Question

"The most widely used stain in microbiology is the Gram stain. Based on differences in the structure of the bacterial cell wall, the Gram stain divides bacteria into two major groups: Gram-positive and Gram-negative. Gram-positive cells have a thick layer of peptidoglycan, a polymer made of amino acids and sugars, in the cell wall. Crystal violet binds to peptidoglycan, rendering the cell purple. Gram-negative cells also have peptidoglycan and initially also stain purple. Since the peptidoglycan layer is much thinner, the crystal violet staining is washed out when the cells are exposed to ethanol. They are then stained by the pink counterstain, commonly safranin or fuchsine. Gram-positive cells also take up safranin, but because of the dark purple staining of the cell wall, the lighter pink color can not be observed. The Gram stain is typically the first step in the identification of unknown bacteria. Peptidoglycan is the target of a class of antibiotics called B-lactam antibiotics. While the peptidoglycan layer is thinner in Gram-negative bacteria, these organisms are less susceptible to B-lactam antibiotics as they have an outer membrane, an additional lipid bilayer that surrounds the cell wall. Gram-positive bacteria have a single lipid bilayer inside the cell wall. Like with all staining methods described here, the basic shape and morphology of the bacterium can also be observed in Gram-stained samples."

The passage above states:  "Gram-positive cells also take up safranin, but because of the dark purple staining of the cell wall, the lighter pink color can not be observed.

but if we can not observe the light pink in gram-positive bacteria how then do we determine if it is gram-positive? can someone please explain what the passage is trying to say. it is confusing.

Answer 1

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.

The passage says that both types of bacteria take up the safranin stain. It clearly means that in a culture of the bacteria where there is a mixture of both types of bacteria when safranin is added, gram-negative bacteria easily takes it up as it is completely decolourized in the previous step by the addition of alcohol but as the gram-positive ones are not decolourized i.e they retain their purple colour, so it is difficult for them to stain red though both types of bacteria take up the red colour. The red colour is clearly visible in the colourless gram-negative ones and not visible in the already violet gram-positive ones.