Combined strain cell positive control wax block and preparation method thereof

Abstract

The invention relates to the technical field of pathology, in particular to a combined strain cell positive control wax block and a preparation method thereof. The preparation method comprises the following steps: strain samples are placed in an inactivation stationary liquid for inactivation treatment to obtain an inactivated strain suspension, the strain samples comprise aspergillus, cryptococcus, talaromyces marneffei and mycobacterium tuberculosis, and the inactivation stationary liquid comprises ethanol and a formaldehyde solution; carrying out centrifugal treatment on the inactivated strain suspension to obtain a precipitate, dissolving the precipitate in an agar solution, and then carrying out solidification treatment to obtain an agar block wrapping the inactivated strain; and sequentially carrying out dehydration treatment and embedding treatment on the agar block to obtain the combined strain cell positive control wax block. According to the method, a large number of positive control wax blocks of various fungi and mycobacterium tuberculosis cells can be rapidly manufactured, the positive bacteria are more, the background is clear, interference is little, misjudgment is not easily caused, positive bacteria can be rapidly observed, and the method can be widely applied to positive control of various morphological experiments.

Description

Technical Field

[0001] This application belongs to the field of pathology technology, and in particular relates to a combined bacterial strain cell positive control paraffin block and its preparation method. Background Technology

[0002] In medical research and clinical diagnosis, the detection and identification of fungal and tuberculosis infections are crucial. Traditional methods for detecting fungi and tuberculosis, such as direct microscopy, culture, and molecular biology techniques, each have their advantages and disadvantages. Among these, pathological diagnostic techniques based on morphological examinations such as various staining methods, immunohistochemistry, and in situ hybridization are the most commonly used pathogen detection techniques. To ensure the accuracy and reliability of the test results, the use of positive controls is essential. When the tissue slide is negative, without internal and external controls, it is difficult to determine whether the tissue was inherently negative or the staining experiment failed. Therefore, establishing external positive controls is a very important quality control measure.

[0003] Based on morphology, fungi can be divided into three categories: yeasts, filamentous fungi, and dimorphic fungi. However, existing fungal positive controls are all for a single type of fungus, such as Aspergillus, resulting in a limited range of observable species and morphologies. In daily work, due to the scarcity of fungal or tuberculosis-positive specimens and the small size of tissues, it is difficult to preserve specimens or use archived paraffin blocks as positive controls, and high-quality positive control tissues are even harder to obtain. There is also a possibility of confusion with the tissue being tested, especially when slides are lent out for consultation, which could lead to misinterpretation. Furthermore, prolonged paraffin exposure can affect staining results. Therefore, in practice, it is difficult to establish a positive control for each pathogenic microorganism on every slide, and currently, few laboratories establish a positive control for every staining case. Therefore, it is essential to prepare high-quality paraffin blocks containing multiple pathogenic microorganisms as positive controls. Summary of the Invention

[0004] The purpose of this application is to provide a combined strain cell positive control wax block and its preparation method, aiming to solve the technical problem of how to produce a high-quality positive control wax block for multiple pathogenic microorganisms.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a method for preparing a combined bacterial strain cell positive control paraffin block, comprising: The strain samples were placed in an inactivation fixative to inactivate them, resulting in an inactivated strain suspension. The strain samples included Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis. The inactivation fixative included ethanol and formaldehyde solution. The inactivated strain suspension was centrifuged to obtain a precipitate, which was then dissolved in an agar solution and solidified to obtain an agar block encapsulating the inactivated strain. The agar blocks were sequentially dehydrated and embedded to obtain positive control wax blocks of the combined bacterial strain cells.

[0006] In some embodiments, the volume ratio of ethanol to formaldehyde solution is (25-35):(5-15). And / or, the formaldehyde solution contains 37-40% formaldehyde by mass; And / or, the inactivation treatment time is 18-24 hours.

[0007] In some embodiments, the centrifugation conditions include: a rotation speed of 1500-2500 r / min and a time of 5-15 min; And / or, the concentration of agar in the agar solution is 15-25 g / L.

[0008] In some embodiments, the dehydration process includes: treating the agar block sequentially with a neutral formalin solution, ethanol, and xylene, followed by a negative pressure wax impregnation process.

[0009] In some embodiments, the neutral formalin solution treatment includes: soaking in a 10% neutral formalin solution for 90-150 minutes at a temperature of 40-45°C. And / or, the ethanol treatment includes: first soaking in a 70-95% ethanol solution at a temperature of 35-37°C, and then soaking in anhydrous ethanol. And / or, the xylene treatment includes: soaking at a temperature of 35-37°C for 60-120 minutes; And / or, the negative pressure wax impregnation process includes: wax impregnation at a temperature of 60-65°C for 90-150 minutes.

[0010] In some embodiments, the embedding process includes: pouring molten paraffin into a mold, then placing the dehydrated agar block containing the inactivated bacterial strain into the mold, and allowing the paraffin to cool and solidify, thereby embedding the tissue in the paraffin block.

