Method for rapid counting of total bacterial count in milk and staining reagent combination

Abstract

The application belongs to the technical field of microorganism detection, and specifically discloses a method for rapidly counting total number of colonies in milk and a staining reagent combination. The method for rapidly counting total number of colonies in milk comprises the following steps: S1, after diluting a milk sample, adding phosphomolybdic acid solution to fully mix, vortexing, and obtaining a mixture; S2, adding an eosin staining solution to the mixture obtained in S1, performing staining treatment at room temperature, and observing and counting the number of microorganisms after staining under a microscope. The application discloses a method for rapidly counting total number of colonies in milk and a staining reagent combination. The method not only improves detection speed and efficiency, but also improves detection accuracy and reliability. This rapid and comprehensive detection technology provides strong technical support for quality control and food safety management of the dairy industry, and has important application value and popularization prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganism detection, and particularly relates to a method for rapidly counting total number of colonies in milk and a staining reagent combination. BACKGROUND

[0002] The determination of total number of colonies in milk is an important indicator for evaluating the hygiene quality and safety of milk. Traditionally, the determination of total number of colonies is mainly carried out by plate counting method, which is a classical and widely used microorganism detection technology. This method first dilutes the milk sample appropriately, and then spreads the diluted sample on a plate, and incubates at 37℃ for 24 to 48 hours. During the incubation process, the cultivable microorganisms in the sample will form independent colonies on the culture medium. By counting the number of these colonies, the number of microorganisms in the original sample can be estimated. The advantage of this method lies in its high sensitivity and quantitative ability, which can effectively detect bacteria, yeast, mold and other microorganism species in milk.

[0003] However, the plate counting method also has obvious limitations. First, this method requires a long incubation time and cannot provide real-time detection results, making it difficult to meet the demand for rapid detection. Second, the plate counting method can only detect cultivable microorganisms and cannot reflect the types of non-culturable microorganisms, which may result in partial loss of data. In addition, the traditional plate counting method is easily disturbed by other biological cells (such as yeast, mold) when detecting complex samples, which affects the accuracy of the detection results. Therefore, it is particularly important to develop a rapid and accurate method for counting total number of colonies. SUMMARY

[0004] The present application aims to provide a method for rapidly counting total number of colonies in milk and a staining reagent combination, which not only improves the detection speed and efficiency, but also improves the accuracy and reliability of the detection, providing more accurate information for the quality control of dairy products and product safety.

[0005] To solve the above technical problems, the technical solution adopted by the present application is as follows:

[0006] A method for rapidly counting total number of colonies in milk, comprising the following steps:

[0007] S1, diluting the milk sample, adding phosphomolybdate solution and fully mixing, and vortexing to obtain a mixture;

[0008] S2, adding eosin staining solution to the mixture obtained in S1, and performing staining treatment at room temperature, and observing and counting the number of microorganisms after staining under a microscope.

[0009] Preferably, in S1, the dilution ratio of the milk sample is 100-1000 times.

[0010] Preferably, in S1, the concentration of the phosphomolybdic acid solution is 10 g / L, and the addition amount of the phosphomolybdic acid solution is 1-10 μL.

[0011] Preferably, in S1, the rotation speed of the vortex is 200-1000 rpm, and the vortex time is 1-3 min.

[0012] Preferably, in S2, the concentration of the eosin staining solution is 10 g / L, and the addition amount of the eosin staining solution is 10-100 μL.

[0013] Preferably, in S2, the staining treatment time is 2 min.

[0014] The application further provides a staining reagent combination for rapidly counting the total number of colonies in milk, comprising:

[0015] a phosphomolybdic acid solution with a concentration of 10 g / L;

[0016] an eosin staining solution with a concentration of 10 g / L;

[0017] Preferably, the addition amount of the phosphomolybdic acid solution is 1-10 μL, and the addition amount of the eosin staining solution is 10-100 μL.

[0018] Compared with the prior art, the application has the following advantages and technical effects:

[0019] 1. The application discloses a method for rapidly counting the total number of colonies in milk, which can complete staining treatment in only 2 minutes and count the total number of colonies within 1 hour through staining and microscopic observation. This rapid detection capability can realize real-time monitoring of milk samples, timely discover potential microbial contamination problems, provide rapid response for the production and processing of dairy products, and effectively reduce food safety risks.

[0020] 2. Plate counting method can only detect cultivable microorganisms, and cannot reflect the types of unculturable microorganisms in milk, which may lead to partial loss of detection results. The present method can cover unculturable microorganisms through staining technology, and provide more comprehensive microbial community data, thereby providing more accurate information for quality control and product safety of dairy products.

