A kind of acid-fast staining solution based on AIE fluorescent probe and its application

Abstract

The present invention discloses an acid-fast staining solution based on an AIE fluorescent probe and an application thereof; the acid-fast staining solution of the present invention comprises an AIE staining solution and an acid decolorizing solution; the AIE staining solution comprises a MASPB molecule and water, and the structural formula of the MASPB molecule is as follows: #imgabs0#. The acid-fast staining solution of the present invention is used for the detection of mycobacteria, and has high accuracy, strong anti-interference and short time.

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule biological diagnosis, and in particular relates to an acid-fast staining solution based on an AIE fluorescent probe and an application thereof. Background Art

[0002] Mycobacterium tuberculosis (M. tuberculosis), commonly known as tuberculosis bacillus, is the pathogen that causes tuberculosis.

[0003] At present, the identification of Mycobacterium tuberculosis mainly includes smear microscopy, tuberculosis immunology, tuberculosis culture, molecular biology and other means. Among them, smear microscopy is the most commonly used basic method, which has the advantages of simple operation, rapid detection, and low requirements for hardware facilities. It is suitable for grassroots implementation. It mainly includes two common methods, Benzene-Nicotiana aurantium acid-fast staining and auramine O fluorescence staining, which use the characteristics of mycobacteria binding to dyes and not being easily decolorized by acidic decolorizing solutions to achieve the staining effect. Among them, the auramine O method is compared with the Benzene-Nicotiana aurantium acid-fast staining method. Due to its fluorescent characteristics, the staining is obvious, the color is bright, and it is easy to observe and identify tuberculosis bacteria. It can be observed through a low-power microscope, which is conducive to improving the efficiency of microscopic screening. However, its staining time is long, and after decolorization with acidic decolorizing solutions, it needs to be restained (using the strong oxidizing property of potassium permanganate to remove background staining and improve contrast). The staining process is usually completed within 15-20 minutes, and auramine O has poor photostability, and usually needs to be operated in the dark. The steps are cumbersome and the time cost is high. Therefore, developing a fast, simple and accurate fluorescent acid-fast staining solution is of great significance to improve identification efficiency. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an acid-fast staining solution based on AIE (Aggregation-induced emission) fluorescent probe and its application in mycobacterium identification. The present invention achieves the purpose of mycobacterium identification through rapid staining of AIE fluorescent molecules and selective decolorization of non-mycobacterium components by decolorizing solution, aiming to overcome the shortcomings of the traditional auramine O method, such as long staining time, cumbersome steps, poor photostability and low accuracy, and provide more options in the field of fluorescent acid-fast staining.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] The present invention provides an acid-fast staining solution based on an AIE fluorescent probe, comprising an AIE staining solution and an acid decolorizing solution; the AIE staining solution comprises a MASPB molecule and water, and the structural formula of the MASPB molecule is as follows:

[0007] .

[0008] Preferably, the concentration of MASPB molecules in the AIE staining solution is 50-70 μM.

[0009] Preferably, the water is ultrapure water.

[0010] Preferably, the acidic decolorizing solution comprises hydrochloric acid and ethanol.

[0011] Further preferably, the volume ratio of hydrochloric acid in the acidic decolorizing solution is 3-6%; the mass concentration of the hydrochloric acid is 30-40%.

[0012] The present invention provides an application of the acid-fast staining solution based on AIE fluorescent probe in detecting mycobacteria.

[0013] The present invention provides a method for detecting mycobacteria, comprising the following steps:

[0014] (1) Spread the sample to be tested on a glass slide and fix it to form a coating;

[0015] (2) Add AIE dye solution until the coating is covered, dye at 50-70 °C for 2-4 min, and rinse;

[0016] (3) Add acidic decolorizing solution until the coating is covered, let stand to decolorize, rinse, and cover with a coverslip;

[0017] (4) Observe the smear through the B channel of the fluorescence microscope; the excitation wavelength of the B channel is 460-490 nm, and the emission wavelength is 510-550 nm;

[0018] Wherein, the AIE staining solution and the acid decolorizing solution are the AIE staining solution and the acid decolorizing solution in the above-mentioned acid-fast staining solution based on the AIE fluorescent probe.

[0019] Preferably, the standing bleaching time in step (3) is 2-3 min.

[0020] Preferably, the fixation in step (1) is performed by heating at 50-70°C.

[0021] Preferably, the dyeing in step (2) is performed by heating the dyeing on a heating plate.

