Carboxymethylated porous reticulated membrane and its use in the decolorization of bacterial stains
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QIANNAN COLLEGE OF ETHNIC MEDICINE
- Filing Date
- 2026-04-28
- Publication Date
- 2026-06-19
AI Technical Summary
Existing bacterial staining techniques suffer from uneven decolorization, cumbersome operation, easy bacterial cell detachment, and high cost of automated equipment, making them difficult to promote in primary healthcare institutions.
A carboxymethylated porous mesh membrane is used. Through modification treatment, the membrane surface is given carboxymethyl functional groups, which are hydrophilic and negatively charged. The porous structure and capillary action are used to achieve uniform diffusion of the decolorizing agent and static operation, thus avoiding the loss of bacteria.
It improves the uniformity and consistency of decolorization, simplifies the operation process, reduces costs, and is suitable for microbial testing in primary healthcare institutions and routine laboratories.
Smart Images
Figure CN122234458A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial detection technology, specifically relating to a carboxymethylated porous mesh membrane and its application in bacterial staining and destaining, especially suitable for the destaining process of Gram staining and acid-fast staining in clinical microbiology testing. Background Technology
[0002] Gram staining and acid-fast staining are fundamental methods for clinical microbial identification, widely used in bacterial classification, infectious disease screening, and etiological diagnosis. The staining results directly determine the accuracy of subsequent biochemical identification, molecular detection, and mass spectrometry analysis. In the complete staining process, the destaining step is the most critical step affecting the interpretation of results, directly determining the ability to distinguish between positive and negative staining.
[0003] Traditional bacterial staining and destaining are performed on ordinary glass slides, using a method of adding destaining agent and repeatedly shaking the slide manually. However, this method has the following inherent technical limitations in long-term clinical application:
[0004] 1. Poor decolorization uniformity: The surface of the glass slide is highly hydrophobic, and after the bacterial film is fixed, it is easy to form an adsorbed water film, which constitutes a contact barrier for the decolorizing agent. This results in the decolorizing agent not being able to spread evenly, leading to local over-decolorization and local under-decolorization, causing misjudgment of the results.
[0005] 2. Cumbersome operation and low standardization: The decolorization effect is highly dependent on the operator's experience and skills. The results vary significantly between different personnel and different batches, making it difficult to achieve uniform quality control.
[0006] 3. Bacterial cells are easily detached and lost: Shaking and rinsing with water will mechanically wash away the bacterial film, resulting in significant loss of bacteria in low-concentration samples, which can easily lead to false negatives and reduce the sensitivity of screening for infectious diseases such as tuberculosis.
[0007] 4. Difficulty in popularizing automated equipment: Existing automated staining instruments achieve decolorization through robotic arms, spraying, or vibration. Although they can improve uniformity, the equipment is expensive, has high maintenance costs, and is bulky, making it impossible to promote its use in primary healthcare institutions, disease control sites, and teaching laboratories.
[0008] There are existing reports on the use of filter membranes for microbial enrichment and staining, but these technologies only use ordinary filter membranes to retain bacteria without modifying or functionalizing the membrane material. The decolorization process still requires shaking or external force assistance, which cannot solve the problems of uneven decolorization and water barrier. At the same time, existing technologies have not achieved the synergistic effect of static decolorization, uniform diffusion and high bacterial retention.
[0009] Therefore, there is an urgent need in this field for a low-cost, easy-to-operate, equipment-free, uniformly decolorized bacterial staining and decolorization technology with high bacterial cell retention rate, in order to solve the bottlenecks of traditional manual operation and existing automated equipment, and meet the actual needs of primary medical institutions and rapid on-site testing. Summary of the Invention
[0010] The purpose of this invention is to overcome the shortcomings of existing bacterial staining and destaining techniques, such as uneven destaining, high operational difficulty, and easy detachment of bacterial cells. It provides a carboxymethylated porous mesh membrane and its application in bacterial staining and destaining. Through material structure and functional modification, it achieves uniform distribution of destaining agent, static operation, and high bacterial cell retention, thereby improving the stability and consistency of bacterial staining, reducing operational difficulty and usage costs, and meeting the testing needs of primary healthcare institutions and routine laboratories.
[0011] A carboxymethylated porous mesh membrane is provided and its application in bacterial staining and destaining.
