Simple and efficient mycoplasma staining detection method
Mycoplasma staining is performed by using a mixture of crystal violet staining solution and sandy yellow staining solution, combined with culture medium activation and electron microscopy observation, the problems of poisoning reagents, expensive instruments and complex operations in the prior art are solved, and simple, efficient and low-cost Mycoplasma detection is achieved.
Patent Information
- Application Number
- CN202411848416.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-04
AI Technical Summary
The existing mycoplasma detection methods have problems such as toxic reagents, harsh operating environments, expensive instruments and complex methods, and it is difficult to provide a simple, convenient, fast and low-cost detection method.
Mycoplasma staining was used to stain mycoplasma by a mixture of crystal violet staining liquid and sandy yellow staining liquid. Through the activation process of liquid and solid culture medium, combined with electron microscopy observation, the efficient detection of mycoplasma was achieved.
Overcoming the toxicity, expensive instruments and complex operation problems of traditional methods, it provides a mycoplasma detection method that is simple to operate, low cost, good repeatability and good dyeing effect.
Smart Images
Figure CN119555468A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of mycoplasma staining, and in particular to a simple and efficient mycoplasma staining detection method. Background Art
[0002] Mycoplasma is a type of minimal prokaryotic cell-type microorganism that has no cell wall, is highly polymorphic, can pass through a bacterial filter, and can be cultured and proliferated in artificial culture medium. It is 0.1 to 0.3 microns in size. Because it can form filamentous and branched shapes, it is called mycoplasma. Mycoplasma is widely present in humans and animals, and most of them are not pathogenic. The mycoplasmas that are pathogenic to humans mainly include pneumonia mycoplasma, human mycoplasma, oral mycoplasma, etc. Mycoplasma has a simple structure and no cell wall. It only has ribosomes and DNA molecules in the plasma membrane and cell body. Because mycoplasma has a tiny structure and can pass through filters, it will interfere with cell culture and other work. Mycoplasma has high nutritional requirements, generally animal serum is added, and it also needs to grow in a microaerobic environment with a carbon dioxide content of 5%-10%. Because mycoplasma has no cell wall, it is insensitive to antibiotics that inhibit cell wall formation. Therefore, penicillin can be added to inhibit the growth of bacteria and avoid contamination of miscellaneous bacteria in mycoplasma culture. The colony morphology of mycoplasma on solid plate culture medium is fried egg-shaped, growing inward to form the yolk part and forming the egg white part on the surface.
[0003] Mycoplasma detection is widely used in both drug testing and clinical practice. At present, the two most commonly used methods for mycoplasma detection and observation are DNA fluorescent staining detection kit method and PCR method. Both staining methods have obvious disadvantages. The staining detection kit method has three major defects: 1. The Hoechst working solution in the kit is toxic to the human body and requires strict protection; 2. The fixative in the kit has a strong irritating odor and must be carried out in a fume hood, which limits the operating environment; 3. When performing mycoplasma detection, culture fluid containing antibiotics cannot be used because it will cause false negative results. Without antibiotics, mycoplasma contamination is likely to occur, which seriously limits the conditions of use. The PCR method mainly includes gel electrophoresis detection and fluorescence quantitative method, which requires the purchase of expensive PCR instruments worth hundreds of thousands of yuan. The principle requires the design of primers in the conserved region of the mycoplasma 16S rRNA gene, and the method is cumbersome and complicated. Therefore, a simple detection method that can directly stain and observe mycoplasmas is needed. Summary of the invention
[0004] In view of the shortcomings of the existing technology, a simple and efficient mycoplasma staining detection method is provided, which provides a simpler, more convenient, faster and lower-cost method for mycoplasma detection.
