CN113528401B - Method for accurately obtaining VBNC-state bacteria - Google Patents
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Abstract
The invention relates to the technical field of microorganisms, in particular to a method for obtaining VBNC bacteria. A method for accurately obtaining VBNC bacteria comprises the following steps: (1) Inducing the bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC bacteria and sublethal damaged bacteria; (2) Removing sublethal damaged bacteria in the mixed system by adopting a selective culture medium plating method; (3) VBNC-state bacteria and dead bacteria were separated by gradient centrifugation. The method can effectively distinguish the sublethal damaged bacteria from the VBNC bacteria, reduce or eliminate the influence of the sublethal damaged bacteria on the precise identification of the physiological characteristics of the VBNC bacteria and the subsequent metabolic research, accurately acquire the pure VBNC bacteria, and provide reliable technical support for the assessment and effective prevention and control of ecological risks and health hazards.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for obtaining VBNC bacteria.
Background
More than 95% of the bacteria in the environment are in a viable, non-culturable (viable but nonculturable, VBNC) state, which is not conducive to people's identification, awareness, exploitation and risk prevention of the bacteria in that state. The conventional evaluation mode of sterilization and disinfection at present, namely plate counting, often ignores the existence of VBNC-state bacteria, so that the number of living bacteria in a detection sample is underestimated. These omitted VBNC bacteria can be transformed into normal culturable bacteria under certain conditions with concomitant recovery of pathogenicity, which presents a safety hazard to people. To enhance the understanding and recognition of VBNC-state bacteria, artificially induced VBNC-state bacteria have received a great deal of attention. However, VBNC bacteria obtained by artificial induction are complex mixed systems, and contain dead bacteria, VBNC bacteria and sublethal damaged bacteria, which interfere with the intensive scientific research of VBNC bacteria.
Different environmental stress-induced VBNC bacteria all contain different proportions of sublethally damaged bacteria. Although both sublethally damaged bacteria and VBNC bacteria have low metabolic activity, unlike VBNC bacteria, sublethally damaged bacteria do not respond adaptively to adverse environmental conditions, but rather are produced by an imbalance in synthesis and catabolism induced by physicochemical stresses. The coexistence of sublethal damaged bacteria and VBNC bacteria not only interferes with the understanding of physiological characteristics of VBNC and the mechanism for maintaining low metabolic activity, but also affects the reasonable use of related removal technologies, thereby increasing ecological risks and health hazards.
Disclosure of Invention
The invention aims to solve the problem that the existing VBNC bacteria induction method can not remove sublethal damaged bacteria and dead bacteria, and provides a method for accurately obtaining VBNC bacteria.
The technical scheme adopted for solving the technical problems is as follows: a method for accurately obtaining VBNC bacteria comprises the following steps:
(1) Inducing the bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC bacteria and sublethal damaged bacteria;
(2) Removing sublethal damaged bacteria in the mixed system by adopting a selective culture medium plating method;
(3) VBNC-state bacteria and dead bacteria were isolated.
Preferably, the inducer is chlorine-containing sterilized water with the concentration of 4-8 mg/L.
Preferably, the inducer is hydrogen peroxide with the concentration of 4-8%.
Preferably, in step (2), a selective agent is added to the LB medium to kill sub-lethal damaged bacteria.
Preferably, the selective agent is sodium chloride solution with the concentration of 15-45 g/L.
Preferably, the selective agent is potassium chloride solution with the concentration of 85-120 g/L.
Preferably, in step (3), the viable bacteria and the dead bacteria are separated by density gradient centrifugation, specifically comprising: suspending the bacterial culture treated in the step (2), and adding 45% -70% Percoll layering liquid; density gradient centrifugation was performed to remove dead bacteria.
Preferably, the bacterium is any one of Escherichia coli, vibrio cholerae, helicobacter pylori, mycobacterium tuberculosis, salmonella typhimurium, and Listeria monocytogenes.