[0012] To achieve the above objectives, the technical solution adopted is as follows:
[0013] First, this invention provides a carboxymethylated porous mesh membrane. The membrane uses a porous mesh membrane as a substrate, which is modified by carboxymethylation to introduce carboxymethyl functional groups (—CH2—COOH) onto its surface, making the membrane surface negatively charged and hydrophilic. The pore size of the porous mesh membrane is 0.22 μm to 0.65 μm. The substrate is selected from mixed cellulose ester membranes or polycarbonate membranes. Further, the pore size of the carboxymethylated porous mesh membrane is preferably 0.45 μm, and the degree of carboxymethylation substitution is preferably 0.4 to 0.6.
[0014] Secondly, the present invention provides a method for preparing the above-mentioned carboxymethylated porous mesh membrane, comprising the following steps: selecting a mixed cellulose ester membrane or polycarbonate membrane with a pore size of 0.22 μm to 0.65 μm as a substrate; placing the substrate in an alkaline solution for activation treatment; adding a carboxymethylating agent to carry out an etherification reaction, so that carboxymethyl functional groups are grafted onto the membrane surface; and obtaining a carboxymethylated porous mesh membrane after washing and drying.
[0015] Furthermore, the present invention provides the application of the above-mentioned carboxymethylated porous mesh membrane in bacterial staining and decolorization, wherein the bacterial staining is preferably Gram staining or acid-fast staining.
[0016] Finally, this invention provides a bacterial staining and decolorization method, comprising the following steps: preparing a dried and fixed bacterial smear; immersing the above-mentioned carboxymethylated porous mesh membrane in a decolorizing agent, allowing the membrane to adsorb the decolorizing agent; covering the bacterial membrane of the smear with the adsorbed decolorizing agent onto the bacterial membrane, and allowing it to stand for decolorization; removing the membrane after decolorization, thus completing the decolorization step. Specifically, when the carboxymethylated porous mesh membrane covers the bacterial membrane, the porous structure of the membrane allows the decolorizing agent to diffuse laterally within the membrane plane and fully contact the bacterial membrane; during the decolorization process, the carboxymethylated porous mesh membrane replaces the adsorbed water layer on the surface of the bacterial membrane through capillary action and hydrophilicity.
[0017] The technical solution of this invention has the following effects:
[0018] 1. A carboxymethylated porous mesh membrane is used, which utilizes its porous structure and strong hydrophilicity to break down the moisture barrier on the surface of the bacterial membrane, allowing the decolorizing agent to diffuse evenly and significantly improving the decolorization uniformity and consistency of results.
[0019] 2. The decolorization process does not require shaking the glass slide, simplifying the operation, reducing labor intensity, and facilitating batch processing and standardized implementation.
[0020] 3. The membrane has a physical retention effect on bacteria and no strong adsorption between it and the bacteria. It reduces the loss of bacteria during decolorization and rinsing, thus improving the reliability of sample detection.
[0021] 4. It is compatible with existing manual staining processes, requires no special equipment, is low-cost and easy to promote, and is suitable for primary healthcare, teaching and routine microbial testing scenarios.
[0022] 5. It is applicable to various bacterial staining systems such as Gram staining and acid-fast staining, and has strong versatility. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of a carboxymethylated porous network membrane structure.
[0024] Figure 2 Image showing the effect of traditional glass slide decolorization;
[0025] Figure 3 This is an image showing the staining and destaining effect of the carboxymethylated porous mesh membrane of the present invention on Gram-negative bacilli.
[0026] Figure 4 This image shows the staining and decolorization effect of the carboxymethylated porous mesh membrane of the present invention on Gram-positive cocci. Detailed Implementation
[0027] The following embodiments are only used to further illustrate the technical solution of the present invention and do not represent a limitation on the scope of protection of the claims; conventional substitutions and adjustments made by those skilled in the art based on the above embodiments without creative effort are all within the scope of protection of the present invention.
[0028] The carboxymethylated porous mesh membrane of this invention is a functionalized membrane material obtained by carboxymethylation modification using a porous mesh membrane as a substrate. Its surface contains carboxymethyl functional groups, exhibiting hydrophilicity and negative charge. It can adsorb decolorizing agents and achieve uniform lateral diffusion, making it suitable for bacterial staining and decolorization processes. The bacterial staining includes conventional microbial staining methods such as Gram staining and acid-fast staining. The decolorizing agents are conventional reagents in the art, including 95% ethanol and hydrochloric acid-alcohol mixtures.
[0029] Example 1: Carboxymethylated porous mesh membrane
[0030] This embodiment provides a carboxymethylated porous mesh membrane product for bacterial staining and decolorization.
