CN111551416A

CN111551416A - Bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining

Info

Publication number: CN111551416A
Application number: CN202010204951.6A
Authority: CN
Inventors: 韦朝海; 陈奔; 吴海珍; 邱光磊; 冯春华; 胡芸
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-03-22
Filing date: 2020-03-22
Publication date: 2020-08-18
Anticipated expiration: 2040-03-22
Also published as: CN111551416B

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

Bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining

Technical Field

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

Background

The balance of decay and growth of bacterial cells in the activated sludge has a great influence on the activity of the activated sludge. In the attenuation of the total activity of the sludge, the attenuation of the metabolic capacity of living cells constitutes a main cause of the decline of the total activity of the sludge. However, the methods of staining for bacteria that are currently widely used do not identify metabolically putrefactive bacterial cells. Considering the relationship between metabolic decay and homology of early apoptosis between eukaryotic cells and between prokaryotes and eukaryotes, the present invention seeks to identify apoptosis-like bacterial cells using Annexin-V-FITC (AVF), whereas AVF dyes are based on eversion of the Phosphatidylserine (PS) cell membrane to detect the early apoptotic state of eukaryotic cells (Raynal P, and Pollard HB.1994. antibodies: the protocol of addressing the biochemical roll for a gene expression of multifuncional calcium-and phospholipd-binding proteins, BiochimBiophys Acta 1197:63-93., Vermes I, Haanen C, Steffens-Nakken H, and Red reagent C.1995. A. non-adsorbed genes for apoptosis test. 184. expressing cell surface protein J.184. the present invention is a method of apoptosis using prokaryotic cell surface protein. Currently, there are numerous staining methods used for studying bacteria, and the methods directly used for staining bacteria include conventional gram staining, spore staining, flagellum staining, nucleic acid staining, and active staining of fluorescent ester substrates (De Clerck L S, Bridts C H, Mertens A M, Moens M, and Stevens W J.1994.use of fluorescent stains in the determination of adherence of human leucocytes to endothel cells and the effect of fluorochromes on cellular functions J.Immunol.172: 115). Among them, staining methods that can be used to identify the physiological state of bacteria generally use nucleic acid dye molecules such as Propidium Iodide (PI) to stain bacteria to distinguish dead cells from live cells. Since PI cannot penetrate the intact cell membrane, only dead cells with damaged cell membranes can be stained because they allow PI to penetrate and interact with DNA. Although PI is thought to stain only dead bacterial cells, as the application proceeds, PI has also been found to have some drawbacks in staining bacterial cells (Netuschil L, Auschill T M, Sculean a, and a bright weiler n.2014. fusion over live/dead staining for the detection of viral microorganisms in biological biolofils-which stain is suitable BMC oralheal 14: 2.). For example, up to 40% of early log phase cells of Sphingomonas sp. and Mycobacteria sp. may allow penetration of PI (Shi L, Gunther S, Hubschmann T, Wick L Y, Harms H, and Muller S.2007. limitations of lipid ingredients a cell viability indicator A71: 592. 598.) while in partially active Saccharomycotes sp. and Shewanella sp. bacteria, the cell membrane may also not prevent PI from entering their cells, indicating that PI may stain partially active bacteria of microorganisms (Davey H M, and Hexley P.2011.Red bud means to strain of microorganism culture J.35. J.F.163. and J.35. of microorganism strain J.35. J.F.F.J.F.J.M.F.F.M.and P.13. strain J.F.F.J.M.F.F.F.J.F.J.J.J.M.F.F.F.A. may be used to allow penetration of PI (III). Thus, the dead/live staining technique based on PI staining is much less able to directly identify viable but intact microbial cells.

