There are many factors that can affect gram staining procedures, some of them are:
1. The age of bacterial culture: bacterial culture with older cells (more than 24 hours) lose Gram positivity and will appear as Gram-negative.
2. Heat: the application of too much heat during fixation can lead to loss of gram positivity. Excessive heat destroys cell wall. Gram-positive begin to appear as gram negative.
3. Overcrowding of cells: overcrowding of cells is also a factor that affects gram staining procedure, it can lead to improper decolorization. When the cell is overcrowded it affects discoloration, thin smears decolorize faster than thick smears.
There are various factors that can affect the gram staining procedure such as the age of culture or type of growth medium. However, the major factor that can influence the grain staining procedure is their different cell walls. It is due to their consistent difference between the cell walls, Gram stain bacteria are divided into two types: Gram-positive bacteria and Gram-negative bacteria.
Gram-positive bacteria have 90% of the cell wall whereas Gram-negative bacteria have 10% of the cell wall.
The main factor that influences the result of a Gram stain (whether an organism is classified as Gram positive or Gram negative) is the structure of their cell wall.
The Gram positive cell wall is much thicker than that of the gram negative organism, thus it retains the dark purple stain. The Gram negative cell wall is much thinner, and it is readily decolorized with alchohol.