A method for extracting intracellular bacteria of ameba protozoa in water environment and environmental application

Abstract

The application belongs to the technical field of microorganism detection, and discloses a method for extracting intracellular bacteria of ameba in a water environment and environmental application; the method comprises the following steps: S1, filtering a water environment sample and ameba enrichment culture; S2, removing extracellular bacteria attached to ameba by coupling treatment of a chelating agent and lysozyme; S3, releasing intracellular bacteria by lysing ameba by using a lysing agent in combination with mechanical suction; and S4, centrifugation and filtration purification to obtain pure intracellular bacteria; the application is simple in operation, low in cost, high in bacterial purity, and can be used for water environment microorganism detection, drinking water safety monitoring, and provides technical support for ameba and intracellular bacteria interaction research.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a method for extracting intracellular bacteria from aquatic amoebas and its environmental applications. Background Technology

[0002] Amoebas are single-celled protozoa widely distributed in natural water bodies and drinking water systems. They can serve as hosts and vectors for various pathogens, and the bacteria that parasitize within their cells can survive and spread in harsh environments such as those requiring disinfection, posing a serious threat to drinking water safety. Accurate and efficient isolation and extraction of intracellular bacteria from amoebas is crucial for studying their symbiotic mechanisms and controlling microbial risks in the aquatic environment.

[0003] Existing methods for extracting intracellular bacteria often suffer from drawbacks such as low separation efficiency, severe extracellular bacterial contamination, and insufficient release of intracellular bacteria. Methods like DNA extraction and metagenomic sequencing struggle to distinguish between intracellular and extracellular bacterial communities, failing to meet the research needs for pure intracellular bacteria. Therefore, developing a simple, highly pure, and widely applicable method for extracting intracellular bacteria from aquatic amoebas is of significant scientific and applied value. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a method for extracting intracellular bacteria from aquatic amoebae and its environmental applications. This method for extracting intracellular bacteria from aquatic amoebae achieves efficient separation of intracellular bacteria through steps of filtration enrichment, removal of extracellular bacteria, release and purification of intracellular bacteria, and has the advantages of high purity, simple operation, and low cost.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for extracting intracellular bacteria from aquatic amoebas includes the following steps: S1. Filtration of water environment samples and enrichment culture of amoebae; S2. Use a chelating agent coupled with lysozyme to remove bacteria attached to the extracellular cells of amoebae. S3. The amoeba is lysed using a lysing agent combined with mechanical suction to release intracellular bacteria; S4. Centrifugation and filtration purification to obtain pure intracellular bacteria.

[0006] Preferably, the specific process of step S1 is as follows: S11. A triple filter is used to connect the water environment sample, and the water sample is filtered with a 1μm PTFE filter membrane; S12, Place the filter membrane on a coating of E. coli ( Escherichia coli Incubate on SM / 5 medium until plaques appear; S13. The cultured amoeba protozoan population was transferred to fresh Escherichia coli (E. coli) Escherichia coliThe amoebas were further cultured on SM / 5 plates to obtain enriched amoebas.

[0007] Preferably, in step S1, the water environment sample is drinking water, with a filtration volume of 50 L; the pH of the SM / 5 culture medium is 7.2, and it is coated with Escherichia coli (…). Escherichia coli The quantity is 10 9 ~10 10 Each individual will continue to be trained for 6 days.

[0008] Preferably, in step S2, the chelating agent is EDTA, and the optimal conditions for the coupling treatment of EDTA and lysozyme are: EDTA concentration of 5 mM and lysozyme concentration of 1 mg / mL.

[0009] Preferably, in step S2, the EDTA treatment conditions are: 29℃, 180 rpm shaking for 15 min, and the treatment is repeated once; the lysozyme treatment conditions are: 29℃, 180 rpm shaking for 1 h, and after treatment, the sample is washed and centrifuged with KK2 buffer.

[0010] Preferably, in step S3, the lysis agent is a 1% Triton X-100 PBS solution, and the mechanical aspiration is performed using a 50mL syringe with a No. 23 needle, with 6 aspirations.

[0011] Preferably, in step S4, the centrifugation conditions are centrifugation at 1800 g for 2 min; the filtration uses a 0.22 μm cellulose acetate membrane.

[0012] Preferably, a method for extracting intracellular bacteria from aquatic amoebas further includes a qualitative and quantitative detection step for intracellular bacteria: qualitative analysis of intracellular bacteria is performed using flow cytometry combined with fluorescent dyes, or genomic DNA is extracted and identified using a soil DNA extraction kit.

[0013] Application of a method for extracting intracellular bacteria from aquatic amoebas in the detection of intracellular bacteria from aquatic amoebas, risk assessment of pathogenic microorganisms in drinking water, or research on the microecology of aquatic bodies.

