CN111551416A - A method for evaluating bacterial apoptosis based on cell membrane phosphatidylserine fluorescence staining - Google Patents

Abstract

The invention discloses a method for evaluating apoptosis-like decline of bacteria by utilizing eversion staining of phosphatidylserine on cell membranes. The method comprises the following steps: centrifuging, resuspending, standing and centrifuging a liquid culture medium containing the bacteria to be detected to obtain immobilized bacteria; adding sterile phosphate buffer solution, and re-suspending to obtain bacterial suspension; adding the lysozyme solution into the bacterial suspension, performing wall dissolving, and centrifuging to obtain bacteria subjected to wall dissolving; uniformly mixing the bacteria subjected to wall dissolution with a sterile phosphate buffer solution to obtain a bacteria diluent; adding the AVF dye and the dyeing binding solution into the bacterial dilution for dyeing; and detecting the fluorescence intensity by using a flow cytometer to obtain a red fluorescence intensity value and a green fluorescence intensity value. The bacterial cell staining method is simple to operate, has obvious staining effect, is a method for firstly improving the AVF staining method used for apoptosis staining in eukaryotic cells to remove staining inhibition, eliminate staining false positive and the like, and is suitable for evaluating apoptosis decline of bacterial cells.

Description

An evaluation of bacterial apoptosis based on cell membrane phosphatidylserine fluorescence staining method

technical field

The invention belongs to the field of cell staining, in particular to a bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining.

Background technique

The balance of decay and growth of bacterial cells in activated sludge has a great influence on the activity of activated sludge. In the decline of total sludge activity, the decline of the metabolic capacity of living cells constituted the main reason for the decline of total sludge activity. However, currently widely used staining methods for bacteria do not identify metabolically spoiled bacterial cells. Considering the relationship between metabolic decay and the homology of early apoptosis in eukaryotic cells and apoptosis in prokaryotes and eukaryotes, the present invention attempts to use Annexin-V-FITC (AVF) to identify apoptosis-like Apoptotic decaying bacterial cells, while AVF dyes are based on phosphatidylserine (PS) cell membrane eversion to detect the early apoptotic state of eukaryotic cells (Raynal P, and Pollard HB. 1994. Annexins: the problem of assessing the biological role for a genefamily of multifunctional calcium-and phospholipid-binding proteins. Biochim Biophys Acta 1197: 63-93., Vermes I, Haanen C, Steffens-Nakken H, and Reutelingsperger C. 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V.J.Immunol.Methods 184:39-51.). At present, there are many staining methods used to study bacteria, and the methods directly used for bacterial staining include traditional Gram staining, spore staining, flagella staining, nucleic acid staining and activity staining of fluorescent ester substrates (De Clerck L S, Bridts C H , Mertens A M, Moens M M, and Stevens W J. 1994. Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function. J. Immunol. Methods 172:115). Among the staining methods that can be used to identify and identify the physiological state of bacteria, nucleic acid dye molecules such as propidium iodide (PI) are commonly used to stain bacteria to distinguish dead cells from living cells. Since PI cannot penetrate intact cell membranes, only dead cells with damaged cell membranes can be stained, as they allow PI to penetrate and interact with DNA. Although PI is considered to only stain dead bacterial cells, with the deepening of its application, it has also been found that PI has some defects in staining bacterial cells (Netuschil L, Auschill T M, Sculean A, and Arweiler N B. 2014. Confusion over live/ dead staining of microorganisms for the detection of vital microorganisms in oral biofilms--which stain is suitable? BMC OralHealth 14:2.). For example, up to 40% of early log phase cells of Sphingomonas sp. and Mycobacterium sp. can allow penetration of PI (Shi L, Gunther S, Hubschmann T, Wick L Y, Harms H, and Muller S. 2007. Limits of propidium iodide as a cell viability indicator for environmental bacteria.Cytometry A 71:592-598.), and in partially viable Saccharomyces sp. and Shewanella sp., the cell membrane also could not prevent PI from entering their cells, indicating that PI is effective against partially viable bacteria Can be dyed (Davey H M, and Hexley P.2011.Red but not dead? Membranes of stressedSaccharomyces cerevisiae are permeable to propidium iodide.Environ.Microbiol.13:163-171.,Yang Y,Xiang Y,and Xu M.2016. From red to green: the propidium iodide-permeable membrane of Shewanella decolorationis S12 isrepairable. Sci.Rep.-UK 5:18583.). Therefore, dead/living staining techniques based on PI staining cannot directly identify microbial cells with intact cell membranes.

