CN113528401A - Method for accurately obtaining VBNC (viable but non-viable) 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 state bacteria. A method for accurately obtaining VBNC bacteria comprises the following steps: (1) inducing bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC state bacteria and sublethal damaged bacteria; (2) removing sublethal damaged bacteria in the mixed system by adopting a selective culture medium plate paving method; (3) VBNC state bacteria and dead bacteria are separated by adopting a gradient centrifugation method. The method can effectively distinguish the sublethal damaged bacteria from the VBNC-state bacteria, reduces or eliminates the influence of the sublethal damaged bacteria on the accurate identification of the physiological characteristics of the VBNC-state bacteria and the subsequent metabolic study, can accurately obtain the pure VBNC-state bacteria, and provides reliable technical support for the evaluation 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 state bacteria.
Background
More than 95% of bacteria in the environment are in a viable but non-culturable (VBNC) state, which is not favorable for people to identify, recognize, develop and utilize the bacteria in the state and prevent risks. The conventional evaluation mode of sterilization and disinfection at present, namely plate counting, usually ignores the existence of VBNC state bacteria, and further underestimates the number of live bacteria in a detected sample. The overlooked VBNC bacteria can be converted into normal culturable bacteria under certain conditions, and the pathogenicity is recovered, so that the potential safety hazard is brought to people. In order to enhance the understanding and recognition of VBNC bacteria, artificial induction of VBNC bacteria has received a great deal of attention. However, VBNC bacteria obtained by artificial induction at present are complex mixed systems, and contain dead bacteria, VBNC bacteria and sublethal damaged bacteria, which interfere with deep scientific research on VBNC bacteria.
VBNC state bacteria induced by different environmental stresses contain sublethal injury bacteria with different proportions. Although both sublethal-injured bacteria and VBNC-state bacteria have low metabolic activity, unlike VBNC-state bacteria, sublethal-injured bacteria do not respond adaptively to adverse environmental conditions, but result from an imbalance in synthesis and catabolism caused by physicochemical stress. The coexistence of sublethal damaged bacteria and VBNC status bacteria not only interferes with people's understanding of VBNC physiological characteristics and mechanisms for maintaining low metabolic activity, but also influences the rational use of related removal technologies, thereby increasing ecological risks and health hazards.
Disclosure of Invention
The invention aims to solve the problem that sublethal damaged bacteria and dead bacteria cannot be removed in the conventional VBNC state bacteria induction method, and provides a method for accurately obtaining VBNC state bacteria.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for accurately obtaining VBNC bacteria comprises the following steps:
(1) inducing bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC state bacteria and sublethal damaged bacteria;
(2) removing sublethal damaged bacteria in the mixed system by adopting a selective culture medium plate paving method;
(3) VBNC-state bacteria and dead bacteria are 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 sublethal-injured bacteria.
Preferably, the selection agent is a sodium chloride solution with the concentration of 15-45 g/L.
Preferably, the selection agent is a potassium chloride solution with the concentration of 85-120 g/L.
Preferably, in the step (3), the density gradient centrifugation method is used for separating live bacteria and dead bacteria, and specifically comprises the following steps: suspending the bacterial culture treated in the step (2), and adding 45% -70% of Percoll layering liquid; density gradient centrifugation is carried out to remove dead bacteria.
Preferably, the bacterium is any one of escherichia coli, vibrio cholerae, helicobacter pylori, mycobacterium tuberculosis, salmonella typhimurium, listeria monocytogenes.
The method for accurately obtaining the VBNC bacteria is simple and quick, and a new method for eliminating the interference of sublethal damaged bacteria and quantitatively detecting the VBNC bacteria is established by using CTC-DAPI staining, heterotrophic plate counting and Selective Medium Plating (SMPT) method coupled density gradient centrifugation technology. The method provided by the invention realizes effective differentiation of sublethal damaged bacteria and VBNC state bacteria, reduces or even eliminates the influence of the sublethal damaged bacteria on accurate identification of physiological characteristics of the VBNC state bacteria and subsequent metabolic research, and provides reliable technical support for environmental health risk assessment and effective prevention and control.