[0031] The carboxymethylated porous mesh membrane uses a mixed cellulose ester porous mesh membrane as the substrate, with a pore size of 0.45 μm, and is a flexible thin film. The membrane material undergoes carboxymethylation modification treatment, introducing carboxymethyl functional groups (—CH2—COOH) onto the surface, giving the membrane a negative charge and strong hydrophilicity. The degree of carboxymethylation substitution is controlled between 0.4 and 0.6. A substitution degree <0.4 indicates insufficient hydrophilicity; a substitution degree >0.6 results in a brittle membrane that is prone to bacterial adhesion.
[0032] Preferably, the substrate has a pore size of 0.45 μm and a degree of substitution of 0.52; the target bacterial species is a mixed bacterial suspension of Escherichia coli and Staphylococcus aureus; the steps are: bacterial film preparation → primary staining and mordant staining simultaneously for 1 min → film covering and decolorization for 15 s → counterstaining → microscopic examination; the effect is: clear distinction between light and dark areas, clean background, and uniform color throughout.
[0033] The membrane has a permeable porous network structure, which can adsorb and store decolorizing agent through capillary action. When it covers the surface of the bacterial film, it can spontaneously wet and expel air bubbles, forming a gapless surface contact with the bacterial film. Through its porous structure and hydrophilic properties, the decolorizing agent can diffuse laterally within the membrane plane. At the same time, it can replace the adsorbed water layer on the surface of the bacterial film, eliminate the moisture barrier, and meet the requirements for static decolorization.
[0034] like Figure 1 This is a schematic diagram of the microstructure of the carboxymethylated porous mesh membrane of the present invention.
[0035] Example 2: Preparation method of carboxymethylated porous mesh membrane
[0036] This embodiment provides a method for preparing the above-mentioned carboxymethylated porous mesh membrane, and the specific steps are as follows:
[0037] 1. Substrate selection: A mixed cellulose ester porous mesh membrane with a pore size of 0.22 μm to 0.65 μm was selected as the substrate and cut into a film size suitable for the coating.
[0038] 2. Activation treatment: The substrate is immersed in an alkaline sodium hydroxide solution and activated at room temperature to improve the hydroxyl reactivity of the film surface;
[0039] 3. Etherification modification: Chloroacetic acid is added as a carboxymethylating agent, and the etherification reaction is carried out at a constant temperature to graft carboxymethyl functional groups onto the surface of the membrane substrate;
[0040] 4. Washing and purification: Rinse the membrane thoroughly with deionized water after the reaction to remove residual reagents and soluble byproducts until the washing solution is neutral;
[0041] 5. Drying and storage: Dry the washed membrane at low temperature to obtain a carboxymethylated porous mesh membrane, and store it in a sealed, light-proof container for later use.
[0042] Using the same preparation process, the substrate can be replaced with a polycarbonate porous mesh membrane to obtain the corresponding carboxymethylated modified membrane product.
[0043] Example 3: Application of carboxymethylated porous mesh membrane in bacterial staining and destaining
[0044] This embodiment provides an application method of carboxymethylated porous mesh membrane in bacterial staining and decolorization.
[0045] The carboxymethylated porous mesh membrane prepared in Example 1 or Example 2 was used in the decolorization step of Gram staining or acid-fast staining.
[0046] In application, the carboxymethylated porous mesh membrane is first immersed in the decolorizing agent to fully wet it, so that the internal pores of the membrane are saturated with adsorption. After being taken out, it is directly covered on the surface of the bacterial membrane that has been initially stained and mordanted. The porous structure and hydrophilicity of the membrane allow the decolorizing agent to diffuse evenly in the membrane and come into full contact with the bacteria, thus completing the static decolorization.
[0047] This application is fully compatible with routine manual staining procedures in clinical microbiology testing. No changes are required to the primary staining, mordant staining, counterstaining, and microscopic examination procedures. No additional instruments or equipment are needed. It can be used for microbial staining detection in clinical testing, primary care screening, teaching experiments, and batch samples.
[0048] Example 4 Bacterial staining and destaining method
[0049] This embodiment provides a bacterial staining and destaining method based on carboxymethylated porous membranes, taking Gram staining as an example:
[0050] 1. Preparation of smears: The bacterial suspension to be tested is evenly spread on a glass slide, dried naturally, and then fixed by flame heat to obtain a standard bacterial smear;
[0051] 2. Initial staining: Add crystal violet staining solution according to the "Standardized Operating Procedures for Clinical Microbiology Laboratory Techniques", let it stand for staining, and rinse gently with running water;
[0052] 3. Mordant: Add Gram-iodine solution as mordant, let stand, and gently rinse with running water;
[0053] 4. Decolorization treatment: Immerse the carboxymethylated porous mesh membrane in 95% ethanol decolorizing agent, remove it after wetting, cover the smeared bacterial film area, and let it stand for decolorization; the decolorizing agent diffuses evenly in the transverse direction along the plane inside the membrane, replacing the adsorbed water layer on the surface of the bacterial film, achieving uniform decolorization without gaps or scouring;
[0054] 5. Counterstaining and microscopic examination: After destaining, gently remove the membrane, add diluted carbolic acid fuchsin staining solution for counterstaining, wash with water, dry, and then examine under an optical microscope.