At the same time, when PI was used for bacterial staining, the permeability of PI to dead bacteria with intact membranes was poor, which resulted in the absence of positive staining. On the other hand, PI also had false positive staining for live bacteria in some environments. Through investigation, it is found that the apoptosis process commonly found in eukaryotic cells may also exist in bacteria, and the apoptosis-like process of bacteria may have an important impact on the physiological state of bacteria. At present, the apoptosis-like staining for bacteria generally directly uses the method of eukaryotic cell apoptosis detection (AVF detection method of phosphatidylserine in outer leaf of cell membrane) for bacteria. Because of the existence of staining barriers such as extracellular polymers and cell walls in bacteria, this method cannot obtain obvious staining effects. The invention discloses a method that can be used for bacterial apoptosis-like staining. The bacteria after pretreatment can be stained with AVF phosphatidylserine and can be used to characterize the bacterial apoptosis-like degree. By comparing the use of PI And AVF staining of starved cells found that AVF has higher specificity for the recognition of starved cells. Apoptosis is a form of eukaryotic cell death characterized by specific morphological and physiological changes such as DNA fragmentation, caspase activation, cell shrinkage, chromatin condensation, and eversion of phosphatidylserine (PS) at the cell membrane. It is energy-dependent and regulated by coordinated gene expression to initiate programmed cell death pathways in response to environmental stress. Early apoptotic cells with metabolic decline can become late apoptotic cells and undergo a process of cell death. Although first discovered in eukaryotic cells, apoptosis-like phenomena have recently been found in functionally homologous prokaryotic cells, suggesting that apoptosis in eukaryotic cells may have evolved from prokaryotic cells. Therefore, established assays for detecting apoptosis in eukaryotic cells can also be used in prokaryotic cells, such as bacteria in sludge. Among all available eukaryotic apoptosis detection methods, AVF staining of PS is the most efficient and sensitive. Since PS is the basic structural molecule that constitutes the lipid bilayer membrane of bacterial cell membranes, PS staining can be widely used in Gram-negative bacteria and Gram-positive bacteria, and is a staining method superior to PI staining. More importantly, AVF staining is mainly used to detect the apoptosis of prokaryotic cells, because PS eversion is a signal of early apoptosis, and the degree of apoptosis can be judged by the degree of PS eversion. However, few studies have used AVF to stain isolated bacteria or bacteria in activated sludge, and the application of staining is inefficient.

SUMMARY OF THE INVENTION

In order to overcome the above deficiencies in the prior art, the purpose of the present invention is to provide a bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining.

Aiming at the deficiencies of cell decay staining techniques in prokaryotic cells or bacteria, one of the main purposes of the present invention is to develop a detection method including necessary bacterial cell fixation, wall lysis treatment and phosphatidylserine eversion on bacterial cell membranes.

In view of the deficiencies in the evaluation method of cell decline of prokaryotic cells or bacteria under the stress of starvation environment, the second main purpose of the present invention is to apply the method of cell membrane phosphatidylserine staining to indicate the degree of apoptosis-like decline in the process of bacterial growth and identify bacteria Starvation time in culture, and validation of apoptotic-like cells in bacterial cell populations under starvation environmental stress.

The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the invention is a method system for evaluating the apoptosis-like decline of bacteria by using the cell membrane phosphatidylserine staining, which is suitable for the field of environmental microorganisms and bacterial activity. recession areas.

The object of the present invention is achieved by at least one of the following technical solutions.

The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the present invention includes: treating the fixed cells with ethanol before staining; The peptidoglycan layer and exposure of the phosphatidylserine on the cell membrane surface, the direct treatment of bacteria with lysozyme will bring additional phosphatidylserine eversion and can be removed by ethanol extraction. After ethanol fixation and lysozyme treatment, bacterial cells maintained the initial state of phosphatidylserine eversion. The initial state of phosphatidylserine on the surface of bacterial cell membrane was positively correlated with the degree of bacterial starvation. Annexin- V-FITC dye was used to stain it, and the staining intensity could just reflect the initial state of bacterial cell membrane phosphatidylserine eversion, and it could be used to evaluate the apoptosis and decline of bacterial cells.