[0014] After adopting the above technical solution, the present invention has the following beneficial effects: The present invention uses EDTA coupled with lysozyme to efficiently remove extracellular bacteria without damaging amoebae; the lysis agent combined with mechanical suction ensures complete lysis without destroying intracellular bacteria, resulting in high extraction purity; the operation is simple and the cost is low, making it suitable for batch detection of water environment samples and supporting research on drinking water safety and microecology. Attached Figure Description

[0015] Figure 1 This is a flowchart of the method of the present invention; Figure 2The effect of EDTA and lysozyme treatment in this invention; Figure 3 The effects of intracellular bacterial release at different culture days are illustrated in this invention. Figure 4 The effect of the number of mechanical suction cycles on the pyrolysis effect is shown in this invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the invention.

[0017] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods. See Figures 1 to 4 . Example 1: Obtaining Entamoeba histolytica from drinking water

[0018] (1) Cultivation of environmental amoebas: A three-stage filter was connected to drinking water, and 50 L of drinking water was filtered using a 1 μm PTFE membrane (Millipore Millex, USA, CA). The membrane was then covered with a layer of 10 9 -10 10 One E. coli ( Escherichia coli, Abbreviation E. coli The amoeba colonies were cultured on SM / 5 medium at pH 7.2 at room temperature until plaques appeared. The first-generation amoeba colonies were then transferred to a new culture containing *E. coli*. Escherichia coli The culture was continued on SM / 5 agar plates. The formulation of the SM / 5 agar plates was as follows: per liter of medium, there were 2.0 g yeast extract, 2.0 g glucose, 2.0 g bacterial peptone, 0.2 g MgSO4, 1.9 g KH2PO4, 1 g K2HPO4 and 15 g agar; the formulation of the KK2 buffer was as follows: per liter of medium, there were 2.2 g KH2PO4 and 0.7 g K2HPO4. The culture was then carried out at 21 °C for 6 days, and the amoebas were obtained by scraping off the medium. Example 2: Removal of bacteria attached to the extracellular cells of amoebas

[0019] The cultured amoebae from Example 1 were suspended in 30 mL of KK2 buffer containing different concentrations of EDTA (0, 3, 5, 10 mM) to achieve an initial amoebae concentration of 10. 7The number of amoebae was determined by centrifugation at 29 °C and 180 rpm for 15 min, followed by quantitative counting of amoebae under different concentrations of EDTA using a hemocytometer. Simultaneously, amoebae were precipitated by centrifugation at 1800 g for 2 min, and the supernatant was then plated on SM / 5 plates for counting. E. coli To determine the changes in the number of free bacteria, all plates were made in triplicate. The centrifuged amoeba pellet was then subjected to another EDTA treatment. The optimal EDTA treatment conditions were determined based on changes in cell morphology and the number of free bacteria observed using a focused ion beam scanning electron microscope (Thermo Fisher Helios 5 UC).

[0020] After obtaining the optimal EDTA treatment conditions, different concentrations of lysozyme (0, 0.5, 1, 3, 5, 10 mg / mL) were added, and the reaction was carried out at 29 °C with shaking at 180 rpm for 1 h. The mixture was then washed twice and resuspended in 30 mL of KK2 solution. The amoebas were counted using a hemocytometer. Simultaneously, the amoebas were precipitated by centrifugation at 1800 g for 2 min. The supernatant was then plated onto SM / 5 plates for counting. E. coli Bacterial count. Example 3: Cell Morphology Analysis of Amoebas and Bacteria

[0021] Samples treated with EDTA (3, 5, 10 mM), and those treated with 5 mM EDTA followed by lysozyme treatment (0.5, 1, 5 mg / mL) and aspirated (3, 6, 15 times) were selected. The samples were fixed overnight in glutaraldehyde, then washed twice with PBS to form a turbid suspension. 4 mm cell slides treated with poly-L-lysine were slowly immersed in the suspension for 1 min. Subsequently, the slides were dehydrated sequentially at 4 °C in 30%, 50%, and 70% ethanol for 5 min, 5 min, and 10 min, respectively, and at room temperature in 80%, 95%, and 100% ethanol for 10, 15, and 15 min, respectively. The slides were then dried in a critical point dryer (Leica CPD300) using liquid CO2 for 2 h. After drying, the slides were sputtered in a metal sputtering machine and the cell morphology was observed under a focused ion beam scanning electron microscope (ThermoFisher Helios 5 UC).

[0022] like Figure 2 The results show that the structure and morphology of Entamoeba histolytica in drinking water were not significantly damaged after EDTA treatment, and the optimal EDTA concentration was 5 mM.

[0023] like Figure 2The results showed that the structure and morphology of Entamoeba histolytica were not significantly damaged after treatment with 5 mMEDT followed by lysozyme treatment, and the optimal lysozyme concentration was 1 mg / mL.

