CN103175768B - Fast detecting biological cell is fluorescent staining kit and the application thereof of state anyway - Google Patents

Abstract

The invention relates to a fluorescent dyeing kit for rapidly detecting the life and death state of biological cells and an application thereof. The kit includes anthocyanin dye and propidium iodide dye. The detection steps are as follows: add the anthocyanin dye and propidium iodide in the kit to the cell suspension, use a fluorescence microscope to observe and count or use a microwell fluorescence plate reader to detect at the emitted light wavelength, according to the fluorescence value of different colors The strength of reflects the living state of the cells in the biological sample liquid. The present invention can quickly observe or detect the number of live cells and dead cells of various cells in the sample, which is intuitive and simple, eliminates the interference of other substances and the shortcomings of long culture period, and is not only applicable to animal cells, but also applicable to large Some Gram-negative and Gram-positive bacteria.

Description

Fluorescent staining kit for rapidly detecting the life and death state of biological cells and its application

technical field

The invention belongs to the field of cell counting, in particular to a fluorescent staining kit for rapidly detecting the life and death state of biological cells and its application.

Background technique

At present, the methods for determining the number of living cells include optical microscope reading counting method, plate coating culture colony counting method, photoelectric turbidimetric method, maximum probability method, and membrane filtration method.

Microscopic direct counting method: Place a small amount of the suspension of the sample to be tested on a special glass slide (also known as a bacteria counter) with a definite area and volume, and directly observe and count under a microscope. The method is simple, fast and intuitive, and can be used for counting suspensions such as yeast, bacteria, mold spores and animal cells. The disadvantage is that it cannot accurately identify the dead and alive state of cells, and the measured results are usually the sum of dead cells and living cells.

Plate colony counting method: Dilute the sample to be tested properly, so that the microbial colonies or animal cells in it are fully dispersed into a single cell state, take a certain amount of diluted sample solution and inoculate it on the plate medium, after cultivation, each single cell Growth and reproduction form colonies or cell communities visible to the naked eye, that is, a single colony should represent a single cell in the original sample. This method is widely applicable to the inspection of biological products (such as live bacteria preparations), and the detection of bacteria index or pollution degree of food, beverages and water (including water sources). The disadvantage is that because the sample to be tested is often not easily dispersed into a single cell, the single colony formed after culture may come from 2-3 or more cells in the sample, or due to the different growth cycles of the bacteria, in the same species Due to the difference in adaptability in the medium, some bacteria need 3 days to grow colonies, and some bacteria need 1 week or even longer, so the results of plate colony counts are often low, and colony formation is usually used. Units (colony-formingunits, cfu) instead of the absolute number of colonies to represent the live bacteria content of the sample.

Contents of the invention

The technical problem to be solved by the present invention is to provide a fluorescent staining kit and its application for rapidly detecting the life and death state of biological cells. It is intuitive and simple, and eliminates the interference of other substances and the shortcomings of long culture period. It is not only suitable for animal cells, but also suitable for most Gram-negative bacteria and Gram-positive bacteria.

A fluorescent staining kit for rapidly detecting the life and death state of biological cells of the present invention, said kit includes anthocyanin dye and propidium iodide dye, the storage concentration of anthocyanin dye is 5-6mM, and propidium iodide dye The storage concentration is 100 μg/mL.

The dilution buffer used for the anthocyanin dye is 1×TE buffer [10 mM Tris-HCl (trishydroxymethylaminomethane-HCl) buffer containing 1 mM EDTA (ethylenediaminetetraacetic acid), pH 8.0]. This dye can penetrate cells in all states, it tends to bind double-stranded DNA in cells, and has less binding to single-stranded DNA and RNA, stains the nucleic acid of cells, and makes cells emit green fluorescence.

The anthocyanin dye is diluted 1000-10000 times when used.

The anthocyanin dye solvent is dimethyl sulfoxide, and the working final concentration when used (the working concentration refers to the actual concentration when staining with the cells) is 5-6 μM.

Anthocyanin dye, its structural formula is:

The solvent and diluent of the propidium iodide dye are ultrapure water. This dye can only specifically pass through the cell membrane of dead cells, bind to DNA or RNA, and make the cells emit red fluorescence, so dead cells can be detected.

