Preparation method of hypha phase induction improved culture medium for malassezia

Abstract

The invention discloses a preparation method of a malassezia hypha phase induction improved culture medium, and relates to the technical field of malassezia culture, and the preparation method comprises the following steps: S1, raw material preparation; s2, treating a water-soluble component and a lipid component; s3, mixing a plurality of components; s4, adjusting the pH value; s5, fixing the volume and sub-packaging; and S6, sterilization and quality detection. According to the method, the raw materials can be accurately weighed through raw material preparation, preliminary preparation of additional additives can be achieved through treatment and mixing of water-soluble components and lipid components, the quality of the culture medium can be guaranteed through pH value adjustment, sterilization and quality detection, the culture medium solution can meet the conversion requirement of malassezia, and the conversion efficiency of malassezia is improved. Therefore, the difficulty of converting the malassezia from the yeast phase to the hypha phase in the in-vitro environment is reduced, the conversion efficiency is ensured, the position switching of the supporting disc and the hopper can be realized through the grooved wheel mechanism and the connecting disc, different solutions are dropwise added according to different pH values of the mixed solution, and the pH adjustment of the mixed solution is completed.

Description

Technical Field

[0001] The invention relates to the technical field of Malassezia culture, and in particular to a method for preparing an improved culture medium for inducing the hyphae phase of Malassezia. Background Art

[0002] Malassezia is a major component of the normal flora of human skin. It is also an opportunistic pathogen and is often considered to be associated with a variety of skin diseases such as pityriasis versicolor, seborrheic dermatitis and Malassezia folliculitis.

[0003] In the prior art, the addition of additional lipids to the culture medium can achieve the isolation and cultivation of Malassezia, but it is difficult for Malassezia to transform from the yeast phase to the mycelium phase in an in vitro environment, and the transformation efficiency is relatively low. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, so as to solve the problem proposed in the above background technology that adding additional lipids to the culture medium can achieve the isolation and cultivation of Malassezia, but it is difficult for Malassezia to transform from the yeast phase to the mycelial phase in an in vitro environment and the transformation efficiency is relatively low.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, comprising the following contents: S1. Raw material preparation: prepare modified mDixon agar, accurately weigh 3-4 parts of 50 mM glycine, 1-1.5 parts of 10.0 mM MgSO4, 3-5 parts of 29.4 mM KH2PO4, 0.1-0.3 parts of 1.0 mM levodopa, 0.8-1.2 parts of kojic acid, 6-10 parts of 10 ml corn oil, 0.1-0.3 parts of yeast extract and 0.5-1.5 parts of squalene; S2. Treatment of water-soluble components and lipid components: put the water-soluble components into a conical flask filled with distilled water, place the conical flask on a magnetic stirrer for stirring, so that the water-soluble components are dissolved, put the lipid components into another container, use a magnetic stirrer or a homogenizer to fully stir and mix, so that the lipid and emulsifier are evenly dispersed to form a stable emulsion; S3. Mixing multiple ingredients: slowly pour the emulsified lipid component into the solution of dissolved water-soluble components, and continue stirring to ensure that the two are fully mixed. Use a pipette to accurately measure a certain amount of induction factor solution, add it to the above mixed solution, and continue stirring for 15-20 minutes to ensure that all ingredients are evenly distributed; S4, pH value adjustment: use a pH meter to measure the pH value of the mixed solution. If the pH value is not within the predetermined range, adjust the pH value of the current mixed solution, and monitor the pH value change in real time during the adjustment until the pH value is within the predetermined range; S5. Volume adjustment and subpackaging: Transfer the pH-adjusted culture medium solution to a volumetric flask, dilute to the required volume with distilled water, and use a sterile pipette or measuring cylinder to dispense the culture medium into sterile culture dishes or conical flasks on a sterile operating table; S6. Sterilization and quality inspection: Place the packaged culture medium into the autoclave. After sterilization is completed, wait for the autoclave to cool naturally and reduce the pressure to a safe range, then take out the culture medium, randomly select some of the sterilized culture medium, place it in a 37°C constant temperature incubator and culture it for 24-48 hours to observe whether there are any bacteria growing, and re-test the content and pH value of the culture medium.