[0011] In some embodiments, inactivation verification is performed before centrifuging the inactivated strain suspension; And / or, the solidification process includes centrifugal solidification at 2-4°C.

[0012] In some embodiments, it also includes: Obtain the positive areas of the combined strain cell positive control wax block, the positive areas including Aspergillus positive areas, Cryptococcus positive areas, Basilella marneffei positive areas and Mycobacterium tuberculosis positive areas; The positive regions are placed into a chip wax mold for arrangement and embedding to produce a bacterial strain positive cell chip wax block.

[0013] In some embodiments, the method further includes: verifying the positive control paraffin block of the combined strain cells by hexamine silver staining, PAS staining, immunofluorescence staining, acid-fast staining, and in situ hybridization.

[0014] Secondly, this application provides a combined strain cell positive control wax block, which is prepared by the preparation method provided in the first aspect of this application.

[0015] The method for preparing combined strain cell positive control paraffin blocks provided in the first aspect of this application involves first inactivating four strain samples (i.e., Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis) with an inactivation fixative. Then, the precipitate obtained by centrifugation is dissolved in an agar solution and solidified to obtain an agar block encapsulating the inactivated strains. Finally, the agar block undergoes dehydration and embedding treatments sequentially to obtain the final product. This preparation method is simple and easy to operate, thus enabling the rapid production of large quantities of positive control paraffin blocks for various fungi and Mycobacterium tuberculosis cells. It not only produces a large number of positive bacteria but also provides clear staining with a clear background and minimal interference, while reducing contamination and minimizing misjudgment. This method is beneficial for rapid observation of positive bacteria and can be widely used as a positive control in various morphological experiments.

[0016] The combined strain cell positive control paraffin block provided in the second aspect of this application is prepared by a method unique to this application. This application verifies the effectiveness of the combined strain cell positive control paraffin block as a positive control, which can be widely used as a positive control in various morphological experiments (special staining, in situ hybridization, etc.), and has good application prospects in positive controls for morphological experiments of fungi and mycobacteria involved in various pathology departments, disease control departments, research institutes, and various medical or microbiological research. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a paraffin image of a combined bacterial strain cell positive control block provided in an embodiment of this application; wherein A is a schematic diagram of the structure of the positive control block, and B is an actual paraffin image of the positive control block; Figure 2 This is a hexamine silver staining image of Aspergillus cell blocks in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 3 This is a hexamine silver staining image of a *Basilella marneffei* cell block in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 4 This is a hexamine silver staining image of Cryptococcus cell blocks in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 5 This is a PAS staining image of Cryptococcus cell blocks in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 6 This is a fluorescent staining image of Cryptococcus cell blocks in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 7 This is an acid-fast staining image of a Mycobacterium tuberculosis cell block in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 8 This is an acid-fast fluorescence staining image of a Mycobacterium tuberculosis cell block in a positive control paraffin block of a combined strain of cells provided in this application embodiment; Figure 9 This is an in situ hybridization staining image of Aspergillus cell blocks in a positive control wax block of a combined strain of cells provided in this application embodiment. Detailed Implementation

[0019] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0020] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0022] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0025] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0026] Currently, archived paraffin blocks are commonly used as positive controls for fungi or acid-fast bacilli. However, positive control tissues are often small specimens, especially lung puncture specimens, with small tissue volume, usually very low bacterial count, and uneven distribution. They are already damaged after routine slide preparation. In addition, subsequent immunohistochemistry, special staining, molecular detection, etc. may be performed, and the integrity of the archived paraffin block tissue must be ensured. Such positive control tissues are difficult to meet the needs of daily work.

[0027] In addition, wax blocks made from a mixture of commercially available highly active yeast and egg white are used as novel positive controls for displaying specific staining of fungi. However, these only represent one fungal morphology, and using egg white as a cell matrix to create a positive control is complex, difficult to control, and prone to contamination.

[0028] Other studies have added samples of Aspergillus flavus strains, identified through culture, to 10% neutral buffered formalin, and then thoroughly mixed the fixed strain samples with pleural effusion sediment cells to prepare cell blocks. However, the introduction of pleural effusion sediment cells makes the procedure more complex, requiring the addition of body fluid samples from other patients; furthermore, the concentration and proportion of body fluid samples are difficult to control, making the method difficult to standardize; additionally, the inclusion of body fluid samples creates a more complex staining background, making positive strains less readily apparent than in this study; there is also a possibility of confusion with the detected tissues, potentially leading to misdiagnosis.

[0029] In summary, most current methods can only prepare positive paraffin blocks for a single strain (a single fungus or Mycobacterium tuberculosis), lacking a technique for preparing positive paraffin blocks for combined strains of different fungi and Mycobacterium tuberculosis, thus limiting their application. Due to the scarcity of fungal or Mycobacterium tuberculosis positive specimens and the small size of most tissues, it is difficult to preserve specimens or use archived paraffin blocks as positive controls. Furthermore, ordinary positive control photographs can be confused with the tissue being tested, especially when slides are lent out for consultation, potentially leading to misinterpretation. Additionally, the age of the paraffin blocks can affect the staining results. Therefore, in practice, it is difficult to establish a positive control for each pathogenic microorganism on every slide, and currently, few laboratories establish a positive control for each staining case. Thus, obtaining reliable, widely available, high-quality positive control paraffin blocks for pathogenic microorganisms is particularly important and urgent.