The method for accurately acquiring the VBNC bacteria is simple and quick, and utilizes CTC-DAPI staining, heterotrophic plate counting and a Selective Medium Plating (SMPT) method to couple density gradient centrifugation technology to establish a novel method for eliminating interference of sublethal damaged bacteria and quantitatively detecting the VBNC bacteria. The method provided by the invention realizes effective differentiation of sublethal damaged bacteria and VBNC bacteria, reduces or even eliminates the influence of the sublethal damaged bacteria and VBNC bacteria on accurate identification of physiological characteristics of the VBNC bacteria and subsequent metabolic research, and provides reliable technical support for environmental health risk assessment and effective prevention and control.
Drawings
FIG. 1 is a graph showing total, viable, culturable, and viable but non-culturable Pseudomonas aeruginosa cell count after 6mg/L chlorine treatment;
FIG. 2 is a photograph taken by an inverted fluorescence microscope after CTC-DAPI staining: (a) Is untreated bacteria (control), (b) is bacteria induced at a chlorine concentration of 6 mg/L;
FIG. 3 is a graph showing sub-lethal injured bacteria count following 6mg/L chlorine treatment;
FIG. 4 is a photograph taken by an inverted fluorescence microscope of the purified sample after CTC-DAPI staining.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples are commercially available unless otherwise specified. The quantitative tests in the examples were all performed in triplicate, and the results were averaged.
The strains used in the examples described below are all commercially available.
The LB liquid medium used in the following examples consisted of a solvent and a solute, the solute and its concentration in the medium were as follows: tryptone 10 g/L, yeast extract 5g/L, sodium chloride 10 g/L, naOH adjust pH to 7.4.
The LB solid medium used in the following examples consisted of a solvent and a solute, the solute and its concentration in the medium were as follows: tryptone 10 g/L, yeast extract 5g/L, sodium chloride 70 g/L, agar powder 15 g/L.
The PBS solution of pH 7.0 was prepared at 0.2 mol/L used in the following examples: and (2) solution A: na (Na) 2 HPO 4 ·12H 2 Adding distilled water into 71.628g of O to reach 1000mL; b, liquid: naH (NaH) 2 PO 4 ·2H 2 Adding distilled water into 31.202g of O to fix the volume to 1000mL; mixing the first solution 72mL and the second solution 28 mL.
Example 1
1. Strain induction treatment
(1) Activation and preparation of pseudomonas aeruginosa thallus
And (3) carrying out activation culture on the pseudomonas aeruginosa freeze-dried powder in an LB liquid culture medium until the pseudomonas aeruginosa freeze-dried powder reaches a logarithmic phase. 5000 Centrifuge at rpm for 10 minutes and wash 3 times with sterile PBS solution. The cells were resuspended in sterile PBS solution and diluted to 10 6 -10 8 Final concentration of CFU mL/L.
(2) Induction of pseudomonas aeruginosa in VBNC state
Sodium hypochlorite sterilized water was added to the medium containing the bacteria activated in the above step to a final chlorine concentration of 6mg/L, and the bacteria were sampled by incubating at 25℃for 90 minutes with stirring at 180 rpm. Before sampling, 100 μl of 20% sodium thiosulfate was added to inactivate residual chlorine. Obtaining VBNC-state bacteria containing sublethal damaged bacteria.
(3) Determination of the number of bacteria that can be cultivated
The number of bacteria which can be cultivated in the bacterial liquid in the low-temperature storage process is measured according to the plate counting method of the national standard 4789.2-2016. And (3) carrying out 10-time gradient dilution on the low-temperature induced bacterial liquid obtained in the steps in a sterile ultra-clean workbench, taking two bacterial liquids with proper dilution gradients of 100 mu L in a sterile LB solid culture medium, uniformly coating each gradient for 3 times, placing the bacterial liquid into an incubator after the bacterial liquid is completely absorbed, inversely culturing at 37 ℃ for 24 hours, and recording the total number of bacterial colonies according to a plate counting principle. When the bacteria culturable number is reduced to zero and the continuous 3d measurement is performed with the bacteria culturable number remaining unchanged to zero, it is considered that the bacteria may enter a viable non-culturable state.