[0006] Preferably, in step S1, glycine, MgSO4, KH2PO4, levodopa, kojic acid, corn oil, yeast extract and squalene are grouped according to water-soluble components and lipid components, respectively, wherein corn oil and squalene are lipid components, and the rest are water-soluble components.

[0007] Preferably, in step S1, when the raw materials are weighed, they are weighed sequentially by an electronic balance, and a pipette is used to transfer the corn oil and squalene during the weighing.

[0008] Preferably, in step S2, when the water-soluble component is stirred by a magnetic stirrer, a heating jacket is installed on the outside of the conical flask to slowly heat the conical flask at a temperature of 50-60° C., and when the lipid component is emulsified, an emulsifier Tween 80 is added to the container containing the lipid component.

[0009] Preferably, in step S3, when adjusting the pH value, the following steps are also included: S31, equipment calibration and testing: calibrate the pH meter, measure the pH value of the current mixed solution after calibration, and obtain the pH value of the current mixed solution; S32, pH value comparison and adjustment: compare the pH value in S31 with a predetermined range, select different solutions for adjustment according to the pH value, slowly add hydrochloric acid solution when the pH value is high, and add sodium hydroxide solution when the pH value is low, and stir the mixed solution with a magnetic stirrer during the adjustment process; S33, pH value monitoring and updating: Use a pH meter to monitor the pH value of the mixed solution during the adjustment process, and transmit the monitoring results to an external controller to achieve real-time updating of the pH value.

[0010] Preferably, in step S6, when observing whether there are any foreign bacteria growing, the colony morphology, changes in culture medium color and changes in culture medium texture are observed with the naked eye. When the colony morphology and culture medium are abnormal, smear microscopy and mycelium morphology are observed under a microscope to obtain the type of foreign bacteria, morphological characteristics and location of occurrence. When detecting the component content of the culture medium, the contents of organic components, lipid components and inorganic components in the culture medium are detected respectively, and the test results are compared with the standard data. The qualification of the culture medium is judged based on the comparison results and in combination with the pH value.

[0011] Preferably, it also includes a magnetic agitator body, one side of the magnetic agitator body is fixedly connected to a base, the top of the base is fixedly connected to a support frame, the top of the support frame is equipped with a groove wheel mechanism, the bottom of the rotating disk of the groove wheel mechanism is rotatably connected to the support frame, the top of the groove wheel of the groove wheel mechanism is fixedly connected to a connecting disk, the outer ring surface of the connecting disk is fixedly connected to a support disk, the bottom of the support disk is overlapped with the support frame, a feeding pipe is inserted through the inside of the support disk, and the top of the feeding pipe is fixedly connected to a hopper.

[0012] Preferably, a motor 1 is fixedly connected to the top of the base, a bevel gear 1 is fixedly connected to one end of the output shaft of the motor 1, a bevel gear 2 is meshed with one side of the bevel gear 1, the top of the bevel gear 2 is fixedly connected to the rotating disk of the grooved wheel mechanism, and the bottom of the bevel gear 2 is rotatably connected to the base.

[0013] Preferably, a fixed block is fixedly connected to one side of the magnetic stirrer body, an electric push rod is fixedly connected to the top of the fixed block, a placement rack is fixedly connected to the top of the electric push rod, and a pH meter body is slidably connected to one side of the placement rack.