[0021] 3. The phosphomolybdic acid-eosin staining combination used in the method can effectively exclude the interference of fat and protein in milk. Phosphomolybdic acid as a mordant can wrap proteins so that they are not stained, while the eosin staining solution can specifically stain microbial cytoplasm. Experimental results show that the method does not stain the cells of the cow, but can successfully stain gram-negative bacteria (such as E. coli) and gram-positive bacteria (such as Staphylococcus aureus), and hardly stains dead bacteria, thereby significantly improving the accuracy and specificity of detection.

[0022] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The milk dyeing results provided for Comparative Example 1 and Examples 1-5 are shown in Table 1, wherein, Figure 1 A in Table 1 is the dyeing result of Comparative Example 1, Figure 1 B in Table 1 is the dyeing result of Example 1, Figure 1 C in Table 1 is the dyeing result of Example 2, Figure 1 D in Table 1 is the dyeing result of Example 3, Figure 1 E in Table 1 is the dyeing result of Example 4, Figure 1 F in Table 1 is the dyeing result of Example 5.

[0024] Figure 2 The milk dyeing results provided for Examples 6-7 are shown in Table 2, wherein, Figure 2 A in Table 2 is the dyeing result of Example 6, Figure 1 B in Table 2 is the dyeing result of Example 7.

[0025] Figure 3 The bovine cell dyeing results are shown in Table 3, wherein, Figure 3 A in Table 3 is the dyeing result of the control group, Figure 3 B in Table 3 is the dyeing result of the experimental group.

[0026] Figure 4 The dyeing results of unsterilized Staphylococcus aureus are shown in Table 4, wherein, Figure 4 A in Table 4 is the dyeing result of the control group, Figure 4 B in Table 4 is the dyeing result of the experimental group.

[0027] Figure 5 The dyeing results of sterilized Staphylococcus aureus are shown in Table 5, wherein, Figure 5 A in Table 5 is the dyeing result of the control group, Figure 5 B in Table 5 is the dyeing result of the experimental group.

[0028] Figure 6 The dyeing results of unsterilized Escherichia coli are shown in Table 6, wherein, Figure 6 A in Table 6 is the dyeing result of the control group, Figure 6 B in Table 6 is the dyeing result of the experimental group.

[0029] Figure 7 The dyeing results of inactivated Escherichia coli are shown in Table 7, wherein, Figure 7 A in Table 7 is the dyeing result of the control group, Figure 7 B in Table 7 is the dyeing result of the experimental group. DETAILED DESCRIPTION

[0030] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples.

[0031] Unless otherwise defined, technical terms or scientific terms used in the present application shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0032] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art, which can be purchased through commercial channels.

[0033] The strain used in the present application is Staphylococcus aureus, numbered as ATCC 25923.

[0034] The strain used in the present application is Escherichia coli, numbered as ATCC 25922. The above-mentioned strains are purchased through commercial channels.

[0035] Phosphomolybdate solution preparation: weigh 1g of phosphomolybdate and put it into a beaker, add about 50mL of deionized water to dissolve, then use a stirrer or glass rod to stir until the phosphomolybdate is completely dissolved. Transfer the solution to a 100mL volumetric flask, use deionized water to constant volume to the calibration line, and mix evenly by gently shaking, to obtain a phosphomolybdate solution with a concentration of 10g / L.

[0036] Eosin staining solution preparation: weigh 1g of eosin and add about 50mL of deionized water, stir with a stirrer or glass rod until it is completely dissolved, then transfer to a volumetric flask after dissolution, use deionized water to constant volume to 100mL, to obtain an eosin staining solution with a concentration of 10g / L.

[0037] Example one

[0038] A method for rapidly counting the total number of colonies in milk, comprising the following steps:

[0039] S1, dilute the milk sample 100 times, then add 1μL of phosphomolybdate solution with a concentration of 10g / L to fully mix, vortex at 1000rpm for 1min to obtain a mixture;

[0040] S2, add 50μL of eosin staining solution with a concentration of 10g / L to the mixture obtained in S1, perform staining treatment at room temperature for 2min, take 20μL of the stained sample and drop it on a glass slide, slowly cover the glass slide from one side to avoid air bubbles, and observe under a 100x objective.

[0041] Example two

[0042] The method is the same as example one, except that the amount of phosphomolybdate solution added is 2μL.

[0043] Example three

[0044] The method is the same as example one, except that the amount of phosphomolybdate solution added is 5μL.

[0045] Example four

[0046] The method is the same as in Example 1, except that the amount of phosphomolybdic acid solution added is 10 μL.