[0005] To achieve the above object, the present invention provides the following technical solution: a simple and efficient mycoplasma staining detection method, comprising the following steps:
[0006] S1, prepare staining solution: mix crystal violet staining solution and safflower staining solution, dilute with sterile distilled water and set aside;
[0007] S2, activation of original liquid culture medium: aspirate the preserved oral mycoplasma stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, and place it in a microaerobic environment for culture;
[0008] S3, secondary activation of mycoplasma solid culture medium: the mycoplasma culture solution in step S2 is absorbed, and the solution is spread and inoculated on a Mycoplasma arginine solid culture medium plate, the tube opening is wrapped with a sealing film, and the plate is placed in a microaerobic environment for culture;
[0009] S4, drop the staining solution prepared in step 1 onto the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, and then rinse off the excess staining solution remaining on the plate with sterile water;
[0010] S5, use a sterilized blade to cut a thin layer of agar on the plate, transfer it to a glass slide, and place the slide on an ordinary electron microscope stage for observation.
[0011] As a preferred embodiment of the present invention, the mixing ratio of the crystal violet staining solution to the safflower staining solution in step S1 is 1:1.
[0012] As a preferred embodiment of the present invention, in step S1, the crystal violet staining solution and the safflower staining solution are mixed and then diluted 10 times with sterile distilled water.
[0013] As a preferred solution of the present invention, the microaerobic environment in step S2 is provided by a carbon dioxide incubator with a carbon dioxide content of 5% to 10%, a culture temperature of 37° C., and a culture time of 3 to 4 days.
[0014] As a preferred solution of the present invention, in step S3, the microaerobic environment is provided by a carbon dioxide incubator with a carbon dioxide content of 5% to 10%, a culture temperature of 37° C., and a culture time of 6 to 10 days.
[0015] As a preferred solution of the present invention, in step S5, if there is sufficient light and the light transmittance of the eyepiece is good, the mycoplasma plate can be directly placed under a microscope for observation.
[0016] As a preferred solution of the present invention, in step S5, observation is first performed under the microscope eyepiece 16 times and the objective lens 4 times field of view, and then the eyepiece remains unchanged and the objective lens is switched to 16 times for observation.
[0017] As a preferred solution of the present invention, the volume of the oral mycoplasma stock solution absorbed in step S2 is 100 ul.
[0018] As a preferred solution of the present invention, the volume of the oral mycoplasma culture fluid absorbed in step S3 is 100 ul.
[0019] The beneficial effects of the present invention are:
[0020] The mycoplasma detection method of the present invention is different from the DNA fluorescent staining detection kit method and the PCR method. The staining solution used is a common Gram staining solution, which is non-toxic and low-priced, and overcomes the limitations and disadvantages of traditional mycoplasma detection methods, such as toxic detection reagents, harsh detection conditions, expensive detection instruments, and complex and cumbersome detection methods. It has the advantages of simple operation, low cost, good repeatability, good staining effect, etc., and provides a new detection method for mycoplasma detection, which is widely used. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a photo of the activated human oral mycoplasma liquid culture medium of Example 1 of the present invention;
[0022] Figure 2 This is a photo of the solid culture medium for human oral mycoplasma after secondary activation according to Example 1 of the present invention;
[0023] Figure 3 This is a photograph of a human oral mycoplasma plate placed under a microscope according to Example 1 of the present invention;
[0024] Figure 4 This is a photograph of the human oral mycoplasma in Example 1 of the present invention in the field of view of a microscope with a 16x eyepiece and a 4x objective lens;
[0025] Figure 5 This is a photograph of the human oral mycoplasma in Example 1 of the present invention in the field of view of a 16x eyepiece and a 16x objective lens of a microscope;
[0026] Figure 6 This is a photograph of the human oral mycoplasma in Example 2 of the present invention in the field of view of a microscope with a 16x eyepiece and a 4x objective lens;
[0027] Figure 7 This is a photograph of the human pneumoniae of Example 3 of the present invention in the field of view of a microscope with a 16x eyepiece and a 4x objective lens;
[0028] Figure 8 This is a photograph of the Mycoplasma hyopneumoniae of Example 4 of the present invention in the field of view of a microscope with a 16x eyepiece and a 4x objective lens. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0030] A simple and efficient mycoplasma staining detection method comprises the following steps:
[0031] S1, prepare staining solution: mix crystal violet staining solution and safflower staining solution, dilute with sterile distilled water and set aside;
[0032] S2, activation of original liquid culture medium: aspirate the preserved oral mycoplasma stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, and place it in a microaerobic environment for culture;
[0033] S3, secondary activation of mycoplasma solid culture medium: the mycoplasma culture solution in step S2 is absorbed, and the solution is spread and inoculated on a Mycoplasma arginine solid culture medium plate, the tube opening is wrapped with a sealing film, and the plate is placed in a microaerobic environment for culture;
[0034] S4, drop the staining solution prepared in step 1 onto the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, and then rinse off the excess staining solution remaining on the plate with sterile water;
[0035] S5, use a sterilized blade to cut a thin layer of agar on the plate, transfer it to a glass slide, and place the slide on an ordinary electron microscope stage for observation.
