Gram-staining is a procedure established by Christian Gram in the late 19th century. It classifies bacteria that remain purple or violet after the staining and washing steps as Gram+, and those that appear pink as Gram- . The difference in coloration is based on the cell wall composition of cells. The cell wall is composed of layers of peptidoglycan, a complex molecule composed of smaller sugar and aminoacid molecules linked alternatively in a chain. Cell walls of Gram+ bacteria have thicker peptidoglycan layers in their cell walls than of Gram- bacteria. So, what happens during Gram-staining is that initially, crystal violet solution stains all cells purple, however, it gets washed off from those bacteria having cell walls with thin peptidoglycan layers. Safranin solution re-stains those cells that lost their purple color in pink while bacteria with thick peptidoglycan layers in their cell walls remain purple. The loss of color during staining attributes the minus sign (-) and we name these bacterial cells Gram – .
Bacteria are unicellular prokaryotic microorganisms. The word prokaryote comes from the Greek πρό (pro) “before” and καρυόν (karyon) “nut or kernel”. “Before kernel” reflects the fact that prokaryotes don’t have a distinct nucleus enclosing the hereditary information of the cell, DNA, that is found in a compact form called chromosome (carrying genes, segments of DNA that encode full proteins or regulatory RNA molecules essential for cell function). Prokaryotes also lack membrane-bound organelles that are specialized compartments where cellular functions such as respiration and protein packaging are carried out. These two major features help us distinguish prokaryotes from eukaryotes (living organisms that have eu “true, genuine” nucleus).