[0022] Preferably, the rinsing in steps (2) and (3) is rinsing with distilled water;

[0023] Preferably, the magnification of the fluorescence microscope in step (4) is 400-1000 times;

[0024] Preferably, in step (4), whether the sample to be tested contains mycobacteria is determined by observing whether it has bright fluorescence.

[0025] Preferably, the mycobacterium is Mycobacterium tuberculosis.

[0026] In the process of detecting mycobacteria, the dye will combine with a variety of other non-mycobacterial components, and therefore will be interfered by other components (such as some cocci, bacilli, and human tissue cells), resulting in false positives. In order to accurately detect mycobacteria and reduce false positives, the selection of dyes is crucial. The dye needs to be stably combined with mycobacteria (to prevent decolorization and reduce accuracy), and cannot be too stably combined with other components (to prevent decolorization and cause false positives). Finally, a low background color is required for easy observation. Although the prior art has a large number of dyes, most of them cannot be used to detect mycobacteria. The present invention has found through experiments that MASPB with aggregation-induced emission characteristics can be applied to acid-fast staining of mycobacteria. An acid-fast staining solution based on the AIE fluorescent probe MASPB is designed, which can quickly combine with mycobacteria. After being treated with an acid decolorizing solution, it can effectively eliminate the color interference of non-mycobacterial components, and can still stably combine with mycobacteria and emit bright fluorescence, and the background is clean and non-fluorescent, and no re-staining operation is required, and the detection time is short and the accuracy is high.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] (1) The acid-fast staining solution of the present invention can complete acid-fast staining in only two steps. Compared with the traditional auramine O acid-fast staining, it effectively simplifies the staining steps and greatly shortens the staining time; and the detection rate of mycobacteria is higher.

[0029] (2) The AIE staining solution used in the present invention is an aqueous solution of AIE molecules, which can eliminate the risk of phenol poisoning in the Zygen-Nicotiana benthamiana staining method and the Auramine O fluorescence staining method, and is safer.

[0030] (3) The present invention uses MASPB to prepare the acid-fast staining solution. Due to its aggregation-induced emission characteristics and good water solubility, the fluorescence after staining is obvious, the photostability is good, and the background fluorescence is low. Compared with the auramine O method, it can effectively avoid photobleaching caused during microscopy.

[0031] (4) The method for detecting mycobacteria of the present invention uses an acid-fast staining solution based on MASPB. By staining at a temperature of 50-70°C, the detection rate of the acid-fast staining solution of the present invention is significantly improved, and the detection rate is higher than that of the auramine O staining method. The staining temperature condition is relatively mild and can be achieved by using a heating plate for fixing smear samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of the steps of AIE acid-fast staining and auramine O acid-fast staining;

[0033] Figure 2This is the fluorescence image of the AIE acid-fast staining of mixed bacteria in Example 1 (the local image is magnified 4 times);

[0034] Figure 3 This is a fluorescence image of the AIE acid-fast staining of Example 2 for a sputum sample (containing Mycobacterium gordonii) (left: fluorescence field, middle: bright field, right: overlay field);

[0035] Figure 4 This is the detection diagram of Mycobacterium gordonii by AIE acid-fast staining in Example 3 (partial diagram magnified 10 times);

[0036] Figure 5 Continuously photographed fluorescence images of Mycobacterium gordonii obtained by AIE acid-fast staining in Example 4;

[0037] Figure 6 This is a fluorescence image of mixed bacteria stained by AIE acid-fast staining without a decolorization step in Comparative Example 1 (partial image magnified 4 times);

[0038] Figure 7 This is the detection diagram of Mycobacterium gordonii by AIE acid-fast staining at room temperature in Comparative Example 2 (partial diagram magnified 10 times);

[0039] Figure 8 This is the fluorescence image of the AIE acid-fast staining of mixed bacteria using DENPB as the AIE molecule in Comparative Example 3 (the local image is magnified 4 times);

[0040] Fig. 9 This is the detection diagram of Mycobacterium gordonii by auramine O acid-fast staining of Comparative Example 4 (partial diagram magnified 10 times);

[0041] Fig.10 This is a continuous fluorescence image of Mycobacterium gordonii obtained by auramine O acid-fast staining of Comparative Example 5. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with examples, but the implementation manner and protection scope of the present invention are not limited to the following examples.

[0043] Comparison of the steps of AIE acid-fast staining and auramine O acid-fast staining of the present invention Figure 1 .

