A method for preparing pathological sections of experimental animals affected by plague

Abstract

This invention provides a method for preparing pathological sections of plague-affected experimental animals. The method includes the following steps: S1: sample pretreatment and fixation; S2: pathogen inactivation verification; S3: tissue dehydration, clearing, and paraffin embedding; S4: paraffin embedding; S5: sectioning and spreading; S6: staining; S7: mounting. The method for preparing pathological sections of plague-affected experimental animals provided by this invention has the advantages of ensuring absolute safety in the preparation process of high-risk pathogen sections through strict biosafety zoning and double inactivation verification, and obtaining high-quality pathological sections.

Description

Technical Field

[0001] This invention relates to the field of pathological experimental technology, and in particular to a method for preparing pathological sections of plague experimental animals. Background Technology

[0002] Plague is a highly contagious and deadly infectious disease caused by Yersinia pestis, a pathogen characterized by high pathogenicity, high infectivity, and high risk. Pathological studies of laboratory animals infected with plague are crucial for understanding its pathogenic mechanisms and pathological changes, and the preparation of pathological sections is a core component of this process.

[0003] However, due to the high-risk characteristics of Yersinia pestis, ensuring the safety of operators and preventing pathogen leakage into the environment during pathological slide preparation remains a challenge that current conventional pathological slide techniques cannot address. Conventional techniques typically focus only on slide quality and lack systematic biosafety control measures. Direct operation in a standard laboratory environment can easily lead to laboratory infections and pathogen spread, causing serious biosafety incidents.

[0004] Therefore, it is necessary to provide a new method for preparing pathological sections of plague experimental animals to solve the above-mentioned technical problems. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a method for preparing pathological sections of plague experimental animals that ensures absolute safety in the process of preparing high-risk pathogen sections and obtains high-quality pathological sections through strict biosafety zoning operations and double inactivation verification.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for preparing pathological sections of plague experimental animals, including: S1: Sample pretreatment and fixation: In the biosafety cabinet in the core area of ​​the ABSL-3 laboratory, experimental animals infected with Yersinia pestis are dissected, and tissues are taken and trimmed into tissue blocks with a thickness of less than 1 cm and a length of less than 2 cm; the tissue blocks are placed in a sterilization fixation box and fixed and sterilized with neutral formaldehyde solution for 10-15 days;

[0007] S2: Pathogen inactivation verification: Take out the tissue block after fixation and sterilization in step S1, and perform Yersinia pestis growth test and Yersinia pestis bacteriophage test until the results of two consecutive tests on the same tissue block are negative.

[0008] S3: Tissue dehydration, clearing and paraffin infusion: In the BSL-2 laboratory, tissue blocks that have been verified as negative in step S2 are washed and then subjected to gradient ethanol dehydration, xylene clearing and paraffin infusion in sequence using a tissue dehydrator.

[0009] S4: Paraffin embedding: The tissue block after being impregnated with paraffin is embedded in paraffin to form a paraffin block;

[0010] S5: Slicing and spreading: Slice the wax block into wax strips, spread them flat on a constant temperature water surface of 40~45℃, pick them up and attach them to a glass slide, and bake and dry them.

[0011] S6: Staining: After dewaxing and hydration of the dried sections, hematoxylin-eosin (H&E) staining is performed;

[0012] S7: Mounting: Apply mounting medium to the stained slide and cover with a coverslip.

[0013] Preferably, the sterilization fixation box in S1 includes a box body and a box lid. The box body is provided with multiple independent specimen receiving cavities formed by longitudinal and transverse partitions. The inner wall of each receiving cavity is provided with an elastic buffer pad. The box body is also provided with a guide channel for distributing and covering the specimen with sterilization liquid.

[0014] Preferably, the time for the dehydration, clearing, and wax impregnation steps in S3 is adjusted according to the type of experimental animal, including mice, rats, and guinea pigs.

[0015] Preferably, when the experimental animal is a mouse, step S3 specifically includes:

[0016] Soak in 60% ethanol for 15 min, 70% ethanol for 15 min, 80% ethanol for 20 min, 90% ethanol for 20 min, 95% ethanol I for 20 min, 95% ethanol II for 20 min, anhydrous ethanol I for 20 min, and anhydrous ethanol II for 20 min in sequence.

