What are the steps of Gram staining

Many people don't know what the Gram staining method is. It is a differential staining method commonly used in cytology. It is a hair re-dyeing method invented by a Danish doctor in 1884. It is divided into four steps: primary staining, mordanting, bleaching, and re-dyeing. Most people don’t know the steps of the Gram staining method. Let me introduce it to you below. Steps of Gram staining.

First, what are the steps of Gram staining? Operation steps: Make smears of different cultured bacteria (note that the smear must not be too thick), dry and fix. When fixing, pass the slide through the flame 1-2 times. Do not overheat it, and the slide should not be too hot to touch.

.Staining (1) For initial staining, add one drop of crystal violet dissolved in oxalic acid for about one minute and then wash with water.

(2) Add iodine solution to the mordant to rinse away any remaining water, cover for about one minute, and then rinse with water.

3) Decolorization: Shake off the water on the slide, and place it against a white background. Wash it with 95% alcohol until the alcohol just stops appearing purple, about 20 to 30 seconds. Rinse off the alcohol immediately with water. (4) Re-stain with safranin solution for 1-2 minutes and rinse with water.

(5) After drying, observe under an oil-immersed microscope. Gram-negative bacteria appear red, while Gram-positive bacteria appear purple. The Gram staining reaction of dispersed bacteria is used as the standard; bacteria that are too densely packed often give false positive results.

6) Using the same method, prepare a mixed slide of Escherichia coli and Bacillus subtilis on a glass slide for Gram staining comparison. The key to Gram staining is to strictly control the degree of alcohol decolorization. If the decolorization is excessive, positive bacteria may be mistakenly stained as negative bacteria; if the decolorization is insufficient, negative bacteria may be mistakenly stained as positive bacteria. In addition, the age of the bacteria also affects the staining results. If the positive bacteria are cultured for too long, or have died or some of the bacteria have dissolved on their own, a negative reaction will often be shown.

Second, the Gram staining reaction is an important trait for bacterial classification and identification . It was founded in 1884 by Danish physician Gr II. Gram staining can not only observe the morphology of bacteria but also distinguish all bacteria into two categories: those with blue-purple staining are called Gram-positive bacteria, represented by G+; those with red staining (counter-staining color) are called Gram-negative bacteria, represented by G-. The different responses of bacteria to Gram staining are due to the different composition and structure of their cell walls.

The steps of the Gram staining method are as follows: the cell wall of Gram-positive bacteria is mainly composed of a network structure formed by peptidoglycan. During the staining process, when treated with ethanol, the pore size in the network structure becomes smaller due to dehydration, and the permeability decreases, so that the crystal violet-iodine complex is retained in the cell and is not easy to decolorize, thus appearing bluish purple; the cell wall of Gram-negative bacteria has a low content of peptidoglycan and a high content of lipid substances. When treated with ethanol, the lipid substances dissolve and the permeability of the cell wall increases, making the crystal violet-iodine complex easily extracted by ethanol and decolorized, and then stained with the color of the counterstain (safranine), thus appearing red.