Browse the corpus

Gram staining is one of the most crucial staining techniques in microbiology. The name comes from the Danish bacteriologist Hans Christian Gram, who first introduced it in 1882 to identify organisms causing pneumonia.[1] Typically, Gram staining is the first test performed, utilizing crystal violet or methylene blue as the primary color.[2] Organisms that retain the primary color and appear purple-brown under a microscope are gram-positive. In contrast, those that do not take up the primary stain and appear red under a microscope are gram-negative.[3] The first step in Gram staining is using crystal violet dye for the slide's initial staining. The next step, also known as fixing the dye, involves using iodine to form a crystal violet-iodine complex to prevent the easy removal of the dye. Subsequently, a decolorizer, often a solvent of ethanol and acetone, is used to remove the dye. The basic principle of Gram staining is the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.[4] Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.[5] Initially, all bacteria take up crystal violet dye; however, with a solvent, the lipid layer from gram-negative organisms is dissolved, causing them to lose the primary stain. In contrast, the solvent dehydrates the gram-positive cell walls, closing the pores and preventing the diffusion of the violet-iodine complex, which results in the bacteria retaining the stain.[6] The duration of decolorization is a critical step in Gram staining, as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.[7] The final step in Gram staining involves using a basic fuchsin stain to give decolorized gram-negative bacteria a pink color for better identification. This process is also known as counterstaining. Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin. Similarly, Haemophilus spp., Legionella spp., and some anaerobic bacteria stain poorly with safranin.[8]

The interpretation of slides can be difficult if the microscopic smear is thick and clumped. Decolorization time should be closely monitored to avoid under-decolorization or over-decolorization. Thicker smears require a longer decolorizing time. Similarly, cultures should undergo evaluation while they are still fresh. Old cultures tend to lose the peptidoglycan cell walls, predisposing gram-positive cells to be gram-negative or gram-variable. Gram stain is not helpful for organisms without a cell wall, such as Mycoplasma species, and for smaller bacteria, such as Chlamydia and Rickettsia species. Gram stain may not falsely reveal organisms in the following scenario: Use of antibiotics before collecting a specimen Inappropriate age of culture (too young or too old) Fixing the smear before it is dry The smear is too thick Low concentration of crystal violet Excessive heat fixation Excessive washing between steps Insufficient exposure to iodine Prolonged decolorization Excessive counterstaining Lack of experience in preparing and reviewing the slide Sometimes, the Gram stain results may not match the final results of cultures and could lead to the inappropriate use of antibiotics.[15]