[0017] Then, it was cleared in xylene I for 5 minutes and in xylene II for 3 minutes.

[0018] Finally, the sample was immersed in paraffin I for 10 minutes, in paraffin II for 20 minutes, and in paraffin III for 30 minutes.

[0019] Preferably, in step S5, the specific steps of baking and drying are as follows: first, dry-bake at 75°C for 10 to 30 minutes, and then bake at 65°C for 0.5 to 1 hour.

[0020] Preferably, the H&E staining step in S6 includes:

[0021] S61: Dewaxing is performed using xylene I and xylene II;

[0022] S62: Wash away xylene with anhydrous ethanol;

[0023] S63: sequentially hydrated with 95%, 90%, and 85% ethanol gradients;

[0024] S64: After rinsing with tap water, perform hematoxylin staining;

[0025] S65: After rinsing with tap water, use 1% hydrochloric acid alcohol to differentiate, and then use dilute ammonia water to turn blue.

[0026] S66: After washing with water, perform eosin staining;

[0027] S67: After rinsing with tap water, it is sequentially dehydrated by passing through 85%, 90%, 95%, 95% ethanol and anhydrous ethanol I and anhydrous ethanol II.

[0028] S68: Use xylene I, xylene II, or xylene III for transparency.

[0029] Compared with related technologies, the method for preparing pathological sections of plague experimental animals provided by the present invention has the following beneficial effects:

[0030] This invention provides a method for preparing pathological sections from experimental animals affected by plague. By organically combining stringent biosafety laboratory requirements, precisely controlled tissue block inactivation procedures, and rigorous pathogen validation processes, it systematically constructs for the first time a safe system for preparing pathological sections targeting highly pathogenic pathogens such as plague. This method not only fundamentally eliminates the risk of pathogen leakage through the principles of "separation of personnel and contaminated areas" and zoned operations, ensuring the absolute safety of operators, but also provides double assurance for the complete inactivation of pathogens by controlling tissue block size, extending the fixation time, and combining it with two consecutive microbiological validations. Furthermore, optimized slide preparation and staining processes for different experimental animals ensure that the final pathological sections possess excellent morphological quality and staining effects, meeting the needs of precise pathological observation. Detailed Implementation

[0031] Example:

[0032] This embodiment uses mice infected with Yersinia pestis as an example to illustrate the specific implementation of the present invention.

[0033] I. Sample pretreatment and fixation (conducted in ABSL-3 laboratory)

[0034] Sampling: In a Class II A2 biosafety cabinet, euthanized mice were fixed onto a corrosion-resistant dissection board. Under aseptic conditions, the mice were rapidly dissected, and tissues from organs with obvious lesions, such as the liver, spleen, and lungs, were harvested in sequence.

[0035] Trimming: Trim the tissue into small pieces about 0.8 cm thick and 1.5 cm long inside the biosafety cabinet, and rinse them with saline solution.

[0036] Fixation: Place the tissue blocks into the individual specimen chambers of a dedicated tissue sterilization fixation box and close the lid. Pour in sufficient 10% neutral formaldehyde fixative (containing a small amount of glacial acetic acid), distributing the fixative evenly into each chamber through the distribution channel to completely submerge the tissue blocks. Elastic cushioning pads prevent tissue damage during handling. Place the entire fixation box in a biosafety cabinet for fixation and sterilization for 10-15 days.

[0037] II. Pathogen inactivation validation (conducted in ABSL-3 laboratory)

[0038] Ten to fifteen days later, a liver tissue block was removed from the biosafety cabinet, rinsed with saline, and the surface was dried with sterile filter paper.

[0039] Cut the center of the tissue block with a sterile scalpel blade and gently press the cut surface onto a bacterial culture plate.

[0040] This plate was used for Yersinia pestis growth culture and Yersinia pestis phage experiments.

[0041] The result was negative after 72 hours. The experiment was repeated once for different parts of the same tissue block.

[0042] After a second 72-hour incubation, the result remained negative. This confirmed that the Yersinia pestis bacteria within the tissue block had been completely inactivated. Other tissue blocks were tested using the same method.

[0043] The dedicated tissue sterilization and fixation box containing the completely inactivated tissue block was then surface-sterilized and transferred from the ABSL-3 laboratory to the BSL-2 laboratory according to the operating procedures.

