4. One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were E. coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why?

Probably, she is not adding the mordant solution (iodine), so I suggest to add iodine before the decoloration step.

Explanation:

In this example, there were used two species of bacteria as controls: E. coli (gram-negative) and Bacillus subtilis (gram-positive). So, if the procedure was correct, E. coli would be pink, and B. subtilis would be purple. However, both controls are pink.

In this case, the most probable explanation is he is not adding the mordant solution (iodine). Gram staining allows distinguishing between gram-negative and positive bacteria. Gram-positive organisms possess a super thick cell wall of peptidoglycan, whereas gram negatives have a thin cell wall with an outer lipidic membrane.

In the procedure of Gram staining is the following:

1. Bacteria are stain with crystal violet: both types of bacteria are stained of purple.

2. Iodine treatment: an iodine solution is added and it reacts with the crystal violet. Iodine is a mordant solution, this means that it creates a larger complex with the crystal violet, and this complex won't go out of cells if the cell wall is thick.

3. Decoloration: in this step, bacteria are bathed in alcohol. The alcohol breaks the outer membrane of Gram-negative bacteria and washes out the solution of crystal violet. However, if there is the complex with the mordant, this bigger molecules won't be able to exit of cells with a thick cell wall or Gram-positive bacterium.

4. Pink staining with safranine or fucshina: Finally, bacteria are stained with a pink solution. If in the previous step, crystal violet went out, they would be pink (gram-negative), but if the purple pigment couldn't go out, bacteria would be purple (gram-positive).

Therefore, if step 2 was not done, both types of bacteria will be pink. So, my suggestion is to add a mordant solution after staining with crystal violet.