[0047] Example 5

[0048] The method is the same as in Example 1, except that the amount of phosphomolybdic acid solution added is 5 μL and the amount of eosin staining solution added is 10 μL.

[0049] Example 6

[0050] The method is the same as in Example 1, except that the amount of phosphomolybdic acid solution added is 5 μL and the amount of eosin staining solution added is 20 μL.

[0051] Example 7

[0052] The method is the same as in Example 1, except that the amount of phosphomolybdic acid solution added is 5 μL and the amount of eosin staining solution added is 100 μL.

[0053] Comparative Example 1

[0054] The method is the same as in Example 1, except that phosphomolybdic acid solution and eosin staining solution are not added. The staining results of Examples 1-7 and Comparative Example 1 are as follows: Figures 1-2 .

[0055] Depend on Figures 1-2 It is known that the combination of phosphomolybdic acid solution and eosin staining agent can successfully stain microorganisms in milk. However, when the amount of phosphomolybdic acid solution added is less than 5 μL, the milk proteins cannot be fully coated by the phosphomolybdic acid, leaving the proteins exposed and easily stained by eosin staining agent. This results in the simultaneous staining of microorganisms and proteins, increasing the number of stained individuals and thus affecting the accuracy of staining. Figure 1 B and Figure 1 The number of stains indicated by C is higher than Figure 1 D and Figure 1 The E in the formula indicates that when 5-10 μL of a 10 g / L phosphomolybdic acid solution and 10-100 μL of a 10 g / L eosin staining agent are added, bacteria in milk can be successfully stained.

[0056] The effectiveness was verified through the following experiments:

[0057] Bovine somatic cell staining: The experiment was divided into a control group and an experimental group. Somatic cell isolation and culture methods from "Establishment and Identification of Bovine Mammary Epithelial Cell Models" were used. Healthy dairy cows were selected, and the udders were disinfected using standard methods. Fresh milk was then collected in sterile bottles and brought back to the laboratory for bovine somatic cell isolation and culture. The cultured bovine somatic cell culture medium was diluted 10... 4After 1 hour, 1 mL was dropped into two 2 mL centrifuge tubes, respectively. The control group was not added with dyeing agent for dyeing, and the experimental group was added with 5 μL of phosphomolybdic acid solution with a concentration of 10 g / L. After fully mixing, it was vortexed uniformly, and then 50 μL of eosin dyeing agent with a concentration of 10 g / L was added. After fully mixing, it was vortexed and dyed at room temperature for two minutes. 20 μL of each group was dropped on a glass slide, and a glass slide was slowly covered from one side to avoid air bubbles. It was observed under a 100 times objective lens. The results are shown in Figure 3 .

[0058] As can be seen from Figure 3 , the bovine somatic cells cannot be dyed by the phosphomolybdic acid solution and the eosin dyeing agent, and the bovine somatic cells have no effect on the dyeing result of the milk.

[0059] 1. Staphylococcus aureus, a gram-positive bacterium, was dyed

[0060] The inactivated Staphylococcus aureus was divided into a control group and an experimental group. The activated Staphylococcus aureus was taken, and the activated Staphylococcus aureus solution was diluted 10 4 times with sterile normal saline. 1.0 mL of the Staphylococcus aureus diluent was dropped into two 2 mL centrifuge tubes, respectively. The control group was not added with dyeing agent for dyeing, and the experimental group was added with 5 μL of phosphomolybdic acid solution with a concentration of 10 g / L. After fully mixing, it was vortexed uniformly, and then 50 μL of eosin dyeing agent with a concentration of 10 g / L was added. After fully mixing, it was vortexed and dyed at room temperature for two minutes. 20 μL of each group was dropped on a glass slide, and a glass slide was slowly covered from one side to avoid air bubbles. It was observed under a 100 times objective lens. The results are shown in Figure 4 .

[0061] As can be seen from Figure 4 , the gram-positive bacterium Staphylococcus aureus can be dyed by the phosphomolybdic acid solution and the eosin dyeing agent.

[0062] The inactivated Staphylococcus aureus was divided into a control group and an experimental group. The activated Staphylococcus aureus was taken, and the activated Staphylococcus aureus solution was diluted 10 4 times with sterile normal saline. 1.0 mL of the Staphylococcus aureus diluent was dropped into two 2 mL centrifuge tubes, respectively, and the two groups were pasteurized and inactivated. The control group was not added with dyeing agent for dyeing, and the experimental group was added with 5 μL of phosphomolybdic acid solution with a concentration of 10 g / L. After fully mixing, it was vortexed uniformly, and then 50 μL of eosin dyeing agent with a concentration of 10 g / L was added. After fully mixing, it was vortexed and dyed at room temperature for two minutes. 20 μL of each group was dropped on a glass slide, and a glass slide was slowly covered from one side to avoid air bubbles. It was observed under a 100 times objective lens. The results are shown in Figure 5 .

