E. coli Gram Stain: Introduction, Principle, Procedure and Result Interpretation
Introduction of E. coli
E. coli in Gram stain as shown above picture. They are bacteria that come under the family Enterobacteriaceae. They are normally found in the environment, foods, and intestines of people and animals. They are a large and diverse group of bacteria. They are normal flora when they are in our intestines. But if they spread to another part of your body, they can cause severe infections like urinary tract infections (UTIs), diarrhea, pyogenic infections, septicemia, respiratory illness and pneumonia, and other illnesses. Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory. The morphology of these bacteria is Gram-negative rods measuring 1-3 µm × 0.4-0.7 µm. Most strains are motile by peritrichate flagella. They are non-sporing and non-encapsulated.
Principle of Gram Stain
The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.
Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.
Requirements for Gram Stain of
a) Compound light microscope
b) Reagents and glasswares
Clean grease-free slides
Crystal violet (Basic dye)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red