[0044] III. Tissue dehydration, clearing, and paraffin infiltration (performed in BSL-2 laboratory)

[0045] The sterility-negative tissue blocks were transferred to the BSL-2 laboratory and placed in a closed tissue dehydrator for automated processing according to the procedure set in the table below (for mice):

[0046] Due to significant individual differences among experimental animals, the required dehydration time varies. See Table 1.

[0047] Table 1: Dehydration, clearing, and paraffin-impregnation times of different experimental animal tissues

[0048] step Operation Project mice rats, guinea pigs 1 60% ethanol 15min 20min 2 70% ethanol 15min 20min 3 80% ethanol 20min 30min 4 90% ethanol 20min 30min 5 95% Ethanol I 20min 30min 6 95% Ethanol II 20min 20min 7 Anhydrous ethanol I 20min 30min 8 Anhydrous ethanol II 20min 30min 9 Xylene I 5min 8min 10 Xylene II 3min 5min 11 Paraffin I 10min 15min 12 Paraffin II 20min 25min 13 Paraffin III 30min 30min

[0049] IV. Paraffin embedding (performed in a standard laboratory)

[0050] Using a paraffin embedding machine, the liver tissue block impregnated with paraffin is placed face down in an embedding frame, molten paraffin is poured in, and it is placed on a cooling table to solidify into a wax block.

[0051] V. Slices and Displays

[0052] After trimming the wax block, it is cut into continuous wax strips 4 micrometers thick using a paraffin slicer.

[0053] Use a brush to lift the wax strip and lay it flat on the water surface of a 42℃ constant temperature sheet spreading machine, allowing it to spread naturally.

[0054] Use a glass slide to lift the flattened wax sheet and pour off any excess water.

[0055] Place the slide on a 75°C slide oven and bake for 15 minutes, then transfer it to a 65°C oven and bake for 45 minutes.

[0056] VI. H&E staining

[0057] Manually stain the dried slices according to the following steps (an automatic staining machine can also be used):

[0058] Xylene I 10 min → Xylene II 5 min (dewaxing)

[0059] Anhydrous ethanol I for 1 min → Anhydrous ethanol II for 1 min (to remove xylene)

[0060] 95% ethanol for 1 min → 90% ethanol for 1 min → 85% ethanol for 1 min → rinse with tap water for 2 min (hydration)

[0061] Stain with hematoxylin for 4 minutes → Rinse with tap water for 1 minute (nuclear staining)

[0062] 1% hydrochloric acid alcohol for 20 seconds → rinse with tap water for 1 minute (differentiation)

[0063] 1% ammonia solution for 30 seconds → rinse with tap water for 1 minute (reverse blue color)

[0064] Stain with eosin solution for 3 minutes → rinse with tap water for 30 seconds (cytoplasmic staining)

[0065] 85% ethanol 20s → 90% ethanol 30s → 95% ethanol I 1min → 95% ethanol II 1min → Anhydrous ethanol I 2min → Anhydrous ethanol II 2min (dehydration)

[0066] Xylene I 2 min → Xylene II 2 min → Xylene III 2 min (clear)

[0067] VII. Sealing

[0068] Remove the tissue section from the xylene solution, wipe away any excess xylene around the edges with filter paper, place a drop of neutral resin in the center of the tissue section, and gently cover with a coverslip, being careful not to create air bubbles. Once the mounting medium has solidified, the tissue can be observed under a microscope.

[0069] Observation results

[0070] The prepared liver tissue pathological sections, when observed under an optical microscope, showed typical plague pathological changes such as disordered hepatocyte cord structure, extensive degeneration and necrosis of hepatocytes, and extensive inflammatory cell infiltration in the portal areas. The sections were structurally intact, with clear contrast between cell nuclei and cytoplasm, and good staining results, meeting the requirements for pathological diagnosis.

