Gram staining is one of the most important microbiology staining procedures. It was initially developed in 1882 by Danish bacteriologist Hans Christian Gram, who used it to detect germs that cause pneumonia. Gram staining, which is often the first test conducted, uses crystal violet or methylene blue as the predominant color.
Gram-positive organisms are organisms that preserve their primary color and look purple-brown under a microscope. Gram-negative organisms are those that do not take up primary stain and look red under a microscope.
The initial staining of the slide with crystal violet dye is the first stage in gram staining. To prevent simple color removal, the following process, often known as fixing the dye, requires employing iodine to generate a crystal violet-iodine complex.
The dye is then removed using a decolorizer, which is usually a solution containing ethanol and acetone. The capacity of the bacterial cell wall to retain the crystal violet dye after solvent treatment is the core concept of gram staining. Gram-positive microbes have more peptidoglycans, whereas gram-negative organisms include more lipids.
Initially, all bacteria take up crystal violet dye; however, the lipid layer of gram-negative organisms is dissolved with the application of solvent. Gram negatives lose their principal stain as the lipid layer dissolves.
Solvent, on the other hand, dehydrates gram-positive cell walls by closing pores, blocking violet-iodine complex transport and leaving bacteria marked. In gram staining, the period of decolorization is important since extended exposure to a decolorizing chemical may eliminate all stains from both kinds of bacteria.
The last stage in gram staining is to apply basic fuchsin stain to decolorized gram-negative bacteria to give them a pink color for easy identification. Counterstain is another name for it.
Safranin is often used as a counterstain, however basic fuchsin stains gram-negative bacteria more powerfully than safranin. Similarly, safranin does not stain Hemophilus spp., Legionella aeruginosa, or several anaerobic bacteria.
Crystal violet, Gram’s iodine, decolorizing agent, and safranin are added to a smear of bacteria on a microscope slide in this order: crystal violet, Gram’s iodine, decolorizing agent, and safranin. Table I shows the color of Gram-negative and Gram-positive bacteria after each reagent was applied.
Gram developed a process that included the use of Crystal Violet (Gentian Violet) as the principal stain, an iodine solution as a mordant, and ethanol as a decolorizer.
Crystal violet stains the cell walls of bacteria. After that, iodine is added as a mordant to produce the crystal violet-iodine complex, which prevents the dye from being removed. Fixing the coloring is what it’s called.
Most bacteria can be stained using basic stains like methylene blue, Gram safranin, or Gram crystal violet. These stains rapidly receive a hydrogen ion or give off a hydroxide ion, resulting in a positively charged stain.
https://bowie1983book.com/ will answer the reagents of the gram stain in order of use are