Meanwhile, when PI is used for bacterial staining, PI has poor permeability to dead bacteria with complete membranes, so that staining positive deletion exists, and on the other hand, PI also has staining false positive to live bacteria in certain environments. Through research, it is found that the apoptosis process commonly existing in eukaryotic cells may also exist in bacteria, and the apoptosis-like process of bacteria may have important influence on the physiological state of bacteria. At present, apoptosis-like staining for bacteria generally directly applies a method for detecting the apoptosis of eukaryotic cells (AVF method for detecting phosphatidylserine outside cell membranes) to bacteria. The method cannot obtain obvious dyeing effect because of the dyeing obstacles such as extracellular polymer and cell wall in the bacteria. The invention discloses a method which can be used for an apoptosis-like staining method of bacteria, the bacteria after pretreatment can stain everted phosphatidylserine by AVF and can be used for characterizing the apoptosis-like degree of the bacteria, and compared with the staining of starvation-treated cells by PI and AVF, the AVF has higher specificity for identifying starvation cells. Apoptosis is a form of eukaryotic cell death characterized by specific morphological and physiological changes such as DNA fragmentation, caspase activation, cell contraction, chromatin condensation, and Phosphatidylserine (PS) eversion across the cell membrane. It is energy-dependent and is regulated by expression of a coordinating gene, initiating the programmed cell death pathway to cope with environmental stress. Early apoptotic cells with metabolic decline may become late apoptotic cells and undergo a process of cell death. Although first discovered in eukaryotic cells, apoptosis-like phenomena were recently discovered in functionally homologous prokaryotic cells, suggesting that apoptosis in eukaryotic cells may have evolved from prokaryotic cells. Thus, established assays for detecting apoptosis in eukaryotic cells may also be used for bacteria in prokaryotic cells, such as sludge. Among all available eukaryotic apoptosis detection methods, AVF staining of PS is the most efficient and sensitive. Since PS is a basic structural molecule constituting a lipid bilayer membrane of a bacterial cell membrane, PS staining can be generally applied to gram-negative bacteria and gram-positive bacteria, and is a staining method superior to PI staining. More importantly, AVF staining is mainly used for detecting apoptosis of prokaryotic cells, because PS eversion is a signal of early apoptosis, and the degree of apoptosis can be judged according to the degree of PS eversion. However, the use of AVF for staining isolated bacteria or bacteria in activated sludge has been rarely studied, and the effectiveness of AVF staining of everted PS in apoptosis-like decay bacteria is further explored by the application of low staining efficiency.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a bacterial apoptosis evaluation method based on cell membrane phosphatidylserine fluorescent staining.

Aiming at the defects of the cell decline staining technology in prokaryotic cells or bacteria, one of the main purposes of the invention is to develop a detection method which comprises necessary bacterial cell fixation, wall-lysing treatment and is used for phosphatidylserine eversion on bacterial cell membranes.

Aiming at the defects of the cell recession evaluation method of prokaryotic cells or bacteria under the pressure of the starvation environment, the invention mainly aims to apply the method for dyeing cell membrane phosphatidylserine to indicate the apoptosis-like recession degree in the growth process of the bacteria, identify the starvation time of the bacteria culture and verify that the apoptosis-like cells exist in the bacterial cell population under the pressure of the starvation environment.

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

The purpose of the invention is realized by at least one of the following technical solutions.

The method for evaluating the apoptosis-like decline of bacteria by utilizing the eversion staining of phosphatidylserine on cell membranes comprises the following steps: treating fixed cells with ethanol before dyeing, treating bacterial cells with lysozyme before dyeing after fixing cells, hydrolyzing peptidoglycan layer of bacterial cell wall by lysozyme and exposing phosphatidylserine on cell membrane surface, turning over additional phosphatidylserine caused by lysozyme direct treatment of bacteria and eliminating phosphatidylserine extra turning over caused by lysozyme lysosomes through ethanol extraction and fixation treatment, the bacterial cells after being fixed by ethanol and treated by lysozyme keep the eversion degree of the phosphatidylserine in the initial state, the eversion initial state of the phosphatidylserine on the surface of the bacterial cell membrane is in positive correlation with the hunger degree of the bacteria, and the bacterial cells are dyed by Annexin-V-FITC dye, so that the dyeing strength can just reflect the eversion initial state of the phosphatidylserine in the bacterial cell membrane, and the bacterial cells can be used for evaluating the apoptosis recession of the bacterial cells.

