What is the significance of Gram staining?

Objective. Kills microorganisms and fixes their cellular structures.

Identify cellular bacterial types.

It is convenient to observe and analyze the pathogenicity of cellular bacteria.

Request. Pay attention to the destaining time, decolorization is the key to the success of Gram staining, insufficient decolorization will cause false positives, and excessive decolorization will cause false negatives.

Staining was performed with strains in the active growth phase.

The smear should not be too thick to avoid false positives caused by incomplete decolorization.

Is that all? Or do you write the test report, the same thing that you wrote at the beginning?

Principle After the initial staining of crystal violet and iodine solution, a complex of crystal violet and iodine insoluble in the cell wall is formed, and the Gram-positive bacteria due to their thick cell wall, peptidoglycan network layers and dense cross-linking, so when they are decolorized with ethanol or acetone, the mesh is reduced due to water loss, and it does not contain lipids, so there will be no gaps in ethanol treatment, so the crystal violet and iodine complex can be firmly left in the wall, so that it is still purple; However, because of its thin cell wall, high lipid content of the outer membrane layer, thin peptidoglycan layer and poor cross-linking, the outer membrane dominated by lipids is rapidly dissolved after encountering the decolorizing agent, and the thin and loose peptidoglycan network cannot prevent the dissolution of crystal violet and iodine complex, so it is still colorless after decolorization by ethanol, and then counterstained by red dyes such as sand yellow, which makes the Gram-negative bacteria red.

The significance of Gram stain is as follows:According to the nature of Gram staining, the identification range of bacteria can be narrowed, which is conducive to further identification of bacterial categories. Therefore, different diagnoses can be made for different diseases.

Since Gram bacteria also exhibit different susceptibility to antibiotics, Gram staining also has significant implications for antibiotic selection.

The principle of Gram stainingGram stain.

The principle is gram-positive bacteria.

It is quite different from the negative bacteria in terms of chemical and physiological properties, and the chromosomal reaction is also different.

The binding of the complex is relatively strong and not easy to decolorize, while the binding of negative bacteria is relatively weak and easy to decolorize, which is the main basis for the staining reaction.

Refer to the above content: Encyclopedia - Gram staining.

The mastery of decolorization time is the key to the success or failure of Gram staining. Filial piety.

There are four basic steps of Gram staining: crystal violet primary staining, iodine liquid mordant staining, ethanol decolorization, and sand yellow counterstaining, among which ethanol decolorization is the key. Because if the destaining is excessive, the banquet may cause false negatives for gram-positive bacteria, and if the decolorization is not sufficient, it may make gram-negative bacteria false-positive.

Gram staining is a widely used identification staining method in bacteriology, through the blind crystal violet primary staining and iodine liquid mordant staining, in the cell wall formed a water-insoluble crystal violet and iodine complex, Gram-positive bacteria due to its cell wall thicker, peptidoglycan network layers and dense cross-linking, Gram staining is a counterstaining method, that is, after the specimen is fixed, first stained with gentian violet, iodine solution mordant with alcohol after mordantization, and then with red counterstaining.

The steps of Gram staining are: primary staining, defloating, mordantization, decolorization, and counterstaining.

1. Fixation: Under sterile conditions, pick a small amount of bacteria with an inoculation loop and coat it on the glass slide.

2. De-floating: Dyeing with ammonium oxalate crystal violet for one minute, and washing with water to remove the floating color.

3. Mordant: mordant with iodine-potassium iodide solution for one minute, and pour out the excess solution.

4. Decolorization: Decolorization with neutral decolorizing agent, Gram-positive bacteria are not faded into purple, and Gram-negative bacteria are faded into colorless.

5. Chafinch counterstaining: 30 seconds of red staining solution or sand yellow counterstaining, Gram-negative bacteria are still colorless, and Gram-negative bacteria are red.

Microscopic examination: After drying, observe with an oil microscope. Gram-negative bacteria are red in color, and Gram-positive bacteria are purple in color. Based on the Gram staining reaction of scattered bacteria, bacteria that are too dense often give false positives.

Dye Composition :

Basic dyes: This has been discussed in simple dyeing, here with crystal violet.

Mordant: Its function is to enhance the affinity between the dye and bacteria, and better strengthen the binding of the dye to the cell. A commonly used mordant is iodine solution.

Destaining agent: Helps the dye decolorize the cells being stained. Bacteria are distinguished by the different degrees of difficulty with which the dye is decolorized.

Gram-positive bacteria are less susceptible to decolorization by poor decolorizers, whereas gram-negative bacteria are susceptible to decolorization. Commonly used decolorizing agents are prochloride or ethanol, which is 95% ethanol used here.

Counterstain solution: also a basic dye whose purpose is to re-stain destained bacteria with another color for comparison with non-depigmented bacteria. An aqueous solution of saffron is used here.

Gram stain.

Generally, it includes four steps, such as primary dyeing, mordant dyeing, decolorization, and counterstaining, and the specific operation methods are:

1) Smear fixation.

2) Ammonium oxalate crystal violet staining for 1 minute.

3) Rinse with distilled water.

4) Cover with iodine solution for about 1 minute.

5) Wash with water and use absorbent paper to absorb the water.

6) Add a few drops of 95% alcohol, shake gently to decolorize, wash with water after 20 seconds to absorb the water.

7) After dyeing cherry jujube with red staining solution (dilute) for 1 minute, rinse with distilled water. Drying, microscopic examination.

Gram stain.

Generally, it includes four steps: primary dyeing, mordant dyeing, decolorization, and counterstaining. Result: Gram-positive bacteria.

It is blue-purple, gram-negative.

Red in color. There are many differences between gram-positive and gram-negative bacteria in terms of chemical composition and physiological properties, and the staining reaction is different. It is now generally believed that gram-positive bacteria contain a special complex of nuclear proteins, magnesium salts, and polysaccharides.

It is associated with iodine and crystal violet.

The complex is very firmly bound and not easy to decolorize, and the negative bacterial complex is bound to the bottom and adsorbs the dye.

Poor, easy to decolorize, which is the main basis for staining reactions.