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FIG. 1 is a graph of total, viable, culturable, and viable but non-culturable Pseudomonas aeruginosa cell counts after 6mg/L chlorine treatment;
FIG. 2 is a photograph taken by an inverted fluorescence microscope after staining CTC-DAPI: (a) untreated bacteria (control), (b) bacteria induced at a chlorine concentration of 6 mg/L;
FIG. 3 is a sublethal damage bacteria count after 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 to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. 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 procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the examples are commercially available unless otherwise specified. In the quantitative tests in the examples, three repeated experiments are set, and the results are averaged.
The strains used in the examples described below are commercially available.
The LB liquid medium used in the following examples consists of a solvent and solutes, the solutes and their concentrations in the medium are as follows: 10 g/L of tryptone, 5g/L of yeast extract, 10 g/L of sodium chloride and pH adjusted to 7.4 by NaOH.
The LB solid medium used in the following examples consisted of a solvent and solutes, the concentrations of which in the medium were as follows: 10 g/L of tryptone, 5g/L of yeast extract, 70 g/L of sodium chloride and 15 g/L of agar powder.
Prepared from 0.2 mol/L of PBS (pH 7.0) solution used in the following examples: liquid A: na (Na)2HPO4·12H2O71.628 g, adding distilled water to a constant volume of 1000 mL; b, liquid B: NaH2PO4·2H2O31.202 g, adding distilled water to a constant volume of 1000 mL; mixing the first solution 72mL and the second solution 28 mL.
The first embodiment,
1. Strain induction treatment
(1) Activation and preparation of pseudomonas aeruginosa thallus
The pseudomonas aeruginosa lyophilized powder is added in the mixture LB, activated culturing in liquid culture medium to logarithmic phase. Centrifuge at 5000 rpm for 10 minutes and wash 3 times with sterile PBS solution. Cells were resuspended in sterile PBS solution and diluted to 106-108Final concentration of CFU mL/L.
(2) VBNC state Pseudomonas aeruginosa induction
To the medium containing the activated bacteria of the above procedure, sodium hypochlorite disinfectant water was added at a final chlorine concentration of 6mg/L, and the bacteria were incubated at 25 ℃ for 90 minutes with stirring at 180 rpm, and sampled. Before sampling, 100. mu.L of 20% sodium thiosulfate was added to inactivate residual chlorine. VBNC-state bacteria containing sublethal injured bacteria were obtained.
(3) Measurement of the culturable number of bacteria
The number of culturable bacteria in the bacterial suspension during cryopreservation was determined according to the plate count method of national standard 4789.2-2016. And (3) carrying out gradient dilution on the low-temperature induced bacterium liquid obtained in the step by 10 times in an aseptic ultra-clean workbench, uniformly coating 100 mu L of two bacterium liquids with appropriate dilution gradients on an aseptic LB solid culture medium, placing each gradient into an incubator after the bacterium liquid is completely absorbed, carrying out inverted culture at 37 ℃ for 24 hours, and recording the total number of colonies according to a flat plate counting principle. When the culturable number of bacteria had decreased to zero and the number of culturable bacteria remained zero after the continuous 3d measurement, it was considered that the bacteria were likely to enter a viable non-culturable state.
(4) Measurement of viable cell 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. CTC was diluted to final concentration of 5mM/L with double distilled water and the mixture was incubated for 4h in microaerophilic 37 ℃. The cells were then filtered through a polymeric black carbon membrane (0.2 μm/well, 25mm diameter).