(4) Determination of viable count
0.5ml of brain heart infusion and 100. Mu.L of 0.05g/L pyruvic acid solution were added to 0.5ml of the bacterial suspension to be analyzed to stimulate cellular respiration. CTCs were diluted with double distilled water to a final concentration of 5mM/L and the mixture was incubated at microaerophilic 37 ℃ for 4h. The cells were then filtered through a polyablack carbon membrane (0.2 μm/well, diameter 25 mm).
(5) Determination of the total number of bacteria
The solution was covered with 5. Mu.g/mL DAPI for 5min for counterstaining. Finally, the stain was vacuum filtered and more than 4 fields were randomly counted per sample under a study-grade inverted fluorescence microscope NIKON TI-E (Japan), three replicates per sample. FIG. 1 is a graph showing the total, viable, culturable and viable but non-culturable Pseudomonas aeruginosa cell count after 6mg/L chlorine treatment. Fluorescence micrographs of 6mg/L chlorine concentration-induced bacteria and control (untreated) after CTC-DAPI staining are shown in FIGS. 2 (a) and (b), respectively.
(2) Quantification and removal of sublethal damaged bacteria
(1) The maximum non-inhibitory concentration (MNIC) of the osmotic molecule NaCl in LB medium was determined: untreated cells were plated on LB agar medium containing different concentrations of NaCl and incubated at 37℃for 48 hours. (MNIC is defined as the highest concentration that inhibits less than 20% of the initial untreated bacterial population). Experiments prove that the maximum inhibition-free concentration of NaCl in the LB culture medium is 45 g/L. NaCl is added into the induced bacterial sample to make the concentration of sodium chloride in the solution 15-45 g/L, and in this concentration range, the living bacteria are not affected, and the sublethal damaged bacteria die.
(2) The resulting bacterial samples were spread on LB agar medium (selection medium) and ordinary LB agar medium (non-selection medium) having NaCl concentration of 40 g/L, respectively, and the plates were cultured at 37℃for 48 hours. The amount of sublethally damaged bacteria is expressed as the difference between the bacterial count on the non-selective medium and the bacterial count on the selective medium, as shown in figure 3.
(3) Isolation of dead bacteria from VBNC bacteria
The VBNC cell culture treated in the step 2 is collected and suspended in 20ml of PBS, 55% Percoll layering liquid is added, and viable bacteria and dead bacteria are separated by a density gradient centrifugation method, wherein the specific process is as follows:
first, the samples were centrifuged at 12000rpm and incubated at 4℃for 1h until two distinct cell layers were visible. The lower part was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ and 4000 rpm for 45min, and taking the lower layer to re-suspend in 50 mu L of PBS to obtain a purified VBNC bacterial sample for removing sublethal injury and dead bacteria.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with heterotrophic plate technology (HPC) found that all viable bacteria in the samples were VBNC bacteria, as shown in fig. 4.
Embodiment II,
1. Strain induction treatment
(1) Activation and preparation of coliform cells
E, activating and culturing the escherichia coli freeze-dried powder in an LB liquid culture medium until the logarithmic phase. 5000 Centrifuge at rpm for 10 minutes and wash 3 times with sterile PBS solution. The cells were resuspended in sterile PBS solution and diluted to 10 6 -10 8 Final concentration of CFU mL/L.
(2) Induction of E.coli in VBNC status
Adding hydrogen peroxide with the concentration of 6% into a culture medium containing the bacteria activated in the steps, incubating the bacteria at 25 ℃ for 95 minutes under stirring at 180 rpm, and sampling to obtain VBNC bacteria containing sublethal injured bacteria.