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

(1) centrifuging liquid culture medium containing bacteria to be detected to remove supernatant and collecting precipitate (centrifugation speed is preferably 5000rpm), mixing ethanol solution with the precipitate, blowing and mixing uniformly by a gun to resuspend cells, standing for 0.5-2h for fixing treatment, centrifuging to collect precipitate (centrifugation speed is preferably 8000rpm), removing supernatant (removing excessive ethanol solution), and treating with EDTA-Na₂Washing the solution, centrifuging to obtain precipitate (retaining thallus), and obtaining the bacteria after the fixation treatment;

(2) uniformly mixing the bacteria subjected to the fixation treatment in the step (1) with a sterile phosphate buffer solution (PBS buffer solution), and carrying out heavy suspension on the bacteria to obtain a bacteria suspension;

(3) adding a lysozyme solution (a solution prepared by using sterile water) into the bacterial suspension in the step (2), performing wall lysis treatment (constant-temperature water bath at 37 ℃), centrifuging to obtain a precipitate (discarding supernatant to remove redundant lysozyme, retaining bacterial cells after wall lysis), removing supernatant to terminate wall lysis reaction, and collecting bacteria after wall lysis;

(4) uniformly mixing the bacteria subjected to wall lysis in the step (3) with a sterile phosphate buffer solution (sterile PBS buffer solution), and diluting the bacteria subjected to wall lysis to obtain a bacteria diluent;

(5) adding an AVF dye and a dyeing binding solution into the bacterial diluent in the step (4), uniformly mixing, and dyeing at room temperature in a dark place to obtain dyed cell sap;

(6) detecting the fluorescence intensity of the stained cell sap in the step (5) by using a flow cytometer to obtain a red fluorescence intensity numerical value and a green fluorescence intensity numerical value, judging that the cell in the apoptosis-like decline state exists in the bacteria to be detected in the step (1) when the log value of the green fluorescence intensity of the bacterial cell is higher than the log value of the green fluorescence intensity of the bacterial cell in the log phase by more than 5%, and judging that the cell in the apoptosis-like decline state does not exist in the bacteria to be detected in the step (1) when the log value of the green fluorescence intensity of the bacterial cell is lower than the log value of the green fluorescence intensity of the bacterial cell in the log phase by less than 5%.

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

Further, the ethanol solution in the step (1) has a volume percentage concentration of 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 fixing treatment is 0.5-2 h.

Preferably, the ethanol solution in the step (1) has a concentration of 70% by volume.

Further, EDTA-Na in the step (1)₂The solution is EDTA-Na₂Adding into water, and mixing to obtain solution; the EDTA-Na₂The concentration of the solution is 200mg/L, and the water is distilled water.

Preferably, in step (1), EDTA-Na is used₂The time for washing the solution is 5-10 min.

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

Further, the lysozyme solution in the step (3) is a solution obtained by uniformly mixing lysozyme and water; 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 30-60min, and the temperature of the wall dissolving treatment is 37 ℃; after the wall lysis treatment, the lysozyme concentration in the cell suspension was 50-200 mg/L.

The lysozyme has the function of preventing the eversion of the phosphatidylserine generated in the lysozyme wall-lysing process.

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

Further, in the bacterial dilution in the step (4), the concentration of the bacteria is 10⁶-10⁷cells/mL。

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

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

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

the binding solution comprises 40mM of hydroxyethylpiperazidine ethanethiosulfonic acid, 600mM NaCl and 10mM of CaCl₂The pH of the binding solution was 7.4.

Preferably, in step (6), after the sample is filled in the flow tube, the green fluorescence intensity of the cell staining is detected by a Beckman Coulter flow cytometer at FL1 fluorescence channel (detection wavelength 480-530nm), wherein the control detection parameters of the flow cytometer are shown in Table 1 below. Table 1 shows the system control parameters for the Beckman Coulter flow cytometer to detect the fluorescence intensity of two bacteria.

TABLE 1

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

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

Carrying out shake flask culture with liquid LB culture solution for 0-48d, and culturing without adding nutrients to starve cells.

Sampling is carried out at different starvation time points, and the fluorescence intensity of cell staining is detected by the method for turning out phosphatidylserine on the bacterial cell membrane, so that the cells with different starvation time can be effectively identified according to the fluorescence intensity.