(5) Determination of the total number of bacteria
Cover with 5. mu.g/mL DAPI solution for 5min for counterstaining. Finally, the stain was filtered off in vacuo and counted randomly over 4 fields per sample under a research grade inverted fluorescence microscope, NIKON TI-E (Japan), three replicates per sample. FIG. 1 is a graph of total, viable, culturable, and viable but non-culturable P.aeruginosa cell counts after 6mg/L chlorine treatment. Fluorescence micrographs of bacteria induced by 6mg/L chloride concentration after CTC-DAPI staining and control (untreated) as shown in FIGS. 2 (a), (b), respectively
2. Quantification and removal of sublethal-damaging bacteria
(1) Determining the maximum uninhibited concentration (MNIC) of the permeation molecule NaCl in LB medium: untreated cells were plated on LB agar medium containing varying concentrations of NaCl and incubated at 37 ℃ for 48 hours. (MNIC is defined as the highest concentration that inhibits less than 20% of the original untreated bacterial population). Experiments prove that the maximum uninhibited concentration of NaCl in LB culture medium is 45 g/L. NaCl is added into the induced bacteria sample to ensure that the concentration of sodium chloride in the solution is 15-45 g/L, and in the concentration range, live bacteria are not influenced, and sublethal injured bacteria die.
(2) The induced bacterial samples were spread on LB agar medium (selection medium) and ordinary LB agar medium (nonselective medium) with a NaCl concentration of 40 g/L, respectively, and the plates were cultured at 37 ℃ for 48 hours. The amount of sublethal damaging bacteria was expressed as the difference between the bacterial count on non-selective medium and the bacterial count on selective medium, as shown in figure 3.
Separation of dead bacteria from VBNC bacteria
Collecting the VBNC cell culture processed in the step 2, suspending the VBNC cell culture in 20ml of PBS, adding 55% Percoll layering liquid, and separating viable bacteria and dead bacteria by using a density gradient centrifugation method, wherein the specific process comprises the following steps:
first, the sample was centrifuged at 12000rpm and incubated at 4 ℃ for 1h until two distinct cell layers were visible. The lower portion was collected and suspended in 70 ml of PBS; and centrifuging for 45min at 4 ℃ and 4000 rpm, and resuspending the lower layer in 50 mu L of PBS to obtain a purified VBNC bacteria sample from which sublethal damage and dead bacteria are removed.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with Heterotrophic Plating (HPC) revealed that all live bacteria in the sample were VBNC bacteria, as shown in fig. 4.
Example II,
1. Strain induction treatment
(1) Activation and preparation of Escherichia coli
And (3) performing activated culture on the escherichia coli freeze-dried powder in an LB liquid culture medium to a logarithmic phase. Centrifuge at 5000 rpm for 10 minutes and wash 3 times with sterile PBS solution. Cells were resuspended in sterile PBS solution and diluted to 106-108Final concentration of CFU mL/L.
(2) Induction of E.coli in VBNC state
Adding 6% hydrogen peroxide into the culture medium containing the activated bacteria, incubating the bacteria at 25 deg.C and 180 rpm under stirring for 95 min, and sampling to obtain VBNC state bacteria containing sublethal injured bacteria.
(3) Measurement of the culturable number of bacteria
Refer to the assay procedure in example 1.
(4) Measurement of viable cell count
Refer to the assay procedure in example 1.
(5) Determination of the total number of bacteria
Refer to the assay procedure in example 1.
Quantification and removal of sublethal-damaging bacteria
(1) Determining the maximum uninhibited concentration (MNIC) of the permeation molecule KCl in LB medium: experiments prove that the maximum uninhibited concentration of KCl in LB culture medium is 120 g/L. KCl is added into the induced bacteria sample to ensure that the concentration of potassium chloride in the solution is 85-120 g/L, and in the concentration range, live bacteria are not affected, but sublethal injured bacteria die.
(2) The induced bacterial samples were spread on LB agar medium (selective medium) and ordinary LB agar medium (non-selective medium) with KCl concentration of 110 g/L, respectively, and the plates were cultured at 37 ℃ for 48 hours. The amount of sublethal damaging bacteria is expressed as the difference between the bacterial count on non-selective medium and the bacterial count on selective medium.