[0036] Then, in step S1, the mixing ratio of the crystal violet staining solution and the safflower staining solution is 1:1.
[0037] Furthermore, in step S1, the crystal violet staining solution and the safflower staining solution are mixed and then diluted 10 times with sterile distilled water.
[0038] Furthermore, the microaerobic environment in step S2 is provided by a carbon dioxide incubator with a carbon dioxide content of 5% to 10%, a culture temperature of 37° C., and a culture time of 3 to 4 days.
[0039] Furthermore, in step S3, the microaerobic environment is provided by a carbon dioxide incubator, the carbon dioxide content is 5% to 10%, the culture temperature is 37° C., and the culture time is 6 to 10 days.
[0040] Then, in step S5, if there is sufficient light and the light transmittance of the eyepiece is good, the mycoplasma plate can be directly placed under a microscope for observation.
[0041] Secondly, in step S5, observation is first performed under the microscope eyepiece 16 times and the objective lens 4 times field of view, and then the eyepiece remains unchanged and the objective lens is switched to 16 times for observation.
[0042] Furthermore, the volume of the oral mycoplasma stock solution aspirated in step S2 is 100 ul.
[0043] Finally, the volume of the oral mycoplasma culture fluid aspirated in step S3 is 100 ul.
[0044] Embodiment 1:
[0045] The human oral mycoplasma type A staining test uses the following 6 steps:
[0046] 1. Prepare the staining solution. Mix crystal violet staining solution and safflower staining solution in a ratio of 1:1, and dilute 10 times with sterile distilled water for later use. The above two staining solutions are common Gram staining solutions, which are non-toxic and inexpensive.
[0047] 2. Activation of mycoplasma liquid culture medium: Absorb 100ul of the preserved human oral mycoplasma stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 3 to 4 days. Figure 1 As shown, it can be found that the culture medium tube is clear and turns red. Clarity indicates that there is no bacterial contamination in the culture medium, and the culture medium turns red because mycoplasma can use arginine in the culture medium to increase the pH value of the culture medium, and the culture medium turns red when it encounters the acid-base indicator phenol red.
[0048] 3. Secondary activation of mycoplasma solid culture medium: Take 100ul of the human oral mycoplasma culture fluid in step 1, spread it on the arginine mycoplasma solid culture medium plate, cover the edge of the plate with sealing film to create a microaerobic environment and prevent external microorganisms from entering and contaminating, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 6 to 10 days. Figure 2 As shown, pinpoint-sized colonies can be seen under light.
[0049] 4. Add the staining solution prepared in step 1 to the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, then rinse off the excess staining solution remaining on the plate with sterile water.
[0050] 5. Use a sterilized blade to cut a thin layer of agar from the plate, transfer it to a glass slide, and place the slide on a common electron microscope stage for observation. Figure 3 As shown, if there is sufficient light and the eyepiece has good light transmittance, the mycoplasma plate can be directly placed under the microscope for observation.
[0051] 6. If Figure 4 As shown in the figure, under the microscope's 16x eyepiece and 4x objective field of view, it can be found that oral mycoplasma type A has a typical poached egg-like morphology. Figure 5 As shown, after switching the objective lens to 16 times for observation, it can be found that the core is thicker and grows downward into the culture medium, surrounded by a thin layer of transparent granular area.
