Live Or Dead Bacterial/Yeast Viability Kit（SYTO-9/PI）

Live or Dead Bacterial Viability kit(SYTO-9/PI) is a convenient kit that uses SYTO-9 green nucleic acid dye and Propidium Iodide (PI)

red fluorescent nucleic acid dye to detect bacterial viability. It can be analyzed by fluorescent analysis, such as Fluorescence Microplate

Readers,flow cytometry and fluorescence microscope. Applicable to a variety of Gram-negative and Gram-positive bacteria, including Escherichia coli, Bacillus subtilis, micrococcus lysoides, etc. In addition, this kit can also be used for staining the vitality of yeast.

The kits contain two fluorescent dyes, SYTO-9 and Propidium Iodide (PI). SYTO-9 is a green nucleic acid fluorescent dye that can stain bacteria with intact and damaged membranes. PI is a red nucleic acid fluorescent dye that stains only dead bacteria with damaged membranes, but PI was added causes a decrease in the fluorescence of SYTO-9 staining. When two dyes were added to the bacterial suspension at the same time, appropriate adjustment of the ratio of SYTO-9 and PI could make the bacteria with intact membrane structure fluoresce green, while the bacteria with damaged membrane structure fluoresce red. The maximum excitation and emission wavelengths of the two dyes combined with nucleic acid were 485/530nm (SYTO-9) and 485/630nm (PI), respectively.

Pack transportation with Wet ice ; Store at -20 ℃ and protected from light for 12 months.

At the recommended reagent dilutions and volumes, kit contains sufficient material to perform:

40 individual tests by flow cytometry assay: 500 μL bacterial suspension was added to 1 μL dye mixture (SYTO 1 μL +PI 1 μL);

200 individual tests by Microplate Readers and fluorescence microscope assay: 100 μL bacteria solution was added to 1 μL diluted dye (SYTO 1 μL+ PI 1 μL+0.85% Nacl 8 μL).

【Note】：Nucleic acids and other media components may bind to SYTO-9 and PI in an unpredictable manner, resulting in a large difference stainning from the expected results. Therefore, it is necessary to remove media residues, and phosphate buffers appear to decrease staining efficiency, 0.85% Nacl solution is recommended.During the experimental process, if it is found that the green fluorescence staining efficiency is too low, or the red fluorescence is too high, the fluorescence intensity can be adjusted to the expected effect by appropriately increasing the volume of the green dye or reducing the volume of the red dye.

Preparation of live/dead bacteria(eg.Escherichia coli, reference for bacterial viability ratio, optional)

(1) Escherichia coli was cultured to late log phase in LB nutrient broth.

(2) The bacteria was divided into two parts with EP tubes, and one part was centrifuged at 8000g for 10 min (the other part was placed at room temperature to prepare live bacteria).

(3) Remove the supernatant and resuspend the pellet in appropriate amount of 0.85% NaCl ,then add an appropriate amount of ethanol to make the final concentration of 70% ethanol mix thoroughly. That need incubate at room temperature for 1 h, mixing every 15 minutes prepare for killed bacteria.

(4) Concentrate both samples by centrifugation at 8000×g for 10 minutes.

(5) Remove the supernatant, add an appropriate of 0.85% NaCl to the two samples to resuspend the bacteria, and centrifuge again as in step 4, and resuspend.

(6) Determine the optical density at 670 nm (OD670) of both samples by spectrophotometer.

(7) The optical density of the two bacterial suspensions (live and killed) at OD670 was 0.03~0.1.

(8) As shown below to obtain the desired living cell/killed cell ratio by mix the two bacterial suspensions .

(1) Centrifuge the bacteria at 8000 g for 10 minutes, remove the supernatant, add an appropriate amount of 0.85% NaCl to resuspend the bacteria, repeat centrifugation again, and resuspend in an appropriate amount of 0.85% NaCl.

(2) Adjust the bacterial optical density, such as E. coli, with OD670 between 0.03 and 0.1.