Separation of dead bacteria from VBNC bacteria
Collecting the VBNC cell culture processed in the step 2, suspending the VBNC cell culture in 20ml of PBS, adding 70% Percoll layering liquid, and separating viable bacteria and dead bacteria by using a density gradient centrifugation method, wherein the specific process comprises the following steps:
first, the sample was centrifuged at 11000rpm and incubated at 4 ℃ for 1h until two distinct cell layers were visible. The lower portion was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ and 5000 rpm for 30min, and resuspending the lower layer in 50 mu L of PBS to obtain the purified VBNC bacteria sample from which the sublethal damage and dead bacteria are removed.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with Heterotrophic Plating (HPC) revealed that all live bacteria in the sample were VBNC bacteria.
EXAMPLE III
1. Strain induction treatment
(1) Activation and preparation of salmonella typhimurium
And (3) carrying out activated culture on the salmonella typhimurium freeze-dried powder in an LB liquid culture medium to logarithmic phase. Centrifuge at 5000 rpm for 10 minutes and wash 3 times with sterile PBS solution. Cells were resuspended in sterile PBS solution and diluted to 106-108Final concentration of CFU mL/L.
(2) Induction of Salmonella typhimurium in VBNC state
Adding sodium hypochlorite disinfectant solution with final chlorine concentration of 7mg/L into the culture medium containing the activated bacteria in the above steps, incubating the bacteria at 25 ℃ for 100 minutes under stirring at 180 rpm, sampling, and adding 100 μ L of 20% sodium thiosulfate to inactivate residual chlorine before sampling to obtain VBNC state bacteria containing sublethal damaged bacteria.
(3) Measurement of the culturable number of bacteria
Refer to the assay procedure in example 1.
(4) Measurement of viable cell count
Refer to the assay procedure in example 1.
(5) Determination of the total number of bacteria
Refer to the assay procedure in example 1.
Quantification and removal of sublethal-damaging bacteria
The bacterial samples obtained by the induction were spread on LB agar medium and ordinary LB agar medium (non-selective medium) respectively, each having a NaCl concentration of 35g/L, and the plates were cultured at 37 ℃ for 48 hours. The amount of sublethal damaging bacteria is expressed as the difference between the bacterial count on non-selective medium and the bacterial count on selective medium.
Separation of dead bacteria from VBNC bacteria
Collecting the VBNC cell culture processed in the step 2, suspending the VBNC cell culture in 20ml of PBS, adding 60% Percoll layering liquid, and separating viable bacteria and dead bacteria by using a density gradient centrifugation method, wherein the specific process comprises the following steps:
first, the sample was centrifuged at 10000rpm and incubated at 4 ℃ for 1h until two distinct cell layers were visible. The lower portion was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ and 4500 rpm for 40min, and resuspending the lower layer in 50 mu L PBS to obtain the purified VBNC bacteria sample from which the sublethal damage and dead bacteria are removed.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with Heterotrophic Plating (HPC) revealed that all live bacteria in the sample were VBNC bacteria.
Example four,
1. Strain induction treatment
(1) Activation and preparation of Vibrio cholerae thallus
The Vibrio cholerae freeze-dried powder is activated and cultured in an LB liquid culture medium to logarithmic phase. Centrifuge at 5000 rpm for 10 minutes and wash 3 times with sterile PBS solution. Cells were resuspended in sterile PBS solution and diluted to 106-108Final concentration of CFU mL/L.
(2) VBNC status Induction of Vibrio cholerae
To the medium containing the bacteria activated in the above step, sodium hypochlorite disinfectant water was added at a final chlorine concentration of 5mg/L, and the bacteria were incubated at 25 ℃ for 100 minutes with stirring at 180 rpm, and sampled. Before sampling, 100. mu.L of 20% sodium thiosulfate was added to inactivate residual chlorine. VBNC-state bacteria containing sublethal injured bacteria were obtained.