The invention provides a method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane, which specifically includes the following steps:

(1) centrifuge the liquid culture medium containing the bacteria to be detected to remove the supernatant and get the precipitate (the centrifugal speed is preferably 5000rpm), then the ethanol solution is mixed with the precipitate, the gun is blowing and mixed evenly, so that the cells are resuspended and left to stand 0.5-2h carry out immobilization treatment, then centrifuge to get the precipitate (the centrifugal speed is preferably 8000rpm), remove the supernatant (that is, remove the excess ethanol solution), wash with the EDTA _- Na solution, and centrifuge to get the precipitate (retain the thalline) to obtain Fixed treated bacteria;

(2) the bacteria after the fixation treatment described in step (1) is mixed with sterile phosphate buffered saline (PBS buffer), and the bacteria are resuspended to obtain a bacterial suspension;

(3) add lysozyme solution (solution prepared with sterile water) to the bacterial suspension described in step (2), carry out wall-dissolving treatment (37°C constant temperature water bath), centrifuge to get the precipitate (discard the supernatant to remove excess The lysozyme, retaining the bacterial cells after wall dissolution), remove the supernatant to terminate the wall dissolution reaction, and collect the bacteria after wall dissolution;

(4) mixing the bacteria after the wall dissolving described in step (3) with sterile phosphate buffer (sterile PBS buffer), diluting the bacteria after the wall dissolving to obtain a bacterial dilution;

(5) adding the AVF dye and the dyeing binding solution to the bacterial diluent described in step (4), mixing evenly, and performing dyeing treatment in the dark at room temperature to obtain the dyed cell fluid;

(6) using a flow cytometer to detect the fluorescence intensity of the dyed cell fluid described in step (5), to obtain a red fluorescence intensity value and a green fluorescence intensity value, when the logarithmic value of the green fluorescence intensity of the bacterial cells is compared to the If the logarithmic value of the green fluorescence intensity of the bacterial cells in several stages is higher than 5%, it is determined that there are cells in a state of apoptosis-like decay in the bacteria to be detected in step (1). Compared with the logarithmic value of the green fluorescence intensity of the bacterial cells in the logarithmic phase lower than 5%, it is determined that the bacteria to be detected in step (1) do not have cells in a state of apoptosis-like decay.

Further, the bacteria to be detected in step (1) are prokaryotic cells.

Further, the volume percentage concentration of the ethanol solution in step (1) is 65-75%; the volume ratio of the ethanol solution to the liquid culture medium containing the bacteria to be detected is 15:1-20:1; the fixed treatment The time is 0.5-2h.

Preferably, the volume percent concentration of the ethanol solution in step (1) is 70%.

Further, the EDTA-Na solution in step ( ₁ ) is a solution obtained by adding EDTA-Na into water and mixing evenly _; the concentration of the EDTA-Na solution is ₂₀₀ mg/L, and the water is distilled water.

Preferably, in step (1), the time of washing with EDTA-Na ₂ solution is 5-10 min.

Further, the concentration of step (2) and step (4) described sterile phosphate buffer is 0.01M; the volume ratio of step (2) described sterile phosphate buffer to step (1) described ethanol solution is 1.3:1-1:1.

Further, the lysozyme solution described in step (3) is a solution obtained by mixing lysozyme and water evenly; the lysozyme is egg white lysozyme; the volume ratio of the lysozyme solution to the bacterial suspension is 1:9-1 : 10; the time of the wall-dissolving treatment is 30-60 min, and the temperature of the wall-dissolving treatment is 37° C.; after the wall-dissolving treatment, the final concentration of lysozyme in the cell suspension is 50-200 mg/L.

The function of the lysozyme is to prevent the extraversion of phosphatidylserine produced during the lysozyme wall lysis.

Preferably, the time of the wall-dissolving treatment in step (3) is 30 min.

Further, in the bacterial dilution solution of step (4), the concentration of bacteria is 10 ⁶ -10 ⁷ cells/mL.

Further, the AVF dye in step (5) is Annexin-V-FITC (Annexin V-fluorescein isothiocyanate); the volume of the AVF dye is 0.5-1.5% of the volume of the bacterial dilution.

Further, the time of the dyeing treatment in step (5) is 15-30 min.

Preferably, the time of the dyeing treatment in step (5) is 15 min.

The volume ratio of the AVF dye and the dyeing binding solution in step (5) is 1:40.

The binding solution contained 40 mM hydroxyethylpiperazine ethanethiosulfonic acid, 600 mM NaCl and 10 mM CaCl ₂ , and the pH of the binding solution was 7.4.

Preferably, in step (6), after the sample is packed in a flow tube, a Beckman Coulter flow cytometer is used to detect the green fluorescence intensity of cell staining under the FL1 fluorescence channel (detection wavelength=480-530nm), wherein the flow cytometer The control and detection parameters of the instrument are shown in Table 1 below. Table 1 is the system control parameter table when the Beckman Coulter flow cytometer detects the fluorescence intensity of the two bacteria.

Table 1

In Table 1, G- represents Ochrobactrum sp., and G+ represents micrococcus sp..

