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The Results of Gram Stain Reactions for E. coli, S. epidermidis and B. subtilis - by Benjamin Lemke

Bio. 112


Escherichia coli, Bacillus subtilis and Staphylococcus epidermidis were analyzed for this lab activity to determine their Gram Stain. After the multi-layered Gram Stain procedure each bacteria were classified as Gram-positive or Gram-negative depending on their cell walls staining color. The results showed that E. coli stained pink and classified as Gram-negative. Both B. subtilis and S. epidermidis stained purple and were classified as Gram-positive. It was determined that E.coli likely stained pink due to having it’s cell walls composed of less peptidoglycan than B. subtilis and S. epidermidis.


Biologists use several methods to classify bacteria. One method, which was used in this lab activity, is called Gram Stain. Gram Stain is a method that uses several staining solutions to interact and bind to the bacteria’s cell wall, which contains a complex polymer made up of glycan strands of repeating disaccharide residues, cross-lined via peptide side chains called peptidoglycan (Hayhurst 2008). It has been determined that the bacterial cell walls will stain purple (Gram positive) or pink (Gram negative) depending on the thickness of the peptidoglycan layer (www.cdc.gov) and (Hoefnagels 2015).

In this lab activity, Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Staphylococcus epidermidis (S. epidermidis) were Gram Stained. It was predicted that only E. coli would be Gram-negative, and both B. Subtilis and S. epidermidis would be Gram-positive.


In order to Gram Stain the bacteria, the following method was used and replicated for each bacterial sample. First, a drop of distilled water was placed onto a glass microscope slide. Next an inoculating loop was flamed for 1-2 seconds using a Bunsen burner, and then allowed to briefly cool in the time it took to pick up a small amount of bacteria culture. The culture was then mixed into the distilled water and let to air dry. This took several minutes. Once dry, the glass slide was heat fixed over Bunsen burner for 5-6 passes.

Five drops of the first stain – crystal violet – was added next to the slide. After allowing the slide to sit undisturbed for one minute, the crystal violet was rinsed using tap water that was trickled over the hand onto the slide for a slow, careful rinse. Bibulous paper was then used to blot dry any excess water so the slide could quickly air dry.

Five to ten drops of the second staining solution – iodine – was added to the slide. After allowing the slide to sit for two minutes, the slide was rinsed again in the same manner as before. Next, ethanol was dripped over the slide held at a downward tipping angle until all the purple dye was no longer visible from the sample. Again, the slide was rinsed with water.

For the final staining solution – safranin – five drops were added to the sample. After letting the sample sit for one minute, the sample was rinsed again with water and dried using bibulous paper and until it was completely air dried.

Lastly, a drop of oil was added to the slide so the sample could be viewed under the microscope using the oil immersion lens.


The E. coli had a Gram Stain reaction color of pink and classified as Gram-negative. Both the S. epidermidis and B. subtilis had a Gram Stain reaction color of purple and then classified as Gram-positive (Table 1).

Table 1: The Outcome of Gram Stain on Three Species of Bacteria

Table 1


The results of the Gram Stain for each bacteria sample supported the hypothesis and were consistent with previous laboratory experiments (Wientjes 1991), (Namvar 2014) and (Silhavy 2010). The thickness of the peptidoglycan layer within the cell wall is a major factor in the staining solutions’ ability to bind to the cell wall and thus the color it stains. It has been shown that the thickness of the peptidoglycan layer in E. coli is near 2.0 nm and up to 5.0 nm (Gumbart 2014). Whereas, in the S. epidermidis and B. subtilis, their peptidoglycan layer are from 50 nm to 5 micrometers (Hayhurst 2008) and about 20 to 40 nm (Dmitriev 2004), respectively. Thus, it was expected that E. coli would Gram Stain pink (Gram-negative) and both the other bacteria would Gram Stain purple (Gram-positive).

One factor that may have influenced the Gram Stain is the time a bacterial cell needs for the stain to bind. First, because cell wall structures and components are variable from bacteria to bacteria, the amount of time needed for a staining solution to bind to the cell wall may be different. Thus, a bacterium that is classically classified as Gram-negative may actually be Gram-positive if allowed the stain to bind longer (Silhavy 2010). A future lab activity that could determine if binding time as a limiting factor would be to vary the binding time of each staining solution.


Biology – Concepts and Investigations. Third Edition. Hoefnagels, Marielle, 2015, McGraw=Hill Education.

Dmitriev, Boris A., 2004. Tertiary Structure of Staphylococcus aureus Cell Wall Murein. J of Bacteriology. 186(21): 7141-7148.

Gumbart, James C., 2014. Escherichia coli Peptidoglycan Structure and Mechanics as Predicted by Atomic-Scale Simulations. PLoS Compt Biol. 10(4): e1003475.

Hayhurst, Emma J., 2008. Cell Wall Peptidoglycan Architecture in Bacillus subtilis. Proc Natl Acad Sci U S A. 105(38): 14603-14608.

Jones, Gilda L., Dever, Stanley M., CDC 1984. The Gram Stain; a new look at an old tool https://stacks.cdc.gov/view/cdc/7646.

Namvar, Amirmorteza E., 2014. Clinical Characteristics of Staphylococcus epidermidis: A Systematic Review. GMS Hyg Infect Control. 9(3): Doc23.

Silhavy, Thomas J., 2010. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5).

Wientjes, F.B., 1991. Amount of Peptidoglycan in Cell Walls of Gram-negative Bacteria. J of Bacteriology. Vol. 173 no 23: 7684-7691.