[0030] Based on this, this application embodiment develops a wax block that simultaneously enables multiple fungi and Mycobacterium tuberculosis to serve as positive controls for pathogens. The specific implementation scheme is as follows.

[0031] In a first aspect, embodiments of this application provide a method for preparing a combined bacterial strain cell positive control paraffin block, comprising: S01: The strain samples are placed in an inactivation fixative to inactivate them and obtain an inactivated strain suspension. The strain samples include Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis. The inactivation fixative includes ethanol and formaldehyde solution. S02: Centrifuge the inactivated strain suspension to obtain a precipitate, dissolve the precipitate in an agar solution, and then perform a coagulation treatment to obtain an agar block encapsulating the inactivated strain. S03: The agar blocks were sequentially dehydrated and embedded to obtain positive control wax blocks of the combined strain cells.

[0032] The preparation method described in this application is simple and easy to operate, thus it can quickly produce a large number of positive control wax blocks of various fungi and Mycobacterium tuberculosis cells. Not only are there many positive bacteria, but the staining effect is also obvious, resulting in a clear background and less interference. At the same time, it reduces contamination and is less likely to cause misjudgment, which is conducive to the rapid observation of positive bacteria. It can be widely used as a positive control for various morphological experiments.

[0033] In some embodiments, the samples in this application can be derived from strains of Aspergillus (filamentous fungus), Cryptococcus (yeast), Bassilago farfara (dimorphic fungus), and Mycobacterium tuberculosis identified by our unit's microbiology laboratory. All four strains are readily available. The sample source is guaranteed, and different strains can be selected according to needs. Strain sampling and strain inactivation treatment are performed, and inactivation verification experiments confirm the effectiveness of strain inactivation. The suspensions of the four inactivated strains are centrifuged separately, heated liquid agar is added, and the mixture is rapidly shaken and mixed. After centrifugation and sedimentation, once the agar has completely solidified, the agar block is carefully removed with tweezers, halved, and placed in an embedding cassette. After dehydration, it is then embedded to prepare strain cell blocks. The strain cell blocks are sectioned and stained. Positive results are verified using hexamine silver, PAS, fungal fluorescence staining, acid-fast staining, acid-fast fluorescence staining, and in situ hybridization.

[0034] Step S01 is the inactivation step.

[0035] Specifically, four bacterial strains—Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis—were placed in an inactivation fixative solution to obtain an inactivated bacterial suspension. The inactivation fixative solution included ethanol and formaldehyde solutions.

[0036] The four bacterial strains mentioned above were identified through culture as Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis. An inactivation fixative solution composed of ethanol and formaldehyde was effective in inactivating and fixing these four bacterial strains.

[0037] In some embodiments, the inactivation fixative includes ethanol and formaldehyde solution. Specifically, the volume ratio of ethanol to formaldehyde solution is (25-35):(5-15). Exemplarily, the volume ratio of ethanol to formaldehyde solution can be 25:5, 25:10, 25:15, 30:5, 30:10, 30:15, 35:5, 35:10, 35:15, etc. For example, ethanol is mixed with 5-15 ml of formaldehyde solution to form an inactivation fixative. Such an inactivation fixative not only improves the inactivation effect but also meets the requirements for specimen fixation, and can better preserve the morphology of pathogenic microorganisms. For example, mixing 30 ml of anhydrous ethanol with 10 ml of formaldehyde solution results in a 75% alcohol concentration, which can effectively inactivate Mycobacterium tuberculosis, killing the bacteria within 2 minutes. The diluted formaldehyde, close to the concentration of commonly used formalin, simultaneously inactivates fungi and Mycobacterium tuberculosis while also providing fixation, effectively preserving the morphology of the pathogenic microorganisms during inactivation. This allows for better inactivation and fixation of the four strains of bacteria mentioned above.

[0038] In some embodiments, the formaldehyde solution contains 37-40% formaldehyde by mass, meaning that formaldehyde dissolves in water to form an aqueous formaldehyde solution with the aforementioned mass percentage. For example, the formaldehyde mass percentage in the formaldehyde solution can be 37%, 38%, 39%, 40%, etc.

[0039] In some embodiments, the inactivation treatment time is 18-24 hours. Specifically, it can be 18 hours, 20 hours, 22 hours, 24 hours, etc. For example, in a biosafety cabinet, Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis are picked up with an inoculation loop and placed in a pre-prepared inactivation fixative solution for overnight inactivation treatment for 18-24 hours.

[0040] In some embodiments, before proceeding to subsequent steps, the inactivated bacterial suspension obtained from the inactivation treatment undergoes an inactivation verification experiment; that is, the inactivation verification is performed on the inactivated bacterial suspension before centrifugation. The inactivation effect of the inactivated bacterial suspension is verified, which ensures more effective inactivation of live bacteria and improves biosafety.

