 µm wide and up to 5 µm in length)[1] is a major factor contributing to the difficulty of fluorescent imaging of living bacterial cultures. This small size means that the genetic modification of bacteria to express GFP (a traditional method of labeling going back decades) can be difficult to impossible due to the size of GFP (25-30 kDa) and its derivatives[1]. Significantly smaller fluorescent probes such as MycoLight™ Green JJ98 or MycoLight™ Red JJ94, with molecular weights of ~.579 kDa and ~.739 kDa, respectively, are a more sizeable option. This vastly decreased size minimizes proteomic interference and damage to cell behavior. Additionally, the ease of labeling and minimized trauma of the simplified protocol allows wider applications to different bacterial strains, as many species are not suitable for genetic manipulation[3]

Many popular nucleic acid (NA) stains including SYTO® 9 (SYTO® 9 is the trademark of Invitrogen™) have an inhibitory effect on cell growth and viability. For endpoint assays, this is an insignificant drawback. However, this common label perturbs cellular processes, skewing results in live cell experiments. Although new bacterial imaging procedures are being continually introduced, such as live cell kinetic imaging and nanoparticle usage, fluorescence remains the dominant methodology in current bacteriological studies[1]. A growing trend within bacteriology is the usage of flow cytometry. As the instrumentation has become more affordable, the efficiency of the procedure has lent itself perfectly to the need for fast, hyper-accurate assays. Other factors include bleaching, variation in fluorescence intensity (binding affinity) in live vs. dead bacterial cells, variations in staining in gram-positive or gram-negative bacteria, and double-staining due to the compound binding to extracellular nucleic acids (eNA), can either increase background signal or obscure accurate readings when attempting multicolor imaging.

The use of cell-permeable NA probes such as SYTO® 9 in conjunction with membrane-impermeable probes such as propidium iodide (PI) is a widely used method in microbiology for testing bacterial cell viability[4]. Ideally, living bacteria will only take in the cell-permeable stains but not the impermeable one, whereas membrane-compromised dead bacteria will be readily accessible to both stains. Fluorescent imaging will then show clear differentiation of living and dead cells. In practice, this technique has three main issues, often due to limitations of the commonly used cell-permeable fluorophore.

PI Is Known to Label Both Living and Dead Adherent Bacterial Cells


In adherent cells (such as biofilms), bacterial viability has been shown[5] to be severely underestimated when assessed by these tests, due to a subpopulation of double-stained cells. Gram-negative E. coli have been shown to fluoresce 96% PI-positive, though 82% of cells were successfully cultivated after imaging, indicating that they were alive and viable. A common gram-positive species, Staphylococcus epidermidis, fluoresced 76% PI-positive, with a full 89% of those cells being subsequently cultivable. Extracellular nucleic acids are often present in adherent colonies and will readily be stained by impermeable probes, forming a false coating-indicating 'dead' cells-surrounding viable bacterial cells[5]. These skewed results may still provide rough information or demonstrate comparative trends when combined by validation via an alternative method, but the additional time and expense of doing so means that the initial, potentially significantly incorrect assessment is accepted as-is.

SYTO® 9 Has Different Binding Affinities for Live and Dead Cells in Gram-Positive and Gram-Negative Bacteria


Gram-positive bacteria such as Staphylococcus aureus show ideal equal signal intensity when staining live and dead cells. However, gram-negative bacteria such as Pseudomonas aeruginosa show 18x higher signal in dead cells over living ones. Even after PI counterstaining, the SYTO® 9 signal for dead cells is still markedly higher than for living bacteria[6]. This variation not only needs to be taken into account in regards to interpretation of viability assay results, but also for counterstain selection.

SYTO® 9 Suffers Severe Bleaching and Diminishing Signal


SYTO® 9 was observed to suffer near-immediate bleaching effects when observed using a laser confocal microscope and fluorescence microscope[6]. For testing small numbers of cells, photobleaching is not as much of a detractor, but for extensive imaging, for high-volume viability assessments, and for ease of use for the end-user, a wider imaging window without diminishing signal is of high importance.

To investigate these variabilities and others, comparisons of MycoLight™ Red JJ94 and SYTO® 9 effects on bacterial growth against a negative control were run. The effectiveness of a bacterial viability kit using MycoLight™ Green JJ98 and PI was tested with each of the bacterial cultures referenced above as well as others. Photostability and brightness of all of the MycoLight™ dyes were explored, and a full spectral comparison of MycoLight™ Green JJ98, SYTO® 9, and PI was made, including peak emission intensities at the popular excitation laser wavelength of 488 nm.

 

Materials and Methods


As with other NA probes, including SYTO® 9, the MycoLight™ dyes have minimal intrinsic fluorescence, which increases exponentially when bound to nucleic acids. MycoLight™ Red JJ94 preferentially labels gram-positive bacteria. MycoLight™ Green JJ98 labels both gram-positive and gram-negative bacteria. For NA probes, avoid using phosphate-containing buffers, as they hinder accurate measurement, due to the DNA phosphate backbone. This is particularly relevant when investigating bacterial cells, due to the variable nature of the DNA structure, such as the phosphorothioate modification, where a phosphate oxygen atom is replaced by a sulfur atom[7]. HEPES or other compatible buffers are recommended.

