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The Gram stain procedure
- First, an inoculum is taken from a culture using an inoculation loop and put on a slide. If the culture is solid, it is diluted by adding a drop of water on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put way too much inoculum at this step.
- The specimen is heat-fixed by passing the slide through a bunsen flame a few times, without allowing the slide to become hot to the touch.
- A basic dye, crystal violet or gentian violet, is used to stain the slide. This dye is taken up by both Gram-positive and Gram-negative bacteria. Allow to stain for 1 minute. The slide should look purple to the unaided eye, and if examined microscopically at this point both Gram-positive and Gram-negative bacteria are purple. Lugol can also be used instead of crystal violet.
- Rinse off with water for a maximum of 5 seconds.
- Add iodine solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
- Rinse with water.
- Apply 95% ethanol or a mixture of acetone and alcohol several times until no more colour appears to come from the sample. This washes away all the unbound basic dye, (usually crystal violet) and leaves Gram-positive organisms stained purple and Gram-negative organisms unstained (colourless).
- Rinse with water.
- Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or fuchsin . This stain is taken up by both Gram-positive and Gram-negative organisms, but does not alter the colour of Gram-positive organism much, as they are already purple. It does, however, make the Gram-negative organisms pinkish-red.
- Blot gently and allow to dry. Do not smear.
Inspect the slide under a microscope
- Gram-positive organisms will appear blue-black or purple.
- Gram-negative organisms will appear red.
- organisms that cannot reliably be differentiated by this staining technique are said to be Gram-variable
Why does this work?
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye/iodine complex. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. In addition to an inner membrane, they also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.
The decolorizing mixture causes dehydration of the multilayered peptidoglycan in the Gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in Gram-negative bacteria, the decolorizing mixture acts as a lipid solvent and dissolves the outer membrane of the Gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the Gram-negative cell is decolorized. The decolorization step is the crucial one, and requires some degree of skill, as Gram-positivity is not an all-or-none phenomenon.
As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" - the human body recognises a foreign body by its antigens; if they are hidden, it becomes harder for the body to detect that bacterium. Often the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane. Lipopolysaccharide is an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan, and in fact the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin.
See also: Staining (biology)
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