[0041] For example, the inactivation verification experiment included: inoculating the inactivated bacterial suspension onto a culture medium using an inoculation loop and culturing it again, observing the results for 14 days (fungi) and 45 days (Mycobacterium tuberculosis). The results showed no bacterial growth in the inactivated bacterial suspension, proving complete inactivation of the live bacteria. The inactivation verification experiment for Mycobacterium tuberculosis used Löwenstein slant agar, incubated at 35-77℃ for 40-42 days. The inactivation verification experiment for fungi used LS0409 Sabouraud agar plates, incubated at 26-28℃ for 12-14 days.

[0042] Step S02 involves obtaining an agar block containing inactivated bacterial strains.

[0043] In some embodiments, the inactivated bacterial strain suspension is centrifuged to obtain a precipitate, which is then dissolved in an agar solution for coagulation to obtain an agar block encapsulating the inactivated bacterial strain. The centrifugation conditions include a rotation speed of 1500-2500 r / min and a time of 5-15 min; these conditions allow for better precipitation of the four types of inactivated bacterial strains. For example, the centrifugation speed can be 1500 r / min, 1800 r / min, 2000 r / min, 2500 r / min, etc., and the centrifugation time can be 5 min, 8 min, 10 min, 12 min, 15 min, etc.

[0044] In some embodiments, the agar concentration in the agar solution is 15-25 g / L; specifically, it can be 15 g / L, 18 g / L, 20 g / L, 22 g / L, 25 g / L, etc. For example, weigh 1.5-2.5 g of agar powder, add 100 ml of distilled water, heat until the agar powder is completely dissolved, and prepare an agar solution of 1.5-2.5% (i.e., 1.5-2.5 g / 100 mL), which is then placed in a water bath at 60-65°C for later use. The agar solution of the above concentration not only disperses the precipitate well but also facilitates subsequent solidification into blocks.

[0045] In some embodiments, the precipitate obtained by centrifuging the inactivated strain suspension is dissolved in agar solution and then centrifuged at 2-4°C to solidify, resulting in an agar block encapsulating the inactivated strain. For example, the precipitate in the strain centrifuge tube is added to 1-2 ml of liquid agar solution, shaken to mix, and then centrifuged at 2-4°C, specifically at 1500-2500 r / min for 2-5 min.

[0046] S03: The agar blocks containing inactivated strains are sequentially dehydrated and embedded.

[0047] In some embodiments, the dehydration treatment includes: sequentially treating the agar block with a neutral formalin solution, ethanol, and xylene, followed by negative pressure paraffin impregnation. Under these conditions, water molecules can be more effectively removed from the agar block containing the inactivated bacterial strain.

[0048] Formalin solution is an aqueous solution containing 37%-40% formaldehyde by mass. Neutral formalin solution is formalin solution diluted with phosphate buffer to prevent oxidation and maintain a pH of around 7.0 for a long time.

[0049] In some embodiments, the neutral formalin solution treatment includes: soaking in a 10% neutral formalin solution [diluted 1:9] for 90-150 minutes at a temperature of 40-45°C. The temperature can be 40°C, 42°C, 45°C, etc., and the time can be 90 minutes, 100 minutes, 120 minutes, 140 minutes, 150 minutes, etc.

[0050] In some embodiments, the ethanol treatment includes: immersing the patient in a 70-95% (v / v) ethanol solution at a temperature of 35-37°C, followed by immersion in anhydrous ethanol. For example, the patient can first immerse in a 70% ethanol solution for 0.5-1.5 hours, then in an 80% ethanol solution for 0.5-1.5 hours, then in a 90% ethanol solution for 0.5-1 hours, then in a 95% ethanol solution for 0.5-1 hours, and finally in anhydrous ethanol for 1-2 hours.

[0051] In some embodiments, xylene treatment includes immersion at a temperature of 35-37°C for 60-120 minutes.

[0052] In some embodiments, the negative pressure wax impregnation process includes: negative pressure wax impregnation at a temperature of 60-65°C for 90-150 minutes. The wax used for impregnation is paraffin wax.

[0053] For example, in one embodiment, the dehydration process includes: soaking in 10% neutral formalin solution for 1 hour (45°C) — soaking in 10% neutral formalin solution for 1 hour (45°C) — soaking in 70% ethanol solution for 1 hour (37°C) — soaking in 80% ethanol solution for 1 hour (37°C) — soaking in 90% ethanol solution for 1 hour (37°C) — soaking in 95% ethanol solution for 50 minutes (37°C) — soaking in anhydrous ethanol for 50 minutes (37°C) — soaking in anhydrous ethanol for 50 minutes (37°C) — soaking in xylene for 25 minutes (37°C) — soaking in xylene for 35 minutes (37°C) — immersion in paraffin wax for 50 minutes (65°C negative pressure) — immersion in paraffin wax for 50 minutes (65°C negative pressure) — immersion in paraffin wax for 50 minutes (65°C negative pressure).