MycoLight™ Red JJ94 and SYTO® 9 Bacterial Cytotoxicity Comparison Methodology


Three healthy, logarithmically growing cultures of E. coli were supplemented with either MycoLight™ Red JJ94 or SYTO® 9 at a concentration of 2.5 µM for 10 minutes, or with a control of 1% DMSO. Both fluorophores use DMSO as a base solvent, so the low dose of DMSO was evaluated to be a fair control. Although DMSO can have a toxic effect, at 1% concentration the bacterial inhibition is minimal3. To measure changes in bacterial populations, optical density (OD) readings at 600 nm were taken using a spectrophotometer over a period of 7 hours. Higher readings demonstrate higher populations of bacteria indicating growth.

Bacterial Viability Kit Using MycoLight™ Green JJ98 Effectiveness Testing Protocol


A logarithmically growing culture of E. coli HST08 was diluted to an average density of ~107 cells/mL in a 0.85% NaCl solution and then divided equally. One culture was left untouched and served as a positive control of 100% living bacterial population. The other culture was treated with ethanol until the population was composed of 70% dead bacteria to serve as a comparison. One third of each of these cultures were removed and combined into a 3rd colony to represent a mixed population. MycoLight Green™ JJ98 and PI were mixed in equal volumes and added to the cultures at a ratio of 1:250 of dye to bacteria (approximately 4 µL of dye per 1 mL of bacterial solution). The solutions were mixed thoroughly and then incubated in the dark at room temperature for 15 minutes before imaging.

Fluorescent Imaging of MycoLight™ Green JJ98 and MycoLight™ Red JJ94 in Live Bacterial Cells


MycoLight™ Green JJ98 was added to a culture of logarithmically growing E. coli at a concentration of 5 µM, vortexed to mix, and then incubated in darkness for 30 minutes prior to imaging. MycoLight™ Red JJ94 was added to a logarithmically growing culture of Rhodococcus qingshengii at a concentration of 2.5 µM, vortexed to mix, and then incubated in darkness for 20 minutes prior to imaging.

Spectrum Comparison of MycoLight™ Green JJ98, SYTO® 9 and Propidium Iodide


The spectrum comparison of the excitation and emission curves of each dye, along with intensity calculations and added excitation sources and filters was generated using the AAT Bioquest Fluorescence Spectrum Viewer tool available here.

 

Results and Discussion


The aggregated results of the four areas of comparison and effectiveness of the MycoLight™ fluorophores are shown and discussed in the same order as they were listed in the methods section above.

MycoLight™ Red JJ94 and SYTO® 9 Bacterial Cytotoxicity Comparison


As shown in Figure 1, SYTO® 9 is toxic to cells over less than half of the experimental time, whereas MycoLight™ Red JJ94 showed very minor variation from the control population. MycoLight™ Red JJ94's compatibility with bacterial growth not only allows further characterization of bacteria, but allows for normal cellular behavior. Particularly in experiments investigating antibiotics and changes in activity over time, this compatibility is essential.


Optical density (OD600 nm) of E. coli LB cultures supplemented with 1% DMSO (control), MycoLight™ Red JJ94 or SYTO® 9 over time. SYTO® 9 inhibits bacterial growth substantially while MycoLight™ Red JJ94 is fully compatible with normal bacterial growth.


Bacterial Viability Kit Effectiveness Using MycoLight™ Green JJ98


The results of the bacterial viability tests using the 3 E. coli colonies were precisely indicative of the known ratios of live and dead bacterial cells, indicating that the potential problems of PI double-labeling were of minimal concern. By employing MycoLight™ Green JJ98 instead of SYTO® 9 as the chosen cell-permeable NA probe, the problems of conflicting fluorescence with PI were minimized.


Fluorescence images of E. coli HST08 stained with MycoLight™ Bacterial Viability Assay Kit. Live bacteria with intact cell membranes showed green fluorescence (Left), while 70% alcohol-killed dead bacteria (Right) with compromised membranes showed red fluorescence. Live and dead E. coli bacterial cells were also visualized in a mixed population (Middle). Images were taken using a Keyence fluorescence microscope equipped with FITC/Texas Red filter sets.


Fluorescent Images of MycoLight™ Green JJ98 and MycoLight™ Red JJ94 in Live Bacterial Cells


The sensitivity of MycoLight™ Green JJ98 is shown well in Figure 3, with the clear outlines of each bacterial cell indicating a good ratio of signal from background noise. Sensitive dyes are useful for quantifying samples which may be low-density, and the brightness is useful for applications which may require more elaborate adjustments and difficult cell lines requiring very low dye concentrations.

