GRAM STAIN

(RRL)
Gram staining is a common technique used to differentiate two large groups of bacteria
based on their different cell wall constituents. The Gram stain procedure distinguishes between
Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive
bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which
retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain
red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet
during the discoloring process. (Monica Z. Bruckner, 2009)

(methodology)
Gram staining involves three processes: staining with a water-soluble dye called crystal violet,
decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a
peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria,
Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the
decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead
stained by the safranin in the final staining process. The process involves three steps:

1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note
that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain
results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Grams iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add drop
by drop to slide until decolorizing agent running from the slide runs clear .
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then
blot dry with absorbent paper.
8. Observe the results of the staining procedure under oil immersion (100x) using a Bright field
microscope.

If the smear stained purple it was reported as gram-positive bacteria but if the smear staine pink
it was reported as gram-negative bacteria. Smears were examined under the microscope using
the oil immersion objective (100x) and checked for the shape of the isolated bacteria (i.e cocci
and bacilli). Cocci is any bacterium that has a spherical, ovoid, or generally round shape and
bacilli is a rod-shaped bacterium.
https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
http://microbeonline.com/gram-staining-principle-procedure-results/