(3) Determination of the number of bacteria that can be cultivated
Reference is made to the measurement method in example 1.
(4) Determination of viable count
Reference is made to the measurement method in example 1.
(5) Determination of the total number of bacteria
Reference is made to the measurement method in example 1.
(2) Quantification and removal of sublethal damaged bacteria
(1) Determining the maximum non-inhibitory concentration (MNIC) of the permeabilizing molecule KCl in LB medium: experiments prove that the maximum inhibition-free concentration of KCl in LB culture medium is 120 g/L. KCl is added to the induced bacterial sample to bring the concentration of potassium chloride in the solution to 85-120 g/L, in which concentration range the living bacteria are not affected and the sublethal damaged bacteria die.
(2) The resulting bacterial samples were spread on LB agar medium (selective medium) and ordinary LB agar medium (non-selective medium) having KCl concentration of 110 g/L, respectively, and the plates were cultured at 37℃for 48 hours. The amount of sublethally damaged bacteria is expressed as the difference between the bacterial count on the non-selective medium and the bacterial count on the selective medium.
(3) Isolation of dead bacteria from VBNC bacteria
The VBNC cell culture treated in the step 2 is collected and suspended in 20ml of PBS, 70% Percoll layering liquid is added, and viable bacteria and dead bacteria are separated by a density gradient centrifugation method, wherein the specific process is as follows:
first, the sample was centrifuged at 11000rpm and incubated at 4℃for 1h until two distinct cell layers were visible. The lower part was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ for 30min at 5000 rpm, and taking the lower layer to re-suspend in 50 mu L of PBS to obtain a purified VBNC bacterial sample for removing sublethal injury and dead bacteria.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with heterotrophic plate technology (HPC) found that all viable bacteria in the samples were VBNC bacteria.
Example III
1. Strain induction treatment
(1) Activation and preparation of salmonella typhimurium
And (3) carrying out activation culture on the salmonella typhimurium freeze-dried powder in an LB liquid culture medium until the salmonella typhimurium freeze-dried powder reaches a logarithmic phase. 5000 Centrifuge at rpm for 10 minutes and wash 3 times with sterile PBS solution. The cells were resuspended in sterile PBS solution and diluted to 10 6 -10 8 Final concentration of CFU mL/L.
(2) Induction of VBNC state Salmonella typhimurium
Adding sodium hypochlorite disinfectant water with the final chlorine concentration of 7mg/L into a culture medium containing the bacteria activated in the steps, incubating the bacteria at 25 ℃ and under stirring at 180 rpm for 100 minutes to sample, and adding 100 mu L of 20% sodium thiosulfate to inactivate residual chlorine before sampling to obtain VBNC bacteria containing sublethal damaged bacteria.
(3) Determination of the number of bacteria that can be cultivated
Reference is made to the measurement method in example 1.
(4) Determination of viable count
Reference is made to the measurement method in example 1.
(5) Determination of the total number of bacteria
Reference is made to the measurement method in example 1.
(2) Quantification and removal of sublethal damaged bacteria
The resulting bacterial samples were spread on LB agar medium and ordinary LB agar medium (non-selective medium) having NaCl concentration of 35g/L, respectively, and the plates were cultured at 37℃for 48 hours. The amount of sublethally damaged bacteria is expressed as the difference between the bacterial count on the non-selective medium and the bacterial count on the selective medium.
(3) Isolation of dead bacteria from VBNC bacteria
The VBNC cell culture treated in the step 2 is collected and suspended in 20ml of PBS, and 60% Percoll layering liquid is added, and viable bacteria and dead bacteria are separated by a density gradient centrifugation method, wherein the specific process is as follows:
first, the sample was centrifuged at 10000rpm and incubated at 4℃for 1h until two different cell layers were visible. The lower part was collected and suspended in 70 ml of PBS; and centrifuging at 4500 rpm for 40min at 4 ℃, and taking the lower layer to re-suspend in 50 mu L of PBS to obtain a purified VBNC bacterial sample for removing sublethal injury and dead bacteria.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with heterotrophic plate technology (HPC) found that all viable bacteria in the samples were VBNC bacteria.