The invention provides a method for evaluating apoptosis-like decline of bacteria by utilizing phosphatidylserine on a cell membrane to perform eversion staining, which is a staining method for bacterial cell membrane Phosphatidylserine (PS) to perform eversion staining. Annexin-V-FITC (AVF) is used to identify bacterial cells that are apoptosis-like in respect of the relationship between metabolic decline and early apoptosis and the homology of apoptosis between prokaryotes and eukaryotes. By detecting two phenolic activated sludge-degrading bacteria separated from the coking wastewater, the invention finds that the dyeing efficiency of directly using the probe is low because the cell wall prevents the dye from dyeing Phosphatidylserine (PS), but the dyeing efficiency can be obviously improved after the cells are pretreated by ethanol fixation and lysozyme hydrolysis. The invention firstly utilizes the improved method to reveal that the cell with apoptosis-like decline shows more PS eversion than the growing cell, and the PS exposure level is in positive correlation with the hunger time. This suggests that starvation can induce apoptosis in apoptotic-like cells and can be assessed by PS eversion. The staining method of the invention must firstly use ethanol to fix cells, then use lysozyme to remove cell walls, and finally use AVF to detect the inherent phosphatidylserine on the bacterial cell membrane. As a result, the specificity of identifying the bacteria starvation time by using the method is better than that of identifying the bacteria death and activity by using Propidium Iodide (PI) in a conventional mode.

The bacterial cell staining method is simple to operate, obvious in staining effect, capable of firstly improving the AVF staining method for eukaryotic cell apoptosis staining in the past to remove staining obstruction, eliminate staining false positive and the like, and suitable for evaluating bacterial cell apoptosis recession.

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

(1) the method for evaluating the apoptosis-like decline of the bacteria by utilizing the eversion staining of the phosphatidylserine on the cell membrane can stain aiming at the phosphatidylserine on the cell membrane of the bacteria;

(2) the method for evaluating the apoptosis-like recession of the bacteria by utilizing the eversion staining of the phosphatidylserine on the cell membrane can promote the combination of AVF and the phosphatidylserine through the lysozyme wall-dissolving treatment, thereby improving the staining efficiency;

(3) the method for evaluating the apoptosis-like decline of bacteria by using the eversion staining of the phosphatidylserine on the cell membrane can eliminate the influence of lysozyme on the eversion of the phosphatidylserine on the cell membrane in an ethanol fixing mode;

(4) the method for evaluating the apoptosis-like decline of the bacteria by utilizing the phosphatidylserine eversion staining on the cell membrane can be used for identifying the starvation time or the starvation degree of the bacterial cells.

Drawings

FIG. 1 is an AVF staining pattern of normal bacterial Ochrobactrum sp. cells after 24h culture under different treatments;

FIG. 2 is a graph showing the fluorescence intensity and corresponding cell number of Ochrobactrum anthropi and Micrococcus cells after 24h culture, stained with the bacterial cell membrane phosphatidylserine staining method and the PI staining method under different treatments;

FIG. 3 is a graph of mean fluorescence intensity of cells of Ochrobactrum anthropi and Micrococcus after PI and AVF staining under different treatment conditions;

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

fig. 5A is a graph of number of viable bacteria (CFU) and bacterial concentration (OD600) versus starvation time for Ochrobactrum sp;

fig. 5B is a graph of the number of viable bacteria (CFU) and the concentration of bacteria (OD600) in Micrococcus sp versus starvation time;

FIG. 5C is a graph of PSE staining fluorescence intensity versus number of viable cells of bacteria of Ochrobactrum sp;

FIG. 5D is a plot of PSE staining fluorescence intensity versus number of viable cells of bacteria;

FIG. 6 is a graph of the fluorescence intensity of PI and AVF staining of two bacterial cells in 0-48d with different starvation times (0-50 d);

FIG. 7 is a graph showing fluorescence intensity peaks of Micrococcus sp. cells stained with ethanol-lysozyme in combination with AVF at different culture times;

FIG. 8 is a conceptual diagram of the determination of phosphatidylserine evagination in bacterial cells by ethanol fixation and lysozyme treatment in combination with AVF staining.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Example 1

Phosphatidylserine eversion dyeing on gram-negative bacterium Ochrobactrum sp

1.5mL (10) of Ochrobactrum sp.culture solution cultured in liquid medium for 24h⁸cells/mL), adding 30mL of ethanol-water solution with the volume percentage content of 70%, wherein the volume ratio of the ethanol solution to the liquid culture medium containing the bacteria to be detected is 20:1, fully and uniformly mixing, fixing for 2h, uniformly mixing the bacteria cells without fixation with sterile water with the same volume, and standing for 2 h;

centrifuging the fixed bacterial cells for 5min under the centrifugal force of 5000rpm, retaining thallus, and removing excessive ethanol-water solution;

preparation of EDTA-Na with sterile Water₂Washing bacterial thallus precipitate with 30mL (200mg/L) solution, centrifuging for 5min again at 5000g, retaining thallus, and removing excessive liquid;

the bacteria were resuspended in sterile Phosphate Buffer (PBS) to a volume of 27 mL.

