How to Do a Gram Stain? – Biology Lab Techniques

Gram staining is used to differentiate
bacterial isolates into 2 large groups based on the properties of their cell
walls. The method was developed in 1884 by Danish scientist Hans Christian Gram. Following staining, Gram-positive bacteria will be colored in purple while Gram-negative bacteria will appear pink. This difference is due to the Gram
negative bacteria thinnest layer of peptidoglycan, which will lose the purple
color during the staining hence being able to display the lighter pink color of the counter stain. To start the procedure, a smear of bacterial culture is applied to a microscope slide and heat-fixed. Heat fixation kills the bacteria and ensures that it won’t rinse out during the various washes. Four reagents are needed to complete the procedure: crystal violet, Lugol’s iodine, either ethanol or acetone, and safranin. The primary stain crystal violet is first
applied to the smear in order to stain all cells. The slide is left for one minute. The excess crystal violet is rinsed out with distilled water. Lugol’s reagent, which contains iodine, binds to crystal violet and traps it within the cell. It is left to bind for one minute. Like before, excess reagent is rinsed
with distilled water. The slide is washed for 10 seconds with
either acetone or ethanol in order to remove the weakly bound crystal violet from the Gram-negative bacteria. Gram-positive bacteria, on the other hand, will remain stained. The slide is promptly rinsed with distilled water to avoid excess decolorization. Safranin is used to counter stain in
pink the Gram-negative cells that lost crystal violet. Gram-positive bacteria are still stained with the darker crystal violet, and therefore, safranin will be
invisible. A last thorough rinsing with distilled water is performed. The slide is then gently dried on tissue paper before observation under the microscope using a 100X immersion oil objective. This is the Gram stain of the
Gram-negative bacilli Escherichia coli. Note its characteristic pink color and
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