Streptococcus pneumoniae in Sputum: Introduction, Principle, Procedure and Result Interpretation
Streptococcus pneumoniae gram stain of sputum as shown above picture. 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.
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 staining of Streptococcus pneumoniae
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