CN114563396A - On-site rapid detection kit and detection method for total number of prokaryotic cell type microorganisms in water - Google Patents

Abstract

The invention belongs to the technical field of bacterial counting, and in particular relates to a kit and a detection method for on-site rapid detection of the total number of prokaryotic cells in water, comprising WST-8, an electronic carrier, α-amantine, prokaryotic cell nutrients and a buffer. The invention can effectively realize on-site rapid detection of the total number of prokaryotic cells in water with high accuracy.

Description

A rapid on-site detection kit and detection method for the total number of prokaryotic microorganisms in water

technical field

The invention belongs to the technical field of bacterial counting, and in particular relates to a rapid on-site detection kit and detection method for the total number of prokaryotic microorganisms in water.

Background technique

Prokaryotic microorganisms include actinomycetes, mycoplasma, chlamydia, spirochetes, rickettsia, and bacteria. The bacteria that people usually say refers to bacteria in the narrow sense. Bacteria are widely distributed in vast rivers, lakes, glaciers and land, as well as underground. Farms are generally distributed in remote mountainous areas, and most of the drinking water sources for livestock breeding are groundwater (self-draining wells) or surface water (mountain spring water). These water sources generally contain certain bacteria. At the same time, during the water supply process, the drinking water is often easily contaminated by a large number of bacteria due to the infiltration and pollution of the water source, the insufficient regular cleaning of the reservoir, or the difficulty in cleaning the inside of the pipeline. The existing national relevant standards for evaluating aquatic pollution are "Sanitation Standard for Drinking Water of the People's Republic of China GB5749-2006", which stipulates that the total number of bacterial colonies in drinking water quality should not exceed 100 CFU/ml; small centralized water supply and decentralized water supply in rural areas. , the total number of colonies in the water should not exceed 500CFU/ml. The above standard detection method is to use the plate colony counting method (CFU counting method) to test the total number of bacterial colonies in the water according to the provisions of "Standard Test Methods for Drinking Water Microbiological Indicators GB/T 5750.12-2006". These regulations refer to the total number of bacteria in human drinking water, and there is currently no relevant standard for animal drinking water. Some aquaculture enterprises refer to human drinking water standards when evaluating the total number of bacteria in drinking water.

This method has the following disadvantages: 1. This method detects the total number of colonies, that is, how many bacterial colonies can be cultivated per milliliter of water, but the index directly related to livestock and poultry diseases is the total number of cells, that is, how many bacteria are contained in each milliliter of water. The difference between the two is that the bacteria that can be cultured in fresh water only account for 0.25%-3.125% of the total number of bacteria (Xiong Yingying, et al, 2021; Stackebrandt et al, 2000). Among these culturable bacteria, different cultures are used. The number of colonies grown from the substrate varied widely. Therefore, it is inaccurate to use the CFU counting method to measure the number of bacteria in water and then evaluate its impact on livestock and poultry health. 2. The existing CFU counting method requires a special laboratory. 3. Special equipment such as ultra-clean workbench, sterilizer and incubator are required. 4. Special culture medium is required. 5. The operator needs to have a certain level of operating technology and operating experience. If the dilution ratio is not well controlled, it will lead to a large number of dense colonies that cannot be counted or too many colonies (more than 300) or too many colonies on the plate containing the solid medium. less (less than 30), which eventually led to the failure of the test. 6, the time is too long. The whole process takes more than 48 hours. For family farms that do not have their own laboratories, it will take 3-4 days for the results to be sent. At the same time, part of the live bacteria will die during transportation, which will reduce the accuracy of the test results. 5. Only some bacteria can be cultivated, and it is more difficult to cultivate mycoplasma, chlamydia, rickettsia and spirochete.

At present, under the background of African swine fever, all farms have increased the frequency of disinfection and expanded the scope of disinfection. How to evaluate the disinfection effect is an issue that the industry pays more and more attention to. At present, the commonly used method to evaluate the disinfection effect of disinfectants is to take samples after disinfection and send them to the laboratory, and use fluorescence quantitative PCR method or cell culture method to detect related viruses. These methods require operators to master certain operating techniques, require specialized professional laboratories, are expensive and cannot be quickly evaluated on site.