Preparing 3mL (2000mg/L) of lysozyme solution by using sterile water, adding the 27mL of bacterial PBS suspension, or adding 3mL of lysozyme solution into 30mL of bacterial PBS suspension, wherein the volume ratio of the lysozyme solution to the bacterial suspension is 1:9, uniformly mixing, and adding the lysozyme suspension into the lysozyme-free treated bacterial cells by using sterile water with the same volume instead of the lysozyme solution;

placing the bacterial suspension liquid treated by adding lysozyme and not adding lysozyme in a constant-temperature water bath at 37 ℃, taking out the suspension liquid after 30min, centrifuging for 5min at 9000rpm, removing the supernatant, removing redundant lysozyme, and retaining bacterial cells after wall dissolving;

the lysed cells were diluted to 10 cell number with sterile PBS⁶cells/mL；

Filtering the cell diluent by using a 200-mesh nylon net to remove cell masses, and reserving filtrate as the cell diluent to be dyed;

adding 5 mul of AVF dye and 200 mul of dyeing binding solution into 800 mul of cell diluent, uniformly mixing, and dyeing for 15min at room temperature in a dark place, wherein the volume of the AVF dye is 0.5 percent of the volume of the total suspension; taking 1000 μ L of cell diluent which is not fixed by ethanol and subjected to wall dissolving treatment, adding 10 μ L of PI dye, mixing uniformly, and dyeing for 15min at room temperature in a dark place;

the flow cytometry AVF staining green fluorescence intensity was measured using FL1 channel for cell, PI staining red fluorescence intensity was measured using FL2 channel for cell, and crossgating was performed using cells without fixation and lysis as negative controls, and AVF staining flow charts for different treated bacterial cells are shown in FIG. 1. FIG. 1 is an AVF staining pattern of normal bacterial Ochrobactrum sp. cells after 24h culture under different treatments, with different AVF staining agent concentrations of 100. mu.g/L, 200. mu.g/L, 300. mu.g/L (A, B, C represent 100. mu.g/L, 200. mu.g/L, 300. mu.g/L, respectively); cells at different starvation periods (1D,3D,10D,30D) were stained with AVF after lysozyme treatment (D, E, F, G for 1D,3D,10D,30D, respectively); after ethanol fixation lysozyme treated PI staining and AVF staining (H, I indicate PI staining, AVF staining, respectively). The results can prove that when no wall-dissolving treatment is adopted, obvious positive staining can not be obtained by using 0.5-1.5% of AVF dye for staining, but a large amount of positive staining can be generated by using bacteria subjected to the wall-dissolving treatment by lysozyme only for AVF staining, and in addition, a large amount of false positive caused by the lysozyme treatment process can be eliminated by using the bacteria subjected to the ethanol fixation for wall-dissolving treatment and staining, so that the eversion degree of the real bacteria cell membrane phosphatidylserine is exposed, and therefore, the ethanol fixation is a necessary step before the wall-dissolving treatment.

EXAMPLES example 2

The phosphatidylserine on the cell membrane under different treatments (lysozyme, ethanol + lysozyme, high-temperature sterilization + lysozyme) of the two strains of phenol degrading bacteria is subjected to outward turning dyeing.

Preparing an LB culture medium, sterilizing at 121 ℃, respectively inoculating Ochrobactrum sp and Micrococcus sp, respectively placing in a shaker at 30 ℃ and 180rpm for culturing for 24 hours, simultaneously sampling when the two bacteria are in logarithmic growth phase, and carrying out treatment on different experimental groups and controls.