The working final concentration of the propidium iodide dye was 10-20 μg/mL.

The solvent of the anthocyanin dye is dimethyl sulfoxide, and the solvent of the propidium iodide dye is ultrapure water.

The application of a fluorescent staining kit for rapidly detecting the life and death state of biological cells of the present invention, the detection steps are as follows: add anthocyanin dye and propidium iodide in the kit to the cell suspension, use a fluorescence microscope to observe and count or use The microwell fluorescence plate reader detects at the wavelength of the emitted light, and reflects the survival status of the cells in the biological sample liquid according to the intensity of the fluorescence values of different colors.

The specific steps of using a fluorescent microscope to observe and count include:

(1) Pipette the cell suspension into a centrifuge tube wrapped with tinfoil;

(2) Add anthocyanin dye and propidium iodide mixed dye with a volume ratio of 1-2:1 at a volume ratio of 7:3 (the ratio of cell suspension to mixed dye), shake up and down, and place 10-30 minutes in the dark; or add anthocyanin dye with a volume ratio of 4:1 (the ratio of cell suspension to dye), shake up and down, and place in the dark for 10-30 minutes to measure the living body; or add volume PI dye with a ratio of 9:1 (the ratio of cell suspension to dye), shake up and down, place in the dark for 10-30 minutes, and measure the dead body;

(3) Filter the stained cell sample solution through a 0.2 μm black filter membrane (Whatman), so that the cells are trapped on the black filter membrane, transfer the filter membrane to a glass slide, drop a drop of non-fluorescent essential oil in the center of the membrane, Cover with a coverslip;

(4) Place the glass slide under a fluorescent microscope, drop a drop of non-fluorescent immersion oil on the center of the cover glass, observe with a 100 times oil lens, and respectively excite light at the optimum excitation light of anthocyanin dye and PI dye (anthocyanin The optimum excitation light of the dye is 488nm, and the optimum excitation light of PI dye is 522nm). The green cells in the eyepiece are living, and the red cells are dead.

The specific steps of using a microwell fluorescence plate reader to detect at the emitted light wavelength include:

(1) Filter the culture medium containing cells through a 0.2 μm filter membrane, and resuspend the collected cells with normal saline;

(2) Add the cell suspension in the microwell plate, and add anthocyanin with a volume ratio of 1-2:1 in the microwell at a volume ratio of 7:3 (the ratio of the cell suspension to the mixed dye) Mix the dye with propidium iodide, shake up and down, and place in the dark for 10-30 minutes; or add anthocyanin dye with a volume ratio of 4:1 (the ratio of cell suspension to dye), shake up and down, and place In the dark for 10-30 minutes, measure the living body; or add PI dye with a volume ratio of 9:1 (the ratio of cell suspension to dye), shake it up and down, and place it in the dark for 10-30 minutes, measure the dead body; microwell The total internal volume does not exceed 300μL, stain for 10-30 minutes, and detect the fluorescence intensity value under the conditions of excitation light of 460nm-500nm and emission light of 500nm-530nm. The intensity of fluorescence reflects the number of living organisms in the cell suspension , under the condition that the excitation light is 460nm-530nm and the emission light is 600nm-640nm, the fluorescence intensity value is detected, and the intensity of fluorescence reflects the number of dead bodies in the cell suspension.

The principle of the present invention is to use two fluorescent dyes to fluorescently label the nucleic acid of the cells. According to the difference in the biological activity of the cells during the culture process, the cells will emit different fluorescences, which reflect the different biological activities of the cells according to the different fluorescence intensities. ratio in the medium. The establishment of this technology provides a general method and technology for in-depth study of the survival status of various biological cells. In particular, it provides a simple and feasible method for the biological characteristics of smaller microbial cells such as Acetobacter xylinum and the synthesis process of bacterial cellulose.