At present, the patented technology that solves the above problems and is the closest to this patented technology is "a method for determining the total number of viable bacteria based on WST-8 color reaction (patent number: CN202111017992.5)". The core of the patented technology is to use WST-8 8 and the chromogenic substrate solution of electron carrier 1-PSM, and adding nutrients to it at the same time to improve bacterial viability, increase the content of dehydrogenase produced by a single bacterium, and improve the detection sensitivity, but this method detects live bacteria. The number of bacteria cannot distinguish between prokaryotes and eukaryotes, and cannot effectively distinguish the number of prokaryotic cells that are more harmful, so the results detected by this method cannot effectively evaluate the degree of harm of bacteria in the environment.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a rapid on-site detection kit and detection method for the total number of prokaryotic microorganisms in water, which can effectively realize the rapid on-site detection of the total number of prokaryotic microorganisms in water with high accuracy.

The content of the present invention includes a kit for rapid on-site detection of the total number of prokaryotic microorganisms in water, comprising WST-8, an electron carrier, α-amantine, prokaryotic cell nutrients and a buffer.

Preferably, the electron carrier is 1-Methoxy PMS, the prokaryotic cell nutrients are beef extract and peptone, and the buffer is disodium hydrogen phosphate.

WST-8 is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfobenzene)-2H-tetrazole mono Sodium salt, 1-Methoxy PMS is 1-methoxy-5-methylphenazine dimethyl sulfate.

Preferably, the concentration of WST-8 is 0.05-0.15mg/ml, the concentration of electron carrier is 0.1-0.3mg/ml, the concentration of α-Amantine is 25-100μg/ml; the concentration of beef extract is 2-10mg/ml, The peptone is 5-15 mg/ml and the buffer concentration is 0.5-2 mg/ml.

Most preferably, the concentration of WST-8 is 0.1mg/ml, the concentration of electron carrier is 0.2mg/ml, the concentration of α-Amantine is 50μg/ml, the concentration of prokaryotic cell nutrients beef extract is 5mg/ml, and the concentration of peptone is 10mg. /ml, the buffer concentration is 1 mg/ml.

The invention provides a detection method using an on-site rapid detection kit for the total number of prokaryotic cell type microorganisms in water. Mixing, after a period of time, the solution changes color to obtain different colors, and the standard colorimetric card is made based on the above color (see Figure 1); mix the sample and the solution of the kit, observe the color change after a period of time, and compare the color with the standard. Compare the color card to determine the total number of prokaryotic microorganisms in the sample.

The bacterial liquid is a mixture of Escherichia coli E.colibacillus, Salmonella Salmonella, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Bacillus coagulans, Staphylococcus aureus and Streptococcus in equal proportions. Bacterial fluid formed later.

Preferably, the detection temperature is 30-60°C.

Preferably, after mixing the sample and the solution of the kit, the standing time is 2-5h.

Preferably, before mixing the visually colored sample and the solution of the kit, centrifugation is performed to remove the color. The sample with color on the eye is the sample before mixing, and the color refers to the various colors of the sample itself, such as red, blue, and so on. The color that appears after mixing refers to the color of the (colorless) sample (if the sample has color such as blue, it is removed by centrifugation to remove the blue) and the color developed after the reaction of the reagent, the result is only yellow or dark yellow.

Preferably, the weight ratio of the solution of the sample to the kit is 3-5:1.

The beneficial effect of the present invention is that the present invention is a technology for rapidly evaluating the concentration of prokaryotic cell type microorganisms in farm water. In the present invention, after the farm water sample is reacted with the reagent of the present invention, the color will change. The dark color indicates that the number of prokaryotic microorganisms is large, the light color indicates that the number of prokaryotic microorganisms is small, and no color indicates that the number of bacteria is very small or no prokaryotic cells. type microorganisms. The basic principle of this set of reagents is that living prokaryotic microorganisms can produce oxidoreductases during the metabolic process, which can catalyze colorless or light-colored substrates into colored products. Within a certain range, the greater the number of living prokaryotic microorganisms, the more enzymes are produced, and the more products, the darker the color. In order to remove the interference of other colored substances and disinfectants in the water, and further increase the linear relationship between the color depth and the concentration of viable bacteria, this method adds a centrifugation to remove the supernatant and a concentration step during sample processing. Centrifuging the supernatant can effectively remove other substances (such as disinfectant ingredients) that contain color and affect the reaction in the water and concentrate the bacteria in the water. Since eukaryotic cells in the environment can also produce oxidoreductase, the present invention adds a reagent component that can inhibit the secretion of oxidoreductase by eukaryotic cells. At the same time, the reagent of the present invention can only enter the body of living bacteria, and the reaction is carried out under the catalysis of oxidoreductase in the body. Dead bacteria have no metabolism and cannot secrete oxidoreductase. In this way, the color depth after the reaction only has a certain linear relationship with the number of living prokaryotic microorganisms. In order to increase the sensitivity of this reagent, bacterial culture medium is added to the reagent formula. During the reaction, bacteria enter the first stage of growth and reproduction (lag period), the metabolism of bacteria is accelerated, the secreted enzymes are increased, and the color displayed is darker, so that the sensitivity of the reagent can be increased. In order to prevent the disorder of the linear relationship caused by the increase in the number of bacteria during the reaction, the reaction time was controlled at 2.5 hours, so that the bacteria were still in the lag phase, and the number of bacteria did not increase but the secreted enzymes increased.

