阅读说明：本技术 一种双染法检测细菌活菌的方法 (Method for detecting live bacteria by double-dyeing method ) 是由 周大祥 李婧怡 于 2019-09-02 设计创作，主要内容包括：本发明公开了一种双染法检测细菌活菌的方法,包括如下步骤：细菌培养、细菌稀释、洗涤重悬、加入反应试剂避光孵育、使用流式细胞仪分析。本发明取得的有益效果为：检测快速、准确,克服了传统qPCR假阳性较高、平板培养检测周期长等缺点,使结果更加有效、可靠。(The invention discloses a method for detecting live bacteria by a double-dyeing method, which comprises the following steps: bacterial culture, bacterial dilution, washing and resuspension, adding reaction reagent and incubating in dark place, and analyzing by using a flow cytometer. The invention has the following beneficial effects: the detection is rapid and accurate, the defects of high false positive, long plate culture detection period and the like of the traditional qPCR are overcome, and the result is more effective and reliable.)

1. A method for detecting live bacteria by a double-staining method is characterized by comprising the following steps:

(1) and (3) bacterial culture: inoculating bacteria into a liquid culture medium, and performing shake culture on a shaking table to a logarithmic phase to obtain a strain liquid;

(2) bacterial dilution: resuspending the strain liquid obtained in the step (1) with sterile distilled water, filtering with a 1000-mesh sterile stainless steel screen, counting with a cell counting plate, and diluting the strain liquid with sterile distilled water to 5 × 10⁵～1×10⁶cfu/ml to obtain bacterial diluent;

(3) washing and resuspending: taking 100 mu l of the bacterial diluent obtained in the step (2) to be placed in a sterile flow tube, washing with deionized water, and then adding 200-400 mu l of annexin VFITC binding solution into the washed bacterial diluent to resuspend bacteria to obtain bacterial resuspension liquid;

(4) sequentially adding 5 mul of Annexin V FITC solution with the concentration of 0.5-10 mu g/ml and 10 mul of PI solution with the concentration of 10-50 mu g/ml into the bacterial resuspension of the step (3), uniformly mixing, and incubating for 10-20 min at room temperature in a dark place;

(5) and (3) analysis: after the incubation was completed, it was immediately analyzed by flow cytometry.

2. The method for detecting the viable bacteria of the bacteria by the double staining method according to claim 1, wherein the culture conditions in the step (1) are as follows: the culture temperature is 25-30 ℃, the culture time is 8-12 h, and the rotating speed is 200-300 r/min.

3. The method for detecting viable bacteria by double staining method according to claim 2, wherein the deionized water washing process in step (3) comprises: centrifuging the bacterial dilution at 4 deg.C for 5min at 3000r/min, collecting precipitate, then resuspending with 1ml deionized water, centrifuging at 4 deg.C for 5min at 3000r/min, collecting precipitate, and repeating above steps for 2 times.

4. A method of detecting viable bacteria according to claim 3 wherein the excitation wavelength of the flow cytometry analysis in step (5) is 490nm and the emission wavelength is 533 nm.

5. The method for detecting viable bacteria by the double staining method according to claim 1 to 4, wherein the bacteria is Ralstonia solanacearum or Escherichia coli.

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for detecting live bacteria by a double-dyeing method.

Background

Bacterial wilt is a devastating disease caused by the bacterium ralstoniae, which is widely distributed in tropical, subtropical and some warm areas of the world. The host range of the ralstonia solanacearum is very wide, hundreds of plants of 44 families are discovered at present and new hosts are discovered continuously, which brings great economic loss to crop production.

Coli is the most predominant and abundant bacterium in the intestine of many animals, mainly parasitic in the large intestine. Coli is propagated in large quantities in the intestine, about 1/3, the dry weight of feces, which can be scattered in the surrounding environment with the feces, affecting the sanitation of the surrounding environment; when this bacterium is detected in water and food, it is presumed that water or food is contaminated with feces, and it is possible to estimate the presence of intestinal pathogenic bacteria, and therefore, Escherichia coli is often used as an index of hygiene in drinking water and food.

Currently, common methods for detecting ralstonia solanacearum and escherichia coli are PCR detection and plate culture methods. However, the PCR detection technology can not only amplify DNA of live bacteria, but also amplify DNA in a free state or DNA of dead bacteria, resulting in the occurrence of false positive; the plate culture method has the disadvantages of long culture period and the like.

