Staining allows us to detect microbes in the microscopic material, determine their number, and quickly study the morphological features of microorganisms. The ratio of bacteria to Gram stain is determined by their ability to retain the complex of gentian violet with iodine formed during the staining process. For coloring, the sample must be applied in a thin layer for accurate staining results. In thick smears, in which cells are in clusters, the stain often precipitates, and gram-negative organisms are more difficult to decolorize. On the contrary, gram-positive organisms in clusters become discolored much faster than singly located cells. Bacteria in the preparation should be at the greatest possible distance from one another. All mixtures before dyeing must be prepared in advance and measured in the amount required. Otherwise, the consequences of dyeing may be distorted or completely incorrect.
All bacteria in relation to the Gram stain are divided into gram-positive – dark purple and gram-negative – red. The ability to stain in one color or another depends on the structure of their cell wall and correlates with many other properties of bacteria. In gram-positive bacteria, aromatic and sulfur-containing amino acids are absent in the cell wall, and low content of lipids is noted, in gram-negative, on the contrary, these substances are contained in large quantities (Tortora, Funke & Case, 2019). In addition, gram-positive bacteria have a magnesium salt of ribonucleic acid, which is absent in gram-negative bacteria. Formed a strong chemical complex with protein, gentian violet, and iodine, which is not destroyed by the short-term action of alcohol. Gram-negative do not form such a complex because they are easily discolored by alcohol. Fuchsin stains gram-negative microorganisms red, while the positive ones become darker. Gram staining of bacteria is based on the different abilities of microorganisms to retain triphenylmethane dyes in the cell – crystal violet or gentian violet. The essence of the method is contingent on the difference in the chemical composition and structure of the bacterial cell wall. As already mentioned, all bacteria on this basis are divided into two groups: gram-positive – Gram-staining, gram-negative – not Gram-staining.
The main mistake made when staining by Gram is over-discoloration or under-discoloration of the smear with alcohol. In the first case, gram-positive bacteria may lose their original color with gentian violet and acquire a red color (characteristic of gram-negative bacteria) as a result of subsequent staining of the smear with fuchsin. In the second case, gram-negative bacteria can retain the blue-violet color of gentian violet.
Certain steps must be clearly followed in a sequence in the Gram stain process. The following solutions should be prepared for staining: 1) an alcoholic solution of crystal violet; 2) Lugol’s iodine solution; 3) a bleaching solution; 4) Safranin. If students cross the primary and secondary dyes and take Safranin as the primary stain and Crystal Violet as the counter stain, then the result will be wrong (Tripathi & Sapra, 2021). The result will be incorrect because of the different structures of Gram-positive and Gram-negative cells. When cells are stained with crystal violet and then stained with Lugol’s solution, peptidoglycan + iodine + gentian violet complex is formed in the CM, which is then decolorized with alcohol. In Gram-negatives, the entire staining complex is washed out of the cell wall, and they become colorless. In Gram positives, the complex remains bound in a thick layer of peptidoglycan. As a result, after the first staining, Gram positives become purple, and Gram-negatives become transparent. Stage 2 of Gram staining – the stage of additional staining with Safranin. After the second step, Gram positives remain purple, and Gram-negatives turn pink.
In conclusion, it follows that if mixing the dyes is allowed and stains first with Safranin and then with crystal violet, then the cells will not discolor and will not stain as they should. Because of this, it will not be possible to determine Gram-positive and Gram-negative cells; in this case, the meaning of staining disappears. Therefore, it is always very important to follow the staining sequence.
Tortora, G. J., Funke, B. R., & Case, C. L. (2019). Microbiology: an introduction. (13th ed.). Pearson. p.77 – 98.
Tripathi, N. Sapra, A. (2021). Gram staining. StatPearls Publishing, Treasure Island (FL).