[0014] Preferably, the pH meter body is electrically connected to a probe, and the probe is inserted into the placement frame. An electric push rod 2 is fixedly connected to one side of the pH meter body, and one end of the electric push rod 2 is fixedly connected to an L-shaped frame, and a motor 2 is fixedly connected to the bottom of the L-shaped frame. One end of the output shaft of the motor 2 is fixedly connected to a flat gear 2, and the bottom of the flat gear 2 is rotatably connected to the L-shaped frame. A flat gear 1 is meshed with one side of the flat gear 2, and the bottom of the flat gear 1 is rotatably connected to the L-shaped frame, and a cleaning strip is fixedly connected to the top of the flat gear 1.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, the raw material preparation can realize accurate weighing of various raw materials, the water-soluble components and lipid components are processed and mixed to realize preliminary preparation of additional additives, and the pH value is adjusted and sterilized and the quality is tested to ensure the quality of the culture medium, so that the culture medium solution meets the conversion requirements of Malassezia, thereby reducing the difficulty of Malassezia conversion from yeast phase to mycelium phase in an in vitro environment and ensuring the conversion efficiency; 2. In the present invention, the top of the electric push rod is fixedly connected to the placement frame, the probe and the pH meter body are electrically connected, the electric push rod drives the placement frame and the probe to move downward, so that the bottom of the probe is inserted into the liquid surface, and the pH value of the mixed solution is detected. The position switching of the support plate and the hopper can be achieved through the groove wheel mechanism and the connecting plate, so that the solutions contained in different hoppers are added according to the different pH values ​​of the mixed solution to complete the pH adjustment of the mixed solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention is a flow chart of a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia; Figure 2 A schematic diagram of the installation of the pH meter body structure of a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia according to the present invention; Figure 3 It is a schematic diagram of the connection between the base and the groove wheel mechanism of a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia according to the present invention; Figure 4 for Figure 2 A local enlarged structural schematic diagram; Figure 5 A schematic diagram of the installation of a motor structure of a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia according to the present invention; Figure 6 This is a schematic diagram of the meshing structure of bevel gear 1 and bevel gear 2 in a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia according to the present invention.

[0017] Legend: 1. pH meter body; 2. Placement rack; 3. Probe; 4. Base; 5. Magnetic stirrer body; 6. Electric push rod 1; 7. Support plate; 8. Groove mechanism; 9. Hopper; 10. Support rack; 11. Motor 1; 12. Bevel gear 1; 13. Feed pipe; 14. Connecting plate; 15. Bevel gear 2; 16. Fixed block; 17. Electric push rod 2; 18. L-shaped rack; 19. Cleaning strip; 20. Flat gear 1; 21. Flat gear 2; 22. Motor 2. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1: Reference Figure 1 As shown: This embodiment provides a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, comprising the following contents: Step 1, raw material preparation: prepare modified mDixon agar, accurately weigh 3 parts of 50 mM glycine, 1 part of 10.0 mM MgSO4, 3 parts of 29.4 mM KH2PO4, 0.1 parts of 1.0 mM L-dopa, 0.8 parts of kojic acid, 6 parts of 10 ml corn oil, 0.1 parts of yeast extract and 0.5 parts of squalene; Glycine, MgSO4, KH2PO4, L-dopa, kojic acid, corn oil, yeast extract and squalene were grouped according to water-soluble components and lipid components, respectively. Among them, corn oil and squalene were lipid components, and the rest were water-soluble components. When weighing the raw materials, they were weighed in sequence using an electronic balance, and a pipette was used to transfer the liquid of corn oil and squalene during weighing.

[0020] Step 2, treatment of water-soluble components and lipid components: put the water-soluble components into a conical flask filled with distilled water, place the conical flask on a magnetic stirrer for stirring to dissolve the water-soluble components, put the lipid components into another container, use a magnetic stirrer or a homogenizer to fully stir and mix, so that the lipid and emulsifier are evenly dispersed to form a stable emulsion. When the magnetic stirrer is stirring the water-soluble components, install a heating jacket on the outside of the conical flask, and slowly heat the conical flask at a heating temperature of 55°C. When emulsifying the lipid components, add emulsifier Tween 80 to the container containing the lipid components.

