HomeHealthHealthGram stain: step by step and what it is for

Gram stain: step by step and what it is for

Gram staining, or simply Gram, is a quick and simple technique that aims to differentiate bacteria according to the characteristics of their cell wall after exposure to different dyes and solutions.

Thus, through Gram staining, it is possible to verify, in addition to the shape of the bacteria, the color they acquire, which is an important result for defining other strategies for identifying the bacterial species and for the doctor to indicate a preventive treatment according to the features observed microscopically.

Gram stain is usually done routinely in the laboratory and is part of the bacterioscopy examination. Understand what bacterioscopy is and how it is done.

How is Gram stain done?

Gram staining is a quick, practical and inexpensive method for identifying bacteria responsible for infections, being useful for doctors to indicate a preventive treatment for the infection that may be occurring, since specific characteristics of these groups of bacteria are known,

Gram staining is done in 7 main steps, however the protocol may vary by laboratory:

  1. Place some colonies of the bacteria on the slide, adding a drop of water to facilitate the homogenization of the colonies;
  2. Allow to dry a little, the blade can pass quickly through the flame to favor drying, however it is important to pay attention to the temperature, since if the temperature is too high it is possible that there is a change in the structure of the bacteria, which can interfere with the result of the exam;
  3. When the slide is dry, cover it with the crystal violet dye and let it act for about 1 minute;
  4. Wash the slide with a stream of running water and cover the slide with lugol, which is intended to fix the blue dye, and let it act for 1 minute. Both types of bacteria can absorb the complex formed by the dye and the lugol, turning blue;
  5. Then, wash the slide with running water and apply 95% alcohol, letting it act for 30 seconds. Alcohol is responsible for dissolving the lipid membrane that forms gram-negative bacteria and, thus, removing the complex formed between the dye and lugol, decolorizing these bacteria. However, in the case of gram-positive bacteria, alcohol dehydrates the cell wall of gram-positive bacteria, causing pores to contract and making them impermeable;
  6. Afterwards, wash it again in running water and cover the slide with the second dye, fuchsin or safranin, and let it act for 30 seconds;
  7. Then, wash the slide with running water and let it dry at room temperature.

Once the slide is dry, it is possible to place a drop of immersion oil and observe the slide under a microscope with a 100x objective, being possible to verify the presence or absence of bacteria, as well as the presence of yeasts and epithelial cells.

what is it for

Gram staining aims to differentiate bacteria according to cell wall characteristics and general morphology. Thus, according to the characteristics observed under the microscope, bacteria can be classified into:

  • Gram-positive bacteria, which are visualized with a blue color due to the fact that they are not discolored by alcohol, since they have a thicker cell wall and their pores contract when exposed to lugol;
  • Gram-negative bacteria, which are visualized as pink/purple due to being bleached by alcohol and stained by safranin or fuchsin.

After viewing the bacteria under the microscope, it is possible that other tests are carried out in the laboratory to identify the species of bacteria. However, through the Gram and association with signs and symptoms presented by the person, the doctor can indicate a preventive treatment until the results of more specific tests are available, as this way it is possible to decrease the bacterial replication rate and prevent complications.

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