[0052] Embodiment 2:
[0053] The human oral mycoplasma type B staining test uses the following 6 steps:
[0054] 1. Prepare the staining solution. Mix crystal violet staining solution and safflower staining solution in a ratio of 1:1, and dilute 10 times with sterile distilled water for later use. The above two staining solutions are common Gram staining solutions, which are non-toxic and inexpensive.
[0055] 2. Activation of mycoplasma liquid culture medium: Absorb 100ul of the preserved human oral mycoplasma stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 3 to 4 days. Figure 1 As shown, it can be found that the culture medium tube is clear and turns red. Clarity indicates that there is no bacterial contamination in the culture medium, and the culture medium turns red because mycoplasma can use arginine in the culture medium to increase the pH value of the culture medium, and the culture medium turns red when it encounters the acid-base indicator phenol red.
[0056] 3. Secondary activation of mycoplasma solid culture medium: Take 100ul of the human oral mycoplasma culture fluid in step 1, spread it on the arginine mycoplasma solid culture medium plate, cover the edge of the plate with sealing film to create a microaerobic environment and prevent external microorganisms from entering and contaminating, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 6 to 10 days. Figure 2 As shown, pinpoint-sized colonies can be seen under light.
[0057] 4. Add the staining solution prepared in step 1 to the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, then rinse off the excess staining solution remaining on the plate with sterile water.
[0058] 5. Use a sterilized blade to cut a thin layer of agar from the plate, transfer it to a glass slide, and place the slide on a common electron microscope stage for observation. Figure 3 As shown, if there is sufficient light and the eyepiece has good light transmittance, the mycoplasma plate can be directly placed under the microscope for observation.
[0059] 6. If Figure 6 As shown, when observed under a microscope with a 16x eyepiece and a 16x objective lens, it can be found that the core of oral mycoplasma type B is thicker and grows downward into the culture medium, surrounded by a thin layer of transparent granular area.
[0060] Embodiment 3:
[0061] The staining test for Mycoplasma pneumoniae in humans uses the following 6 steps:
[0062] 1. Prepare the staining solution. Mix crystal violet staining solution and safflower staining solution in a ratio of 1:1, and dilute 10 times with sterile distilled water for later use. The above two staining solutions are common Gram staining solutions, which are non-toxic and inexpensive.
[0063] 2. Activation of mycoplasma liquid culture medium: Absorb 100ul of the stored human pneumoniae stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 3 to 4 days. Figure 1 As shown, it can be found that the culture medium tube is clear and turns red. Clarity indicates that there is no bacterial contamination in the culture medium, and the culture medium turns red because mycoplasma can use arginine in the culture medium to increase the pH value of the culture medium, and the culture medium turns red when it encounters the acid-base indicator phenol red.
[0064] 3. Secondary activation of mycoplasma solid culture medium: Take 100ul of the human pneumonia mycoplasma culture fluid in step 1, spread it on the arginine mycoplasma solid culture medium plate, cover the edge of the plate with sealing film to create a microaerobic environment and prevent external microorganisms from entering and contaminating, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 6 to 10 days. Figure 2 As shown, pinpoint-sized colonies can be seen under light.
[0065] 4. Add the staining solution prepared in step 1 to the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, then rinse off the excess staining solution remaining on the plate with sterile water.
[0066] 5. Use a sterilized blade to cut a thin layer of agar from the plate, transfer it to a glass slide, and place the slide on a common electron microscope stage for observation. Figure 3 As shown, if there is sufficient light and the eyepiece has good light transmittance, the mycoplasma plate can be directly placed under the microscope for observation.
[0067] 6. If Figure 7As shown, under the 16x eyepiece and 4x objective field of view of the microscope, it can be found that human pneumoniae Mycoplasma has a typical fried egg-like morphology.
[0068] Embodiment 4:
[0069] The staining test for Mycoplasma hyopneumoniae uses the following 6 steps:
[0070] 1. Prepare the staining solution. Mix crystal violet staining solution and safflower staining solution in a ratio of 1:1, and dilute 10 times with sterile distilled water for later use. The above two staining solutions are common Gram staining solutions, which are non-toxic and inexpensive.