[0021] Step 3: Mixing multiple ingredients: slowly pour the emulsified lipid component into the solution of dissolved water-soluble components, and continue stirring to ensure that the two are fully mixed. Use a pipette to accurately measure a certain amount of induction factor solution and add it to the above mixed solution. Continue stirring for 20 minutes to ensure that all ingredients are evenly distributed. When adjusting the pH value, the following steps are also included: 31. Equipment calibration and testing: Calibrate the pH meter, measure the pH value of the current mixed solution after calibration, and obtain the pH value of the current mixed solution; 32. Comparison and adjustment of pH value: Compare the pH value in 31 with the predetermined range, and select different solutions for adjustment according to the pH value. When the pH value is high, slowly add hydrochloric acid solution, and when the pH value is low, add sodium hydroxide solution for adjustment. During the adjustment process, use a magnetic stirrer to stir the mixed solution; 33. pH value monitoring and updating: Use a pH meter to monitor the pH value of the mixed solution during the adjustment process, and transmit the monitoring results to an external controller to achieve real-time updating of the pH value.

[0022] Step 4: pH adjustment: Use a pH meter to measure the pH of the mixed solution. If the pH is not within the predetermined range, adjust the pH of the current mixed solution. During the adjustment, monitor the pH change in real time until the pH is within the predetermined range.

[0023] Step 5. Make up the volume and dispense: Transfer the pH-adjusted culture medium solution to a volumetric flask, make up the volume to the required volume with distilled water, and dispense the culture medium into sterile culture dishes or Erlenmeyer flasks using a sterile pipette or measuring cylinder on a sterile operating table.

[0024] Step 6, sterilization and quality inspection: put the packaged culture medium into the autoclave. After the sterilization is completed, wait for the autoclave to cool down naturally and reduce the pressure to a safe range, then take out the culture medium, randomly extract part of the sterilized culture medium, and place it in a 37°C constant temperature incubator for 48 hours to observe whether there are any bacteria growing, and re-test the composition content and pH value of the culture medium. When observing whether there are any bacteria growing, observe the colony morphology, the color change of the culture medium and the texture change of the culture medium with the naked eye. When the colony morphology and culture medium are abnormal, use a microscope to perform smear microscopy and observe the hyphae morphology to obtain the type, morphological characteristics and location of the bacteria. When performing smear microscopy, pick a small amount of suspicious growth from the culture medium to make a smear. After staining with Gram staining or other staining methods, observe the cell morphology, size, arrangement and staining characteristics under a microscope. Gram-positive bacteria are purple after Gram staining, and Gram-negative bacteria are red. The type of bacteria can be preliminarily determined by observation; When testing the component content of the culture medium, the contents of organic components, lipid components and inorganic components in the culture medium are tested respectively, and the test results are compared with the standard data. The qualification of the culture medium is judged based on the comparison results and in combination with the pH value. If both the component content and the pH value meet the standard, the culture medium is qualified; if one or more of the component content or the pH value do not meet the standard, it is judged as unqualified, and the unqualified culture medium is marked and recovered, and the recovered culture medium is processed. When the component content and pH value of the culture medium deviate from the standard by a small amount, it can be corrected by adjusting its component content and pH value so that the treated culture medium meets the standard. When the component content and pH value of the culture medium deviate seriously from the standard and it is difficult to meet the qualified standard through adjustment, the current culture medium is discarded and re-prepared.

[0025] In this example, 3.75 g of 50 mM glycine, 1.203 g of 10.0 mM MgSO4, 4.0 g of 29.4 mM KH2PO4, 0.2 g of 1.0 mM L-dopa, 1 g of kojic acid, 108 g of corn oil, 0.2 g of yeast extract and 1 ml of squalene were added to the modified mDixon agar and mixed to form a culture medium. The presence of glycine and yeast extract provided a variety of essential amino acids for the growth of Malassezia hyphae. Kojic acid and L-dopa also stimulated the transformation of Malassezia to the hyphae phase in vitro. Mg 2+ Isosalt ions and squalene can be utilized by Malassezia, and additional additives can provide favorable conditions for Malassezia to transform into the mycelial phase, ensuring the effect and efficiency of the transformation of Malassezia to the mycelial phase.