[0044] Embodiment 1:

[0045] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0046] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0047] Sample preparation: select colonies of Escherichia coli, Staphylococcus aureus and Mycobacterium gordonii and shake them in a culture medium to obtain bacterial liquids of the above three bacteria, and mix the above three bacterial liquids and shake them evenly to obtain a mixed bacterial sample;

[0048] Smear: Dip a cotton swab into the mixed bacterial sample and smear it on a glass slide, and heat it on a heating plate at 50°C for 1 minute to fix it;

[0049] Staining: Add AIE staining solution to cover the coating, heat and dye on a heating plate at 60°C for 2-4 minutes, and then rinse carefully with distilled water;

[0050] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0051] Observation results: Direct microscopic examination was performed under a fluorescence microscope with a 10× eyepiece and a 100× objective lens under the B channel (excitation: 460-490nm; emission: 510-550nm). Figure 2 shown.

[0052] Embodiment 2:

[0053] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0054] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0055] Sample preparation: Pick up Mycobacterium gordonii colonies and shake them in a culture medium to obtain a bacterial liquid, add the Mycobacterium gordonii bacterial liquid to a sputum sample, mix well, and obtain a sputum sample containing Mycobacterium gordonii;

[0056] Smear: Use a cotton swab to pick up the above sputum sample and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0057] Staining: Add AIE staining solution to cover the coating, heat and dye on a heating plate at 60°C for 2-4 minutes, and then rinse carefully with distilled water;

[0058] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0059] Observation results: Direct microscopic examination was performed under a fluorescence microscope with a 10× eyepiece and a 40× objective lens under the B channel (excitation: 460-490nm; emission: 510-550nm). Figure 3 shown.

[0060] Embodiment 3:

[0061] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0062] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0063] Sample preparation: Pick a colony of Mycobacterium gordonii and shake it in a culture medium to obtain a bacterial solution;

[0064] Smear: Dip a cotton swab into the above bacterial solution and smear it on a glass slide, and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0065] Staining: Add AIE staining solution to cover the coating, heat and dye on a heating plate at 60°C for 2-4 minutes, and then rinse carefully with distilled water;

[0066] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0067] Observation results: Direct microscopic examination was performed under the fluorescence microscope B channel (excitation: 460-490nm; emission: 510-550nm), 10× eyepiece, 40× objective lens, and the results were statistically analyzed. Figure 4 shown.

[0068] Embodiment 4:

[0069] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0070] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0071] Sample preparation: Pick a colony of Mycobacterium gordonii and shake it in a culture medium to obtain a bacterial solution;

[0072] Smear: Dip a cotton swab into the above bacterial solution and smear it on a glass slide, and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0073] Staining: Add AIE staining solution to cover the coating, heat and dye on a heating plate at 60°C for 2-4 minutes, and then rinse carefully with distilled water;

[0074] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0075] Observation results: Under the fluorescence microscope B channel (excitation: 460-490nm; emission: 510-550nm), 10× eyepiece, 40× objective lens observation, under laser irradiation, continuous fluorescence images were taken. The results are as follows Figure 5 shown.

[0076] Comparative Example 1:

[0077] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0078] Sample preparation: select colonies of Escherichia coli, Staphylococcus aureus and Mycobacterium gordonii and shake them in a culture medium to obtain bacterial liquids of the above three bacteria, and mix the above three bacterial liquids and shake them evenly to obtain a mixed bacterial sample;

[0079] Smear: Dip a cotton swab into the mixed bacterial sample and smear it on a glass slide, and heat it on a heating plate at 50°C for 1 minute to fix it;

[0080] Staining: Add AIE staining solution to cover the coating, heat and stain at 60℃ on a heating plate for 2-4 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0081] Observation results: Direct microscopic examination was performed under a fluorescence microscope with a 10× eyepiece and a 100× objective lens under the B channel (excitation: 460-490nm; emission: 510-550nm). Figure 6 shown.

[0082] Comparative Example 2:

[0083] Preparation of AIE staining solution: Dissolve MASPB solid in ultrapure water to prepare a 10 mM stock solution, and dilute the MASPB stock solution to 60 μM with ultrapure water;

[0084] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0085] Sample preparation: Pick a colony of Mycobacterium gordonii and shake it in a culture medium to obtain a bacterial solution;

[0086] Smear: Dip a cotton swab into the above bacterial solution sample and smear it on a glass slide, and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0087] Staining: Add AIE staining solution until the coating is covered, stain at room temperature for 2-4 minutes, and then rinse carefully with distilled water;

[0088] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0089] Observation results: Direct microscopic examination was performed under a fluorescence microscope with a 10× eyepiece and a 40× objective lens under the B channel (excitation: 460-490nm; emission: 510-550nm). Figure 7 shown.