The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the present invention can be applied to identify the starvation degree or starvation time of bacteria. The starvation time is 0-48d. The dye molecule AVF will mark the phosphatidylserine on the cell membrane of the bacteria to be tested in the process of identifying the starvation degree or starvation time of the bacteria.

Shake flasks were cultured with bacterial liquid LB medium for 0-48 days, and no nutrients were added during the culture to form cell starvation.

Sampling at different starvation time points and detecting the fluorescence intensity of cell staining by the above-mentioned method of phosphatidylserine on the bacterial cell membrane, can effectively identify cells at different starvation times according to the fluorescence intensity.

The method for evaluating bacterial apoptosis-like decline by phosphatidylserine eversion staining on cell membrane provided by the present invention is a staining method for bacterial cell membrane phosphatidylserine (PS) eversion. Considering the relationship between metabolic decay and early apoptosis and the homology of apoptosis between prokaryotes and eukaryotes, Annexin-V-FITC (AVF) was used to identify apoptotic decay-like bacterial cells. Through the detection of two phenolic-degrading activated sludge bacteria isolated from coking wastewater, the present invention found that the direct use of this probe has a lower staining efficiency because the cell wall prevents the dye from staining phosphatidylserine (PS), but when The staining efficiency was significantly improved after cells were pretreated with ethanol fixation and lysozyme hydrolysis. The present invention utilizes this improved method to reveal for the first time that apoptosis-like declining cells show more PS eversion than growing cells, and the PS exposure level is positively correlated with starvation time. This suggests that starvation induces apoptosis in apoptotic-like cells and can be assessed by PS eversion. The staining method described in the present invention must first fix the cells with ethanol, then use lysozyme to remove the cell wall, and finally use AVF to detect the inherent phosphatidylserine on the bacterial cell membrane. The results showed that the specificity of identifying the starvation time of bacteria by this method was better than that of using propidium iodide (PI) to identify the life and death of bacteria.

The bacterial cell staining method of the invention is simple to operate and has obvious staining effect. It is the first time that the AVF staining method used for eukaryotic cell apoptosis staining has been improved to remove staining obstacles, eliminate staining false positives, etc., and make it suitable for bacterial cells. The method for evaluating apoptosis decline. Through this method, the present invention also verifies for the first time that the apoptosis-like decline of bacteria exists in the bacterial population under the pressure of starvation environment, and finds that it has strong practicability and wide universality. It can be used in the evaluation of cell decay in mixed bacteria samples (such as sludge) in the environment.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the present invention can stain the phosphatidylserine on the bacterial cell membrane;

(2) The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the present invention can promote the combination of AVF and phosphatidylserine through lysozyme wall-dissolving treatment, and improve the staining efficiency;

(3) The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membrane provided by the present invention can eliminate the influence of lysozyme on cell membrane phosphatidylserine eversion by means of ethanol fixation;

(4) The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on the cell membrane provided by the present invention can be used to identify the starvation time or starvation degree of bacterial cells.

Description of drawings

Fig. 1 is the AVF staining diagram of normal bacterial Ochrobactrum sp. cells under different treatments after culturing for 24h;

Fig. 2 is the distribution diagram of the fluorescence intensity and the corresponding cell number of the bacterial cell membrane phosphatidylserine staining method and PI staining method under different treatments of Bacillus pallidum and Micrococcus cells after culturing for 24 hours;

Figure 3 is a graph of the mean fluorescence intensity of Bacillus pallidum and Micrococcus cells stained with PI and AVF under different treatment conditions;

Figure 4 is a graph showing the degradation curves of Ochrobactrum sp. and Micrococcus sp. to different concentrations of phenol in a period of 0-60 h under aerobic conditions;

Fig. 5A is the change curve of surviving bacterial count (CFU) and bacterial concentration (OD600) of Ochrobactrum sp. to starvation time;

Fig. 5B is the change curve of the surviving bacterial count (CFU) and bacterial concentration (OD600) of Micrococcus sp. to starvation time;

Figure 5C is a graph showing the relationship between the fluorescence intensity of PSE staining of Ochrobactrum sp. and the number of viable bacterial cells;

Figure 5D is a graph showing the relationship between the fluorescence intensity of PSE staining of Micrococcus sp. and the number of viable bacterial cells;

Figure 6 is a graph of the fluorescence intensity of two bacterial cells stained by PI and AVF with different starvation times (0-50 d) within 0-48 d;

Figure 7 is a graph of the fluorescence intensity peaks of Micrococcus sp. cells stained with ethanol-lysozyme combined with AVF for different incubation times;

Figure 8 is a conceptual schematic diagram of ethanol fixation and lysozyme treatment combined with AVF staining to measure phosphatidylserine eversion in bacterial cells.