[0026] Example 2: Reference Figure 1 As shown: This embodiment provides a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, comprising the following contents: Step 1, raw material preparation: prepare modified mDixon agar, accurately weigh 3 parts of 50 mM glycine, 1 part of 10.0 mM MgSO4, 3 parts of 29.4 mM KH2PO4, 0.1 parts of 1.0 mM L-dopa, 0.8 parts of kojic acid, 6 parts of 10 ml corn oil, 0.1 parts of yeast extract and 0.5 parts of squalene; Glycine, MgSO4, KH2PO4, L-dopa, kojic acid, corn oil, yeast extract and squalene were grouped according to water-soluble components and lipid components, respectively. Among them, corn oil and squalene were lipid components, and the rest were water-soluble components. When weighing the raw materials, they were weighed in sequence using an electronic balance, and a pipette was used to transfer the liquid of corn oil and squalene during weighing.

[0027] Step 2, treatment of water-soluble components and lipid components: put the water-soluble components into a conical flask filled with distilled water, place the conical flask on a magnetic stirrer for stirring to dissolve the water-soluble components, put the lipid components into another container, use a magnetic stirrer or a homogenizer to fully stir and mix, so that the lipid and emulsifier are evenly dispersed to form a stable emulsion. When the magnetic stirrer is stirring the water-soluble components, install a heating jacket on the outside of the conical flask, and slowly heat the conical flask at a heating temperature of 50°C. When emulsifying the lipid components, add emulsifier Tween 80 to the container containing the lipid components.

[0028] Step 3: Mixing multiple ingredients: slowly pour the emulsified lipid component into the solution of dissolved water-soluble components, and continue stirring to ensure that the two are fully mixed. Use a pipette to accurately measure a certain amount of induction factor solution and add it to the above mixed solution. Continue stirring for 15 minutes to ensure that all ingredients are evenly distributed. When adjusting the pH value, the following steps are also included: 31. Equipment calibration and testing: Calibrate the pH meter, measure the pH value of the current mixed solution after calibration, and obtain the pH value of the current mixed solution; 32. Comparison and adjustment of pH value: Compare the pH value in 31 with the predetermined range, and select different solutions for adjustment according to the pH value. When the pH value is high, slowly add hydrochloric acid solution, and when the pH value is low, add sodium hydroxide solution for adjustment. During the adjustment process, use a magnetic stirrer to stir the mixed solution; 33. pH value monitoring and updating: Use a pH meter to monitor the pH value of the mixed solution during the adjustment process, and transmit the monitoring results to an external controller to achieve real-time updating of the pH value.

[0029] Step 4: pH adjustment: Use a pH meter to measure the pH of the mixed solution. If the pH is not within the predetermined range, adjust the pH of the current mixed solution. During the adjustment, monitor the pH change in real time until the pH is within the predetermined range.

[0030] Step 5. Make up the volume and dispense: Transfer the pH-adjusted culture medium solution to a volumetric flask, make up the volume to the required volume with distilled water, and dispense the culture medium into sterile culture dishes or Erlenmeyer flasks using a sterile pipette or measuring cylinder on a sterile operating table.

[0031] Step 6. Sterilization and quality inspection: Place the packaged culture medium into the autoclave. After sterilization is completed, wait for the autoclave to cool naturally and reduce the pressure to a safe range, then take out the culture medium, randomly select some of the sterilized culture medium, place it in a 37°C constant temperature incubator for 24 hours, observe whether there is any bacteria growing, and re-test the content and pH value of the culture medium. When testing the content of the culture medium, test the content of organic components, lipid components and inorganic components in the culture medium respectively, compare the test results with the standard data, and judge whether the culture medium is qualified based on the comparison results and the pH value.

[0032] In this embodiment, the heating temperature of the water-soluble components, the stirring time of the mixture of multiple components, and the culturing time of the sterilized culture medium in the constant temperature incubator are adjusted, and the conversion efficiency of Malassezia from the yeast phase to the mycelium phase is lower than that in Example 1.