[0055] This method requires no shaking of the glass slide during the decolorization process, is simple to operate, produces uniform decolorization, and preserves the integrity of the bacterial cells. It is suitable for the detection of microbial staining in routine and low-concentration samples.
[0056] like Figure 2 The effect of traditional slide staining method; Figure 3 This is a staining effect image of Gram-negative bacilli on the carboxymethylated porous mesh membrane of the present invention; Figure 4 This image shows the staining effect of the carboxymethylated porous mesh membrane of this invention on Gram-positive cocci. Traditional slide staining achieves a destaining uniformity of approximately 75% and takes 5–6 minutes. This invention utilizes a carboxymethylated porous mesh membrane, leveraging its porous structure and strong hydrophilicity to break down the moisture barrier on the bacterial membrane surface, allowing for uniform diffusion of the destaining agent. This results in a destaining uniformity approaching 100%, requiring only 3.5 minutes, significantly improving destaining uniformity and operational efficiency. The destaining process of this invention eliminates the need to shake the slide, simplifying operation, reducing labor intensity, and facilitating batch processing and standardized implementation. The membrane of this invention has a physical retention effect on bacteria and exhibits no strong adsorption between the membrane and the bacterial cells, reducing bacterial loss during destaining and rinsing, and improving sample detection reliability. This invention is compatible with existing manual staining procedures, requires no specialized equipment, is low-cost, easy to promote, and suitable for primary healthcare, teaching, and routine microbiological testing scenarios. This invention is applicable to various bacterial staining systems, including Gram staining and acid-fast staining, demonstrating strong versatility.
Claims
1. A carboxymethylated porous mesh membrane, characterized in that: Using a porous mesh membrane as the substrate, the membrane is modified by carboxymethylation, and its surface contains carboxymethyl functional groups (—CH2—COOH), which are negatively charged and hydrophilic; the pore size of the porous mesh membrane is 0.22μm to 0.65μm; the substrate is a mixed cellulose ester membrane or a polycarbonate membrane.
2. The carboxymethylated porous mesh membrane according to claim 1, characterized in that: The pore size is 0.45 μm.
3. The carboxymethylated porous mesh membrane according to claim 1, characterized in that: The degree of carboxymethylation is 0.4–0.
6.
4. A method for preparing a carboxymethylated porous mesh membrane according to any one of claims 1 to 3, characterized in that, include: 1) Select a mixed cellulose ester membrane or polycarbonate membrane with a pore size of 0.22μm~0.65μm as the substrate; 2) Activate the substrate in an alkaline solution; 3) Add carboxymethylating agents to carry out etherification reaction, so that carboxymethyl functional groups are grafted onto the membrane surface; 4) Wash and dry to obtain a carboxymethylated porous mesh membrane.
5. The application of the carboxymethylated porous mesh membrane according to any one of claims 1 to 3 in bacterial staining and decolorization.
6. The application according to claim 5, characterized in that: The bacterial staining is Gram staining or acid-fast staining.
7. A method for bacterial staining and destaining, characterized in that, Includes the following steps: 1) Preparation of dried and fixed bacterial smears; 2) Immerse the carboxymethylated porous mesh membrane according to any one of claims 1 to 3 in a decolorizing agent, so that the membrane adsorbs the decolorizing agent; 3) Cover the bacterial film of the smear with a carboxymethylated porous mesh membrane adsorbed with decolorizing agent, and let it stand for decolorization; 4) Remove the film after decolorization to complete the decolorization process.
8. The method according to claim 7, characterized in that: When the carboxymethylated porous mesh membrane is applied to the bacterial film, the decolorizing agent diffuses laterally within the membrane plane through the porous structure of the membrane, and replaces the adsorbed water layer on the surface of the bacterial film through capillary action and hydrophilicity.
9. The method according to claim 7, characterized in that: The membrane reduces bacterial loss by repelling bacteria through its negative surface charge and physically trapping bacteria through its porous structure.
10. The method according to claim 7, characterized in that: During Gram staining, primary staining and mordant staining can be performed simultaneously. A carboxymethylated porous mesh membrane is placed over the bacterial membrane, and primary staining solution and mordant solution are added at the same time. After staining, the mixture is allowed to stand and then washed with water.