Detection principle: When two dyes are added to the cell suspension at the same time, the anthocyanin dye can enter all cells and make the cells emit green fluorescence. When the cells are in a living state, the cell membrane only allows the anthocyanin dyes to enter the cells, and the PI dyes cannot enter the cells, and the cells in the living state only show green fluorescence. When the cell is in a dying state and the permeability of the cell membrane increases, PI will partially enter the cell, embed into the nucleic acid part of the cell that has been stained with green fluorescence, cover part of the green fluorescence, and make the cell emit part of the red fluorescence. Detected under the microscope are cells that fluoresce orange. When the cells are in a dead state, PI can pass through the cell membrane in large quantities, embed into the nucleic acid of the cells, and cover the green fluorescence of anthocyanins, and the dead cells will display red fluorescence as a whole.

Beneficial effect

The invention can observe or detect the quantity of live cells and dead cells of various cells in the sample quickly and in real time, which is intuitive and convenient. Moreover, the anthocyanin dyes are derived from nature, have low toxicity and are easy to use. Since the dye can only specifically bind to the double-stranded nucleic acid to emit fluorescence, the interference of other substances and the disadvantages of long culture period are eliminated. This staining method is not only suitable for animal cells, but also for most Gram-negative and Gram-positive bacteria.

Description of drawings

Fig. 1 is to detect the Acetobacter xylinum of same system with fluorescent microscope inspection and smear plate method, the contrast of detection result;

Figure 2 is the optimal dye concentration of anthocyanin dyed cells, RFU=total fluorescence intensity of stained cells-cell blank fluorescence-fluorescence of dye blank.

Fig. 3 is the optimal dye concentration of PI dye staining thalli, RFU=staining thalline total fluorescence intensity-thalline blank fluorescence-fluorescence of dye blank;

Figure 4 is the ratio change of the RFU cumulative intensity of the Staphylococcus aureus suspension mixed with different ratios of dead bacteria and live bacteria detected by the kit, RFU=total fluorescence intensity of stained bacteria-cell blank fluorescence-dye blank fluorescence;

Figure 5 is the ratio change of the RFU cumulative intensity of the E. coli suspension mixed with different ratios of dead bacteria and live bacteria detected by the kit, RFU=total fluorescence intensity of stained bacteria-cell blank fluorescence-fluorescence of dye blank ;

Figure 6 is the ratio change of the RFU cumulative intensity of the Acetobacter xylinum suspension mixed in different proportions of dead bacteria and live bacteria detected by the kit, RFU=total fluorescence intensity of stained bacteria-cell blank fluorescence-dye blank fluorescence;

Fig. 7 is to detect the standard curve of Acetobacter xylinum viable bacteria with kit;

Fig. 8 is the standard curve of using the kit to detect the dead bacteria of Acetobacter xylinum.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

(1) Anthocyanin dyes are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 5-6mM, which is the prepared concentrated solution of anthocyanin dyes for storage.

(2) PI dye was dissolved in ultrapure water to 100 μg/mL, and stored away from light

(3) Pipette 1mL of Acetobacter xylinum solution into a 2mL centrifuge tube wrapped with tinfoil.

(4) Add 300 μL of anthocyanin dye diluted 1000 times with buffer and 200 μL of 100 μg/mL PI dye, shake up and down several times, and place in the dark for 10-30 minutes. The dilution buffer is 1×TEbuffer (containing 1 mM EDTA) 10 mM Tris-HCl buffer, pH 8.0).

(5) Filter the stained bacterial solution through a 0.2 μm black filter membrane (Whatman), trap the bacteria on the black filter membrane, transfer the filter membrane to a glass slide, drop a drop of non-fluorescent essential oil in the center of the membrane, Cover with a coverslip.

(6) Observe the slide under the 100X objective lens of a fluorescence microscope. Since different dyes have different optimal excitation bands, it is necessary to perform detection under the optimal excitation light of anthocyanin dyes and PI dyes. Green cells are live cells, and red cells are dead cells. Take 1mL of Acetobacter xylinum from the same system, dilute it 10 times, 100 times, 1000 times, 10000 times, spread the diluted bacteria on the plate respectively, wait for colonies to grow, and compare the calculated number of colonies with a fluorescence microscope The experimental results are compared (experimental results are shown in Figure 1). The experimental results show that the number of bacteria measured by the fluorescence microscope is 1-2 orders of magnitude higher than the detection results obtained by the plate count method, which is the same as reported in the literature, because the plate count method is not tested Samples are often difficult to completely disperse into single cells, so the single colonies formed after culture may come from 2-3 or more cells in the sample, so the results of plate colony counts are often low. However, it has a good linear relationship with the results detected by fluorescence microscopy, R ² =0.9872.