[0033] Example 3: Reference Figure 2-Figure 6 As shown: This embodiment provides a method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, which also includes a magnetic stirrer body 5, a base 4 is fixedly connected to one side of the magnetic stirrer body 5, a support frame 10 is fixedly connected to the top of the base 4, a groove wheel mechanism 8 is installed on the top of the support frame 10, the bottom of the rotating disk of the groove wheel mechanism 8 is rotatably connected to the support frame 10, a connecting disk 14 is fixedly connected to the top of the groove wheel of the groove wheel mechanism 8, a support disk 7 is fixedly connected to the outer ring surface of the connecting disk 14, the bottom of the support disk 7 overlaps the support frame 10, a feed pipe 13 is inserted through the inside of the support disk 7, and a hopper 9 is fixedly connected to the top of the feed pipe 13, a motor 11 is fixedly connected to the top of the base 4, a bevel gear 12 is fixedly connected to one end of the output shaft of the motor 11, a bevel gear 15 is meshed with one side of the bevel gear 12, and the top of the bevel gear 15 is fixedly connected to the rotating disk of the groove wheel mechanism 8, The bottom of bevel gear 2 15 is rotatably connected to the base 4, one side of the magnetic stirrer body 5 is fixedly connected to a fixed block 16, the top of the fixed block 16 is fixedly connected to an electric push rod 1 6, the top of the electric push rod 1 6 is fixedly connected to a placement rack 2, one side of the placement rack 2 is slidably connected to a pH meter body 1, the pH meter body 1 is electrically connected to a probe 3, the probe 3 is inserted through the placement rack 2, one side of the pH meter body 1 is fixedly connected to an electric push rod 2 17, one end of the electric push rod 2 17 is fixedly connected to an L-shaped frame 18, the bottom of the L-shaped frame 18 is fixedly connected to a motor 22, one end of the output shaft of the motor 22 is fixedly connected to a flat gear 21, the bottom of the flat gear 21 is rotatably connected to the L-shaped frame 18, one side of the flat gear 21 is meshed with a flat gear 1 20, the bottom of the flat gear 20 is rotatably connected to the L-shaped frame 18, and the top of the flat gear 20 is fixedly connected to a cleaning strip 19.

[0034] In this embodiment, the magnetic stirrer body 5 can be used to support and reinforce one side of the base 4, and the base 4 can be used to support the top of the support frame 10 to ensure the safety of the support frame 10 when in use. The motor 11 can provide power for the rotation of the bevel gear 12. Through the meshing of the bevel gear 12 and the discharge pipe 13, the discharge pipe 13 is driven to rotate when the bevel gear 12 rotates, thereby realizing the operation of the groove wheel mechanism 8, driving the support plate 7 and the connecting plate 14 to rotate intermittently, and changing the position of the hopper 9 through the rotation of the support plate 7 and the connecting plate 14, so that different solutions contained in the two hoppers 9 can be discharged into the container through the discharge pipe 13. The magnetic stirrer body 5 can accelerate the mixing between the mixed liquid in the container and the dripping solution, which is convenient for the uniformity of pH value detection and the speed of pH adjustment. Rod 16 can adjust the use height of the placement rack 2 and the probe 3, so that the probe 3 can be inserted into the liquid surface to detect the pH value of the mixed solution. After the solution is detected, the probe 3 is moved up by the electric push rod 16. At this time, the placement rack 2 and the probe 3 are moved up synchronously, so that the placement rack 2 slides along one side of the pH meter body 1, and the fixed block 16 stably supports the bottom of the pH meter body 1. The pH meter body 1 and the electric push rod 2 17 remain stationary, and then the electric push rod 2 17 is used to drive the L-shaped rack 18 to move, so that one side of the flat gear 20 overlaps with the probe 3, and then the motor 22 is used to drive the flat gear 21 to rotate, and the flat gear 21 drives the flat gear 20 to rotate. The flat gear 20 and the cleaning strip 19 rotate synchronously, and the bottom and outer ring surface of the probe 3 are wiped by the cleaning strip 19 to achieve surface cleaning of the probe 3.