Detailed ways

The specific implementation of the present invention will be further described below with reference to examples, but the implementation and protection of the present invention are not limited thereto. It should be pointed out that, if there are any processes that are not described in detail below, those skilled in the art can realize or understand them with reference to the prior art. If the reagents or instruments used do not indicate the manufacturer, they are regarded as conventional products that can be purchased in the market.

Example 1

Phosphatidylserine eversion staining on the cell membrane of a phenol-degrading Gram-negative bacterium Ochrobactrum sp.

Take 1.5mL (10 ⁸ cells/mL) of Ochrobactrum sp. culture solution cultured in liquid medium for 24h, add 30mL of ethanol-water solution with a volume percentage of 70%, and the volume ratio of the ethanol solution to the liquid medium containing the bacteria to be detected For 20:1, mix well, fix for 2h, and mix the unfixed bacterial cells with the same volume of sterile water and let stand for 2h;

Centrifuge the fixed bacterial cells for 5 min under the action of a centrifugal force of 5000 rpm, retain the bacterial cells, and remove the excess ethanol-water solution;

Prepare 30mL (200mg/L) of EDTA _- Na solution with sterile water to wash the bacterial cell pellet, centrifuge again at 5000g for 5min, retain the cell body, and remove excess liquid;

Resuspend the bacteria in sterile phosphate buffered saline (PBS) to a volume of 27 mL.

Prepare 3mL (2000mg/L) of lysozyme solution with sterile water, add above-mentioned 27mL bacterial PBS suspension, or add 3mL lysozyme solution to 30mL bacterial PBS suspension, the volume ratio of lysozyme solution and bacterial suspension is 1: 9. Mix well, and add the same volume of sterile water to the lysozyme solution without lysozyme treatment into the PBS suspension;

The bacterial suspensions treated with lysozyme and without lysozyme were placed in a constant temperature water bath at 37°C, and after 30 minutes, the suspension was taken out and centrifuged at 9000 rpm for 5 minutes, the supernatant was discarded to remove excess lysozyme, and the bacterial cells after wall dissolution were retained;

Dilute the wall-lysed cells to 10 ⁶ cells/mL with sterile PBS;

Filter the cell dilution with a 200-mesh nylon mesh to remove cell clumps, and retain the filtrate as the cell dilution to be stained;

Take 800 μL of cell dilution solution, add 5 μL of AVF dye, and 200 μL of dye binding solution. After mixing, the volume of AVF dye is 0.5% of the total suspension volume, and stain at room temperature for 15 minutes in the dark. Add 10 μL of PI dye to 1000 μL of the treated cell dilution, mix well, and stain at room temperature for 15 minutes in the dark;

The FL1 channel of flow cytometry was used to detect the green fluorescence intensity of AVF staining, and the FL2 channel was used to detect the red fluorescence intensity of PI staining of cells, and the unfixed and wall-lysing treated cells were used as negative controls to draw the gate. The staining flow chart is shown in Figure 1. Figure 1 shows the AVF staining diagram of normal bacteria Ochrobactrum sp. cells under different treatments after 24 hours of culture. The concentration of different AVF dyes is 100μg/L, 200μg/L, 300μg/L (A, B, C represent 100μg/L, respectively , 200μg/L, 300μg/L); AVF staining of cells treated with lysozyme at different starvation periods (1d, 3d, 10d, 30d) (D, E, F, G represent 1d, 3d, 10d, 30d, respectively) ; PI staining and AVF staining treated with lysozyme after ethanol fixation (H, I indicate PI staining and AVF staining, respectively). The results can prove that when the lysozyme treatment is not used, no obvious positive staining can be obtained by using 0.5-1.5% AVF dye, while only using the lysozyme-treated bacteria for AVF staining will produce a large number of positive staining. In addition, using ethanol-fixed bacteria for wall-dissolving treatment and staining can eliminate a large number of false positives caused by the lysozyme treatment process, thereby exposing the real bacterial cell membrane phosphatidylserine eversion. Therefore, ethanol fixation is a wall-dissolving treatment necessary steps before.

Implementation Example 2

Phosphatidylserine eversion staining on the cell membrane of two phenol-degrading bacteria under different treatments (lysozyme, ethanol + lysozyme, high temperature sterilization, high temperature sterilization + lysozyme).

Prepare LB medium, sterilize at 121 °C, inoculate Ochrobactrum sp. and Micrococcus sp. respectively, and place them at 30 °C for 24 hours on a shaker at 180 rpm. Both bacteria are in the logarithmic growth phase. At the same time sampling, different experimental groups and control treatment.