Example 2

(1) Dilute the concentrated solution of anthocyanin dye storage by 1000 times to 20000 times, and use the dye with the same volume but different dilution times to stain the bacteria according to the steps 1-3 in Example 1, and use the MolecularDevicesFlexstationII type calcium flow fluorescence instrument to perform It is found that the fluorescence intensity of the bacteria decreases with the increase of the dilution factor of the dye. When the dilution is 5000 times, the dyeing effect is better and the amount of the dye is relatively low, which is more economical. If the dilution factor increases, the detected fluorescence value is close to the range of the error limit, and the error of the detection value increases (the experimental results are shown in Figure 2).

(2) The concentration of the dye PI was prepared from 1 μg/mL to 100 μg/mL, and the dyes with the same volume but different dilution times were used to stain the bacteria. It was found that the dyeing effect was the best when the concentration was 10 μg/mL (the experimental results are shown in Figure 3 ).

Example 3

(1) Cultivate Staphylococcus aureus until the turbidity OD value of the bacterial concentration is 0.7, take 25mL of the bacterial liquid and centrifuge at 12000r/min for 10-15 minutes;

(2) The collected bacteria were resuspended in 2 mL of normal saline;

(3) Dissolve 1mL of bacterial solution in 20mL of normal saline, and another 1mL in 20mL of 70% isopropanol;

(4) Stir every 15 minutes and incubate at room temperature for 1 hour;

(5) Centrifuge at 12000r/min for 10-15 minutes;

(6) Resuspend in 20mL of normal saline, then centrifuge and wash, the operation steps are the same as above;

(7) Resuspend with 10mL of normal saline respectively, and mix live bacteria and dead bacteria according to the ratio in Table 1;

Table I

Ratio of live bacteria to dead bacteria The volume of viable bacteria (mL) The volume of dead bacteria (mL) 0:100 0 2 10:90 0.2 1.8 50:50 1 1 100:0 2 0

(8) Add 140 μL of the mixed bacterial solution to the microwell plate, add 40 μL of 5 mM anthocyanin dye and 20 μL of 100 μg/mL PI dye under dark room conditions, and excite under the excitation light of 488 nm, respectively at 522 nm and 622 nm The detection is performed under the detection light, and the fluorescence intensity measured at 522nm is divided by the fluorescence intensity measured at 622nm to obtain the ratio of RFU. Then plot the RFU ratio with the proportion of living bacteria, and then get the regression equation and R2 value by regression. ^The experimental results are shown in Figure 4. The results showed that the linearity of the regression equation was very good, R ² =0.9599, indicating that after the dyes in the kit stained the cells, the fluorescence intensity excited at the relevant excitation wavelengths could well correspond to the dead or alive state of the cells.

Example 4

(1) Cultivate Escherichia coli until the turbidity OD value of the bacterial concentration is 1.2, take 25mL of the bacterial liquid and centrifuge at 12000r/min for 10-15 minutes;

(2) The collected bacteria were resuspended in 2 mL of normal saline;

(3) Dissolve 1mL of bacterial solution in 20mL of normal saline, and another 1mL in 20mL of 70% isopropanol;

(4) Stir every 15 minutes and incubate at room temperature for 1 hour;

(5) Centrifuge at 12000r/min for 10-15 minutes;

(6) Resuspend in 20mL of normal saline, then centrifuge and wash, the operation steps are the same as above;

(7) Resuspend with 10mL of normal saline respectively, and mix live bacteria and dead bacteria according to the ratio in Table 1;

(8) Add 140 μL of the mixed bacterial solution to the microwell plate, add 40 μL of 5 mM anthocyanin dye and 20 μL of 100 μg/mL PI dye under dark room conditions, and excite under the excitation light of 488 nm. The detection is performed under the detection light, and the fluorescence intensity measured at 522 nm is divided by the fluorescence intensity measured at 622 nm to obtain the ratio of RFU. Then plot the RFU ratio with the ratio of living bacteria, and then get the regression equation and R2 value by regression. ^The experimental results are shown in Figure 5. The results showed that the linearity of the regression equation was very good, R ² =0.9804, indicating that after the dyes in the kit stained the cells, the fluorescence intensity excited at the relevant excitation wavelengths could well correspond to the dead or alive state of the cells.