[0054] In some embodiments, the embedding process includes: pouring molten paraffin into a mold, then placing a dehydrated agar block containing inactivated strains into the mold, allowing the paraffin to cool and solidify, thus embedding the tissue in the paraffin block, and finally obtaining a positive control paraffin block of the combined strain cells.

[0055] In this application, the prepared combined strain cell positive control paraffin blocks were verified by hexamine silver staining, PAS staining, immunofluorescence staining, acid-fast staining, and in situ hybridization.

[0056] The hexamine silver staining procedure includes: sectioning positive control paraffin blocks of Aspergillus, Cryptococcus, and Bassilago farfara cells, dewaxing to water, and then performing hexamine silver staining according to the kit instructions. The PAS (Periodic Acid-Schiff stain) staining procedure includes: sectioning positive control paraffin blocks of Aspergillus, Cryptococcus, and Bassilago farfara cells, dewaxing to water, and then performing PAS staining according to the kit instructions. The fungal immunofluorescence staining procedure includes: sectioning positive control paraffin blocks of Aspergillus, Cryptococcus, and Bassilago farfara cells, dewaxing to water, and then performing fungal immunofluorescence staining according to the kit instructions. The acid-fast staining procedure includes: sectioning positive control paraffin blocks of Mycobacterium tuberculosis cells, dewaxing to water, and then performing acid-fast staining according to the kit instructions. The acid-fast immunofluorescence staining procedure includes: sectioning positive control paraffin blocks of Mycobacterium tuberculosis cells, dewaxing to water, and then performing acid-fast immunofluorescence staining according to the kit instructions. The in situ hybridization steps include: sectioning the positive control paraffin block of Aspergillus strain cells, dewaxing to water, and then performing in situ hybridization staining according to the kit instructions.

[0057] In some embodiments, the preparation method of this application further includes: obtaining positive regions (including Aspergillus-positive regions, Cryptococcus-positive regions, Basilaria marneffeis-positive regions, and Mycobacterium tuberculosis-positive regions) from the combined strain cell positive control wax block; arranging and embedding the positive regions in a chip wax mold to prepare a strain-positive cell chip wax block. Specifically, the positive regions of Aspergillus, Cryptococcus, Basilaria marneffeis-positive regions, and Mycobacterium tuberculosis strains in the combined strain cell positive control wax block are observed and marked under a microscope; the positive regions are extracted using a tissue microarray gun and injected into the chip wax mold, arranged in order; the chip wax mold is baked until slightly melted, and then embedded to prepare a strain-positive cell chip wax block. Further, the strain-positive cell chip wax block is verified: after slicing the strain-positive cell chip wax block, hexamine silver and acid-fast staining are performed again to observe the colony morphology.

[0058] In some embodiments, positive areas of cell blocks can be marked under a microscope, the positive areas can be extracted with a tissue microarray gun and injected into a chip mold, arranged in order, and the chip mold can be baked until slightly melted before embedding to produce a strain-positive cell chip block, thus creating a combined strain block. One small block can meet the positive control requirements of three types of fungi and Mycobacterium tuberculosis, and each positive control is relatively independent in the chip.

[0059] This application employs tissue microarray technology, placing three types of fungi and Mycobacterium tuberculosis within the same wax block, eliminating the need to distinguish between different wax blocks. Each type of fungus and Mycobacterium tuberculosis is represented as a separate dot on the microarray. Figure 1As shown, each positive control is not mixed together, making the pathogen morphology easier to identify and thus more efficient and convenient to use as a positive control. This application establishes a stable, easily collected, convenient, and widely applicable method for preparing combined strain cell positive control paraffin blocks (containing fungi and a type of Mycobacterium tuberculosis). Tissue microarray technology is used within the same paraffin block to simultaneously provide positive controls for three major fungal groups and Mycobacterium tuberculosis. Furthermore, the entire process avoids the introduction of difficult-to-master procedures such as egg white or other patient fluid samples, facilitating standardization. Subsequent inactivation experiments verify the inactivation effect, and various staining and in situ hybridization methods validate the effectiveness of the positive controls and clarify their application scope. Since microbiology laboratories in various institutions have sufficient strain samples, this method can rapidly produce large quantities of fungal and Mycobacterium tuberculosis positive control cell blocks, which can be widely used in morphological and molecular biology techniques.

[0060] Secondly, embodiments of this application provide a combined bacterial strain cell positive control paraffin block. Specifically, the combined bacterial strain cell positive control paraffin block of this application embodiment is prepared by the preparation method provided in the first aspect of this application embodiment.

[0061] The combined strain cell positive control paraffin blocks in this application embodiment were prepared using a unique preparation method specific to this application embodiment. This application embodiment verifies the effectiveness of the combined strain cell positive control paraffin blocks as positive controls. They can be widely used as positive controls in various morphological experiments (special staining, in situ hybridization, etc.) and have excellent application prospects in positive controls for morphological experiments involving fungi and mycobacteria in various pathology departments, disease control departments, research institutes, and various medical or microbiological research fields.