The operation of the invention is simple, and the whole operation process only needs to take out the drinking water and inject it into the scale of the reagent tube, and then directly put it at 37°C for 0-2.5 hours. It can be operated on site, no special laboratory is required, and the professional requirements for operators are not high. Visualization, the operation results can be judged by visual inspection of the color changes, without the need for instruments and equipment, and no absorbance detection device. The speed is fast, the time is short, and the result can be obtained in 0-2.5h during the whole process.

The invention can be used to detect the total number of prokaryotic cell type microorganisms in water, and can realize rapid on-site detection. The invention adopts the method of mixing 7 kinds of microorganisms in equal proportion to prepare a standard colorimetric card. When actually detecting the total number of prokaryotic cell type microorganisms in water, the result is It is more accurate and the error is small; the present invention can also quickly evaluate the environment, such as the disinfecting effect of the breeding farm.

Description of drawings

FIG. 1 is a schematic diagram of a standard colorimetric card of the present invention.

Fig. 2 is the reaction color development result of the reagent of the present invention and three kinds of prokaryotic cells.

Fig. 3 is the reaction color development result of the reagent of the present invention and three kinds of eukaryotic cells.

Fig. 4 is the reaction color development result of the reagent of the present invention and 7 kinds of prokaryotic cell type microorganisms (bacteria).

Fig. 5 is the reaction color development result of the reagent of the present invention and three kinds of prokaryotic microorganisms (bacteria).

Fig. 6 is the reaction color development result of the reagent of the present invention and three kinds of mixed bacteria.

Fig. 7 is the reaction color development result of the reagent of the present invention and three kinds of mixed bacteria.

Detailed ways

Example 1

Mix WST-8, electron carrier PMS, α-Amantine, prokaryotic cell nutrients (beef extract and peptone) and buffer solution disodium hydrogen phosphate, the solvent is water, and the additions of the above components are respectively, WST- 8 is 5mg, PMS is 10mg, α-Amantine 2.5mg, beef extract 0.2g, peptone 0.5g, disodium hydrogen phosphate 50mg, after adding water to dissolve each component, the final total volume is 100ml. A reaction solution was obtained.

Example 2

Compared with Example 1, the addition amounts of the above components are respectively, WST-8 is 15mg, PMS is 30mg, α-Amanita 10.0mg, beef extract 1.0g, peptone 1.5g, disodium hydrogen phosphate 200mg, Others are the same as in Example 1.

Example 3

A detection method using an on-site rapid detection kit for the total number of prokaryotic cells in water, the steps are:

Make standard colorimetric cards, and combine Escherichia coli E.colibacillus, Salmonella Salmonella, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Bacillus coagulans, Staphylococcus aureus, and Streptococcus together. The bacterial liquids of 7 kinds of bacteria were respectively configured into different concentration gradients (concentration gradients were 10 ³ /ml ^* , 10 ⁴ /ml, 10 ⁵ /ml, 10 ⁶ /ml, 10 ⁷ /ml, 10 ⁸ standard samples/ml).

Note: The calculation basis of */ml: the total number of colonies (CFU/ml) is obtained by the plate counting method. For a culturable bacteria, the total number of colonies per milliliter (CFU/ml) is approximately the total number of bacteria per milliliter. (pieces/ml).