Example 5

(1) Filter 25 mL of Acetobacter xylinum with a 0.22 μm filter membrane and resuspend in 2 mL of normal saline;

(2) Dissolve 1mL of the bacterial solution in 1mL of normal saline, and fix another 1mL of the bacterial solution with 1mL of 5% glutaraldehyde;

(3) Stir once every 15 minutes and place at room temperature for 1 hour;

(4) Filter with a 0.22 μm filter membrane, and wash the bacterial cells retained on the filter membrane twice with normal saline.

(5) Resuspend the washed live bacteria and dead bacteria in 10 mL of normal saline, and mix the live bacteria and dead bacteria according to the ratio in Table 2.

(6) Add 140 μL of the mixed bacterial solution into the microwell plate, add 5 mM anthocyanin dye and 20 μL of 100 μg/mLPI dye under dark room conditions, excite under the excitation light of 488 nm, and detect the light at 522 nm and 622 nm Under the detection, the fluorescence intensity measured at 522nm was divided by the fluorescence intensity measured at 622nm to obtain the ratio of RFU. Then the ratio of live bacteria is plotted against the RFU ratio, and the regression equation and R2 value are obtained. ^The experimental results are shown in Figure 6. The results showed that the linearity of the regression equation was very good, R ² =0.977, indicating that after the dyes in the kit stained the cells, the fluorescence intensity excited at the relevant excitation wavelengths could well correspond to the dead or alive state of the cells.

Table II

Ratio of live bacteria to dead bacteria The volume of viable bacteria (mL) The volume of dead bacteria (mL) 0:100 0 2 20:80 0.4 1.6 50:50 1 1 80:20 1.6 0.4

Example 6

(1) Filter Acetobacter xylinum with a 0.22 μm filter membrane, and resuspend it with physiological saline to make a bacterial suspension. Add anthocyanin dye and PI dye to the bacterial suspension, take a part from the same system for fluorescence microscope detection, and a part for fluorescence plate reader detection, and compare the number of bacteria stained in the fluorescence microscope with the number read in the plate reader The fluorescence intensity values were compared and plotted to obtain the corresponding relationship between the number of Acetobacter xylinum viable bacteria and the fluorescence intensity (the experimental results are shown in Figure 7), and the corresponding relationship between the number of Acetobacter xylinum dead bacteria and the fluorescence intensity obtained by PI staining (see Figure 7). 8).

Figure 7 shows that the correlation coefficient R ² of the bacterial concentration of Acetobacter xylinum stained with anthocyanin dye and the fluorescence intensity RFU is 0.9379, indicating that the linearity is very good.

Figure 8 shows that the correlation coefficient R ² between the dead bacteria concentration and the fluorescence intensity RFU of Acetobacter xylinum stained with PI dye is 0.9723, indicating that the linear relationship between the fluorescence intensity generated after PI staining and the dead bacteria concentration is also very good.

(2) The detection sensitivity of anthocyanin dye and PI staining was 10 ⁵ bacterial concentration, and the highest detection limit was 10 ⁹ bacterial concentration.

(3) There are errors in the detection of the fluorescent plate reader. When the number of bacteria in the fluorescent microscope is too large, there will be errors in the counting results, and there will also be errors in the dilution of the bacterial solution, and it is often not accurate step by step according to the order of magnitude. dilution.

