草酸铵分子式为C₂H₁₀N₂O₅，分子量为142.11，结晶紫分子式为C₂₅H₃₀ClN₃，分子量为407.99。结晶紫染色液(Crystal Violet Staining Solution)是一种组织或细胞染色时常用的可以把细胞核染成深紫色的染色液。用于革兰氏染色。结晶紫是一种碱性染料，可以和细胞核中的DNA结合，从而产生细胞核染色。
阿拉丁草酸铵结晶紫染色液是革兰氏染色法中的一种成分，主要由草酸铵、结晶紫、乙醇等组成。在革兰阳性菌染色中，细胞经草酸铵结晶紫染色后再经Gram碘液处理，形成不溶性复合物，该复合物不能通过细胞壁，不易被脱色，所以保持紫色。其染色结果为pH0.5（绿）～pH2.0（蓝），染色后细菌与环境形成鲜明对比，因此可以清楚地观察到细菌的形态、排列及某些结构特征，而用以分类鉴定。该产品仅适用于科研实验，不可做他用。
自备材料：接种环戒挑取细菌的其他工具、酒精灯、载玻片、光学显微镜。
操作步骤(仅供参考)：
1、按实验具体要求操作或参考下列革兰氏染色的方法。
2、涂片：取待检细菌，于载玻片中央涂成薄层或者在载玻片上滴加少许无菌水，取菌液与水混合均匀，涂成薄层。
3、干燥：涂片后在室温下自然干燥，也可在酒精灯上略加温，使之迅速干燥。
4、固定：手持载玻片一端，标本面朝上，在酒精灯的火焰外侧快速来回移动3~5次，每次1s，温度不宜过高，防止菌体蛋白变性，放置待凉后染色。也可以用甲醇戒乙醇固定。
5、初染：滴加结晶紫染色液染色1-2min，清水冲洗去染色液。
6、媒染：滴加Gram碘液，并覆盖载玻片室温放置1-2min，水洗。
7、脱色：滴加脱色液摇动，摇动10～30s，直至流下的脱色液不出现紫色为止，立即用水冲去脱色液，终止反应。
8、复染：滴加沙黄染色液染色，30~60s，水洗。
9、干燥。镜检：置油镜观察。
染色结果：
注意事项：
1、涂片之前，应事先在背面做好圆圈标记，以便判断后续试验的位置。
2、取细菌时，应注意自我防护，拔试管塞时，应将试管口通过火焰略加烧灼，最后将接种环在火焰上烧灼灭菌。
3、加热固定涂片时，应注意玻片勿太靠近火焰，一般要求玱片温度不超过60°C，以玱片背面触及手背皮肤不觉过烫为宜。
4、革兰氏染色的关键在于严格掌握脱色程度，脱色时间应根据经验判断。脱色过度，阳性菌可被误染为阴性菌；脱色不够，阴性菌可被误染为阳性菌。
5、待检细菌培养时间会影响染色，阳性菌培养时间过长或已死亡戒细菌溶解，常呈阴性。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
The molecular formula of ammonium oxalate is C₂H₁₀N₂O₅ with a molecular weight of 142.11, while the molecular formula of crystal violet is C₂₅H₃₀ClN₃ with a molecular weight of 407.99. Crystal Violet Staining Solution is a commonly used staining reagent for tissue or cell samples, which stains cell nuclei dark purple. It is primarily applied in Gram staining. As a basic dye, crystal violet can bind to DNA in the cell nucleus, thereby achieving nuclear staining.
Aladdin Ammonium Oxalate Crystal Violet Staining Solution is a key component of the Gram staining method, consisting mainly of ammonium oxalate, crystal violet, and ethanol. In the staining of Gram-positive bacteria, the cells are first stained with ammonium oxalate crystal violet and then treated with Gram's iodine solution, forming an insoluble complex. This complex cannot penetrate the bacterial cell wall and is not easily decolorized, thus retaining the purple color. The staining exhibits a color transition from green (at pH 0.5) to blue (at pH 2.0). After staining, bacteria form a sharp contrast against the background, allowing for clear observation of their morphology, arrangement, and certain structural characteristics, which facilitates bacterial classification and identification.
Materials to be prepared by the user: Inoculation loops, other tools for picking bacteria, alcohol lamps, glass slides, and light microscopes.
Operating Procedures (For Reference Only):
1. Operate according to the specific requirements of the experiment or refer to the following Gram staining method.
2. Smear Preparation: Take the bacteria to be tested and spread them into a thin layer at the center of a glass slide. Alternatively, place a drop of sterile water on the glass slide, mix the bacterial suspension thoroughly with the water, and then spread it into a thin layer.
3. Drying: Air-dry the smear at room temperature naturally. You may also slightly heat the slide over an alcohol lamp to accelerate drying.
4. Fixation: Hold one end of the glass slide with the specimen side facing up, and move it quickly back and forth 3–5 times over the outer flame of an alcohol lamp (1 second per pass). Avoid excessive temperature to prevent denaturation of bacterial proteins. Allow the slide to cool before staining. Methanol or ethanol can also be used for fixation.
5. Primary Staining: Cover the specimen with crystal violet staining solution and stain for 1–2 minutes. Rinse the slide with clean water to remove the staining solution.
6. Mordanting: Add Gram's iodine solution to cover the entire glass slide, leave it at room temperature for 1–2 minutes, and then rinse with water.
7. Decolorization: Apply decolorizing solution and shake the slide continuously for 10–30 seconds until no purple color runs off with the decolorizing solution. Immediately rinse the slide with water to terminate the reaction.
8. Counterstaining: Stain the slide with safranin staining solution for 30–60 seconds, followed by a water rinse.
9. Drying and Microscopic Examination: Dry the slide and observe under an oil immersion lens.
Staining Results:
Gram-positive bacteria
| Blue to purple
|
Gram-negative bacteria
| Red
|
Precautions:
1. Before preparing the smear, mark a circle on the back of the glass slide in advance to determine the position for subsequent tests.
2. Pay attention to personal protection when collecting bacteria. When removing the test tube stopper, pass the mouth of the test tube through an alcohol lamp flame for slight flaming sterilization. Finally, sterilize the inoculation loop by flaming it over the alcohol lamp.
3. When heat-fixing the smear, make sure the glass slide is not too close to the flame. Generally, the temperature of the slide should not exceed 60°C, which can be judged by touching the back of the slide to the back of your hand— it should not feel excessively hot.
4. The key to Gram staining is strictly controlling the decolorization degree, and the decolorization time should be determined based on experience. Over-decolorization may cause Gram-positive bacteria to be mistakenly stained as Gram-negative; insufficient decolorization may lead to Gram-negative bacteria being misidentified as Gram-positive.
5. The culture time of the bacteria to be tested will affect the staining result. Gram-positive bacteria that have been cultured for an excessively long time, or are already dead or lysed, often show a Gram-negative staining pattern.
6. For your safety and health, wear a lab coat and disposable gloves during the operation.
ZJ25F1230687
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