[0071] 2. Activation of mycoplasma liquid culture medium: Take 100ul of the stored Mycoplasma hyopneumoniae stock solution and add it to the arginine mycoplasma broth culture medium. Wrap the tube mouth with sealing film and place it in a carbon dioxide incubator for cultivation. The carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 3 to 4 days. Figure 1 As shown, it can be found that the culture medium tube is clear and turns red. Clarity indicates that there is no bacterial contamination in the culture medium, and the culture medium turns red because mycoplasma can use arginine in the culture medium to increase the pH value of the culture medium, and the culture medium turns red when it encounters the acid-base indicator phenol red.
[0072] 3. Secondary activation of mycoplasma solid culture medium: Take 100ul of the culture medium of Mycoplasma hyopneumoniae in step 1, spread it on the solid culture medium plate of Mycoplasma arginine, cover the edge of the plate with sealing film to create a microaerobic environment and prevent external microorganisms from entering and contaminating, place it in a carbon dioxide incubator for cultivation, the carbon dioxide content is 5% to 10%, the culture temperature is 37℃, and the culture time is 6 to 10 days. Figure 2 As shown, pinpoint-sized colonies can be seen under light.
[0073] 4. Add the staining solution prepared in step 1 to the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, then rinse off the excess staining solution remaining on the plate with sterile water.
[0074] 5. Use a sterilized blade to cut a thin layer of agar from the plate, transfer it to a glass slide, and place the slide on a common electron microscope stage for observation. Figure 3 As shown, if there is sufficient light and the eyepiece has good light transmittance, the mycoplasma plate can be directly placed under the microscope for observation.
[0075] 6. If Figure 8 As shown, under the 16x eyepiece and 4x objective field of view of the microscope, it can be found that Mycoplasma hyopneumoniae has a typical poached egg-like morphology.
[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A simple and efficient mycoplasma staining detection method, characterized in that: The following steps are involved: S1, prepare staining solution: mix crystal violet staining solution and safflower staining solution, dilute with sterile distilled water and set aside; S2, activation of original liquid culture medium: aspirate the preserved oral mycoplasma stock solution, add it to the arginine mycoplasma broth culture medium, wrap the tube mouth with sealing film, and place it in a microaerobic environment for culture; S3, secondary activation of mycoplasma solid culture medium: the mycoplasma culture solution in step S2 is absorbed, and the solution is spread and inoculated on a Mycoplasma arginine solid culture medium plate, the tube opening is wrapped with a sealing film, and the plate is placed in a microaerobic environment for culture; S4, drop the staining solution prepared in step 1 onto the mycoplasma plate to be observed, leave it at room temperature for 10 minutes to wait for the staining solution to combine with the mycoplasma and dry naturally, and then rinse off the excess staining solution remaining on the plate with sterile water; S5, use a sterilized blade to cut a thin layer of agar on the plate, transfer it to a glass slide, and place the slide on an ordinary electron microscope stage for observation.
2. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: In step S1, the mixing ratio of the crystal violet staining solution and the safflower staining solution is 1:
1.
3. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: In step S1, the crystal violet staining solution and the safflower staining solution are mixed and then diluted 10 times with sterile distilled water.
4. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: The microaerobic environment in step S2 is provided by a carbon dioxide incubator with a carbon dioxide content of 5% to 10%, a culture temperature of 37° C., and a culture time of 3 to 4 days.
5. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: In step S3, the microaerobic environment is provided by a carbon dioxide incubator with a carbon dioxide content of 5% to 10%, a culture temperature of 37° C., and a culture time of 6 to 10 days.
6. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: In step S5, if there is sufficient light and the light transmittance of the eyepiece is good, the mycoplasma plate can be directly placed under a microscope for observation.
7. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: In step S5, the common electron microscope uses either or both of a 16x eyepiece and a 4x objective lens combination or a 16x eyepiece and a 16x objective lens combination.
8. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: The volume of the oral mycoplasma stock solution aspirated in step S2 is 100 ul.
9. A simple and efficient mycoplasma staining detection method according to claim 1, characterized in that: The volume of the mycoplasma culture solution absorbed in step S3 is 100 ul.