GRAM STAINING IN BACTERIA
Bacteria are microorganisms that are mostly free living, prokaryotic and contain only one cell. Bacteria
are classified depending on various characteristics such as shape, cell wall composition, mode of
respiration, and mode of nutrition. Gram staining technique classifies bacteria on basis of cell wall
composition; gram negative and gram positive bacteria. The ability of the bacterial cell wall to hold onto
the crystal violet dye after solvent treatment is the fundamental idea behind gram staining.
Gram positive bacteria are those that retain the primary stain; crystal violet during staining. This is
because it possesses a thick peptidoglycan layer on the cell wall according to the research conducted.
These bacteria are also characterized by; absence of an outer membrane, lower lipid content and move
around with aid of cilia and flagella. Teichoic acids and lipoids are present, forming lipoteichoic acids,
which serve as chelating agents, and also for certain types of adherence.
Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-transpeptidase.
A substantially lesser volume of periplasm than that in gram-negative bacteria.
Gram negative bacteria are those that do not retain the crystal violet during staining. Gram negative
bacteria have a thin peptidoglycan layer. There is a cytoplasmic inner cell membrane present. Has an
outer membrane with phospholipids in the inner leaflet and lipopolysaccharides, which are made up of
lipid A, core polysaccharide, and O antigen) in the outer leaflet. The outer membrane contains porins,
which function as pores for specific molecules. Periplasm is a concentrated gel-like material that fills the
gap between the cytoplasmic and outer membranes. Rather than adhering to the peptidoglycan, the S-
layer is directly linked to the outer membrane.
GRAM STAINING STEPS
Components
Crystal violet (primary stain)
Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall)
Decolorizer (ethanol)
Safranin (secondary stain)
Water
STEPS
i. Make a slide of bacteria from a pure culture sample to be stained. Heat fix the sample to
the slide by carefully passing the slide with a drop or small piece of sample on it through
a Bunsen burner three times.
ii. After adding crystal violet, the principal stain, to the sample or slide, let it sit for one
minute. For no more than five seconds, rinse the slide under a mild water stream to get
rid of any loose crystal violet.
iii. As a mordant or an agent that adheres the crystal violet to the bacterial cell wall, add
Gram's iodine and let it sit for one minute.