[0024] like Figure 4 The results showed that the optimal number of aspirations for Entamoeba histolytica in drinking water should be 6. After the 6th aspiration, intracellular... B. agricolaris The release of bacteria reached its maximum and the number no longer increased significantly. After six aspirations, the amoebas were severely damaged, but the bacteria remained almost intact. Example 4: Quantitative analysis of the release extent of intracellular bacteria from amoebas

[0025] 10 7 Entamoeba per mL and 10 4 cells / mL E. coli The mixture was then inoculated onto cultures that had been cultured two days prior. B. agricolaris On SM / 5 plates, culture for another 4, 6, 9, and 12 days to obtain [products / items]. B. agricolaris Amoeba protozoa.

[0026] To quantify the extent of intracellular bacterial release from amoebae, forward scattered light (FSC) was used to identify larger amoebae, using 488 nm laser emission for identification. B. agricolaris The bacteria's inherent red fluorescence allows them to appear as amoebas on flow cytometry scatter plots. E. coli and B. agricolaris Scattered clusters of isolated bacteria were delineated to distinguish them. B. agricolaris Bacteria. Use optimal EDTA and lysozyme to target bacteria. B. agricolaris The amoeba was treated using the same steps as in Example 3. After centrifugation, the precipitate was added to 30 mL of 1% Triton X-100 PBS solution. The precipitate was then aspirated using a 50 mL syringe with a 23-gauge needle to obtain the desired intracellular amoeba bacteria. 3 mL of the aspirated sample was centrifuged and washed twice. Flow cytometry was used to detect the different amounts of bacteria collected. B. agricolaris Changes in bacterial count.

[0027] like Figure 3 The results showed that when culturing *Entamoeba histolytica* in drinking water, the number of *Entamoeba histolytica* reached its maximum after 6 days of culture. B. agricolaris The bacteria reach their peak release level after 6 days.

[0028] like Figure 4 The results showed that intracellular... E. coli To release the bacteria without damaging them, the optimal number of aspirations is 6.

[0029] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for extracting intracellular bacteria from aquatic amoebas, characterized in that, Includes the following steps: S1. Filtration of water environment samples and enrichment culture of amoebae; S2. Use a chelating agent coupled with lysozyme to remove bacteria attached to the extracellular cells of amoebae. S3. The amoeba is lysed using a lysing agent combined with mechanical suction to release intracellular bacteria; S4. Centrifugation and filtration purification to obtain pure intracellular bacteria.

2. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 1, characterized in that, The specific process of step S1 is as follows: S11. A triple filter is used to connect the water environment sample, and the water sample is filtered with a 1μm PTFE filter membrane; S12, Place the filter membrane on a coating of E. coli ( Escherichia coli Incubate on SM / 5 medium until plaques appear; S13. The cultured amoeba protozoan population was transferred to fresh Escherichia coli (E. coli) Escherichia coli The amoebas were further cultured on SM / 5 plates to obtain enriched amoebas.

3. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 2, characterized in that: In step S1, the water environment sample is drinking water, with a filtered volume of 50 L; the pH of the SM / 5 culture medium is 7.2, and it is coated with Escherichia coli (E. coli). Escherichia coli The quantity is 10 9 ~10 10 Each individual will continue to be trained for 6 days.

4. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 1, characterized in that, In step S2, the chelating agent is EDTA, and the optimal conditions for the coupling treatment of EDTA and lysozyme are: EDTA concentration of 5 mM and lysozyme concentration of 1 mg / mL.

5. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 4, characterized in that, In step S2, the EDTA treatment conditions are: 29℃, 180 rpm shaking for 15 min, and the treatment is repeated once; the lysozyme treatment conditions are: 29℃, 180 rpm shaking for 1 h, and after treatment, the sample is washed and centrifuged with KK2 buffer.

6. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 1, characterized in that: In step S3, the lysis agent is a 1% Triton X-100 PBS solution, and mechanical aspiration is performed using a 50 mL syringe with a No. 23 needle, with 6 aspirations.

7. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 1, characterized in that: In step S4, the centrifugation conditions are centrifugation at 1800 g for 2 min; the filtration uses a 0.22 μm cellulose acetate membrane.

8. The method for extracting intracellular bacteria from aquatic amoebas as described in claim 1, characterized in that, It also includes qualitative and quantitative detection steps for intracellular bacteria: qualitative analysis of intracellular bacteria is performed using flow cytometry combined with fluorescent dyes, or genomic DNA is extracted and identified using a soil DNA extraction kit.

9. The method for extracting intracellular bacteria from aquatic amoebas as described in any one of claims 1 to 8 is used in the detection of intracellular bacteria from aquatic amoebas, risk assessment of pathogenic microorganisms in drinking water, or research on the microecology of aquatic bodies.

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