Spore fluorescent staining solution and application thereof

Abstract

The invention discloses a spore fluorescent staining solution and application thereof. The fluorescent whitening agent 220 is used for detecting spores; the spore fluorescent staining solution provided by the invention comprises a fluorescent whitening agent 220. The spore fluorescent staining solution disclosed by the invention is simple in component, low in cost, capable of accurately detecting spores in a sample to be detected, low in toxicity and small in influence on the environment; the detection method disclosed by the invention is simple and convenient to operate and rapid in determination and can be completed by one-step operation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a spore fluorescent staining solution and an application thereof. Background Art

[0002] Some bacteria (such as Bacillus, Clostridium, a few cocci, etc.) form a dormant structure in the cell in the late stage of their growth and development that is round or oval, thick-walled, low in water content, and highly resistant to adversity. This structure is called a spore. In different bacteria, the location of the spore is different, some are in the middle, some are at the end, and some are at the top. Spores are generally round, oval, or cylindrical. In some bacteria, the diameter of the spore is smaller than the diameter of the bacterial body. These bacteria are called Bacillus and are aerobic bacteria; in other bacteria, the diameter of the spore is larger than the diameter of the bacterial body, making the entire bacterial body spindle-shaped or drum-shaped. These bacteria are called Clostridium and are anaerobic bacteria. The spores of Clostridium are located in the middle of the bacterial body. The spores of Clostridium tetani are located at one end of the bacterial body, making the bacterial body drumstick-shaped. All bacteria of the aerobic genus Bacillus and the anaerobic genus Clostridium have spores. Among cocci and spirilla, only a few species have spores, and among cocci, only the genus Sporosarcina produces spores. The structure of spores is quite complex. Spores have multiple layers of thick and dense cell membranes, which are core, inner membrane, spore wall, cortex, outer membrane, spore shell and spore coat from the inside to the outside. In particular, the spore shell is impermeable and has a protective effect, which can prevent chemicals from penetrating. When spores are formed, some special enzymes can be synthesized, which have stronger heat resistance than the enzymes in the reproductive body. The core and cortex of the spore contain a large amount of dipicolinic acid (DPA), which accounts for 5-15% of the dry weight of the spore. It is a unique component of the spore and is not found in bacterial reproductive bodies and other biological cells. The salt formed by the combination of DPA and calcium can improve the thermal stability of various enzymes in the spores. Since spores are different from vegetative cells in structure and chemical composition, spores also have many characteristics different from vegetative cells. The main feature of spores is their strong resistance to high temperature, ultraviolet rays, dryness, ionizing radiation and many toxic chemicals. Therefore, spores are difficult to be killed during food processing and medical device sterilization. After food processing, the remaining unkilled spores can quickly germinate into bacterial nutrients when the external environment is suitable, causing food corruption and deterioration. While causing economic losses, they will also produce diarrheal and vomiting toxins, causing outbreaks of foodborne diseases. Therefore, spore detection is one of the main items for sterilization effect testing in the fields of food and medical devices.

[0003] However, spores are highly refractive, with dense outer membranes and low permeability, making coloring and decolorization difficult, and conventional staining is not possible. At present, there are two main methods for traditional spore detection: fuchsin-methylene blue staining solution or malachite green-safranin O staining solution. Both methods have certain limitations. The fuchsin-methylene blue staining solution is complicated to operate. When staining, the smear must be placed on an alcohol lamp to heat it, and the staining solution must be kept steaming but not boiling for 4 to 5 minutes. After that, it is necessary to use alcohol to decolorize, wash with water, re-stain, and wash with water, which places high demands on the testers. The malachite green-safranin O staining solution is simpler to operate than the fuchsin-methylene blue staining solution, but it also requires four steps: heating and staining the smear, washing with water, re-staining, and washing with water. The median lethal dose of the key dye malachite green is 80 mg / kg (mice, oral), which is highly toxic to the environment.

[0004] Therefore, a spore detection method that is easy to operate, safe, environmentally friendly, accurate and efficient is needed. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a spore fluorescent staining solution and an application thereof; compared with the existing staining solution, the staining solution of the present invention has a simple composition, only requires one fluorescent dye to achieve high-brightness fluorescent staining of spores, has a low cost, and the key raw material, fluorescent whitening agent 220, has a median lethal dose of 5300 mg / kg (rat, oral), has low toxicity, and has little impact on the environment.