[0071] Compared with related technologies, the method for preparing pathological sections of plague experimental animals provided by the present invention has the following beneficial effects:

[0072] This invention provides a method for preparing pathological sections from laboratory animals affected by plague. By organically combining stringent biosafety laboratory requirements, precisely controlled tissue block inactivation procedures, and rigorous pathogen validation processes, it systematically constructs for the first time a safe system for preparing pathological sections targeting highly pathogenic pathogens such as Yersinia pestis. This method not only fundamentally eliminates the risk of pathogen leakage through the principles of "separation of personnel and contaminated areas" and zoned operations, ensuring the absolute safety of operators, but also provides double assurance for the complete inactivation of pathogens by controlling tissue block size, extending the fixation time, and combining it with two consecutive microbiological validations. Furthermore, optimized slide preparation and staining processes for different laboratory animals ensure that the final pathological sections possess excellent morphological quality and staining effects, meeting the needs of precise pathological observation.

[0073] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing pathological sections of plague experimental animals, characterized in that, Includes the following steps: S1: Sample pretreatment and fixation: In the biosafety cabinet in the core area of ​​the ABSL-3 laboratory, the experimental animals that died specifically from Yersinia pestis were dissected, and the tissues were taken and trimmed into tissue blocks with a thickness of less than 1 cm and a length of less than 2 cm; the tissue blocks were placed in a sterilization fixation box and fixed and sterilized with neutral formaldehyde solution for 10-15 days. S2: Pathogen inactivation verification: Take out the tissue block after fixation and sterilization in step S1, and perform Yersinia pestis growth test and Yersinia pestis bacteriophage test until the results of two consecutive tests on the same tissue block are negative. S3: Tissue dehydration, clearing and paraffin infusion: In the BSL-2 laboratory, tissue blocks that have been verified as negative in step S2 are washed and then subjected to gradient ethanol dehydration, xylene clearing and paraffin infusion in sequence using a tissue dehydrator. S4: Paraffin embedding: The tissue block after being impregnated with paraffin is embedded in paraffin to form a paraffin block; S5: Slicing and spreading: Slice the wax block into wax strips, spread them flat on a constant temperature water surface of 40~45℃, pick them up and attach them to a glass slide, and bake and dry them. S6: Staining: After dewaxing and hydration of the dried sections, hematoxylin-eosin (H&E) staining is performed; S7: Mounting: Apply mounting medium to the stained slide and cover with a coverslip.

2. The method for preparing pathological sections of plague experimental animals according to claim 1, characterized in that, The sterilization fixation box in S1 includes a box body and a box lid. The box body has multiple independent specimen receiving cavities formed by longitudinal and transverse partitions. The inner wall of each receiving cavity is provided with an elastic buffer pad. The box body also has a guide channel for distributing and covering the specimen with sterilization liquid.

3. The method for preparing pathological sections of plague experimental animals according to claim 1, characterized in that, The time for the dehydration, clearing, and wax impregnation steps in S3 is adjusted according to the type of experimental animal, including mice, rats, and guinea pigs.

4. The method for preparing pathological sections of plague experimental animals according to claim 3, characterized in that, When the experimental animal is a mouse, step S3 specifically involves: Soak in 60% ethanol for 15 min, 70% ethanol for 15 min, 80% ethanol for 20 min, 90% ethanol for 20 min, 95% ethanol I for 20 min, 95% ethanol II for 20 min, anhydrous ethanol I for 20 min, and anhydrous ethanol II for 20 min in sequence. Then, it was cleared in xylene I for 5 minutes and in xylene II for 3 minutes. Finally, the sample was immersed in paraffin I for 10 minutes, in paraffin II for 20 minutes, and in paraffin III for 30 minutes.

5. The method for preparing pathological sections of plague experimental animals according to claim 1, characterized in that, In step S5, the specific steps of baking and drying are as follows: first, dry bake at 75°C for 10 to 30 minutes, and then bake at 65°C for 0.5 to 1 hour.

6. The method for preparing pathological sections of plague experimental animals according to claim 1, characterized in that, The H&E staining step in S6 includes: S61: Dewaxing is performed using xylene I and xylene II; S62: Wash away xylene with anhydrous ethanol; S63: sequentially hydrated with 95%, 90%, and 85% ethanol gradients; S64: After rinsing with tap water, perform hematoxylin staining; S65: After rinsing with tap water, use 1% hydrochloric acid alcohol to differentiate, and then use dilute ammonia water to turn blue. S66: After washing with water, perform eosin staining; S67: After rinsing with tap water, it is sequentially dehydrated by passing through 85%, 90%, 95%, 95% ethanol and anhydrous ethanol I and anhydrous ethanol II. S68: Use xylene I, xylene II, or xylene III for transparency.