甲基红 (Methyl Red) 是一种常用的弱酸性 pH 指示剂，属于偶氮类染料，变色范围在 pH4-6 之间 (由红变黄)，分子式为 C₁₅H₁₅N₃O₂，分子量为 269.30，CAS 号为 493-52-7。 甲基红在酸性环境中以质子化形式存在，呈现红色；在碱性环境中以去质子化形式存在，呈现黄色。由于植物细胞液泡通常呈酸性反应 (pH 约 4.5-5.5)，恰处于甲基红的变色范围之内，因此可通过甲基红的显色变化指示植物细胞及液泡的酸度。液泡酸度越高 (pH 越低)，红色越深；酸度越低 (pH 越接近 6)，颜色越偏黄色。植物细胞酸度染色液 (甲基红指示法) 主要由甲基红、乙醇、水等组成，用于指示植物组织或细胞内的酸碱度。与中性红指示法不同，甲基红属于弱酸性偶氮染料，其变色机制为直接的酸碱平衡指示，颜色变化反映组织或细胞环境的 pH 值。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、显微镜、载玻片、盖玻片、镊子、吸水滤纸
2、蒸馏水或去离子水、植物材料 (新鲜取材)
操作步骤 (仅供参考)：
1、取新鲜植物组织，用镊子撕取一层表皮细胞，置于洁净载玻片上。
2、滴加植物细胞酸度染色液 (甲基红指示法) 完全覆盖样本，室温染色 3-10min。
3、用吸水滤纸沿玻片边缘吸去多余染色液，滴加 1 滴蒸馏水，盖上盖玻片，用滤纸从盖玻片侧面吸去多余水分。
4、立即置于显微镜下观察或拍照。
5、(备选) 如需对比酸度差异，可同时取不同组织或不同处理条件的样本平行染色，比较颜色红黄深浅。
染色结果：
1、pH 为 2-3 细胞内呈樱桃红色，pH 为 4 细胞内呈橙红色，pH 为 5 细胞内呈橙黄色，pH 为 6 细胞内呈黄色。
2、活细胞液泡呈红色至橙红色 (颜色越红表明液泡酸度越高，pH 越低)；酸度较低的细胞或区域趋向黄色。细胞壁一般不着色或呈浅黄色。
注意事项：
1、本品用于指示植物细胞酸度，取材和染色过程中应保持样本新鲜，避免长时间暴露或干燥。
2、初次使用本方法染色，应先做预实验。染色时间可根据组织类型和细胞活性适当调整。
3、如发现溶液颜色明显变化或产生沉淀，应更换新液。
4、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Methyl Red is a commonly used weakly acidic pH indicator belonging to azo dyes. Its color transition range is pH 4–6 (red to yellow). Its molecular formula is C₁₅H₁₅N₃O₂, molecular weight 269.30, and CAS No. 493‑52‑7.Methyl Red exists in a protonated form and appears red under acidic conditions; under alkaline conditions, it exists in a deprotonated form and appears yellow. Plant cell vacuoles are generally acidic (pH approx. 4.5–5.5), which falls within the color transition range of Methyl Red. Therefore, the color change of Methyl Red can be used to indicate the acidity of plant cells and vacuoles. The higher the vacuolar acidity (the lower the pH value), the deeper the red color; the lower the acidity (the closer the pH is to 6), the more yellow the color.
Plant Cell Acidity Staining Solution (Methyl Red Indicator Method) is mainly composed of Methyl Red, ethanol and water. It is used to indicate the pH inside plant tissues or cells. Unlike the Neutral Red indicator method, Methyl Red is a weakly acidic azo dye. Its color-changing mechanism relies on direct acid-base equilibrium, and the color change reflects the pH value of the tissue or cellular microenvironment. This reagent is for research use only and is not intended for clinical diagnosis or other purposes.
Materials to Be Prepared:
1. Microscope, glass slides, coverslips, forceps, absorbent filter paper
2. Distilled or deionized water, fresh plant materials
Operating Procedures (For Reference Only):
1. Take fresh plant tissue, peel off a layer of epidermal cells with forceps and place them on a clean glass slide.
2. Add Plant Cell Acidity Staining Solution (Methyl Red Indicator Method) to fully cover the sample, and stain at room temperature for 3–10 min.
3. Blot off excess staining solution from the edge of the slide with absorbent filter paper. Add one drop of distilled water, place the coverslip, and remove surplus water from the side of the coverslip using filter paper.
4. Immediately observe or take photographs under a microscope.
5. (Optional) To compare acidity differences, stain samples from different tissues or treatment groups in parallel and compare the intensity of red and yellow color.
Staining Results:
1. At pH 2–3, a cherry-red color is observed inside cells; at pH 4, an orange-red color; at pH 5, an orange-yellow color; at pH 6, a yellow color.
2. Vacuoles of living cells appear red to orange-red (redder color indicates higher vacuolar acidity and lower pH). Cells or regions with low acidity tend to show yellow color. Cell walls are generally unstained or pale yellow.
Precautions:
1. This product is used to indicate plant cell acidity. Keep samples fresh during sampling and staining; avoid prolonged exposure or desiccation.
2. A preliminary trial test is recommended for first-time use. Staining duration may be adjusted according to tissue type and cell viability.
3. Replace with fresh reagent if obvious color change or precipitate occurs in the solution.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
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