Bacterial cells of Ochrobactrum sp. and Micrococcus sp. treated with lysozyme were only subjected to wall lysis treatment. Lysozyme with a final concentration of 200 mg/L was added to the bacterial culture solution diluted 20 times with PBS, and the temperature was kept in a water bath at 37 °C for 1 h, and then discarded after centrifugation. clear;

In the experimental control group of Ochrobactrum sp. and Micrococcus sp. treated with ethanol + lysozyme, the log-phase bacterial cells in 2 mL of bacterial culture solution were collected by centrifugation, and then the cells were resuspended in 70% ethanol, and the supernatant was removed by centrifugation after fixation for 2 hours. , and then carry out lysozyme wall-dissolving treatment, add lysozyme with a final concentration of 200 mg/L to the bacterial culture solution diluted 20 times with PBS, keep the temperature in a water bath at 37 °C for 1 h, and discard the supernatant after centrifugation;

For the experimental control group of Ochrobactrum sp. and Micrococcus sp. treated with high-temperature steam sterilization, 2ml of bacterial solution was taken and kept at 121°C for 20 minutes for high-temperature steam sterilization to obtain a post-sterilization control. control experiment;

Finally, high-temperature steam sterilization, high-temperature steam sterilization + wall-dissolving treatment, ethanol fixation + wall-dissolving treatment, lysozyme wall-dissolving treatment and untreated bacteria were stained with PI and AVF respectively. The fluorescence intensity and bacterial cell distribution are shown in the figure. 2, and the average fluorescence intensity of each treatment group is shown in Figure 3. In Figure 3, Nt is no treatment, L is lysozyme treatment, A is high temperature sterilization treatment, Al is high temperature sterilization + lysozyme treatment, and El is ethanol fixation + lysozyme treatment. Bacterial cells treated with lysozyme were then stained with AVF and showed significantly positive staining than those without lysozyme treatment (Figure 2). The bacteria treated with ethanol + lysozyme can eliminate the positive staining after lysozyme treatment. However, the bacteria after high-temperature steam sterilization have all died, and the fluorescence intensity of AVF staining directly without lysozyme wall-dissolving is only slightly higher than that of the untreated group. The increase in the staining fluorescence intensity proved that after the removal of the cell wall, the phosphatidylserine could indeed be exposed, the staining barrier was removed, and the AVF could be successfully stained. The PI staining of bacteria treated with high-temperature steam sterilization was significantly different from that of ethanol-fixed + lysozyme-treated PI staining (Figure 2, Figure 3), indicating that PI did not completely enter the sterilized dead bacterial cells, proving that PI There are flaws as a conventional method of bacterial live staining. However, there are also significant differences in AVF staining between the cells treated with high temperature steam sterilization and the cells treated with high temperature steam sterilization and then lysed, which proves that the presence of cell walls hinders the binding of AVF and phosphatidylserine. Enzyme lysate treatment is a key step to ensure the effectiveness of staining.

Example 3

The relationship between the fluorescence intensity of two phenol-degrading bacteria under starvation environment stress and the starvation time of bacterial cells under the two staining methods was verified.

Two strains of bacteria were screened into the sludge of the coking wastewater treatment reactor, including one gram-negative bacteria and one gram-positive bacteria, both of which have good phenol degradation effects. The inoculum amount was controlled at 2%, and the degradation experiment with phenol as the only carbon source was carried out. It was found that both strains could achieve complete degradation of 750 mg/L phenol within 0-60 h (Fig. 4). A in Figure 4 is Ochrobactrum sp., B is Micrococcus sp. Subsequently, the 16S rRNA full-length sequencing of the two phenol-degrading bacteria was performed and compared with the BLAST tool of NCBI, and it was found that the two bacteria belonged to the genera Ochrobactrum and Micrococcus, respectively.

Take the slants of Ochrobactrum sp. and Micrococcus sp., inoculate them into liquid LB medium for shake flask culture at 30°C, 150 rpm, and culture time 0-48 d. During the culture process, no nutrients are added to constitute cell nutrient starvation. The colony forming unit (CFU) and bacterial concentration (OD600) were measured in the medium, and the bacterial liquid treated with different starvation time was sampled (3h, 6h, 9h, 12h, 15h, 18h, 21h, 1d, 2d, 3d, 5d , 8d, 10d, 12d, 17d, 20d, 24d, 26d, 30d, 36d, 48d). The correlation between the CFU and OD600 of the two phenol-degrading bacteria within 0-48d on the starvation time is shown in Fig. 5A and Fig. 5B. FIG. 5A is the result graph of Ochrobactrum sp., and FIG. 5 is the result graph of Micrococcus sp. It can be found that the two bacteria enter the logarithmic growth phase rapidly after inoculation 3-6h, and reach the highest CFU until 18-24h, and then remain in the stable growth phase within 1-5d, keeping the total CFU basically constant, and the CFU begins to gradually decrease after 5d. Entering the dying stage, Figure 5C and Figure 5D show a negative correlation between the number of viable cells and the fluorescence intensity of AVF staining, and the distribution trend can be fitted with a logarithmic model. FIG. 5C is the result graph of Ochrobactrum sp., and FIG. 5D is the result graph of Micrococcus sp.