[0006] The advantages of the spore fluorescence staining method of the present invention are mainly reflected in the simple operation. Imaging can be completed in one step without complicated water washing steps. After staining, the spores emit bright blue fluorescence, which can conveniently and quickly determine the presence or absence of spores.

[0007] The purpose of the present invention is achieved through the following technical solutions: The present invention provides an application of a fluorescent whitening agent 220 in detecting spores. The structural formula of the fluorescent whitening agent 220 is as follows: .

[0008] The present invention provides a spore fluorescent staining solution, comprising a fluorescent whitening agent 220, the structural formula of the fluorescent whitening agent 220 is as follows: .

[0009] The spore fluorescent staining liquid of the present invention adopts fluorescent brightener 220, which has fluorescent characteristics, can emit blue light, and has the advantage of good light stability. It can combine with spores to emit bright blue fluorescence, and can observe whether spores exist in the bacteria to be tested, thereby improving the spore detection rate.

[0010] Preferably, the spore fluorescent staining solution also includes a buffer solution or water.

[0011] Further preferably, the concentration of the fluorescent whitening agent 220 in the spore fluorescent staining solution is 10-100 μM.

[0012] More preferably, the concentration of the fluorescent whitening agent 220 in the spore fluorescent staining solution is 20-40 μM.

[0013] Further preferably, the buffer solution is a phosphate buffer solution with a pH of 7.2-7.4.

[0014] More preferably, the phosphate is anhydrous sodium dihydrogen phosphate and disodium hydrogen phosphate dodecahydrate, the mass concentration of anhydrous sodium dihydrogen phosphate is 0.1-50 g / L, and the mass concentration of disodium hydrogen phosphate dodecahydrate is 0.1-200 g / L.

[0015] The present invention provides an application of the spore fluorescent staining solution in detecting spores.

[0016] The present invention provides a method for detecting spores, comprising the following steps: (1) Smear the sample to be tested on a glass slide to form a smear; (2) Add the above-mentioned spore fluorescent staining solution and heat and stain for 2 to 10 minutes; (3) Cover with a coverslip and remove excess staining solution; (4) Examination under a fluorescence microscope.

[0017] Preferably, the temperature of the heat dyeing is 40-70°C.

[0018] More preferably, the temperature of the heat dyeing is 40°C.

[0019] Preferably, the heating dyeing time is 2 to 4 min.

[0020] Preferably, the microscopic examination determines whether the sample contains spores by observing whether it has bright fluorescence.

[0021] Preferably, the excitation wavelength of the fluorescence microscope is 330-380 nm.

[0022] Further preferably, the excitation wavelength of the fluorescence microscope is 365 nm.

[0023] Preferably, the emission wavelength of the fluorescence microscope is 410nm~490nm.

[0024] Preferably, the objective lens magnification of the fluorescence microscope is 40×~100×.

[0025] The present invention uses a spore fluorescent staining solution containing a fluorescent brightener 220 to specially stain the spores in the sample to be tested, thereby realizing rapid fluorescent detection of the spores; the operation process of the present invention is very simple, and only needs to evenly smear the bacterial sample on a cover glass, add a drop of fluorescent staining solution and heat for about 3 minutes until the staining solution is slightly dry, and then cover the cover glass to directly observe the results.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The fluorescent brightener 220 of the present invention can achieve specific bright staining of spores, while not staining or staining the bacteria at a low brightness, and has a low fluorescence background, so that the spores in the sample to be tested can be detected quickly and accurately.

[0027] (2) The spore fluorescent staining solution of the present invention has simple components and low cost, can accurately detect spores in the sample to be tested, and the key raw material is less toxic than malachite green and has little impact on the environment.

[0028] (3) The detection method of the present invention is easy to operate, fast to measure, and can be completed in one step; it is more convenient and faster to operate than the fuchsin-methylene blue staining method and the malachite green-safranin O method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing the staining effect of spore-producing Bacillus subtilis using spore fluorescent staining solutions of different concentrations in Example 1.

[0030] Figure 2 This is a diagram showing the staining effect of Bacillus subtilis that can produce spores after incubation with spore fluorescent staining solution for different time periods in Example 2.

[0031] Figure 3 This is a diagram showing the staining effect of using spore fluorescent staining solution on Bacillus subtilis that can produce spores at different time periods in Example 3.

[0032] Figure 4 This is a diagram showing the staining effect of non-spore-producing Staphylococcus aureus and Salmonella using a spore staining solution in Example 4.

