Ammonium Oxalate crystal violet staining solution is a component of Gram staining. In Gram-positive bacteria staining, cells are stained with ammonium oxalate crystal violet and then treated with Gram iodine solution to form an insoluble complex, which cannot pass through the cell wall and is not easily decolorized, so it remains purple. After staining, the bacteria are in sharp contrast with the environment, and the morphology, arrangement and certain structural characteristics of the bacteria can be clearly observed, which can be used for classification and identification. Operation steps (for reference only): 1. Operate according to the specific requirements of the experiment 2. Smear: Take the bacteria to be tested, apply a thin layer on the center of the glass slide or drop a little sterile water on the glass slide, mix the bacteria and water evenly, and apply a thin layer. 3. Drying: After smearing, let it dry naturally at room temperature, or warm it slightly on an alcohol lamp to make it dry quickly. 4. Fixation: Hold one end of the glass slide with the specimen facing up, and move it back and forth quickly 3 to 5 times on the outside of the flame of the alcohol lamp, each time for 1 s. It can also be fixed with methanol or ethanol. 5. Initial dyeing: add crystal violet staining solution dropwise to dye for 1-2 minutes, rinse with water to remove the dyeing solution. 6. Mordant: Add Gram iodine solution dropwise, and cover the slides for 1-2 minutes at room temperature, then wash with water. 7. Decolorization: dropwise add the decolorizing solution and shake for 10-30s, until the decolorizing solution flowing down does not appear purple, immediately rinse the decolorizing solution with water to terminate the reaction. 8. Counterstaining: add sand yellow staining solution dropwise for dyeing, 30~60s, and wash with water. 9. Dry. Microscopic examination: observe with oil mirror. Staining result: Gram-positive bacteria: blue to purple; Gram-negative bacteria: red. Precautions: 1. Before smearing, circle marks should be made on the back in order to judge the position of subsequent tests. 2. When taking bacteria, you should pay attention to self-protection. When removing or plugging the stopper of the test tube, the mouth of the test tube should be slightly cauterized by flame, and finally the inoculation ring should be cauterized on the flame. When heating and fixing the smear, it should be noted that the glass slide should not be too close to the flame. Generally, the temperature of the glass slide should not exceed 60 °C. 3. The key to Gram staining is to strictly control the degree of decolorization, and the decolorization time should be judged according to experience. Excessive decolorization, positive bacteria can be mistakenly infected as negative bacteria; insufficient decolorization, negative bacteria can be mistakenly infected as positive bacteria. 4. The culture time of the bacteria to be tested will affect the staining. If the culture time of the positive bacteria is too long or the dead bacteria are dissolved, it is often negative. 5. For your safety and health, please wear a lab coat and disposable gloves.
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