(3) 1 volume of SYTO-9 and 1 volume of PI were mixed thoroughly in a microfuge tube.Then 8 volumes of 0.85% NaCl solution were added to obtain 100× dye solution.

(4) For every 100 μL bacterial suspension, add 1 μL of 100× dye solution.

(5) Mix thoroughly and incubate at room temperature for 15 minutes, away from light.

(6) Take 5μL of stained bacterial suspension drops on the slide, and then gently cover it with an 18 mm square cover slide.

(7) The live bacteria (green fluorescence) and dead bacteria (red fluorescence) can be observed and imaged using the FITC and Cy3 channels under a fluorescence microscope, respectively.

(1) Centrifuge the bacteria at 8000 g for 10 minutes, remove the supernatant, add an appropriate amount of 0.85% NaCl to resuspend the bacteria, repeat centrifugation again, and resuspend in an appropriate amount of 0.85% NaCl.

(2) Adjust the bacterial optical density, such as E. coli, with OD670 between 0.03 and 0.1.

(3) 1 volume of SYTO-9 and 1 volume of PI were mixed thoroughly in a microfuge tube.Then 8 volumes of 0.85% NaCl solution were added to obtain 100× dye solution.

(4) Add 1 μL of 100× dye solution to every 100 μL of bacterial suspension in a flat-bottom 96-well plate.

(5) Mix thoroughly and incubate at room temperature for 15 minutes away from light.

(6) Fluorescence measurement and data analysis

① Measure the fluorescence of each well using an excitation wavelength of ~485 nm and an emission wavelength of ~530 nm (emission 1, green);

② Measure the fluorescence of each well using an excitation wavelength of ~485 nm and an emission wavelength of ~630 nm (emission 2, red);

③ Alculate the fluorescence ratio = emission 1 / emission 2;

④ Plot a linear graph with the proportion of live E. coli cells in the suspension as the x-axis and the emission 1/emission 2 ratio as the y-axis.

(1) Centrifuge the bacteria at 8000 g for 10 minutes, remove the supernatant, add an appropriate amount of 0.85% NaCl to resuspend the bacteria, repeat centrifugation again, and resuspend in an appropriate amount of 0.85% NaCl.

(2) Adjust the bacterial optical density, such as E. coli, with OD670 between 0.03 and 0.1.

(3) 1 volume of SYTO-9 and 1 volume of PI were mixed thoroughly in a microfuge tube.Then 8 volumes of 0.85% NaCl solution were added to obtain 100× dye solution.

(4) Add 5 μL of the dye mixture to every 500 μL of bacterial suspension.

(5) Mix thoroughly and incubate at room temperature for 15 minutes, away from light.

(6) For flow cytometry analysis: Use the FITC channel to detect the SYTO-9 fluorescence (green, live cells) and Use the PE/PC5.5 channel to detect the propidium iodide (PI) fluorescence (red, dead cells).

1. Since the SYTO-9 and propidium iodide (PI) components are limited, be sure to centrifuge at low speed before opening the cap to avoid loss.

2. After first-time use, you can aliquot the dyes into smaller portions for storage to avoid repeated freeze-thaw cycles and contamination.

3. SYTO-9 and PI are both storage solutions with a capacity of 1000×, which have been optimized to be suitable for most bacteria. However, in order to obtain more satisfactory results, please conduct certain concentration tests for different types of bacteria. The final concentration for the use of SYTO-9 and PI is generally 0.5-2×, with the highest recommended final concentration being 1×. For example, when yeast staining, SYTO-9 recommends a final concentration of 1× and PI recommends a final concentration of 2×.

4. For E. coli, the recommended optical density (OD670) is 0.03-0.1. For Bacillus subtilis spores, the recommended OD670 is 0.01-0.1. For Micrococcus luteus, the recommended OD670 is 0.01-0.1. For yeast, the recommended OD670 is 0.1-0.3.

5. For your safety and health, please wear a lab coat and disposable gloves when operating.

For Research Use Only!