Gram staining is the most important differential coloring method for highlighting bacterial species. The method is characterized by the use of 2 dyes in succession: cristalviolet and safranin.
Initially the chrystalerate is precipitated by an oxidation with the iodine solution (lugol). The resulting complex is fixed on the bacterial cellular partetewith bonds of different nature and intensity. The differentiating solution eliminates the chrystalviolet complex – iodine from the capsule of some bacteria but does not act on others that remain colored and define themselves as positive gram. The decolored bacteria are then highlighted with a red contrast dye and are called negative Grams. The properties of Gram positive bacteria are attributed to retaining the dye complex – iodine to the bond that forms between the complex and a molecule found only in positive Grams: ribonucleolate magnesium.
Gram Procedure Coloring
- Bring the section to the distilled water.
- Pour the Safranina into a vertical tub for histology, put the slide in and incubate at 56-58 degrees C for 15 minutes;
- Wash in distilled water.
- Place on section 10 drops of the Violet: leave to act 3 minutes.
- Drain the slide and, without washing it, give it 10 drops of iodine: leave for 3 minutes.
- Wash in distilled water and dry the slide first in filter paper, then in the air for 10 minutes.
- Pour the contents of the Decoloring into a vertical tub for histology: shake the slide for 1 minute;
GRAM COLORING RESULTS
Blue Gram-Positive Bacteria
Red Gram-negative bacteria
Gram coloring relationship with the cell wall
The bacterial wall is a very important structure for the physiology of the bacterium and often also for its pathogenic action, located outside the cell membrane and can sometimes be surrounded by a mucous capsule. Its functions range from nutrient filter to osmotic resistance. The key component is a huge polymer called peptidoglycan, the structural unit of which consists of two carbohydrates: N-acetyl-glucosammine and N-acetyl-muramic acid (NAG and NAM) chained together (beta 1-6 and beta 1-4 bonds). The NAM is also bound a tetrapeptide in position 3. Let's imagine a chain of NAG-NAM-NAG-NAM… arranged as you see it, the polypeptide chain would be tied to the NAM down. In a different plan (let's case in front of that row) there would be another chain with the same characteristics. What connects you? Very often it is a chain of wisteria (addressed in this case perpendicular to the screen). This is the essential scaffolding of the wall. In the three dimensions, in the positive Grams (G) this chain forms a compact layer, multiplied by many layers held together by other molecules such as theic acid.
The result is to have a very thick wall, which fixes the violet cristal of the coloring and that does not allow its elimination with ethanol (click here for the full wall, here for a bacterium G) ). The G's are often referred to as monoderms. In negative Gram bacteria (G-), on the other hand, the peptideglican layer is very thin, and divides two layers of phosfolipids: that of the plasma membrane and that of the outer membrane, and the space between the peptidoglycan and the plasma membrane is called periplasmic space. Proteins, such as porines, and bacterial lipoposaccaride (LPS) are present in the outer membrane. Sometimes the saccharine chain (antigen-O) is missing or reduced, in which case we talk about lipooligosaccaride (LOS).