Gram-positive R. qingshengii was shown with a good signal-to-noise ratio when labeled by MycoLight™ Red JJ94. Other bacterial strains may require dye-loading protocol adjustments, but MycoLight™ Red JJ94 was compatible with all of the strains tested, with minor alterations based on instrumentation, cell density, and other factors.


Left: E. coli were stained with 5 µM of MycoLight™ Green JJ98 for 30 minutes and imaged with FITC channel with a Keyence fluorescence microscope at 20x. Right:Rhodococcus qingshengii was stained with 2.5 µM of MycoLight™ Red JJ94 for 20 minutes. Image was taken by a Keyence fluorescence microscope with a Cy5 filter set.


Spectrum Comparison of MycoLight™ Green JJ98, SYTO® 9 and PI


Excitation laser at 488 nm included, as well as the common Green Channel filter as referenced by Stocks et al.[8] in their paper on spectral overlap and DNA binding affinities of SYTO® 9 and PI. Excitation curves are shown as dotted lines and emission curves are shown as solid lines filled in with each fluorophore color.


Excitation and emission curves of MycoLight™ Green JJ98, SYTO® 9, and PI.


Table 1. Peak intensity percentage comparisons of fluorophores.
Fluorophore Type Peak (nm) Peak Intensity (Ex max) Peak Intensity (488 nm laser) Spillover (530/30 filter)
Propidium Iodide excitation 537      
Propidium Iodide emission 618 100% 55% 0%
MycoLight™ Green JJ98 excitation 482      
MycoLight™ Green JJ98 emission 512 100% 91% 51%
SYTO® 9 excitation 483      
SYTO® 9 emission 500 100% 87% 42%

Demonstrating a mild improvement of 4% fluorescence intensity, MycoLight™ Green JJ98 more importantly is more thoroughly within the wavelength range of the green filter commonly installed on flow cytometers and fluorescence microscopes. This ~10% improvement (51% versus SYTO® 9's 42%) represents a dependable, stable signal even in low-density samples. When MycoLight™ Green JJ98 is used as a standalone probe, this sensitivity allows the end-user to optimize dye concentrations for ideal signal levels. This flexibility allows for easier multiparameter imaging, where targets may be of varying levels of expression and mismatched dye intensities may swamp weaker signals.

 

Conclusion


SYTO® 9 is useful for applications as an NA probe in pure bacterial cultures and samples with low density (Applied and Environmental Microbiology citation, Sensitive determination), but its cytotoxicity, variable binding affinities for differing bacterial groups, and high bleaching necessitate the use of alternative cell-permeable fluorophores for bacterial viability assays and other experiments with live bacterial cells. For flow cytometers or fluorescence microscopes, MycoLight™ Green JJ98 is a useful alternative fluorophore for bacterial viability testing in combination with traditional PI counterstaining. MycoLight™ Green JJ98's improved excitation by the 488 laser and resistance to bleaching allow for better and easier imaging with a wider useful visualization window. For flow cytometry especially, steady signal is crucial for generating dependable counts. Bacterial viability tests using PI and permeable NA probes are the workhorse of microbiology, and an updated version that doesn't require extensive validation are key to preserving the efficacy of the test. For customized tests and experiments with gram-positive bacterial species such as Staphylococcus epidermidis referenced earlier in this paper, MycoLight™ Red JJ94 can be incubated long-term with no negative effects on bacterial growth, and generates excellent signal. Both of these aspects minimize hands-on time, and the red color emission spectrum allows pairing with common green fluorescent probes for multiparameter imaging.

 

References


  1. Schneider Johannes P. & Basler Marek (2016) Shedding light on biology of bacterial cells Phil. Trans. R. Soc. B, 371. https://doi.org/10.1098/rstb.2015.0499
  2. International Bureau of Weights and Measures (2006), The International System of Units (SI) (PDF) (8th ed.), p. 126, ISBN 92-822-2213-6 https://www.bipm.org/utils/common/pdf/si_brochure_8_en.pdf
  3. Petra Hildebrandt, Kristin Surmann , Manuela Gesell Salazar, Nicole Normann, Uwe Völker, Frank Schmidt. (2016) Alternative Fluorescent Labeling Strategies for Characterizing Gram-Positive Pathogenic Bacteria: Flow Cytometry Supported Counting, Sorting, and Proteome Analysis of Staphylococcus aureus Retrieved from Infected Host Cells. International Society for Advancement of Cytometry, Journal of Quantitative Cell Science, 89 (10), 932-940. https://doi.org/10.1002/cyto.a.22981
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  7. Wang, L., Jiang, S., Deng, Z., Dedon, P. C., & Chen, S. (2019). DNA phosphorothioate modification-a new multi-functional epigenetic system in bacteria. FEMS microbiology reviews, 43(2), 109–122. https://doi.org/10.1093/femsre/fuy036
  8. S. M. Stocks. (2004) Mechanism and use of the commercially available viability stain, BacLight. International Society for Advancement of Cytometry, Journal of Quantitative Cell Science, 61A (2), 189-195. https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.20069