Therefore, it is an urgent need to solve the problem of the art to provide a method for rapidly and accurately detecting viable bacteria.

Disclosure of Invention

In view of this, the present invention provides a method for rapidly and accurately detecting the number of viable bacteria in bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting live bacteria by a double-staining method comprises the following steps:

(1) and (3) bacterial culture: inoculating bacteria into a liquid culture medium, and performing shake culture on a shaking table to a logarithmic phase to obtain a strain liquid;

(2) bacterial dilution: resuspending the strain liquid obtained in the step (1) with sterile distilled water, filtering with a 1000-mesh sterile stainless steel screen, counting with a cell counting plate, and diluting the strain liquid with sterile distilled water to 5 × 10⁵～1×10⁶cfu/ml to obtain bacterial diluent;

the diameter of the bacteria is 0.5-5 mul, and a 1000-mesh stainless steel screen is added for filtering before obtaining the single cell strain diluent so as to remove the bacteria aggregated into clusters.

(3) Washing and resuspending: taking 100 mu l of the bacterial diluent obtained in the step (2) to a sterile flow tube, washing with deionized water, and adding 200-400 mu l of Annexin VFITC binding solution into the washed bacterial diluent to resuspend bacteria to obtain bacterial resuspension;

(4) adding 5 mul of annexin V FITC solution with the concentration of 0.5-10 mu g/ml and 10 mul of PI solution with the concentration of 10-50 mu g/ml into the bacterial heavy suspension in the step (3), uniformly mixing, and incubating for 10-20 min at room temperature in a dark place;

(5) and (3) analysis: after the incubation was completed, it was immediately analyzed by flow cytometry.

The principle of Annexin V FITC and PI (propidium iodide) double staining method is that PS (phosphatidylserine) is exposed outside a cell membrane of a bacterium at the early death stage due to the change of phospholipid symmetry of the cell membrane, the PS can be specifically combined with a linker V (Annexin V) labeled with Fluorescein Isothiocyanate (FITC), but the bacterium still maintains the integrity of the cell membrane, so that the fluorescent dye PI stained by denatured chromatin can not enter the bacterium; however, bacteria in late death stage can be labeled with Annexin V FITC and PI at the same time, and the number of labeled dead bacteria can be quantitatively analyzed by FCM (flow cytometry).

Preferably, the culture conditions in step (1) are: the culture temperature is 25-30 ℃, the culture time is 8-12 h, and the rotating speed is 200-300 r/min.

Preferably, the deionized water washing process in the step (3) specifically comprises: centrifuging the bacterial dilution at 4 deg.C for 5min at 3000r/min, collecting precipitate, then resuspending with 1ml deionized water, centrifuging at 4 deg.C for 5min at 3000r/min, collecting precipitate, and repeating above steps for 2 times.

Preferably, the excitation wavelength analyzed by flow cytometry in step (5) is 490nm and the emission wavelength is 533 nm.

Preferably, the bacterium is ralstonia solanacearum or escherichia coli.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the detection is rapid and accurate. The defects of high false positive, long plate culture detection period and the like of the traditional qPCR are overcome, and the result is more effective and reliable.

Drawings

FIG. 1 shows the results of the test of example 1, wherein: the first quadrant represents mechanically damaged cells, the second quadrant represents late apoptotic and necrotic cells, the third quadrant represents viable cells, and the fourth quadrant represents early apoptotic cells;

FIG. 2 shows the results of the test of example 4, wherein: the first quadrant represents mechanically damaged cells, the second quadrant represents late apoptotic and necrotic cells, the third quadrant represents viable cells, and the fourth quadrant represents early apoptotic cells;

FIG. 3 shows the results of the test of example 7, wherein: the first quadrant represents mechanically damaged cells, the second quadrant represents late apoptotic and necrotic cells, the third quadrant represents viable cells, and the fourth quadrant represents early apoptotic cells;

FIG. 4 shows the results of the test of example 8, wherein: the first quadrant represents mechanically damaged cells, the second quadrant represents late apoptotic and necrotic cells, the third quadrant represents viable cells, and the fourth quadrant represents early apoptotic cells.

Detailed Description