Mix 7 kinds of bacterial liquids with a concentration of 10 ⁸ /ml in equal volumes to make a mixed standard sample of this concentration, take 80 μl of it and 20 μl of the reaction solution of Example 1 to react at 37 ° C for 2.5 hours, the obtained color Converted to paper, a color bar corresponding to 10 ⁸ /ml is formed. The remaining concentrations are 10 ³ /ml, 10 ⁴ /ml, 10 ⁵ /ml, 10 ⁶ /ml, 10 ⁷ /ml, operate according to this method, and get the corresponding color bar. A standard color chart was made based on the above color bar, and the result is shown in Figure 1.

Escherichia coli bacterial liquid, Staphylococcus aureus bacterial liquid and Bacillus bacterial liquid are configured into different concentration gradients (concentration gradients are respectively 10 ³ /ml, 10 ⁴ /ml, 10 ⁵ /ml, 10 ⁶ /ml) , 10 ⁷ /ml, 10 ⁸ /ml) standard samples, mix 80 μl of the standard sample with 20 μl of the reaction solution of Example 1, and let stand at 37° C. for 2.5 hours to observe the color of the solutions of different standard samples. At the same order of magnitude concentration, the colors of the three are closer to the color value of the standard colorimetric card at the same concentration (Fig. 5), indicating that the oxidoreductase activities of different prokaryotic microorganisms are similar, and the same standard comparison can be used. The color chart compares the concentration of different bacteria.

Example 4

Rapid on-site determination of the total number of prokaryotic microorganisms in drinking water in farms

Take an appropriate amount of drinking water with a syringe and inject it into a reagent tube (containing the reaction solution described in Example 1), stop the injection when the water level rises to the mark, and shake well. The above shaken sample was allowed to stand at 25°C, and the color change was observed.

Qualitative judgment. Judgment standard: If the color of the sample changes (compared to the negative control tube) at any time point within 0-150 minutes, it can be judged that the total number of colonies in the sample exceeds the standard.

Preparation of negative control tube: Take a detection tube containing the reagent (that is, the reaction solution described in Example 1), and inject the reagent in the negative control tube into the reagent tube. The slight pink color of the negative control tube represents the color of the reagent itself .

Relative quantitative determination. The above shaken sample was allowed to stand at 37°C for 2.5 hours, and the color change was observed. Judgment standard: Compare the sample test tube left standing for 2.5 hours with the standard colorimetric card to determine the bacterial concentration in the sample.

Example 5

On-site rapid assessment of disinfection effect of farm disinfectant

Sample handling and testing prior to disinfection. Liquid samples were drawn directly with a syringe to 1.5ml. If a solid sample is to be evaluated on the plane of a carriage, it can be wiped in a certain area with a wet glove containing sterile normal saline, and the water can be squeezed out for testing or the sewage after rinsing with water can be used for testing. The above samples were centrifuged at 5000 rpm for 10 minutes, and 1/10 of the total volume (800 μL) of sterile physiological saline was mixed and precipitated, and the total volume was 80 μL. Then, the detection was carried out according to the relative quantitative determination method of Example 4.

Sample processing and testing after disinfection are the same as above.

Evaluation of the effect of disinfectant

If there is a color change before disinfection, but there is no color change after disinfection, the disinfectant is determined to be effective, and other tests such as African swine fever virus detection can be further carried out. If there is a color change before disinfection, but also after disinfection (even if the color is lighter), it is determined that the disinfection is invalid or the effect is better, and it is necessary to re-sterilize, and then the next test can be carried out after there is no color change, such as the detection of African swine fever virus Wait.

Comparative Example 1

Compared with Example 1, in Comparative Example 1, α-Amantine was removed, and the others were the same as Example 1.

Example 6

The reaction solution of Example 1 was mixed with 80 μL of Escherichia coli (10 ⁶ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and the color changed (Fig. 2A, left 3).

The reaction solution of Comparative Example 1 was mixed with 80 μl of Escherichia coli (10 ⁶ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and the color changed (right 1 in FIG. 2A ).

The reaction solution of Example 1 was mixed with 80 μl of Staphylococcus aureus (10 ⁶ /ml), and it was allowed to stand at 37° C. for 2.5 hours, and the color changed (Fig. 2B, left 3).

The reaction solution of Comparative Example 1 was mixed with 80 μl of Staphylococcus aureus (10 ⁶ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and there was a color change (right 1 in FIG. 2B ).