[0033] Figure 5 This is a comparison chart of the staining effects of using spore fluorescent staining solution, malachite green safranin O staining solution and safranin O staining solution in Example 5. DETAILED DESCRIPTION

[0034] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0035] Embodiment 1: A spore fluorescent staining solution and a staining method thereof.

[0036] (1) Dissolve fluorescent brightener 220 in purified water to obtain spore fluorescent staining solutions with concentrations of 20 μM and 40 μM.

[0037] (2) Select Bacillus subtilis that has been cultured for one day, pick out a single colony and dissolve it in a small amount of water. Take 10 μL and smear it on a glass slide to make a bacterial smear of appropriate thickness. Take 200 μL of the above spore fluorescent staining solution and drop it directly on the sample. Heat it on a heating table at 40°C for 3 minutes until the stain is slightly dry. Cover it with a coverslip and use absorbent paper to absorb the excess stain. Select an excitation light wavelength of 365nm and an emission wavelength of 410nm~490nm. Place it under a fluorescence microscope objective (40×) for direct microscopic examination. The staining effect is as follows: Figure 1 shown.

[0038] Embodiment 2: A spore fluorescent staining solution and a staining method thereof.

[0039] (1) Dissolve fluorescent brightener 220 in purified water to obtain a spore fluorescent staining solution with a concentration of 20 μM.

[0040] (2) Select Bacillus subtilis that has been cultured for one day, pick out a single colony and dissolve it in a small amount of water. Take 10 μL and smear it on a glass slide to make a bacterial smear of appropriate thickness. Take 200 μL of the above spore fluorescent staining solution and directly drop it on the sample. Heat it on a heating table at 40°C for 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, and 10 minutes until the stain is slightly dry. Cover with a coverslip and use absorbent paper to absorb excess stain. Select an excitation light wavelength of 365 nm and an emission wavelength of 410 nm~490 nm. Place it under a fluorescence microscope objective (40×) for direct microscopic examination. The staining effect is as follows: Figure 2 shown.

[0041] Embodiment 3: A spore fluorescent staining solution and a staining method thereof.

[0042] (1) Dissolve fluorescent brightener 220 in purified water to obtain a spore fluorescent staining solution with a concentration of 20 μM.

[0043] (2) Select Bacillus subtilis that have been cultured for one, two, and three days, pick out single colonies and dissolve them in a small amount of water. Take 10 μL and smear it on different glass slides to make bacterial smears of appropriate thickness. Take 200 μL of the above spore fluorescent staining solution and drop it on the sample. Heat it on a heating table at 40°C for 3 minutes until the stain is slightly dry. Cover it with a coverslip and use absorbent paper to absorb the excess stain. Select an excitation light wavelength of 365 nm and an emission wavelength of 410 nm~490 nm. Place it under a fluorescence microscope objective (40×) for direct microscopic examination. The staining effect is as follows: Figure 3 shown.

[0044] Embodiment 4: A spore fluorescent staining solution and a staining method thereof.

[0045] (1) Dissolve fluorescent brightener 220 in purified water to obtain a spore fluorescent staining solution with a concentration of 20 μM.

[0046] (2) Select Staphylococcus aureus and Salmonella that have been cultured for one day, pick out single colonies and dissolve them in a small amount of water, take 10 μL and smear them on different glass slides to make bacterial smears of appropriate thickness, take 200 μL of the above spore fluorescent staining solution and drop them on the samples, heat them on a heating table at 40°C for 3 minutes until the staining solution is slightly dry, cover with a coverslip, and use absorbent paper to absorb the excess staining solution. Select an excitation light wavelength of 365 nm and an emission wavelength of 410 nm to 490 nm, place it under a fluorescence microscope objective (40×) for direct microscopic examination. The staining effect is as follows: Figure 4 shown.

[0047] Embodiment 5: A spore fluorescent staining solution and a staining method thereof.

[0048] (1) Dissolve fluorescent brightener 220 in purified water to obtain a spore fluorescent staining solution with a concentration of 20 μM.

[0049] (2) Select Bacillus subtilis cultured for one day, pick a single colony and dissolve it in a small amount of water, take 10 μL and smear it on different slides to make bacterial smears of appropriate thickness, and stain them with spore fluorescent staining solution, malachite green safranin O staining solution and safranin O staining solution. Compare the detection steps, time and effects of different staining methods. The staining effect is as follows: Figure 5 ,The comparison of detection steps and time is shown in Table 1.