For the different starved bacterial solutions, ethanol-lysozyme treatment combined with AVF staining and PI were used to distinguish between dead and alive cells. Flow cytometry was used to detect the green fluorescence of AVF and the red fluorescence of PI. The average intensity of green fluorescence of cell staining was detected by the above-mentioned method of phosphatidylserine on the bacterial cell membrane, and the bacterial cell dilution solution that was not fixed with ethanol and wall-dissolved was stained with PI, and the obtained bacterial culture time was Scatter plots and linear and sigmoidal model fits of the stained mean fluorescence intensity are shown in Figure 6. In Fig. 6, panel A shows PI staining of P. pallidum cells, panel B shows AVF staining of Bacillus pallidum cells, panel C shows PI staining of Micrococcus, and panel D shows AVF staining of Micrococcus. AVF staining was carried out after ethanol and lysozyme treatment and no special treatment was performed on cells before PI staining.

The results showed that the correlation of phosphatidylserine staining of bacterial cell membrane with AVF on the time of cell starvation was significantly higher than that of bacterial DNA staining with PI on the time of cell starvation. At the same time, in the treatment of Micrococcus sp. bacterial cells with ethanol-lysozyme combined with AVF staining, it was found that in the starvation time of 0-36 days, the fluorescence intensity of AVF staining gradually increased with time, which also indicated that the PS valgus of the cells gradually increased. In particular, it can be seen that obvious apoptotic cells began to appear around 10d (Fig. 7), and the distribution of apoptotic cells reached the highest after 36 days, which can prove that apoptotic cells did appear, and the ethanol-lysozyme The method combined with AVF staining was successfully identified.

Based on the research results of the present invention, in order to clarify the principle of PS staining of ethanol-fixed bacterial cells, the present inventors conducted the following analysis and proposed a hypothetical mechanism of PS staining of bacterial cells (refer to FIG. 8 ). Since the cell walls of Gram-negative bacteria and positive bacteria both cover the outside of the cell membrane, and the cell membrane is mainly composed of lipid bilayers, the peptidoglycan layer is the common primary structure of bacterial cells in these two bacterial cell walls. In ordinary eukaryotic cells, there is no cell wall preventing AVF from binding to PS, whereas for bacterial cells, the pore size of a typical cell wall-permeable material is about 2 nm. In addition, the crystal unit cell size of AVF is more than 4 nm (a=155.87a, c=37.34a), indicating that the maximum pore size allowed by the peptidoglycan layer of the prokaryotic cell wall is smaller than the molecular size of AVF, which leads to the obstruction of AVF. Therefore, hydrolysis of peptidoglycan in the cell wall is necessary for PS staining of bacterial cells. In eukaryotic cells, there are two intracellular enzymes that maintain PS balance on the cell membrane. Among them, promiscuous enzymes transfer PS from the inner leaflet to the outer leaflet, and phospholipase transfers PS from the outer leaflet to the inner leaflet. In prokaryotic cells, the eversion of PS may also be catalyzed by similarly functional intracellular enzymes. When lysozyme hydrolyzes the cell wall, the bacteria initiate the PS eversion reaction. According to the results of previous studies of the present invention, ethanol fixation can effectively remove the extra eversion of PS brought about by lysozyme-treated cells. There are two possible explanations for this effect of ethanol fixation. The first is that ethanol may prevent the binding of PS and AVF. The second is that ethanol may rapidly stop all metabolism within the cell, including the PS eversion response. Subsequently, the effect of ethanol-fixed PS staining was verified with starved cells. After ethanol lysozyme treatment of starved cells PS staining, Micrococcus sp. staining was obviously positive (Figure 6, Figure 7). Since ethanol-fixed starved cells still stained positive for PS, ethanol did not affect the binding of PS to AVF. However, when bacterial cells were fixed with ethanol, all enzymes were inactivated. There was no additional PS eversion during lysozyme hydrolysis. Therefore, ethanol helps to maintain the original state of PS on the cell membrane caused by cellular decay, preventing any PS eversion caused by lysozyme hydrolysis. Therefore, the PS everted state after ethanol fixation represents the original PS everted state of bacterial decline. This result shows that lysozyme hydrolysis treatment after ethanol fixation is an effective method to detect the pro-PS state of bacterial cell membrane eversion. Combined with the results of previous starvation treatment experiments, this method can also be further used to effectively evaluate bacterial apoptosis-like degree of decline.

