Gram staining technique is an express method that allows you to determine the type of bacteria. The pathogen detection technology was developed by the bacteriologist Hans Gram. He was an unusual person, modest. In his work, he said: “I publish the painting technique, knowingly knowing that it has some drawbacks, but I do not lose hope that my method will be finalized and become useful.” And so it happened. His technology has become one of the leading in determining the type of bacteria. The Gram stain technique is based on the structure of prokaryotes, which is why bacteria acquire one or another color.
Recommendations
Before proceeding with staining, it is necessary to prepare the workplace and sample. The quality of the latter determines how good the result will be. For example, a sputum of deep cough, and not just saliva, is taken for analysis.
When working with samples, safety rules must be followed. During work, gloves should be on hand. You should also be careful when working with alcohol, acetone. These substances are flammable and flammable when heated.
When working with glass slides, they are carefully taken with tweezers or two fingers on the side edges so as not to touch the smear.
Preparation for work
The Gram stain technique begins with preparing oneself for the upcoming work. Sterile gloves are always worn on the hands, long hair should be removed, tied. The workplace should be located in a sterile, well-ventilated area. Before the manipulation, the performance of the microscope must be checked.
The next step is to prepare the slides. If they are not clean enough, then the glasses are washed under running water with soap. This will help remove dirt, grease. The surface must be treated with alcohol or another method used in the laboratory.
Sample preparation
Gram stain technique involves sample preparation. To do this, take a slide and gently place a sample on its center. The technique of staining Gram smears makes it possible to see the type of bacteria cultivated in a Petri dish.
To begin work, a thin layer of the test liquid is applied to the glass. You should not wait more than a day from the start of the sampling of the material to the analysis, since many pathogens break down the cell wall, and the reaction to staining becomes completely unpredictable. If it is supposed to begin the study of tissues, then a couple of drops of the material should be applied to the glass slide. The liquid is distributed over the glass evenly with an edge of another glass. The sample is allowed to dry.
If a sample is taken from a Petri dish for coloring, then the procedure for taking material is carried out with a loop. And when examining a smear, it is pressed against a glass slide with another glass.
Sample fixation
The Gram stain technique involves specimen fixation. Under the influence of heat, bacteria adhere to the glass and do not wash off.
In order to fix the smear, it is necessary to slide a glass slide over the burner. Do not overheat the glass so that no changes appear in the sample.
You can fix the sample with methanol. To do this, apply a couple drops of methyl alcohol to the glass, and drain the excess. Glass is dried in a natural way. This method is preferable due to the fact that it damages the studied cells less and shows a cleaner background.
Dyeing
The technique for preparing a smear of Gram stain preparations involves staining in the container in which the sample will be placed. Usually a tray or glass saucer is used for this. A slide with the preparation is placed in it so that the liquid flows into the tray.
First, a solution of crystalline violet or gentian violet is applied to the sample. It is collected in a pipette and a few drops are applied to the drug. After a minute, the result is evaluated. All gram-positive pathogens turn purple.
Then the dye is washed off with water so that it slightly washed the smear. Next, iodine is applied to the sample and left for a minute. After that, it is also washed off gently. After this procedure, a bleaching agent is applied to the glass. It can be a solution of acetone and alcohol. The smear is treated with the solution until the violet hue disappears.
A contrast dye is applied to the smear. It can be fuchsin or safran. It allows you to create a contrast between gram-negative and gram-positive bacteria. As a result of the effect of the drug, gram-negative pathogens become fuchsia. The dye is washed off with water.
The final step is to dry the slide. After this, the Gram stain technique (it is used quite often in microbiology) is considered completed.
Evaluation of the results
A glass slide is placed under the microscope. Gram-positive bacteria in the smear will be purple. Gram-negative bacteria will turn pink or red. This diversity arises as a result of the structural features of the walls of pathogens. In some, only the violet dye penetrates, while in others it is fuchsian.
The erroneous result may be caused by the prolonged exposure of the glass slide under the influence of a bleaching liquid. Therefore, the procedure is carried out twice to make sure the diagnosis is correct. Repeated analysis allows you to verify that the color is correct.
When working with pathogenic microorganisms, always be careful, as your health depends on it.