Understanding the Gram Stain: A Fundamental Microbiological Technique

The Gram stain is a very simple technique that is based on the use of a dye that allows the bacteria to be observed better than under the microscope.

The bacteria are single-celled microorganisms that habitually coexist with the human being. In many cases they are part of our usual flora and that of our environment without causing any health hazard. In other situations, bacteria can be pathogenic, that is, they produce infections of various types: skin, abdominal, bone, etc. In order to identify and treat them, it is necessary classify them. The different classifications meet various criteria and bacterial characteristics: morphology, metabolism, presence of cell wall, etc.

One of the ways to differentiate bacteria is based on the presence of a cell wall. Thus, there are bacteria with a thick wall (made up of peptidoglycans) and others with a much thinner wall.

What is the Gram stain?

The Gram stain has been used in microbiology since the late 19th century. It is a very simple technique that is based on the use of a Colorant (staining) and that is very useful in medicine.

Gram stain is a type of staining what is done about bacteria to better observe them under the microscope. According to the distribution of peptidoglycan of the cell wall that surrounds them, they stain in one way or another. Thus, the bacteria that do not stain through this technique they are called Gram negative. They are formed by a thinner wall formed by fewer layers of peptidoglycan and a second membrane rich in lipids (which repels Gram stain), under the microscope they appear colorless.

The Gram positive have a much thicker cell wall, formed by a large number of peptidoglycan layers between which the Gram stain is inserted, giving a violet color intense under the microscope and are classified as Gram +.

The great practical contribution of the Gram stain is that it allows to determine the type of as well as its efficacy. The antibiotic of choice must be able to cross the bacterial wall, depending on whether the bacterial wall is gram positive or negative, the most effective antibiotic will be selected.

How is the Gram stain done?

The process is very simple, and the steps to follow would be:

• Collect sample of bacteria to study using a swab (sterile cotton stick).
• To extend said sample on a slide and let it dry.
• Pin up the sample using an alcohol (methanol).
• Apply the dye of gentian violet on the slide and wait one minute.
• Rinse the sample with water and apply a fixative gentian violet (lugol). Lugol and gentian violet form a water-insoluble complex capable of penetrating the wall of bacterial cells.
• Wash the slide again with a mixture of alcohol and acetone for a few seconds.
• Optionally you can add a safranin or fuchsin stain to distinguish gram negatives that will appear under the microscope (rather than colorless) with a pink or red hue. Wash off with water.
• You can now see the sample under a microscope where they will be visualized in color The gram-positive ones are purple and the gram-negative ones are pink-reddish in color.

In the case of gram positive bacteria the insoluble complexes they remain trapped between the peptidoglycan layers of the bacterial wall, undissolved and giving the characteristic purple coloration when observed under the microscope. On the other hand, in gram-negative bacteria, these compounds doe dissolve and lose their color. They will appear colorless or pink-red under the microscope, depending on whether fuchsin has been added at the end of the Gram stain technique.

Between the The most frequent gram positive bacteria are found Staphylococcus, and Enterococcus. Examples of gram-negative bacteria are and Pseudomonas aeuruginosa. The limitations of the technique are given by the characteristics of the sample, the staining protocol, and also the fact that some bacteria do not have a cell wall.

• It is a very simple technique that is based on the use of a dye (staining) and that is very useful in medicine and that allows us to observe bacteria better than under the microscope.
• The great practical contribution of gram staining is that it allows determining the type of antibiotic as well as its efficacy.
• The process for staining is very simple.

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Intensive Medicine Specialist.