Content
Gram stain, 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, by means of Gram staining, it is possible to check, in addition to the shape of the bacteria, the color they acquire, and this result is important for defining other strategies for identifying the bacterial species and for the doctor to indicate preventive treatment according to the microscopically observed characteristics.
Gram staining is usually done routinely in the laboratory and is part of the bacterioscopy exam. Understand what bacterioscopy is and how it is done.
How Gram stain is done
Gram stain is a fast, 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 depending on the laboratory:
- Place some colonies of the bacteria on the slide, adding a drop of water to facilitate homogenization of the colonies;
- Let it dry a little, and 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 the exam;
- When the slide is dry, cover with the violet crystal dye and let it act for about 1 minute;
- Wash the slide with a stream of running water and cover the slide with the lugol, which has the objective of fixing 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;
- Then, wash the slide with running water and apply 95% alcohol, leaving it to 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 the lugol, discoloring these bacteria. However, in the case of gram-positive bacteria, alcohol dehydrates the cell wall of gram-positive bacteria, causing the pores to contract and making them impermeable;
- Then, wash in running water again and cover the slide with the second dye, fuchsin or safranin and let it act for 30 seconds;
- Then, wash the slide under running water and let it dry at room temperature.
As soon as the slide is dry, it is possible to put a drop of immersion oil and observe the slide under the microscope with a 100x objective, being possible to check the presence or absence of bacteria, as well as the presence of yeasts and epithelial cells.
What is it for
Gram staining has the main objective of differentiating bacteria according to the characteristics of the cell wall 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 constrict when exposed to lugol;
- Gram-negative bacteria, which are visualized with a pink / purple color due to the fact that they are discolored by alcohol and stained by safranin or fuchsin.
After viewing the bacteria under the microscope, it is possible that further tests will be carried out in the laboratory to identify the species of the bacterium. However, through the Gram and association with signs and symptoms presented by the person, the doctor can indicate a preventive treatment until the result of more specific exams is available, as it is thus possible to decrease the rate of bacterial replication and prevent complications.