[0063] As can be seen from Figure 5 , the dead gram-positive bacterium Staphylococcus aureus cannot be dyed by the phosphomolybdic acid solution and the eosin dyeing agent.

[0064] 3. Staining of Gram-negative bacteria Escherichia coli

[0065] Unactivated E. coli: The experiment was divided into a control group and an experimental group. E. coli was activated, and the activated E. coli solution was diluted 10% with sterile physiological saline. 4 1.0 mL of diluted *E. coli* solution was added to two 2 mL centrifuge tubes for each group. The control group received no staining, while the experimental group received 5 μL of 10 g / L phosphomolybdic acid solution. After thorough mixing and vortexing, 50 μL of 10 g / L eosin staining was added, and the mixture was thoroughly mixed and vortexed for two minutes at room temperature. 20 μL of each solution was placed on a glass slide, and the slide was slowly covered from one side to avoid air bubbles. The results were observed under a 100x objective lens. Figure 6 .

[0066] Depend on Figure 6 It is known that Gram-negative bacteria Escherichia coli can be stained by phosphomolybdic acid solution and eosin staining agent.

[0067] Inactivated E. coli: The experiment was divided into a control group and an experimental group. E. coli was activated, and the activated E. coli solution was diluted 10% with sterile physiological saline. 4 To inactivate Staphylococcus aureus, 1.0 mL of diluted Staphylococcus aureus solution was added to two 2 mL centrifuge tubes, and both tubes were pasteurized. The control group was stained without staining. The experimental group was treated with 5 μL of 10 g / L phosphomolybdic acid solution, thoroughly mixed, vortexed, and then 50 μL of 10 g / L eosin staining was added, thoroughly mixed, vortexed, and stained at room temperature for two minutes. 20 μL of each solution was placed on a glass slide, and the slide was slowly covered from one side to avoid air bubbles. The results were observed under a 100x objective lens. Figure 7 .

[0068] Depend on Figure 7 It is known that dead Gram-negative Escherichia coli will not be stained by phosphomolybdic acid solution and eosin staining agent.

[0069] The *Staphylococcus aureus* and *Escherichia coli* experimental groups in Example 5 were examined and counted under a microscope. 20 μL samples were then subjected to a plate count test, which was conducted according to the national standard GB 4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". Three replicate experiments were performed, and the results are shown in Table 1.

[0070] Table 1. Colony Count Results

[0071]

[0072]

[0073] From Table 1, it can be seen that the number of S. aureus and E. coli and the number of microscopic count in milk is slightly greater than the number of plate count, which can be attributed to the overlapping colonies in plate culture and the presence of unculturable microorganisms in milk, but compared to the traditional plate count which needs to consume longer time, the eosin plus phosphomolybdate staining method used in the present application can reflect the total number of colonies in the sample more quickly.

[0074] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: it can still modify or equivalently replace the technical solutions of the present application, and these modifications or equivalent replacements also cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for rapid enumeration of the total number of colonies in milk, characterized in that, The method comprises the following steps: S1, after diluting the milk sample, adding phosphomolybdic acid solution to fully mix, vortexing to obtain a mixture; S2, adding eosin staining solution to the mixture obtained in S1, performing staining treatment at room temperature, taking samples for microscopic observation and counting the number of microorganisms after staining; The concentration of the phosphomolybdic acid solution is 10 g / L, and the addition amount of the phosphomolybdic acid solution is 5-10 μL; The concentration of the eosin staining solution is 10 g / L, and the addition amount of the eosin staining solution is 10-100 μL.

2. The method of claim 1, wherein, In S1, the dilution multiple of the milk sample is 100-1000 times.

3. The method of claim 1, wherein, In S1, the vortexing rotation speed is 200-1000 rpm, and the vortexing time is 1-3 min.

4. The method of claim 1, wherein, In S2, the staining treatment time is 2 min.

5. A staining reagent combination for rapid enumeration of the total number of colonies in milk, characterized in that It comprises: phosphomolybdic acid solution with a concentration of 10 g / L; eosin staining solution with a concentration of 10 g / L; wherein the addition amount of the phosphomolybdic acid solution is 5-10 μL, and the addition amount of the eosin staining solution is 10-100 μL.

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