Claims (9)

1. A fluorescent staining kit for rapidly detecting the dead and alive state of biological cells, characterized in that: the kit includes anthocyanin dye and propidium iodide dye, the storage concentration of anthocyanin dye is 5-6mM, iodide The storage concentration of propidium dye is 100 μg/mL; the structural formula of anthocyanin dye is: 2. A fluorescent staining kit for rapidly detecting the life and death state of biological cells according to claim 1, characterized in that: the dilution buffer used for the anthocyanin dye is 1×TEbuffer, and the propidium iodide dye is The diluent used is ultrapure water. 3. A fluorescent staining kit for rapidly detecting the life and death state of biological cells according to claim 1, wherein the anthocyanin dye is diluted 1000-10000 times when used. 4 . A fluorescent staining kit for rapidly detecting the life and death of biological cells according to claim 1 , wherein the working final concentration of the anthocyanin dye is 5-6 μM. 5. a kind of fluorescence staining kit of rapid detection biological cell life and death state according to claim 1 is characterized in that: the solvent of described anthocyanin dye is dimethyl sulfoxide, and the solvent of propidium iodide dye is Ultra-pure water. 6 . A fluorescent staining kit for rapidly detecting the dead or alive state of biological cells according to claim 1 , wherein the working final concentration of the propidium iodide dye is 10-20 μg/mL. 7. The application of a fluorescent dyeing kit for rapidly detecting the life and death state of biological cells as claimed in claim 1, characterized in that: the detection step is as follows: add anthocyanin dye and iodide in the kit to the biological sample liquid Propidium, use a fluorescence microscope to observe and count or use a microwell fluorescence plate reader to detect at the emitted light wavelength, and reflect the survival status of the cells in the biological sample according to the intensity of the fluorescence values of different colors. 8. the application of a kind of fluorescent staining kit of rapid detection biological cell life and death state according to claim 7, it is characterized in that: described concrete steps of utilizing fluorescence microscope observation and counting comprise: (1) Draw the cell suspension into a centrifuge tube wrapped with tinfoil; (2) Add a mixed dye of anthocyanin dye and propidium iodide at a volume ratio of 1-2:1, the volume ratio of the cell suspension to the mixed dye is 7:3, shake up and down, and place in the dark for 10-30 minutes; or add anthocyanin dye with a volume ratio of 4:1 to the cell suspension, shake up and down, and place it in the dark for 10-30 minutes to measure the living body; or add PI dye with a volume ratio of 9:1 to the cell suspension , shake up and down, place in the dark for 10-30 minutes, measure the dead body; (3) Filter the dyed biological sample solution through a 0.2 μm black filter membrane to trap cells on the black filter membrane, transfer the filter membrane to a glass slide, drop a drop of non-fluorescent essential oil in the center of the membrane, and cover it Slide; (4) Place the glass slide under a fluorescent microscope, drop a drop of non-fluorescent immersion oil in the center of the cover glass, observe with a 100-fold oil immersion lens, and detect under the optimal excitation light of anthocyanin dye and PI dye respectively, Green cells in the eyepiece are live, red cells are dead. 9. The application of a fluorescent staining kit for rapidly detecting the life and death state of biological cells according to claim 7, wherein the specific steps of utilizing a micropore fluorescence plate reader to detect at the emitted light wavelength include: (1) Filtrate the medium containing the cells through a 0.2 μm filter membrane, and resuspend the collected cells with physiological saline; (2) Add the cell suspension in the microwell plate, add the mixed dye of anthocyanin dye and propidium iodide that the volume ratio is 1-2:1 in the microwell, the volume ratio of the cell suspension and the mixed dye is 7:3, shake up and down, place in the dark for 10-30 minutes; or add anthocyanin dye with a volume ratio of 4:1 to the cell suspension, shake up and down, place in the dark for 10-30 minutes, measure the living body; or Add PI dye with a volume ratio of 9:1 to the cell suspension, shake it up and down, place it in the dark for 10-30 minutes, and measure the dead body; the total volume in the microwell does not exceed 300 μL, stain for 10-30 minutes, under the excitation light of 460nm -500nm, the emission light is 500nm-530nm, and the fluorescence intensity value is detected. The intensity of fluorescence reflects the number of living bodies in the cell suspension. Under the condition of excitation light of 460nm-530nm and emission light of 600nm-640nm Detect the value of the fluorescence intensity, the intensity of the fluorescence reflects the number of dead bodies in the cell suspension.