The preparation of sterile PBS used in the above method: potassium dihydrogen phosphate: 0.27g, disodium hydrogen phosphate: 1.42g, sodium chloride: 8g, potassium chloride 0.2g, add about 800mL of deionized water and fully stir to dissolve, Then add dilute hydrochloric acid to adjust pH to 7.4, make up to 1L, pH=7.6, and use after sterilization.

The dyes AVF and PI used in the above method were provided by Shanghai Sangong "Annexin V Apoptosis Detection Kit, FITC/PI double staining method".

The above examples are only preferred embodiments of the present invention, and are only used to explain the present invention, but not to limit the present invention. Changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention shall belong to the present invention. the scope of protection of the invention.

Claims (9)

1. a method for evaluating the apoptosis-like decline of bacteria with phosphatidylserine eversion staining on cell membrane, is characterized in that, comprises the steps: (1) Centrifuge the liquid culture medium containing the bacteria to be tested to remove the supernatant to obtain the precipitate, mix the ethanol solution with the precipitate, and mix evenly to resuspend the cells. - Washing with Na ₂ solution, centrifuging to get the precipitate, to obtain the bacteria after immobilization; (2) uniformly mixing the immobilized bacteria in step (1) with sterile phosphate buffered saline, and resuspending the bacteria to obtain a bacterial suspension; (3) adding the lysozyme solution to the bacterial suspension described in step (2), performing wall-dissolving treatment, and centrifuging to obtain the precipitate to obtain the bacteria after wall-dissolving; (4) uniformly mixing the wall-dissolved bacteria and sterile phosphate buffer in step (3), and diluting the wall-dissolved bacteria to obtain a bacterial dilution; (5) adding the AVF dye and the dyeing binding solution to the bacterial dilution solution in step (4), mixing evenly, and performing dyeing treatment in the dark to obtain the dyed cell fluid; (6) Use a flow cytometer to detect the fluorescence intensity of the stained cell fluid in step (5), and obtain the red fluorescence intensity value and the green fluorescence intensity value. When the fluorescence intensity of AVF staining exceeds the fluorescence intensity of the log phase cultured cell control Intensity, it is determined that the liquid medium to be detected in step (1) has bacterial cells in a state of apoptosis-like decline, and when the fluorescence intensity of AVF staining does not exceed the fluorescence intensity of the log-phase cultured cell control, it is determined that In step (1), the liquid medium to be tested does not have bacterial cells in a state of apoptosis-like decline. 2. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1, wherein the volume percent concentration of the ethanol solution in step (1) is 65-75%; The volume ratio of the ethanol solution to the liquid culture medium containing the bacteria to be detected is 15:1-20:1; the time of the fixation treatment is 0.5-2 h. 3. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1, wherein the solvent of the EDTA-Na solution in step ( ₁ ) is water; the The concentration of EDTA-Na ₂ solution is 100-500 mg/L. 4. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1, characterized in that, the sterile phosphate buffer solution of the step (2) and the step (4) is performed. The concentration is 0.01-0.1 M; the volume ratio of the sterile phosphate buffer in step (2) to the ethanol solution in step (1) is 1.3:1-1:1. 5. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1, wherein the lysozyme solution in step (3) is obtained by mixing lysozyme and water evenly solution; the lysozyme is egg white lysozyme; the volume ratio of the lysozyme solution to the bacterial suspension is 1:9-1:10; the time of the wall-dissolving treatment is 30-60 min, and the temperature of the wall-dissolving treatment is 30-60 min. 37°C; after wall lysis treatment, the final concentration of lysozyme in the cell suspension is 50-200 mg/L. 6 . The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1 , wherein, in the bacterial dilution solution of step (4), the concentration of bacteria is 10 ⁶ - 10 ⁷ cells/mL. 7. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1, wherein the AVF dye in step (5) is Annexin-V-FITC; the AVF The volume of dye is 0.5-1.5% of the volume of bacterial dilution. 8 . The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1 , wherein the volume of the staining binding solution in step (5) is 25% of the volume of the bacterial dilution solution. %. 9 . The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membranes according to claim 1 , wherein the staining treatment time in step (5) is 15-30 min. 10 .

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