1. Overview

1.1 Purpose and scope

This SOP is intended for the standardized application of SYTO 9 and PI combined live/dead staining in microbial viability evaluation. It is suitable for suspended bacterial samples such as Escherichia coli and Staphylococcus aureus, and can also be extended to membrane integrity stratification analysis in biofilm samples, complex matrix samples, and yeast/fungal samples. This method is appropriate for antimicrobial evaluation, comparison of disinfection treatments, antibacterial material research, and rapid status screening of environmental or food samples, but it should not be used alone as a substitute for culture-based, metabolic, or proliferation-based assays for absolute viability determination.

1.2 Principle

(1) SYTO 9 can enter membrane-intact cells and also penetrate some membrane-damaged cells. It is therefore better regarded as a marker of overall cellular nucleic acid signal or the background of the membrane-intact population, rather than a strictly live-cell-specific dye.

(2) PI mainly enters cells after membrane permeability increases or membrane structure is disrupted and then binds nucleic acids. It is therefore more suitable as an indicator of the membrane-damaged population rather than being simply equated with irreversible death.

(3) During dual staining, both SYTO 9 and PI bind nucleic acids. After PI enters damaged cells, it usually weakens or displaces the local signal dominance of SYTO 9 on nucleic acids, so membrane-damaged cells often appear red-dominant. Green-dominant signals are more consistent with the membrane-intact population, red-dominant signals are more consistent with the membrane-damaged population, and mixed red-green signals more often indicate transitional damage populations or heterogeneous subpopulations.

2. Materials and reagents

2.1 Core dyes

(1) SYTO 9 dye component A.

(2) PI dye component B.

2.2 Basic reagents

(1) Nutrient broth medium.

(2) 0.85% NaCl solution or an appropriate buffer.

(3) 70% isopropanol, for preparation of dead-cell controls.

(4) Sterile dH2O.

(5) Optional: BSA, for reducing nonspecific adsorption in low-concentration samples.

2.3 Instruments and consumables

(1) Benchtop centrifuge capable of reaching 10,000 × g.

(2) Fluorescence microscope.

(3) Fluorometer.

(4) Fluorescence microplate reader.

(5) Glass or acrylic cuvettes with a 1 cm path length.

(6) 16 × 125 mm borosilicate glass culture tubes.

(7) Flat-bottom 96-well plates.

(8) Microcentrifuge tubes, 30–40 ml centrifuge tubes, glass slides, and 18 mm square coverslips.

3. Preparation of standard live-cell and dead-cell control samples

3.1 Bacterial culture

(1) Culture 30 ml of Escherichia coli or Staphylococcus aureus in nutrient broth until the late logarithmic growth phase.

(2) Centrifuge 25 ml of the bacterial culture at 10,000 × g for 10–15 min to concentrate the cells.

(3) Remove the supernatant and resuspend the pellet in 2 ml of 0.85% NaCl or an appropriate buffer.

3.2 Preparation of live-cell and dead-cell controls

(1) Transfer 1 ml of the resuspended bacterial suspension into a 30–40 ml centrifuge tube containing 20 ml of 0.85% NaCl or an appropriate buffer as the live-cell control.

(2) Transfer another 1 ml of the resuspended bacterial suspension into a 30–40 ml centrifuge tube containing 20 ml of 70% isopropanol as the dead-cell control.

(3) Incubate both tubes at room temperature for 1 h, inverting and mixing every 15 min.

(4) Centrifuge both samples at 10,000 × g for 10–15 min.

(5) Resuspend the pellets in 20 ml of 0.85% NaCl or an appropriate buffer and centrifuge again under the same conditions to remove residual isopropanol.

(6) Finally, resuspend the two samples separately in 10 ml of 0.85% NaCl or an appropriate buffer.

3.3 Concentration standardization

(1) Measure the optical density at 670 nm (OD670) using 3 ml of each bacterial suspension in a glass or acrylic cuvette with a 1 cm path length.

(2) Before staining, the live-cell group, dead-cell group, and mixed groups should be adjusted to the same cell concentration or a similar OD level to avoid interference from differences in total cell number during subsequent red/green signal comparison.

(3) If a methodological calibration curve is required, mixed samples with different live/dead proportions may be further prepared. This step is particularly valuable during assay establishment for new strains, initial instrument optimization, or validation of a new dye lot.

4. Staining system and control setup

4.1 Control setup

At least the following five categories of controls are recommended for each experimental batch:

(1) Blank matrix control, for identifying autofluorescence and particulate background.

(2) SYTO 9 single-stain control, for assessing green background and spectral spillover.

(3) PI single-stain control, for assessing red background and spectral spillover.

(4) Live-cell dual-stain control, for establishing the green-dominant region.

(5) Dead-cell dual-stain control, for establishing the red-dominant region.

For complex samples, these controls should not be omitted. Among them, the single-stain controls and blank control are particularly important for threshold setting, spillover assessment, and identification of particle interference.

