First, the basic principle Gram staining reaction is the importance of bacterial classification and identification. It was founded in 1884 by the Danish physician Gram. Gram stain not only observes the morphology of bacteria but also distinguishes all bacteria into two categories: the gram-positive bacteria called blue-purple, called G+; the staining reaction is red (Re-stained color) is called Gram-negative bacteria and is represented by G-. The different responses of bacteria to Gram staining are due to the different composition and structure of their cell walls. The cell wall of Gram-positive bacteria is mainly composed of a network structure formed by peptidoglycan. In the dyeing process, when treated with ethanol, the pore size in the network structure is reduced due to dehydration, and the permeability is lowered. The crystal violet-iodine complex is retained in the cells and is not easily discolored, and thus exhibits a blue-violet color; the cell wall of the Gram-negative bacteria has a low peptidoglycan content and a high lipid content, and when treated with ethanol, the lipid The substance dissolves, and the permeability of the cell wall increases, so that the crystal violet-iodine complex is easily decolorized by ethanol extraction, and then dyed with the color of the counterstain (saffron), and thus appears red.
Gram staining requires four different solutions: a basic dye initial dye; a mordant; a decolorising agent and a counterstain. The basic dyeing solution of the basic dye acts as described in the basic principle of the single staining method of bacteria, and the initial dyeing solution for Gram staining is generally crystal violet. The role of mordant is to increase the affinity or adhesion between the dye and the cell, that is, to help the dye to be fixed on the cell in a certain way, so that it is not easy to fall off, and iodine is a commonly used mordant. The decolorizing agent is to decolorize the stained cells. Different types of cells have different decolorization reactions, some can be decolored, some can not, and the decolorizing agent is usually 95% alcohol. The counterstaining solution is also a basic dye, the color of which is different from the initial dyeing solution. The purpose of counterstaining is to make the decolored cells stain with a different color than the initial dyeing solution, while the undecolored cells still retain the initial dyed color. Thus, the cells are divided into two major groups, G+ and G-, and the commonly used counterstaining solution is safranin.
Second, the equipment E. coli, Bacillus subtilis;
Gram stain, slides, microscopes, etc.
Third, the operation steps
1. The smear was smeared with Bacillus subtilis cultured for 14-16 hours and E. coli cultured for 24 hours (note that the smear should not be too thick), dried and fixed. When it is fixed, it can pass the flame 1-2 times, and it can't be overheated. It is better to use the slides without hot hands.
2. Dyeing (1) Initial dyeing Add a drop of ammonium oxalate crystal violet for about one minute and wash with water.
(2) mordant Drip iodine solution to wash away residual water, and cover for about one minute, wash.
(3) Decolorization The water on the glass slide is cleaned and lined with a white background. It is dripped with 95% alcohol until the outflow of alcohol just does not appear purple. For about 20-30 seconds, immediately flush the alcohol with water.
(4) Counterstaining Dyeing with blush solution for 1-2 minutes, washing with water.
(5) Microscopic examination After drying, observe the oil mirror. Gram-negative bacteria are red, and Gram-positive bacteria are purple. Based on the Gram staining reaction of dispersed bacteria, too dense bacteria are often false positives.
(6) The same method was used to prepare a mixture of Escherichia coli and Bacillus subtilis on a glass slide for Gram staining.
The key to Gram staining is to strictly control the degree of alcohol decolorization. For example, if the discoloration is excessive, the positive bacteria can be mistakenly dyed as negative bacteria; when the discoloration is insufficient, the negative bacteria can 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 and some of the bacteria dissolve themselves, they are often negative.
Fourth, the experimental report
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