[0062] The embodiments of this application can simultaneously use the above three fungi and Mycobacterium tuberculosis as positive control wax blocks for pathogens. Moreover, the wax blocks are made of agar, which eliminates the need to add pleural effusion to precipitate cells. On the one hand, the operation is simpler, and on the other hand, there is no need to constantly adjust the ratio of pleural effusion to agar, making it easier to standardize.

[0063] In some embodiments, the combined strain cell positive control wax block of this application includes the strain positive cell chip wax block prepared above. The positive cell chip wax block control can have various combinations, and different tissue chip arrangements and different positive control combinations can be made according to needs to meet various requirements.

[0064] This application employs tissue-on-a-chip technology. Positive sites are precisely located and then perforated using a chip gun to create a bacterial microarray. This allows each positive control strain to be relatively independent within the chip, while simultaneously satisfying the needs of multiple positive controls within a single positive control chip block. Because tissue-on-a-chip technology requires less tissue and precisely locates each small point, the bacterial positive cell microarray block of this application is easier to identify various positive control bacteria, saves more tissue, and has a longer lifespan. This achieves a widely applicable, efficient, and economical method for producing positive controls for fungi and Mycobacterium tuberculosis.

[0065] The following description is based on specific embodiments.

[0066] Example 1 1. Materials and Methods.

[0067] 1.1 Sample source: Samples of strains identified as Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis by culture in our unit's microbiology laboratory.

[0068] 1.2 Sampling and inactivation of bacterial strains: In a biosafety cabinet, Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis were inoculated into a pre-prepared inactivation fixative using an inoculation loop and inactivated overnight for 18 hours to obtain an inactivated bacterial suspension. The inactivation fixative consisted of 30 ml of anhydrous ethanol and 10 ml of formaldehyde aqueous solution (the formaldehyde aqueous solution contained 40% formaldehyde by mass).

[0069] 1.3 Inactivation verification experiment: The inactivated strain suspension was inoculated into the culture medium with an inoculation loop and cultured again. The results were observed for 14 days (fungi) and 45 days (tuberculosis).

[0070] The tuberculosis bacillus was cultured on Löwenstein slant agar at 37°C for 42 days. The fungi were cultured on LS0409 Sabouraud agar plates at 28°C for 14 days. Results confirmed that both bacteria were inactivated.

[0071] 1.4 Method for preparing strain cell wax blocks.

[0072] 1.4.1 Place the inactivated strain suspension in a centrifuge tube and centrifuge at 2000 r / min for 10 min. Discard the supernatant and keep the bottom precipitate for later use.

[0073] 1.4.2 Weigh 1g of agar powder, add 50ml of distilled water, heat until the agar powder is completely dissolved, prepare an agar solution of 2g / 100mL, and place it in a 65℃ water bath for later use.

[0074] 1.4.3 Add 2 ml of the above liquid agar solution to the precipitate in the centrifuge tube and shake to mix.

[0075] 1.4.4 Centrifugation and precipitation: After mixing the sample, centrifuge at 4℃, 2000r / min, for 5min.

[0076] 1.4.5 After the agar has completely solidified, an agar block containing the inactivated strain is obtained; the agar block is carefully removed with tweezers, cut in half, placed in an embedding cassette, dehydrated, and then embedded to prepare a positive control wax block for the combined strain cells.

[0077] Dehydration procedure: Soak in 10% neutral formalin solution for 1 hour (45℃) — Soak in 10% neutral formalin solution for 1 hour (45℃) — Soak in 70% ethanol solution for 1 hour (37℃) — Soak in 80% ethanol solution for 1 hour (37℃) — Soak in 90% ethanol solution for 1 hour (37℃) — Soak in 95% ethanol solution for 50 minutes (37℃) — Soak in anhydrous ethanol for 50 minutes (37℃) — Soak in anhydrous ethanol for 50 minutes (37℃) — Soak in xylene for 25 minutes (37℃) — Soak in xylene for 35 minutes (37℃) — Wax immersion for 50 minutes (65℃ negative pressure) — Wax immersion for 50 minutes (65℃ negative pressure) — Wax immersion for 50 minutes (65℃ negative pressure).

[0078] Embedding: Prepare an embedding mold, pour molten paraffin into the mold, and then quickly place the dehydrated agar block containing the inactivated bacterial strain into the mold, adjusting the position and orientation of the tissue. After the paraffin cools and solidifies, the tissue is embedded in the paraffin block.

[0079] 1.5 Verification of the effect of combined strain cell positive control paraffin blocks.

[0080] 1.5.1 Special dyeing.