4.2 General staining requirements

(1) SYTO 9 and PI should be prepared at a fixed ratio. The ratio should not be arbitrarily changed during the experiment simply to obtain a brighter signal in one channel, because ratio changes directly affect signal dominance after dual staining.

(2) The staining procedure should be performed under light-protected conditions.

(3) Incubation for 15 min at room temperature in the dark may be used as the default starting condition. If the target strain, sample density, or detection platform changes, optimization should be performed through pilot experiments. Incubation that is too short may underestimate the membrane-damaged population, whereas incubation that is too long may increase background, diffusion, and signal drift.

(4) After staining, detection should be completed within a fixed time window as far as possible to avoid inter-sample variation caused by differences in holding time.

5. Workflow A: Fluorescence microscopy detection

5.1 Applicable scenarios

This workflow is suitable for suspended bacteria, biofilms, adherent samples, and analysis of local damage patterns. Microscopic detection is more suitable for observing morphology, aggregation state, spatial distribution, and surface-to-depth heterogeneity.

5.2 Procedure

(1) In a microcentrifuge tube, combine equal volumes of component A (SYTO 9) and component B (PI), and mix well.

(2) Add 3 µl of the premixed dye solution to each 1 ml bacterial suspension. Under this recommended dilution condition, the final DMSO content in the staining working solution is approximately 0.3%.

(3) Mix gently and incubate for 15 min at room temperature in the dark.

(4) Pipette 5 µl of the stained bacterial suspension onto a glass slide and cover with an 18 mm square coverslip.

(5) Observe using filter sets suitable for the green channel, red channel, and dual-channel imaging. If needed, the green and red channels may be acquired separately.

(6) Exposure time, gain, and acquisition parameters should be fixed across groups. For biofilm or adherent samples, multiple fields of view or different focal/depth positions should be recorded rather than relying on only a single representative region.

6. Workflow B: Fluorometric detection

6.1 Applicable scenarios

This workflow is suitable for establishing the relationship between the green/red fluorescence ratio and the proportion of live cells, and for batch comparison of membrane integrity changes before and after treatment.

6.2 Sample concentration adjustment

(1) Adjust live and dead Escherichia coli suspensions to 1 × 10^8 bacteria/ml, corresponding to approximately 0.03 OD670.

(2) Adjust live and dead Staphylococcus aureus suspensions to 1 × 10^7 bacteria/ml, corresponding to approximately 0.15 OD670.

(3) For fluorometric detection, the concentration of Staphylococcus aureus suspensions is usually 10-fold lower than that of Escherichia coli suspensions.

6.3 Preparation of live/dead ratio samples

Prepare five bacterial suspensions with different live/dead ratios in 1 cm glass, acrylic, or quartz fluorescence cuvettes. The total volume of each sample is 3 ml.

6.4 Staining procedure

(1) In a microcentrifuge tube, add 30 µl of component A (SYTO 9) and 30 µl of component B (PI), and mix well to prepare the dye premix.

(2) Add 9 µl of the dye premix to each bacterial suspension with a different live/dead ratio.

(3) Gently pipette several times to mix and incubate for 15 min at room temperature in the dark.

6.5 Fluorescence measurement and data analysis

(1) Measure the fluorescence emission spectrum of each bacterial suspension using a fluorometer, with excitation at 470 nm and an emission scan range of 490–700 nm.

(2) Integrate the green fluorescence over 510–540 nm and the red fluorescence over 620–650 nm.

(3) Calculate the green/red fluorescence ratio: RatioG/R = Fcell,em1 / Fcell,em2.

(4) Plot the proportion of live cells on the x-axis and RatioG/R on the y-axis to establish a reference curve or compare integrity stratification changes in treated samples.

7. Workflow C: Fluorescence microplate reader detection

7.1 Applicable scenarios

This workflow is suitable for high-throughput comparison in flat-bottom 96-well plates and is well suited for pre/post-treatment difference analysis, condition screening, and repeated-experiment statistics.

7.2 Sample concentration adjustment

(1) Adjust live and dead Escherichia coli suspensions to 2 × 10^8 bacteria/ml, corresponding to approximately 0.06 OD670.

(2) Adjust live and dead Staphylococcus aureus suspensions to 2 × 10^7 bacteria/ml, corresponding to approximately 0.3 OD670.

(3) For fluorescence microplate reader detection, the concentration of Staphylococcus aureus suspensions is usually 10-fold lower than that of Escherichia coli suspensions.

7.3 Preparation of live/dead ratio samples

Prepare five bacterial suspensions with different live/dead ratios in 16 × 125 mm borosilicate glass culture tubes. The total volume of each sample is 2 ml.

7.4 Staining procedure

(1) In a microcentrifuge tube, add 6 µl of component A (SYTO 9) and 6 µl of component B (PI), and mix well.

(2) Add the above 12 µl dye premix into 2 ml sterile dH2O to prepare a 2× staining mixture.