[0050] For spore fluorescence staining, take 200 μL of the above spore fluorescence staining solution and drop it on the sample, heat it on a heating table at 40°C for 3 minutes until the staining solution is slightly dry, cover it with a coverslip, and use absorbent paper to absorb the excess staining solution. Select the excitation light wavelength of 365nm and the emission wavelength of 410nm~490nm, and place it under the biological (fluorescence) microscope objective (40×) for direct microscopic examination.

[0051] For Malachite Green Safranin O staining, take 200 μL of Malachite Green staining solution and drop it on the sample, heat it on a heating table at 40°C for 10 minutes, wash the sample with water until no staining solution is removed for about 1 minute, then add 200 μL of Safranin O staining solution and stain the sample at room temperature for 3 minutes, wash it with water again until no staining solution is removed for about 1 minute, cover it with a coverslip, and use absorbent paper to absorb excess water. Place it under the objective of a biological (fluorescence) microscope and perform direct microscopic examination using bright field.

[0052] For Safranin O staining, add 200 μL of Safranin O staining solution to stain the sample at room temperature for 3 minutes, wash with water until no staining solution is removed for about 1 minute, cover with a coverslip, and use absorbent paper to absorb excess water. Place under the objective of a biological (fluorescence) microscope, and perform direct microscopic examination using bright field.

[0053] Table 1 Comparison of staining steps and time

[0054] Result analysis: (1) From Figure 1 It can be seen that different concentrations of spore staining solution can dye spores into bright blue fluorescence, and bacterial cells (non-spores) are dyed into light blue (20μM in the left picture and 40μM in the right picture).

[0055] (2) From Figure 2 It can be seen that after incubation with the staining solution for different time periods, the spores can be stained with bright blue fluorescence after 2 minutes of incubation.

[0056] (3) From Figure 3 It can be seen that the spores cultured at different times were all stained with bright blue fluorescence.

[0057] (4) From Figure 4 It can be seen that Gram-positive Staphylococcus aureus and Gram-negative Salmonella that do not produce spores will not be stained fluorescently by the spore fluorescent stain.

[0058] (5) From Figure 5It can be seen from the table that both the spore fluorescent staining solution and the malachite green safranin O staining solution can stain spores, while when the more toxic malachite green is not used, safranin O can only stain the bacteria, and the spores are not colored, making it difficult to observe. However, it can be seen from Table 1 that the malachite green safranin O staining solution is more complicated and takes longer than the spore fluorescent staining solution.

[0059] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. The use of fluorescent whitening agent 220 in detecting spores is characterized in that: The structural formula of the fluorescent brightener 220 is as follows: 。 2. A spore fluorescent staining solution, characterized in that: Including fluorescent whitening agent 220, the structural formula of fluorescent whitening agent 220 is as follows: 。 3. The spore fluorescent staining liquid according to claim 2, characterized in that The spore fluorescent staining solution also includes a buffer solution or water.

4. The spore fluorescent staining liquid according to claim 3, characterized in that The concentration of the fluorescent brightener 220 in the spore fluorescent staining solution is 10-100 μM.

5. The spore fluorescent staining liquid according to claim 3, characterized in that: The buffer solution is a phosphate buffer solution with a pH of 7.2-7.

4.

6. The spore fluorescent staining solution according to claim 5, characterized in that: The phosphates are anhydrous sodium dihydrogen phosphate and disodium hydrogen phosphate dodecahydrate, the mass concentration of anhydrous sodium dihydrogen phosphate is 0.1-50 g / L, and the mass concentration of disodium hydrogen phosphate dodecahydrate is 0.1-200 g / L.

7. A method for detecting spores, characterized in that: The following steps are involved: (1) Smear the sample to be tested on a glass slide to form a smear; (2) adding the spore fluorescent staining solution according to any one of claims 2 to 6 dropwise, and heating and staining for 2 to 10 minutes; (3) Cover with a coverslip and remove excess staining solution; (4) Examination under a fluorescence microscope.

8. The method for detecting spores according to claim 7, characterized in that: The temperature for heating dyeing is 40~70℃.

9. The method for detecting spores according to claim 7, characterized in that: Microscopic examination determines whether the sample contains spores by observing whether it has bright fluorescence.

10. The method for detecting spores according to claim 7, characterized in that: The excitation wavelength of the fluorescence microscope is 330~380nm.

Images