The reaction solution of Example 1 was mixed with 80 μl of Bacillus (10 ⁶ cells/ml), and it was allowed to stand at 37° C. for 2.5 hours, and there was a color change (left 3 in FIG. 2C ).

The reaction solution of Comparative Example 1 was mixed with 80 μl of Bacillus (10 ⁶ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and there was a color change (right 1 in FIG. 2C ).

The reaction solution of Example 1 was mixed with 80 μl of porcine alveolar macrophages (10 ⁷ cells/ml), and left at 37° C. for 2.5 hours, with no color change (left 3 of FIG. 3A ).

The reaction solution of Comparative Example 1 was mixed with 80 μl of porcine alveolar macrophages (10 ⁷ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and there was a color change (right 1 in FIG. 3A ).

The reaction solution of Example 1 was mixed with 80 μl of Pk-15 cells (10 ⁷ cells/ml), and left at 37° C. for 2.5 hours, with no color change (left 3 in FIG. 3B ).

The reaction solution of Comparative Example 1 was mixed with 80 μl of Pk-15 cells (10 ⁷ cells/ml), and allowed to stand at 37° C. for 2.5 hours, and there was a color change (right 1 in FIG. 3B ).

The reaction solution of Example 1 was mixed with 80 μl of Vero cells (10 ⁷ cells/ml), and left to stand at 37° C. for 2.5 hours, with no color change (left 3 in FIG. 3C ).

The reaction solution of Comparative Example 1 was mixed with 80 μl of Vero cells (10 ⁷ cells/ml), and left at 37° C. for 2.5 hours, and there was a color change (right 1 in FIG. 3C ).

The pictures of the above tests are shown in Figures 2 and 3. It can be seen from the figures that the concentration of the microorganisms represented by the color results matches the concentration of the microorganisms themselves, indicating that the results of the present application are relatively accurate.

Through the above test, it can be effectively proved that the reaction solution of the present application effectively inhibits the microorganisms of eukaryotic cells, and only detects the microorganisms of prokaryotic cells.

Example 7

Take 1ml of Bacillus (concentration of 10 ⁶ /ml), Staphylococcus aureus 1ml (concentration of 10 ⁵ /ml) and Escherichia coli 1ml (concentration of 10 ⁵ /ml) and mix, the reaction of Example 1 The solution was added to the mixture of the above-mentioned bacteria, and was allowed to stand at 37°C for 2.5 hours, and the color change was observed as shown in Figure 6 (results of three repeated tests). 10 ⁶ /ml, and the actual.

Example 8

Take 1 ml of Escherichia coli (concentration of 10 ⁶ cells/ml), 1 ml of Staphylococcus aureus (concentration of 10 ⁵ cells/ml) and 1 ml of porcine alveolar macrophages (10 ⁶ cells/ml) and mix them. The reaction solution was added to the mixture of the above bacteria, and stood at 37°C for 2.5 hours to observe the color change (as shown in Figure 7, the results of three repeated tests) and compared with the standard colorimetric card, showing the total concentration value of prokaryotic cell type microorganisms. It is 10 ⁶ /ml, which is consistent with the actual.

Example 9

The present invention adopts WST-8, electron carrier and α-Amanita to coordinate. In order to screen out the optimal dosage of α-Amantine, α-Amantine was diluted into 8 concentration gradients, so that the final concentrations were 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, respectively. ml, 6.25μg/ml, 3.125μg/ml, 1.506μg/ml and 0.00μg/ml, a total of 8 reagents (other raw materials are 5mg of WST-8, 10mg of PMS, 0.2g of beef extract, 0.5g of peptone, disodium hydrogen phosphate 50mg). The above-mentioned 8 reagents were reacted with 3 kinds of prokaryotic cell type bacteria and 3 kinds of eukaryotic cell type bacteria respectively (the bacterial concentration of 3 kinds of prokaryotic cell type bacteria was 10 ⁶ /ml, and the bacterial concentration of 3 kinds of eukaryotic cell type bacteria was 10 6 /ml. 10 ⁷ /ml). The results showed that all 8 reagents and 3 kinds of prokaryotic cells had color reaction (Fig. 2). The minimal inhibitory concentrations for the three eukaryotic cells were 25 μg/ml, 12.5 μg/ml and 12.5 μg/ml, respectively ( FIG. 3 ). After the reagent combination was depleted of α-Amantine (ie, Comparative Example 1), three kinds of prokaryotic cells and three kinds of eukaryotic cells had color reactions (Fig. 2, the rightmost tube in Fig. 3).