Performing wall-dissolving treatment on the lysozyme-treated Ochrobactrum sp and Micrococcus sp bacterial cells, adding lysozyme with the final concentration of 200mg/L into a bacterial culture solution diluted by 20 times by PBS, performing water bath at 37 ℃ for 1h, centrifuging, and removing the supernatant;

firstly, centrifugally collecting logarithmic phase bacterial thallus in 2mL of bacterial culture solution in an ethanol + lysozyme treated Ochrobactrum sp and Micrococcus sp experiment control group, then resuspending the thallus by 70% ethanol, centrifugally removing supernatant after fixing for 2h, then carrying out lysozyme wall-dissolving treatment, adding lysozyme with the final concentration of 200mg/L into the bacterial culture solution diluted by 20 times by PBS, carrying out water bath at 37 ℃ for constant temperature for 1h, and discarding the supernatant after centrifugation;

performing high-temperature steam sterilization on 2ml of bacterial liquid at 121 ℃ for 20min to obtain sterilized control, and performing high-temperature steam sterilization and lysozyme treatment control experiments;

and finally, PI and AVF are used for respectively dyeing bacteria subjected to high-temperature steam sterilization treatment, high-temperature steam sterilization + wall lysis treatment, ethanol fixation + wall lysis treatment, lysozyme wall lysis treatment and no treatment, the fluorescence intensity and the bacterial cell distribution are shown in figure 2, and the average fluorescence intensity of each treatment group is shown in figure 3. In FIG. 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. The lysozyme-treated bacterial cells were stained with AVF, and showed a significantly positive stain compared to the bacterial cells not treated with lysozyme (FIG. 2). The bacteria treated by the ethanol and the lysozyme can eliminate positive staining after the lysozyme treatment. The bacteria sterilized by high-temperature steam are all dead, the fluorescence intensity of AVF dyeing directly without lysozyme wall lysis is only slightly improved for a non-treated group, and the obvious improvement of the fluorescence intensity of AVF dyeing can be still observed after lysozyme wall lysis, which proves that the phosphatidylserine can be really exposed after cell walls are removed, the dyeing obstruction is removed, and the AVF dyeing can be smoothly carried out. PI staining of bacteria subjected to high-temperature steam sterilization is obviously different from PI staining of ethanol fixation + lysozyme treatment (figure 2, figure 3), which shows that PI does not completely enter sterilized dead bacterial cells, and the defects of PI serving as a conventional method for dying and alive are proved. The AVF staining of the cells subjected to high-temperature steam sterilization and the cells subjected to re-wall lysis after high-temperature steam sterilization are also obviously different, and the fact that the cell walls have a barrier effect on the combination of the AVF and phosphatidylserine is proved, so that the wall lysis by lysozyme is a key step for ensuring the effectiveness of staining.

Example 3

And (3) verifying the relation between the fluorescence intensity of the two phenol degrading bacteria under the starvation environmental pressure and the starvation time of the bacterial cells under the two staining methods.

The two strains are screened into the sludge of the coking wastewater treatment reactor, and comprise a gram-negative strain and a gram-positive strain, and both the gram-negative strain and the gram-positive strain have better phenol degradation effect. The inoculation amount is controlled to be 2%, degradation experiments with phenol as the sole carbon source are carried out, and the complete degradation of 750mg/L phenol can be realized by both strains within 0-60h (figure 4). In fig. 4, a is ochrobastrum sp and B is Micrococcus sp. Subsequently, 16S rRNA full-length sequencing is carried out on the two phenol degrading bacteria, and comparison is carried out by a BLAST tool of NCBI (national center of Biotechnology information) to find that the two bacteria belong to the genera Ochrobactrum and Micrococcus respectively.

Taking inclined planes of Ochrobactumsp and Micrococcus sp, inoculating the inclined planes to a liquid LB culture solution, performing shake flask culture at 30 ℃, 150rpm and the culture time of 0-48d, not supplementing nutrient substances in the culture process to form cell nutrition starvation, measuring Colony Forming Units (CFU) and bacteria concentration (OD600) in the culture process, and sampling the bacteria solution treated at different starvation times (3h,6h,9h,12h,15h,18h,21h,1d,2d,3d,5d, 8d, 10d,12d,17d,20d,24d,26d,30d,36d and 48 d). The correlation between CFU and OD600 of two phenol-degrading bacteria in 0-48d versus starvation time is shown in FIG. 5A and FIG. 5B. Fig. 5A is a graph showing the results of ochrobastrum sp, and fig. 5 is a graph showing the results of Micrococcus sp. It can be seen that both strains rapidly enter the logarithmic growth phase 3-6h after inoculation until reaching the highest CFU at 18-24h, and then are in the growth stationary phase within 1-5D, keeping the total CFU substantially constant, and the CFU starts to gradually decline after 5D and enters the death phase, and the negative correlation between the number of active cells and the fluorescence intensity of AVF staining is shown in FIG. 5C and FIG. 5D, and the distribution trend can be fitted by a logarithmic model. Fig. 5C is a graph showing the results of ochrobastrum sp, and fig. 5D is a graph showing the results of Micrococcus sp.