(3) Add 100 µl of each bacterial suspension mixture to each well. It is recommended to set three replicate wells for each preparation.

(4) Edge wells of the 96-well plate should preferably be left empty or filled with blank solution to reduce false readings caused by edge effects.

(5) Add 100 µl of the 2× staining mixture to each well and pipette up and down thoroughly to mix.

(6) Incubate for 15 min at room temperature in the dark.

7.5 Fluorescence measurement and data analysis

(1) Measure green fluorescence at an excitation wavelength of approximately 485 nm and an emission wavelength of approximately 530 nm.

(2) Measure red fluorescence at an excitation wavelength of approximately 485 nm and an emission wavelength of approximately 630 nm.

(3) Calculate the green/red fluorescence ratio: RatioG/R = Fcell,em1 / Fcell,em2.

(4) Plot the proportion of live cells on the x-axis and RatioG/R on the y-axis to establish a calibration relationship or compare integrity changes before and after treatment.

8. Result interpretation

8.1 Basic interpretive logic

(1) Green-dominant: more appropriately interpreted as predominance of the membrane-intact population.

(2) Red-dominant: more appropriately interpreted as predominance of the membrane-damaged population.

(3) Mixed red-green: more often indicates transitional damage populations, local heterogeneity, or coexistence of different damage stages.

(4) Overall weak signal: first review sample amount, pretreatment, dye ratio, incubation time, and background interference.

8.2 Considerations for interpretation in different sample types

(1) In Gram-positive bacteria, early damage stages may not immediately generate strong red signals, so short-term assessments should focus more on changes in the transitional population.

(2) Gram-negative bacteria usually show clearer red-green stratification, but outer membrane damage still does not necessarily indicate irreversible death.

(3) In yeasts and fungi, cell size, cell wall composition, and budding status all affect threshold setting, so bacterial conditions should not be directly applied.

(4) In biofilm samples, greater emphasis should be placed on damage distribution across superficial/deep layers and local regions rather than relying only on the overall red-green ratio.

(5) In complex matrix samples, relative changes before and after treatment are more appropriate than direct absolute viability conclusions. Such samples often contain particles, autofluorescence, and non-microbial nucleic acid contamination, and should therefore preferably undergo washing, low-speed impurity removal, filtration, or moderate centrifugation enrichment before detection.

9. Quality control points

9.1 Dye and incubation control

(1) The ratio of SYTO 9 to PI should remain consistent throughout the same experimental batch.

(2) All samples should be kept under the same light-protection duration, incubation time, and detection interval.

(3) Do not attempt to obtain brighter signals simply by increasing the proportion of one dye, as this will alter signal dominance after dual staining.

9.2 Sample consistency control

(1) The live-cell group, dead-cell group, and mixed groups should be standardized to the same bacterial concentration before staining.

(2) Sample aggregation affects both microscopic observation and quantitative platform readouts, and uniform dispersion should therefore be maintained as much as possible.

(3) Particle and debris background in complex samples may increase false-positive signals and should be identified preferentially through pretreatment and blank controls.

9.3 Instrument parameter control

(1) In microscopic analysis, exposure, gain, and field acquisition strategy should be fixed.

(2) In fluorometer and microplate reader detection, excitation/emission windows and integration ranges should be fixed.

(3) When establishing curves, it is recommended to use the same dye batch, the same batch of live/dead bacterial preparations, and the same type of cuvette or plate format.

10. Common problems and cause analysis

10.1 Green signal is too strong and the red population is not obvious

(1) The SYTO 9 ratio is too high.

(2) PI is insufficient or the incubation time is too short.

(3) The dead-cell control was not prepared adequately and membrane damage was insufficient.

10.2 Red background is too high

(1) The PI ratio is too high.

(2) There is excessive sample debris or obvious background nucleic acid contamination.

(3) The complex matrix was not sufficiently cleaned before analysis.

10.3 The mixed red-green population is excessively large

(1) A true transitional damage population may be present.

(2) Different damage stages may coexist.

(3) This may also be related to unstable dye ratios, inconsistent sample concentrations, or inappropriate threshold settings.

10.4 Overall signal is weak

(1) The bacterial amount is insufficient.

(2) Staining is inadequate or the incubation time is inappropriate.

(3) Prolonged sample holding time caused signal decay.

(4) Pretreatment, staining conditions, and detection parameters should be reviewed first.

11. Safety and operational specifications

11.1 Personal protection

(1) Laboratory coats and disposable gloves should be worn throughout the experiment.

(2) Reagents such as isopropanol, ethanol, and SDS should be handled in accordance with chemical safety regulations.

11.2 Sample and waste disposal

(1) Waste containing bacterial samples, dyes, and organic treatment solutions should be collected separately.

(2) All waste should be disposed of according to laboratory biosafety and chemical safety requirements.

12. Product table related to SYTO 9 and PI combined live/dead staining

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