[0081] 1.5.1.1 Hexamine silver staining: Positive control paraffin blocks of Aspergillus, Cryptococcus, and Bassilago farfara cells were sectioned, dewaxed to water, and then stained with hexamine silver according to pathological procedures: oxidize with 8% chromic acid aqueous solution for 20 min, rinse briefly with tap water, treat with 5% sodium metabisulfite solution for 1 min, rinse with running water for 5 min, wash twice with distilled water, place in hexamine silver working solution in an incubator (58-60℃) for 60-90 min, until the sections turn yellowish-brown (under a microscope, molds appear dark brown), wash with distilled water. Add 0.1% gold chloride solution to adjust the color for 2-34 min, rinse briefly with running water. Treat with 2% sodium thiosulfate solution for 2-5 min, rinse with running water for 5 min. Counterstain with eosin for 1-2 seconds, wash quickly with water, dehydrate with 95% ethanol and anhydrous ethanol, clear with xylene, mount, and examine under a microscope. 1.5.1.2 PAS staining: The positive control paraffin blocks of Aspergillus, Cryptococcus and Bassilago farfara cells were sectioned, dewaxed to water, and then PAS staining was performed according to the kit (pathological glycogen staining solution: BA4080A, Zhuhai Beso Biotechnology Co., Ltd.).

[0082] 1.5.1.3 Fungal immunofluorescence staining: The positive control paraffin blocks of the combined strains of Aspergillus, Cryptococcus, and Bassilago farfara were sectioned, dewaxed to water, and then subjected to fungal immunofluorescence staining according to the kit (immunochromogenic reagent: GYL-CFW01, Jiangsu Gongyinglian Biotechnology Co., Ltd.).

[0083] 1.5.1.4 Acid-fast staining: The positive control paraffin block of the combined strain of Mycobacterium tuberculosis was sectioned, dewaxed to water, and then acid-fast stained according to the procedure of the kit (pathological acid-fast staining solution: BA4090B, Zhuhai Beso Biotechnology Co., Ltd.).

[0084] 1.5.1.5 Acid-fast immunofluorescence staining: The positive control paraffin block of the combined strain of Mycobacterium tuberculosis was sectioned, dewaxed to water, and then subjected to acid-fast immunofluorescence staining according to the procedure of the kit (acid-fast staining solution, Guangdong Medical Device Registration No. 20210528, Guangzhou Kehan ​​Biotechnology Co., Ltd.).

[0085] 1.5.2 In situ hybridization.

[0086] 1.5.2.1 Aspergillus in situ hybridization: The positive control paraffin block of the combined strain of Aspergillus cells was sectioned, dewaxed to water, and then in situ hybridization staining was performed according to the procedure of the kit (tissue section RNA in situ detection kit: JZKH0001, Guangzhou Kehan ​​Biotechnology Co., Ltd.).

[0087] 1.6. Preparation of positive wax blocks for bacterial cell microarray.

[0088] 1.6.1 Observe and mark the positive areas of Aspergillus, Cryptococcus, Basilaria marneffei and Mycobacterium tuberculosis strains in the positive control wax block of the combined strain cells under a microscope.

[0089] 1.6.2 Use a tissue microarray gun to extract positive areas and insert them into a microarray wax mold. Arrange them in order, bake the microarray wax mold until it is slightly melted, and then embed them to make a microarray wax block of positive cells of the bacterial strain.

[0090] 1.6.3 Verification of positive cell chip blocks: After slicing the cell chip blocks, the colony morphology was observed again after hexamine silver and acid-fast staining.

[0091] 2. Results.

[0092] 2.1 Inactivation verification experiment: The inactivated strain suspension was inoculated into the culture medium with an inoculation loop and cultured again. The results were observed for 14 days (fungi) and 45 days (tuberculosis). No strains grew in the inactivated strain suspension, which proved that the live bacteria were completely inactivated.

[0093] 2.2 Verification of the effectiveness of strain cell paraffin blocks as a positive control.

[0094] 2.2.1 Special dyeing.

[0095] 2.2.1.1 Hexamine silver staining: as shown Figure 2 As shown, after staining with hexamine silver, Aspergillus cell blocks revealed the formation of black or brownish-black hyphae of varying sizes. The hyphae were rod-shaped and extended radially or coral-like. Chains of conidia were continuously produced by the vesicles, clustering around the vesicles in a radial arrangement (resembling "Aspergillus heads"). Figure 3 As shown, *Basilaria marneffei* exhibits black, round, oval, or sausage-shaped mycelial bodies with visible septa and some hyphae formation. Figure 4 As shown, Cryptococcus cells stained with hexamine silver appear as black, roundish bacteria with thick-walled capsules.

