Gram Stain
It gets its name from the Danish bacteriologist Hans Christian Gram, who first introduced it in 1882, mainly to identify organisms causing pneumonia
INDICATION - whenever a bacterial infection is suspected for easy and early diagnosis
Gram’s stain classifies the bacteria into two major groups based on the cell wall of the bacteria
Gram- positive bacteria retain the gentian violet-iodine complex and appear blue-purple, whereas the Gram- negative bacteria lose their gentian violet-iodine complex with decolorization step and appear pink when counterstained with safranin.
A 5-minute Gram staining procedure as well as a 5-second Gram staining procedure is also available although the 5 minute procedure is preferred
Overall, Gram’s stain is accurate in 61 percent of cases of bacterial keratitis.
If performed correctly, Gram’s stain identifies the organism correctly in upto 75 percent of the cases caused by a single organism and in 37 percent cases of polymicrobial keratitis.
DIFFICULTIES IN GRAM STAINING:
-Sometimes only small number of organisms is present in the smear, which may be difficult to identify as these are usually present in areas which contain necrotic epithelial cells and numerous large polymorphonuclear cells.
-Gram-negative organisms are more difficult to identify than the Gram-positive organism due to their lighter color
-Gram-negative organism may appear gram-positive if decolorization is inadequate.
-Caution is also mandatory against various artifacts, which may accompany Gram’s stain such as stained deposits, carbon particles, talcum powder, sodium chloride, crystals, melanin and granules.
-Precipitated gentian violet may mimic gram-positive cocci.
STEPS:
-Crystal violet stain is added over the fixed culture.
-After 10 to 60 seconds, the stain is poured off, and the excess stain is rinsed with water. The goal is to wash off the stain without losing the fixed culture.
-Iodine solution is used to cover the smear for 10 to 60 seconds. This step is known as "fixing the dye." The iodine solution is poured off, and the slide is rinsed with running water. Excess water from the surface is shaken off.
-A few drops of decolorizer are added to the slide. Decolorizers are often the mixed solvents of ethanol and acetone. This step is known as "solvent treatment." The slide is rinsed with water for 5 seconds. To prevent excess decolorization in the gram-positive cells, stop adding decolorizer as soon as the solvent is not colored as it flows over the slide.
-The smear is counterstained with basic fuchsin solution for 40 to 60 seconds. The fuchsin solution is washed off with water, and excess water is blotted with the bibulous paper. The slide can also be air-dried after shaking off excess water.
-The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.
-Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content
-Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer from gram-negative organisms is dissolved
-With the dissolution of the lipid layer, gram negatives lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure of pores, preventing diffusion of violet-iodine complex, and thus, bacteria remain stained.
-The length 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
EXAMPLES:
Examples of gram-positive organisms are:
-Cocci: Staphylococcus species, and Streptococcus species
-Bacilli: Corynebacterium species, Clostridium species, Clostridioides species, and Listeria species
Examples of gram-negative organisms are:
-Cocci: Neisseria gonorrhoeae, Neisseria meningitidis, and Moraxella species
-Bacilli: Escherichia coil, Pseudomonas species, Proteus species, and Klebsiella species
Examples of gram-variable organisms include: Actinomyces species
Image from Rajan Eye Care Hospital
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