This stain is the most basic stain and can be used on specimen smears or colony smears. The result of staining will classify the bacteria into Gram-positive and Gram-negative. This experiment is from Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory testing majors.
Operation method
Gram stain preparation and staining method experiment
Principle
This stain is the most basic stain and can be used on specimen smears or colony smears. The results of the stain classify the bacteria into Gram-positive and Gram-negative categories. Move I. Experimental reagents: For more product details, please visit Aladdin Scientific website.
1. Crystalline violet solution:
A solution: crystal violet: 2g, 95% ethanol: 20ml.
Liquid B: ammonium oxalate: 0.8g, distilled water: 80ml.
It is necessary to mix liquid A and liquid B 24 hours before use. Liquid A and B should be mixed 24 hours before use, filtered and put into reagent bottle for spare.
2. Iodine solution:
Iodine: 1g, potassium iodide: 2g Distilled water: 300ml.
Mix and grind iodine with potassium iodide, add a few milliliters of water to dissolve it gradually, then grind it, and continue to add small amounts of distilled water until it is completely dissolved, and finally make up the amount of water.
Alternatively, a small amount of distilled water can be used to completely dissolve the potassium iodide, then add the iodine tablets, and when completely dissolved, add water to 300 ml.
3. Decolorizing solution: 95% ethanol.
4. Re-dyeing solution:
A. Storage solution: sand yellow: 2.5g, 95% ethanol: 100ml.
B. Application being: liquid A: 10ml, distilled water: 90ml.
Bacterial slice preparation: the first step of staining is to make a smear. When smearing with bacterial liquid, dip the inoculating ring into the bacterial liquid and point it on the slide, the specimen can be coated directly on the slide, when smearing with colonies, first take 1 drop of saline, place it on the slide, pick the colonies with an inoculating needle, and smear them in the saline. Care must be taken to operate gently when smearing. Violent action will change the original arrangement of the bacterial cells, or cause the bacterial flagellum to fall off, affecting the accuracy of the results. The prepared smear should be dried naturally and fixed by flame, the fixation temperature is not easy to be too high, subject to the back of the slide touching the back of the hand is not hot, otherwise the cell morphology may be changed. The fixed smear should be stained.
Second, staining method:
l. The smears were fixed by flame, and stained with crystal violet solution for 1min, and the staining solution was washed away with water.
2. Add iodine solution for 1min, wash with water.
3. Add decolorizing solution, shake from time to time for about 30 seconds, until no purple color comes off, wash with water.
4. Add complex solution, dye for 30 seconds, wash.
5. Dried and examined by microscope.