(3) Measurement of the culturable number of bacteria
Refer to the assay procedure in example 1.
(4) Measurement of viable cell count
Refer to the assay procedure in example 1.
(5) Determination of the total number of bacteria
Refer to the assay procedure in example 1.
Quantification and removal of sublethal-damaging bacteria
The bacterial samples obtained by the induction were spread on LB agar medium and ordinary LB agar medium (non-selective medium) respectively, each having a NaCl concentration of 20 g/L, and the plates were cultured at 37 ℃ for 48 hours. The amount of sublethal damaging bacteria is expressed as the difference between the bacterial count on non-selective medium and the bacterial count on selective medium.
Separation of dead bacteria from VBNC bacteria
Collecting the VBNC cell culture processed in the step 2, suspending the VBNC cell culture in 20ml of PBS, adding 50% Percoll layering liquid, and separating viable bacteria and dead bacteria by using a density gradient centrifugation method, wherein the specific process comprises the following steps:
first, the sample was centrifuged at 12000rpm and incubated at 4 ℃ for 1h until two distinct cell layers were visible. The lower portion was collected and suspended in 70 ml of PBS; and centrifuging at 4 ℃ and 4000 rpm for 30min, and resuspending the lower layer in 50 mu L of PBS to obtain a purified VBNC bacteria sample from which sublethal damage and dead bacteria are removed.
Taking a purified VBNC sample for detection: CTC-DAPI staining combined with Heterotrophic Plating (HPC) revealed that all live bacteria in the sample were VBNC bacteria.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (8)
1. A method for accurately obtaining VBNC bacteria is characterized by comprising the following steps:
(1) inducing bacteria by using an inducer to obtain a mixed system containing dead bacteria, VBNC state bacteria and sublethal damaged bacteria;
(2) removing sublethal damaged bacteria in the mixed system by adopting a selective culture medium plate paving method;
(3) VBNC-state bacteria and dead bacteria are isolated.
2. The method for accurately obtaining VBNC bacteria, as claimed in claim 1, wherein said inducing agent is sterilized water containing chlorine at a concentration of 4-8 mg/L.
3. The method for accurately obtaining VBNC bacteria of claim 1, wherein the inducing agent is hydrogen peroxide with a concentration of 4-8%.
4. The method for accurately obtaining VBNC bacteria, as claimed in claim 1, wherein in step (2), the selective medium plating method comprises the following steps: a selective agent was added to LB medium to kill sublethal-injured bacteria.
5. The method for accurately obtaining VBNC bacteria, as claimed in claim 4, wherein the selection agent is sodium chloride solution with concentration of 15-45 g/L.
6. The method for accurately obtaining VBNC bacteria, as claimed in claim 4, wherein the selection agent is potassium chloride solution with concentration of 85-120 g/L.
7. The method for accurately obtaining VBNC bacteria, as claimed in claim 1, wherein in step (3), the density gradient centrifugation method is adopted to separate live bacteria and dead bacteria, and comprises the following steps: suspending the cell culture treated in the step (2), and adding 45% -70% of Percoll layering liquid; density gradient centrifugation is carried out to remove dead bacteria.
8. The method for obtaining bacteria in VBNC state accurately as claimed in any one of claims 1-7, wherein the bacteria is any one of Escherichia coli, Vibrio cholerae, helicobacter pylori, Mycobacterium tuberculosis, Salmonella typhimurium, Listeria monocytogenes.
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| CN115094017A (en) * | 2022-06-30 | 2022-09-23 | 江南大学 | Method for inducing yeast to enter VBNC state |
| CN115232779A (en) * | 2022-07-18 | 2022-10-25 | 浙江省农业科学院 | Method for inducing salmonella enteritidis to enter VBNC state by utilizing sodium hypochlorite |
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| CN115232779A (en) * | 2022-07-18 | 2022-10-25 | 浙江省农业科学院 | Method for inducing salmonella enteritidis to enter VBNC state by utilizing sodium hypochlorite |
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