What would account for the following observation From a lab

What would account for the following observation? (From a lab focusing on gram stains, simple stains, and identification methods)

You cannot find any cells on the slide of a simple stain of an overnight culture of Bacillus megaterium. Please explain.

Solution

What would account for the following observation? (From a lab focusing on gram stains, simple stains, and identification methods)

Answer:

Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.

Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. The process involves three steps:

You cannot find any cells on the slide of a simple stain of an overnight culture of Bacillus megaterium. Please explain.

There are may be two-three probable reasons

a) may not be properly stained

b) may not be bacillus fixed to the slide and at the time washing became washout