[0035] In the present invention, firstly, a variety of raw materials are accurately weighed, the raw materials are divided into water-soluble components and lipid components and processed separately, the processed various components are mixed and stirred, and after stirring evenly, the pH value of the mixed solution is detected and adjusted, and after adjustment, the prepared culture medium solution is transferred to a volumetric flask for volume determination and packaging, and the packaged culture medium is sterilized and quality tested to complete the overall preparation of the culture medium, wherein when the pH value of the mixed solution is adjusted, the mixed solution is poured into a container above the magnetic stirrer body 5, the magnetic stirrer body 5 is used to stir the mixed solution, the use height of the placement frame 2 and the probe 3 is adjusted by the electric push rod 6, the bottom of the probe 3 is inserted into the liquid surface, the pH value of the current mixed solution is detected, and the detection result It is displayed on the display screen of the pH meter body 1 that the type of solution to be added to the mixed liquid is selected according to the pH value. The two hoppers 9 contain hydrochloric acid and sodium hydroxide solutions respectively. The motor 11 drives the bevel gear 12 to rotate. The bevel gear 12 drives the bevel gear 2 15 and the rotating disk of the groove wheel mechanism 8 to rotate. The rotating disk drives the groove wheel to rotate intermittently. When the groove wheel rotates, the connecting disk 14 and the supporting disk 7 rotate synchronously. The positions of the two hoppers 9 can be changed to move the corresponding hopper 9 to the top of the container. The solenoid valve on the discharge pipe 13 is opened, and the corresponding solution can be dripped into the container. The magnetic stirrer body 5 is used to stir while dripping until the pH value of the mixed liquid is within the predetermined range, thereby completing the pH adjustment of the mixed liquid.

[0036] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an improved culture medium for inducing the mycelial phase of Malassezia, characterized in that: Includes the following: S1. Raw material preparation: prepare modified mDixon agar, accurately weigh 3-4 parts of 50 mM glycine, 1-1.5 parts of 10.0 mM MgSO4, 3-5 parts of 29.4 mM KH2PO4, 0.1-0.3 parts of 1.0 mM levodopa, 0.8-1.2 parts of kojic acid, 6-10 parts of 10 ml corn oil, 0.1-0.3 parts of yeast extract and 0.5-1.5 parts of squalene; S2. Treatment of water-soluble components and lipid components: put the water-soluble components into a conical flask filled with distilled water, place the conical flask on a magnetic stirrer for stirring, so that the water-soluble components are dissolved, put the lipid components into another container, use a magnetic stirrer or a homogenizer to fully stir and mix, so that the lipid and emulsifier are evenly dispersed to form a stable emulsion; S3. Mixing multiple ingredients: slowly pour the emulsified lipid component into the solution of dissolved water-soluble components, and continue stirring to ensure that the two are fully mixed. Use a pipette to accurately measure a certain amount of induction factor solution, add it to the above mixed solution, and continue stirring for 15-20 minutes to ensure that all ingredients are evenly distributed; S4, pH value adjustment: use a pH meter to measure the pH value of the mixed solution. If the pH value is not within the predetermined range, adjust the pH value of the current mixed solution, and monitor the pH value change in real time during the adjustment until the pH value is within the predetermined range; S5. Volume adjustment and subpackaging: Transfer the pH-adjusted culture medium solution to a volumetric flask, dilute to the required volume with distilled water, and use a sterile pipette or measuring cylinder to dispense the culture medium into sterile culture dishes or conical flasks on a sterile operating table; S6. Sterilization and quality inspection: Place the packaged culture medium into the autoclave. After sterilization is completed, wait for the autoclave to cool naturally and reduce the pressure to a safe range, then take out the culture medium, randomly select some of the sterilized culture medium, place it in a 37°C constant temperature incubator and culture it for 24-48 hours to observe whether there are any bacteria growing, and re-test the content and pH value of the culture medium.

2. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 1, characterized in that: In step S1, glycine, MgSO4, KH2PO4, L-dopa, kojic acid, corn oil, yeast extract and squalene are grouped according to water-soluble components and lipid components, respectively, wherein corn oil and squalene are lipid components, and the rest are water-soluble components.

3. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 1, characterized in that: In step S1, when the raw materials are weighed, they are weighed sequentially by an electronic balance, and a pipette is used to transfer the liquid of corn oil and squalene during the weighing.

4. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 1, characterized in that: In step S2, when the water-soluble component is stirred by a magnetic stirrer, a heating jacket is installed on the outside of the conical flask to slowly heat the conical flask at a temperature of 50-60° C. When the lipid component is emulsified, an emulsifier Tween 80 is added to the container containing the lipid component.

5. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 4, characterized in that: In step S3, when adjusting the pH value, the following steps are also included: S31, equipment calibration and testing: calibrate the pH meter, measure the pH value of the current mixed solution after calibration, and obtain the pH value of the current mixed solution; S32, pH value comparison and adjustment: compare the pH value in S31 with a predetermined range, select different solutions for adjustment according to the pH value, slowly add hydrochloric acid solution when the pH value is high, and add sodium hydroxide solution when the pH value is low, and stir the mixed solution with a magnetic stirrer during the adjustment process; S33, pH value monitoring and updating: Use a pH meter to monitor the pH value of the mixed solution during the adjustment process, and transmit the monitoring results to an external controller to achieve real-time updating of the pH value.

6. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 1, characterized in that: In step S6, when observing whether there are any foreign bacteria growing, the colony morphology, changes in culture medium color and changes in culture medium texture are observed with the naked eye. When the colony morphology and culture medium are abnormal, smear microscopy and mycelium morphology are observed under a microscope to obtain the type of foreign bacteria, morphological characteristics and location of occurrence. When detecting the component content of the culture medium, the content of organic components, lipid components and inorganic components in the culture medium is detected respectively, and the test results are compared with the standard data. The qualification of the culture medium is judged based on the comparison results and the pH value.

7. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 1, characterized in that: The invention also comprises a magnetic stirrer body (5), wherein one side of the magnetic stirrer body (5) is fixedly connected to a base (4), the top of the base (4) is fixedly connected to a support frame (10), the top of the support frame (10) is provided with a grooved wheel mechanism (8), the bottom of the rotating disk of the grooved wheel mechanism (8) is rotatably connected to the support frame (10), the top of the grooved wheel of the grooved wheel mechanism (8) is fixedly connected to a connecting disk (14), the outer annular surface of the connecting disk (14) is fixedly connected to a supporting disk (7), the bottom of the supporting disk (7) is overlapped with the supporting frame (10), a feeding pipe (13) is inserted through the inside of the supporting disk (7), and the top of the feeding pipe (13) is fixedly connected to a hopper (9).

8. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 7, characterized in that: The top of the base (4) is fixedly connected to a motor 1 (11); one end of the output shaft of the motor 1 (11) is fixedly connected to a bevel gear 1 (12); one side of the bevel gear 1 (12) is meshed with a bevel gear 2 (15); the top of the bevel gear 2 (15) is fixedly connected to a rotating disk of the grooved wheel mechanism (8); and the bottom of the bevel gear 2 (15) is rotationally connected to the base (4).

9. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 7, characterized in that: A fixed block (16) is fixedly connected to one side of the magnetic stirrer body (5); an electric push rod (6) is fixedly connected to the top of the fixed block (16); a placement rack (2) is fixedly connected to the top of the electric push rod (6); and a pH meter body (1) is slidably connected to one side of the placement rack (2).

10. The method for preparing a Malassezia mycelial phase induction improved culture medium according to claim 9, characterized in that: The pH meter body (1) is electrically connected to a probe (3), the probe (3) being inserted through the placement frame (2), one side of the pH meter body (1) being fixedly connected to an electric push rod (17), one end of the electric push rod (17) being fixedly connected to an L-shaped frame (18), the bottom of the L-shaped frame (18) being fixedly connected to a motor (22), one end of the output shaft of the motor (22) being fixedly connected to a flat gear (21), the bottom of the flat gear (21) being rotatably connected to the L-shaped frame (18), one side of the flat gear (21) being meshed with a flat gear (20), the bottom of the flat gear (20) being rotatably connected to the L-shaped frame (18), and the top of the flat gear (20) being fixedly connected to a cleaning strip (19).

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