This reagent combination (WST-8 is 10mg, PMS is 20mg, α-Amanita 5mg, beef extract 0.5g, peptone 1g, disodium hydrogen phosphate 100mg, after adding water to dissolve each component, the final total volume is 100ml, At this time, α-amantine is 50 μg/ml), which can make prokaryotic microorganisms develop color, such as Salmonella (2.2×10 ⁸ /ml), Staphylococcus (7.9×10 ⁷ /ml), Bacillus ( 8.8×10 ⁷ /ml), Streptococcus (1.55×10 ⁶ /ml), Haemophilus parasuis (2.3×10 ⁷ /ml), Actinobacillus pleuropneumoniae (3.6×10 ⁷ /ml) and Escherichia coli (4.0×10 ⁶ /ml) ( FIG. 4 ). The combination of the above reagents and eukaryotic cells PK-15 cells, porcine alveolar macrophages (PAM) and Vero cells showed no color reaction (the second tube on the left in Figure 3). The above results prove that the reagent combination can effectively inhibit the secretion of oxidoreductase by eukaryotic cells, but does not inhibit the secretion of oxidoreductase by prokaryotic cells. It should be understood by those of ordinary skill in the art that the discussion of any of the above embodiments is only exemplary, and is not intended to imply that the protection scope of the present application is limited to these examples; Technical features can also be combined, steps can be carried out in any order, and there are many other variations of the different aspects of one or more of the embodiments in the application as described above, which are not provided in detail for the sake of brevity.

The embodiment or embodiments in this application are intended to cover all such alternatives, modifications and variations that fall within the broad scope of this application. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of one or more embodiments in this application should be included within the protection scope of this application.

Claims (10)

1. An on-site rapid detection kit for the total number of prokaryotic cell type microorganisms in water is characterized by comprising WST-8, an electron carrier, alpha-amanitine, prokaryotic cell nutrients and buffer solution.

2. The kit for rapidly detecting the total number of the prokaryotic cell type microorganisms in water on site as claimed in claim 1, wherein the electron carrier is 1-Methoxy PMS, the prokaryotic cell nutrients are beef extract and peptone, and the buffer solution is disodium hydrogen phosphate.

3. The kit for the on-site rapid detection of the total number of the prokaryotic cell type microorganisms in water as claimed in claim 1, wherein the concentration of WST-8 is 0.05-0.15mg/ml, the concentration of electron carriers is 0.1-0.3mg/ml, and the concentration of alpha-amanitine is 25-100 μ g/ml.

4. The kit for rapidly detecting the total number of the prokaryotic cell type microorganisms on site in water as claimed in claim 2, wherein the concentration of beef extract is 2-10mg/ml, the concentration of peptone is 5-15mg/ml, and the concentration of buffer solution is 0.5-2 mg/ml.

5. The kit for rapidly detecting the total number of the prokaryotic cell type microorganisms in water on site as claimed in claim 3 or 4, wherein the concentration of WST-8 is 0.1mg/ml, the concentration of an electron carrier is 0.2mg/ml, the concentration of alpha-amastatin is 50 μ g/ml, the concentration of prokaryotic cell nutrient substances is 5mg/ml, the concentration of peptone is 10mg/ml, and the concentration of a buffer solution is 1 mg/ml.

6. A method for on-site rapid detection of total number of microorganisms of prokaryotic cell type in water using the kit as defined in any one of claims 1 to 5, wherein a standard colorimetric card is prepared, a bacterial solution is prepared as a standard sample with different concentration gradients, the standard sample and a solution of the kit are mixed, after a period of time, the solution is discolored to obtain different colors, and the standard colorimetric card is prepared based on the colors; and mixing the sample with the solution of the kit, observing color change after a period of time, comparing with a standard colorimetric card, and judging the total number of the prokaryotic cell type microorganisms in the sample.

7. The method according to claim 6, wherein the detection temperature is 30 to 60 ℃.

8. The detection method according to claim 6, wherein the sample is allowed to stand for 2 to 5 hours after being mixed with the solution of the kit.

9. The method according to claim 6, wherein the sample having the visually colored appearance is centrifuged before being mixed with the solution of the kit.

10. The assay of claim 6 wherein the weight ratio of sample to solution in the kit is 3-5: 1.

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