[0090] Comparative Example 3:

[0091] Preparation of AIE dye solution: DENPB solid was dissolved in ultrapure water to prepare a 10mM stock solution, and the DENPB stock solution was diluted with ultrapure water to 60μM (structural formula: );

[0092] Preparation of acidic decolorizing solution: prepare anhydrous ethanol and concentrated hydrochloric acid (37wt%), with the volume ratio of concentrated hydrochloric acid being 5%;

[0093] Sample preparation: select colonies of Escherichia coli, Staphylococcus aureus and Mycobacterium gordonii and shake them in a culture medium to obtain bacterial liquids of the above three bacteria, and mix the above three bacterial liquids and shake them evenly to obtain a mixed bacterial sample;

[0094] Smear: Dip a cotton swab into the mixed bacterial sample and smear it on a glass slide. Let it dry for 5 minutes or heat it on a heating plate at 50°C for 1 minute to fix it.

[0095] Staining: Add AIE staining solution to cover the coating, heat and dye on a heating plate at 60°C for 2-4 minutes, and then rinse carefully with distilled water;

[0096] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, rinse carefully with distilled water, and cover with a coverslip;

[0097] Observation results: Direct microscopic examination was performed under a fluorescence microscope with a 10× eyepiece and a 100× objective lens under the B channel (excitation: 460-490nm; emission: 510-550nm). Figure 8 shown.

[0098] Comparative Example 4:

[0099] Acid-fast staining was performed using a traditional auramine O acid-fast staining kit (G1217 Acid-fast Staining Kit (Auramine O Method) from Beijing Solebow Technology Co., Ltd.).

[0100] Sample preparation: Pick a colony of Mycobacterium gordonii and shake it in a culture medium to obtain a bacterial solution;

[0101] Smear: Dip a cotton swab into the above bacterial solution and smear it on a glass slide, and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0102] Staining: Add auramine O staining solution until the coating is covered, stain for 10-15 minutes away from light, and rinse carefully with distilled water;

[0103] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, and then rinse carefully with distilled water;

[0104] Re-staining: Add re-staining solution until the coating is covered, wait for 2 minutes, and then rinse carefully with distilled water;

[0105] Direct microscopic examination was performed under the fluorescence microscope B channel (excitation: 460-490nm; emission: 510-550nm), 10× eyepiece, 40× objective lens, and the results were statistically analyzed. Fig. 9 shown.

[0106] Comparative Example 5:

[0107] Acid-fast staining was performed using the traditional auramine O acid-fast staining kit.

[0108] Sample preparation: Pick a colony of Mycobacterium gordonii and shake it in a culture medium to obtain a bacterial solution;

[0109] Smear: Dip a cotton swab into the above bacterial solution and smear it on a glass slide, and heat it on a heating plate at 50℃ for 1 minute to fix it;

[0110] Staining: Add auramine O staining solution until the coating is covered, stain for 10-15 minutes away from light, and rinse carefully with distilled water;

[0111] Decolorization: Add acidic decolorizing solution until the coating is covered, wait for 2-3 minutes, and then rinse carefully with distilled water;

[0112] Re-staining: Add re-staining solution until the coating is covered, wait for 2 minutes, and then rinse carefully with distilled water;

[0113] Under the fluorescence microscope B channel (excitation: 460-490nm; emission: 510-550nm), 10× eyepiece, 40× objective lens observation, laser irradiation, continuous fluorescence images were taken, the results are as follows Fig.10 shown.

[0114] Result analysis:

[0115] (1) From Figure 1 It can be seen that the AIE acid-fast staining of the present invention has fewer steps (no need for re-staining) and takes less time to stain, which is conducive to rapid detection and saves time costs;

[0116] (2) From Figure 2 It can be seen that the AIE acid-fast staining of the present invention has a good detection effect on mixed bacteria samples: the short rod-shaped Escherichia coli and the spherical Staphylococcus aureus show almost no fluorescence, the overall background fluorescence is low, and the branched Mycobacterium gordonii shows bright fluorescence, with clear morphological structure and easy to distinguish, which can be used for the identification of mycobacteria;

[0117] (3) From Figure 3 It can be seen that the AIE acid-fast staining of the present invention is suitable for mycobacterium detection in actual sputum samples. Non-mycobacterium components (cells, cocci, etc.) in sputum hardly show fluorescence, the overall background fluorescence is low, and the branched Mycobacterium gordonii shows bright fluorescence. When observed under a 40× objective lens, the morphological structure of mycobacteria is clear and easy to distinguish, which is conducive to rapid screening during microscopic examination.