Fourth embodiment,
1. Strain induction treatment
(1) Activation and preparation of vibrio cholerae thallus
And (3) activating and culturing the vibrio cholerae freeze-dried powder in an LB liquid culture medium to a logarithmic phase. 5000 Centrifuge at rpm for 10 minutes and wash 3 times with sterile PBS solution. The cells were resuspended in sterile PBS solution and diluted to 10 6 -10 8 Final concentration of CFU mL/L.
(2) Induction of Vibrio cholerae in VBNC state
Sodium hypochlorite sterilized water was added to the medium containing the bacteria activated in the above step to a final chlorine concentration of 5mg/L, and the bacteria were sampled by incubating at 25℃for 100 minutes with stirring at 180 rpm. Before sampling, 100 μl of 20% sodium thiosulfate was added to inactivate residual chlorine. Obtaining VBNC-state bacteria containing sublethal damaged bacteria.
(3) Determination of the number of bacteria that can be cultivated
Reference is made to the measurement method in example 1.
(4) Determination of viable count
Reference is made to the measurement method in example 1.
(5) Determination of the total number of bacteria
Reference is made to the measurement method in example 1.
(2) Quantification and removal of sublethal damaged bacteria
The resulting bacterial samples were spread on LB agar medium and ordinary LB agar medium (non-selective medium) having NaCl concentration of 20 g/L, respectively, and the plates were cultured at 37℃for 48 hours. The amount of sublethally damaged bacteria is expressed as the difference between the bacterial count on the non-selective medium and the bacterial count on the selective medium.
(3) Isolation of dead bacteria from VBNC bacteria
The VBNC cell culture treated in the step 2 is collected and suspended in 20ml of PBS, and 50% Percoll layering liquid is added, and viable bacteria and dead bacteria are separated by a density gradient centrifugation method, wherein the specific process is as follows:
first, the samples were centrifuged at 12000rpm and incubated at 4℃for 1h until two distinct cell layers were visible. The lower part was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ and 4000 rpm for 30min, and taking the lower layer to re-suspend in 50 mu L of PBS to obtain a purified VBNC bacterial sample for removing sublethal injury and dead bacteria.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with heterotrophic plate technology (HPC) found that all viable bacteria in the samples were VBNC bacteria.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present invention should be covered by the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (2)
1. The method for accurately obtaining the VBNC bacteria is characterized by comprising the following steps of:
(1) Inducing the bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC bacteria and sublethal damaged bacteria;
(2) A selective culture medium plating method is adopted, a selective agent is added into an LB culture medium, and sub-lethal damage bacteria in the mixed system are removed;
(3) Separating VBNC-state bacteria and dead bacteria;
the bacteria are any one of pseudomonas aeruginosa, escherichia coli, salmonella typhimurium and vibrio cholerae;
the inducer of the pseudomonas aeruginosa, the salmonella typhimurium or the vibrio cholerae is chlorine-containing disinfectant water with the concentration of 4-8mg/L, and the selection agent is sodium chloride solution with the concentration of 15-45 g/L;
the inducer of the escherichia coli is hydrogen peroxide with the concentration of 4-8%, and the selector is potassium chloride solution with the concentration of 85-120 g/L.
2. The method for accurately obtaining VBNC-state bacteria according to claim 1, wherein in the step (3), viable bacteria and dead bacteria are separated by a density gradient centrifugation method, specifically comprising: suspending the cell culture treated in the step (2), and adding 45% -70% Percoll layering liquid; density gradient centrifugation was performed to remove dead bacteria.
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Inventor after: Liu Chunguang Inventor after: Qi Zheng Inventor before: Liu Chunguang |
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