For different starvation treated bacteria liquids, ethanol-lysozyme treatment and AVF dyeing and PI conventional dyeing for cell death and survival resolution are respectively adopted, and a flow cytometer is used for detecting the green fluorescence of AVF and the red fluorescence of PI by fluorescence intensity. The average intensity of the green fluorescence of the cell staining was measured by the above-described method of phosphatidylserine eversion on the bacterial cell membrane, and the bacterial cell dilution without ethanol fixation and lysis treatment was stained with PI, and the scattering point of the obtained bacterial culture time to the stained average fluorescence intensity and the fitting of the linearity and S-type pattern are shown in FIG. 6. FIG. 6A shows Ochrobactrum pallidum cell PI staining, B shows Ochrobactrum pallidum cell AVF staining, C shows Micrococcus PI staining, and D shows Micrococcus AVF staining. AVF staining was performed after ethanol and lysozyme treatment whereas no special treatment was given to the cells before PI staining.

The results show that the correlation of AVF on bacterial cell membrane phosphatidylserine staining on cell starvation time is obviously higher than that of PI on bacterial DNA staining. Meanwhile, in the treatment of ethanol-lysozyme and AVF combined staining of Micrococcus sp bacterial cells, the AVF staining fluorescence intensity is gradually enhanced along with the time extension in the starvation time of 0-36d, which also indicates that the PS eversion of the cells is gradually increased, particularly obvious apoptosis-like cells begin to appear in about 10d (figure 7), the distribution of the apoptosis-like cells reaches the highest after 36d, and the apoptosis-like cells can be proved to be really appeared and successfully identified by the method of ethanol-lysozyme and AVF combined staining.

According to the results of the present invention, in order to clarify the principle of the PS staining of ethanol-immobilized bacterial cells, the present invention carried out the following analysis and proposed a hypothetical mechanism of the PS staining of bacterial cells (see FIG. 8). Since the cell walls of both gram-negative and positive bacteria are covered outside the cell membrane, which is mainly composed of lipid bilayers, the peptidoglycan layer is a common bacterial cell primary structure in both bacterial cell walls. In normal eukaryotic cells, there is no cell wall to prevent AVF from binding to PS, whereas for bacterial cells, the typical pore size of the cell wall permeable material is about 2 nm. In addition, the crystal unit cell size of the AVF is more than 4nm (a is 155.87a, c is 37.34a), which shows that the highest pore size allowed by the prokaryotic cell muropeptide layer is smaller than the molecular size of the AVF, thus causing the obstruction to the AVF. Thus, hydrolysis of peptidoglycan in the cell wall is a necessary condition for PS staining of bacterial cells. In eukaryotic cells, there are two intracellular enzymes that maintain PS balance on the cell membrane. Wherein promiscuous enzyme transfers PS from the inner leaflet to the outer leaflet and phospholipid transferase transfers PS from the outer leaflet to the inner leaflet. In prokaryotic cells, however, 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 previous research results of the invention, the ethanol fixation can effectively remove the extra eversion of PS brought by lysozyme treatment of 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 halt all metabolism within the cell, including the PS valgus response. Subsequently, starved cells were used to verify the effect of ethanol-fixed PS staining. After the starved cells were treated with ethanol lysozyme and stained with PS, Micrococcus sp. Since the ethanol fixed starved cells were positive for PS staining, ethanol did not affect the binding of PS to AVF. However, when bacterial cells were fixed with ethanol, all enzymes lost activity. During lysozyme hydrolysis, there was no additional PS eversion. Thus, ethanol helps to maintain the original state of the PS on the cell membrane caused by cell decay, preventing any eversion of the PS caused by lysozyme hydrolysis. Thus, the PS eversion state after ethanol fixation represents the PS eversion primitive state of bacterial decay. The result shows that the lysozyme hydrolysis treatment after the ethanol fixation is an effective method for detecting the PS protostate of the bacterial cell membrane eversion, and the method can be further used for effectively evaluating the apoptosis-like decline degree of the bacteria by combining the previous starvation treatment experiment result.