[0096] 2.2.1.2 PAS staining: PAS staining of Aspergillus cell blocks showed that Aspergillus formed purplish-red hyphae of varying sizes, with the hyphae extending radially or in a coral-like pattern. Chains of conidia were continuously produced by the phialpiniophores, clustered around the vesicles in a radial arrangement (resembling "Aspergillus heads"). Cryptococcus cell blocks, after PAS staining, showed purplish-red, nearly circular mycelia with birefringence and thick-walled capsules. Figure 5 As shown, *Basilella marneffei* presents as purplish-red, round, oval, or sausage-shaped cells with visible septa and some hyphae formation. 2.2.1.3 Fungal Immunofluorescence Staining: After immunofluorescence staining of Aspergillus cell blocks, Aspergillus showed pale blue, refractive hyphae of varying sizes. The hyphae were rod-shaped and extended radially or coral-like. Chains of conidia were continuously produced by the vesicles, clustered around the vesicles in a radial arrangement (resembling "Aspergillus heads"). Cryptococcus cell blocks, after immunofluorescence staining, showed pale blue, refractive, roundish fungal bodies with thick-walled capsules. Figure 6 As shown, *Basilaria marneffei* consists of pale blue, refractive, round, oval, or sausage-shaped cells with visible septa and some hyphae formation. 2.2.1.4 Acid-fast staining: After acid-fast staining, Mycobacterium tuberculosis cell blocks show clusters and scattered distributions of short, purple-red stained bacilli, such as... Figure 7 As shown. 2.2.1.5 Acid-fast immunofluorescence staining: After acid-fast immunofluorescence staining, Mycobacterium tuberculosis cell blocks showed clusters and scattered distribution of short, red-stained bacilli, such as... Figure 8 As shown. 2.2.2 In situ hybridization.

[0097] In situ hybridization of Aspergillus strains: After in situ hybridization staining of Aspergillus cell blocks, positive staining signals are shown as red dots or patches. Hyphae distribution morphology is partially visible. The red signal is distinct, clearly located, and contrasts sharply with the background, making it very easy to identify. Figure 9 As shown.

[0098] 2.3 Positive control of combined strain cell wax blocks.

[0099] This application successfully prepared a combined strain cell block positive control, including three major categories of fungi (yeast, filamentous fungi, and dimorphic fungi) and Mycobacterium tuberculosis, which greatly expands the application scope compared with existing single strains. The effectiveness of this combined strain cell block positive control (fungi and mycobacteria) as a positive control (fungi and mycobacteria) was verified by using morphological staining (fungal and acid-fast bacillus staining) and molecular biology (in situ hybridization) methods. It can be widely used as a positive control for various morphological experiments (special staining, in situ hybridization techniques, etc.) and can be used in pathology departments, disease control departments, research institutes, and various medical or microbiological research involving morphological experiments on fungi and mycobacteria.

[0100] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a combined bacterial strain cell positive control paraffin block, characterized in that, include: The strain samples were placed in an inactivation fixative to inactivate them, resulting in an inactivated strain suspension. The strain samples included Aspergillus, Cryptococcus, Basilaria marneffei, and Mycobacterium tuberculosis. The inactivation fixative included ethanol and formaldehyde solution. The inactivated strain suspension was centrifuged to obtain a precipitate, which was then dissolved in an agar solution and solidified to obtain an agar block encapsulating the inactivated strain. The agar blocks were sequentially dehydrated and embedded to obtain positive control wax blocks of the combined bacterial strain cells.

2. The preparation method according to claim 1, characterized in that, The volume ratio of ethanol to formaldehyde solution is (25-35):(5-15). And / or, the formaldehyde solution contains 37-40% formaldehyde by mass; And / or, the inactivation treatment time is 18-24 hours.

3. The preparation method according to claim 1, characterized in that, The centrifugation conditions include: a rotation speed of 1500-2500 r / min and a time of 5-15 min; And / or, the concentration of agar in the agar solution is 15-25 g / L.

4. The preparation method according to claim 1, characterized in that, The dehydration process includes: treating the agar block sequentially with neutral formalin solution, ethanol, and xylene, followed by negative pressure wax impregnation.

5. The preparation method according to claim 4, characterized in that, The neutral formalin solution treatment includes: soaking in a 10% neutral formalin solution for 90-150 minutes at a temperature of 40-45℃. And / or, the ethanol treatment includes: first soaking in a 70-95% ethanol solution at a temperature of 35-37°C, and then soaking in anhydrous ethanol. And / or, the xylene treatment includes: soaking at a temperature of 35-37°C for 60-120 minutes; And / or, the negative pressure wax impregnation process includes: wax impregnation at a temperature of 60-65°C for 90-150 minutes.

6. The preparation method according to claim 1, characterized in that, The embedding process includes: pouring molten paraffin into a mold, then placing the dehydrated agar block containing the inactivated bacterial strain into the mold, and waiting for the paraffin to cool and solidify so that the tissue is embedded in the paraffin block.

7. The preparation method according to any one of claims 1-6, characterized in that, The inactivation of the strain suspension was verified before centrifugation. And / or, the solidification process includes centrifugal solidification at 2-4°C.

8. The preparation method according to any one of claims 1-6, characterized in that, Also includes: Obtain the positive areas of the combined strain cell positive control wax block, the positive areas including Aspergillus positive areas, Cryptococcus positive areas, Basilella marneffei positive areas and Mycobacterium tuberculosis positive areas; The positive regions are placed into a chip wax mold for arrangement and embedding to produce a bacterial positive cell chip wax block.

9. The preparation method according to any one of claims 1-6, characterized in that, Also includes: The positive control paraffin blocks of the combined strain cells were verified by hexamine silver staining, PAS staining, immunofluorescence staining, acid-fast staining, and in situ hybridization.

10. A combined bacterial strain cell positive control paraffin block, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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