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How to Gram Stain

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1884 by the Danish scientist H. Gram. This method is based on the different ability of microorganisms to retain triphenylmethane dyes - crystalline violet or gentian violet - in the cell.

Gram stain is the most versatile of complex painting methods.

1. Fix. smear cover filter boom. and a carbolic solution of gentian violet is applied to it. Staining is carried out for 1 to 2 minutes. 2. The paper is removed, the dye is drained and, without rinsing the preparation with water, it is treated for 1 to 2 minutes with a Lugol solution until blackening. 3. Lugol's solution is drained, the stained smear is discolored with 96º ethyl alcohol for no more than 30 s. 4. The drug is washed with water. 5. The smear is additionally stained for 1 to 2 minutes

aqueous solution of fuchsin. 6. The dye is drained, the drug is washed with water, dried

with filter paper and microscope with immersion system. Gram-positive bacteria turn purplecolor, gram-negative - in pink.

Gram stain

Gram stain (or Gram method) - an empirically derived method for distinguishing bacteria by staining them with a specific method into two large groups: Gram-positive and Gram-negative), which differ in the chemical and physical properties of their cell wall.

Using

Gram stain is one of the most useful staining procedures in laboratory research in microbiology. The procedure is widely used as a tool to distinguish between Gram-negative and Gram-positive bacteria, usually the first step in determining the identity of a specific bacterial sample.

In medicine, Gram stain is performed on a blood test or biopsy when an infection is suspected. This method is much faster than culture, and is especially important when determining the type of infection is necessary for the prognosis and choice of treatment method. For example, in the diagnosis of cerebrospinal fluid with meningitis and joint fluid for septic arthritis.

When analyzing, for example, Coca and spore-bearing forms of bacteria, as well as yeast, are gram-positive and stain in blue-black (dark blue) color, most other bacteria are gram-negative and stain in red, the nuclei of eukaryotic cells become bright red, and the cytoplasm is pink.

Staining Technique

Gram staining refers to a complex method of staining, when two dyes affect the smear, one of which is the main, and the other is optional. In addition to dyes, for complex coloring methods, non-disintegrating substances are used: alcohol, acids, and others. For Gram staining, triphenylmethane group dyes are often used: gentian, methyl violet or a violet crystal. Gram-positive (gram (+)) microorganisms give a strong connection with the indicated dyes and iodine. However, they do not discolor when exposed to alcohol, as a result of which, with additional fuchsin staining with Gram (+), microorganisms do not change, the violet color is first attached.

Gram-negative (gram (-)) microorganisms form compounds with the main dyes and iodine, it is easily destroyed by the action of alcohol. As a result, microbes become discolored and then stained with fuchsin, acquiring a red color.

Preparation of material for painting

The material of the test is distributed in a thin layer on the surface of well-defatted glass. The prepared smear is dried in air and, after complete drying, is fixed.

When fixing, the smear is fixed on the surface of the glass, and therefore, with further staining of the drug, the cells are not washed off. In addition, killed microbial cells stain better than living ones. There are physical methods of fixation, which are based on the action of high temperature on the cell, and chemical methods involving the use of chemical agents that cause coagulation of cytoplasmic proteins.

Physical method of fixing Glass with the drug take tweezers and smooth movement is carried out 2-3 times over the upper part of the burner flame. The entire fixation process should take no more than 2 s. The reliability of fixation is checked by this method: a glass-free surface of the glass is applied to the back surface of the left hand. With proper fixation, the glass should be hot, but not cause a burn sensation (70-80 ° C).

Chemical Fixation Method For fixations of smears are used methyl alcohol, acetone, a mixture of Nikiforov (a mixture of ethyl alcohol 96% and anesthetic ether in a ratio of 1: 1), Carnoy liquid (ethyl alcohol 96% - 60%, chloroform 30%, glacial acetic acid 10%). A glass with a dried smear is immersed in a glass with a fixing substance for 10-15 minutes and then dried in air.

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