[0118] (4) From Figure 4 It can be seen that the AIE acid-fast staining of the present invention has a good detection rate for Mycobacterium gordonii, with a detection rate of more than 80%;

[0119] (5) From Figure 5 It can be seen that the AIE acid-fast staining of the present invention has good light stability, and there is no obvious fluorescence decay after continuous shooting for 10 minutes;

[0120] (6) From Figure 6 It can be seen that without the decolorization step, after staining with the AIE staining solution, Escherichia coli, Staphylococcus aureus and Mycobacterium gordonii all showed bright fluorescence. Compared with the results of the embodiment, it shows that after decolorization treatment with the acidic decolorizing solution, the color interference of non-mycobacterial components can be effectively eliminated, while the mycobacteria can still be stably combined and show bright fluorescence;

[0121] (7) From Figure 7 It can be seen that for the detection of Mycobacterium gordonii by the AIE acid-fast staining solution, the staining effect of the comparative example 2 under room temperature is poor compared with the staining under 60°C on the heating plate of Example 3, and the mycobacterium detection rate is only about 50%, indicating that the staining under 50-70°C of the present invention is beneficial to improve the detection rate of mycobacteria by AIE acid-fast staining;

[0122] (8) From Figure 8 It can be seen that the acid-fast staining solution prepared by the AIE molecule DENPB was used to stain the sample. After decolorization with an acid decolorizing solution, Escherichia coli and Staphylococcus aureus still showed bright fluorescence, but there was a large color interference from non-mycobacterial components, and it could not be used for the identification of mycobacteria.

[0123] (9) From Fig. 9 It can be seen that the detection rate of auramine O acid-fast staining for Mycobacterium gordonii is poorer than that of AIE acid-fast staining, and the detection rate is only about 70%;

[0124] (10) From Fig.10 It can be seen that the photostability of auramine O acid-fast staining is poor, and there is obvious fluorescence decay after continuous shooting for 3 minutes.

[0125] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. An acid-fast staining solution for mycobacterium identification based on AIE fluorescent probe, characterized in that: It includes an AIE staining solution and an acidic decolorizing solution; the acidic decolorizing solution includes hydrochloric acid and ethanol; the AIE staining solution includes a MASPB molecule and water, and the structural formula of the MASPB molecule is as follows: 。 2. The acid-fast staining solution for mycobacterium identification based on AIE fluorescent probe according to claim 1, characterized in that: The concentration of MASPB molecules in the AIE staining solution is 50-70 μM.

3. The acid-fast staining solution for mycobacterium identification based on AIE fluorescent probe according to claim 1, characterized in that: The volume ratio of hydrochloric acid in the acidic decolorizing solution is 3-6%; the mass concentration of the hydrochloric acid is 30-40%.

4. Use of the mycobacterium identification acid-fast staining solution based on AIE fluorescent probe according to any one of claims 1 to 3 in identifying mycobacteria.

5. A method for identifying mycobacteria, characterized in that: For non-disease diagnosis fields, the following steps are included: (1) Spread the sample to be tested on a glass slide and fix it to form a coating; (2) Add AIE dye solution until the coating is covered, dye at 50-70°C for 2-4 min, and rinse; (3) Add acidic decolorizing solution until the coating is covered, let stand to decolorize, rinse, and cover with a coverslip; (4) Observe the smear through the B channel of the fluorescence microscope; judge whether the sample contains mycobacteria by observing whether there is bright fluorescence; the excitation wavelength of the B channel is 460-490 nm, and the emission wavelength is 510-550 nm; Wherein, the AIE staining solution and the acid decolorizing solution are the AIE staining solution and the acid decolorizing solution in the acid-fast staining solution for mycobacterium identification based on AIE fluorescent probe according to any one of claims 1 to 3.

6. The method for identifying mycobacteria according to claim 5, characterized in that: The standing bleaching time in step (3) is 2-3 min.

7. The method for identifying mycobacteria according to claim 5, characterized in that: The fixation described in step (1) is heating fixation at 50-70°C.

8. The method for identifying mycobacteria according to claim 5, characterized in that: The magnification of the fluorescence microscope in step (4) is 400-1000 times.

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