Preparation of sterile PBS used in the above method: potassium dihydrogen phosphate: 0.27g, disodium hydrogen phosphate: 1.42g, sodium chloride: 8g and 0.2g of potassium chloride, and about 800mL of deionized water are added and fully stirred to be dissolved, then diluted hydrochloric acid is added to adjust the pH to 7.4, the volume is adjusted to 1L, the pH is 7.6, and the mixture is used after being sterilized.

The dyes AVF and PI used in the method are provided by Shanghai's "Annexin V apoptosis detection kit" and FITC/PI double staining method ".

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Claims

1. A method for evaluating the apoptosis-like decline of bacteria by using phosphatidylserine evagination staining on cell membranes, which is characterized by comprising the following steps:

(1) centrifuging a liquid culture medium containing bacteria to be detected to remove supernatant and take precipitate, mixing an ethanol solution with the precipitate, uniformly mixing to resuspend cells, standing for fixing treatment, centrifuging to take precipitate, and using EDTA-Na₂Washing the solution, centrifuging and taking the precipitate to obtain the bacteria after the fixation treatment;

(2) uniformly mixing the bacteria subjected to the fixation treatment in the step (1) with a sterile phosphate buffer solution, and re-suspending the bacteria to obtain a bacteria suspension;

(3) adding a lysozyme solution into the bacterial suspension obtained in the step (2), performing wall-dissolving treatment, centrifuging and taking precipitates to obtain bacteria subjected to wall dissolving;

(4) uniformly mixing the bacteria subjected to wall dissolving in the step (3) with a sterile phosphate buffer solution, and diluting the bacteria subjected to wall dissolving to obtain a bacteria diluent;

(5) adding an AVF dye and a dyeing binding solution into the bacterial diluent in the step (4), uniformly mixing, and dyeing in a dark place to obtain dyed cell sap;

(6) and (3) detecting the fluorescence intensity of the cell fluid after dyeing in the step (5) by using a flow cytometer to obtain a red fluorescence intensity value and a green fluorescence intensity value, judging that the bacterial cells in the apoptosis-like decline state exist in the liquid culture medium to be detected in the step (1) when the fluorescence intensity of AVF dyeing exceeds the fluorescence intensity of logarithmic phase culture cell contrast, and judging that the bacterial cells in the apoptosis-like decline state do not exist in the liquid culture medium to be detected in the step (1) when the fluorescence intensity of AVF dyeing does not exceed the fluorescence intensity of logarithmic phase culture cell contrast.

2. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the ethanol solution of step (1) has a concentration of 65-75% by volume; 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 fixing treatment is 0.5-2 h.

3. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the EDTA-Na of step (1)₂The solvent of the solution is water; the EDTA-Na₂The concentration of the solution is 100-500 mg/L.

4. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine eversion staining on cell membrane according to claim 1, wherein the concentration of the sterile phosphate buffer solution of step (2) and step (4) is 0.01-0.1M; the volume ratio of the sterile phosphate buffer solution in the step (2) to the ethanol solution in the step (1) is 1.3:1-1: 1.

5. The method for evaluating the apoptosis-like recession of bacteria by the eversion staining of phosphatidylserine on the cell membrane according to claim 1, wherein the lysozyme solution in the step (3) is a solution obtained by uniformly mixing lysozyme with water; 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 30-60min, and the temperature of the wall dissolving treatment is 37 ℃; after the wall lysis treatment, the lysozyme concentration in the cell suspension was 50-200 mg/L.

6. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the concentration of bacteria in the bacterial dilution of step (4) is 10⁶-10⁷cells/mL。

7. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the AVF dye of step (5) is Annexin-V-FITC; the volume of the AVF dye is 0.5-1.5% of the volume of the bacterial dilution.

8. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the volume of the staining conjugate in step (5) is 25% of the volume of the bacterial dilution.

9. The method for evaluating the apoptosis-like decline of bacteria by phosphatidylserine evagination staining on cell membrane according to claim 1, wherein the staining time of step (5) is 15-30 min.