PCAT Microbiology – Gram Staining
By Andrew Dombrowski
PCAT Microbiology is a very high-yield biology topic for the exam, accounting for up to 20% of content in the Biological Processes section. This is because a thorough knowledge of microbes is essential for practicing pharmacists, as many prescriptions are dispensed to treat microbial infections.
An important distinction is made between Gram-positive and Gram-negative bacteria. Students generally memorize the fact that Gram-positive bacteria have a cell wall containing a thick layer of peptidoglycan, while Gram-negative bacteria have a thin layer of peptidoglycan surrounding their cell wall, which in turn is surrounded by an outer membrane.
This is all important information, but you can take your knowledge to the next level by carefully reviewing the actual Gram staining procedure itself.
The basic idea of Gram staining is to leverage this structural difference in terms of the peptidoglycan components of cell walls in a way that can be easily visualized.
The first step is to take a slide with heat-fixed cells and to flood it with crystal violet (CV) dye, which in aqueous solution dissociates into CV+ and Cl− ions. The CV+ ions interact with the cell walls and membranes of both Gram-positive and Gram-negative cells. Then the slide is gently washed with water to remove excess dye.
The next step is to flood the slide with iodine. Iodine in the form of I− ions binds with the CV+ ions to form CV-iodine complexes. These larger complexes are trapped within the thick peptidoglycan layer of Gram-positive bacteria, and also adhere gently to the thin peptidoglycan layer of Gram-negative bacteria. As in the previous step, the slide is gently washed with water to remove excess dye.
The third step is where the action really happens. In this step, the slide is rinsed with a “decolorizer,” which is usually 95% ethanol, although other options are possible. The idea here is that the ethanol washes away the outer cell membrane of Gram-negative cells and strips away the CV-iodine complex, while the dehydrating effect of the ethanol tightens the peptidoglycan cross-links of the Gram-positive cells to trap the CV-iodine complexes even more effectively. After this step is performed, we have good news and bad news. The good news is that we’ve managed to dye the Gram-positive cells, but not the Gram-negative cells. The bad news is that we can’t actually see the Gram-negative cells.
In order to see the Gram-negative cells, we need a final counterstain. Usually, a dye called safranin is used for this purpose, although fuchsin can be used too. The counterstain is used to stain cells pink. This actually applies to both Gram-positive and Gram-negative cells, but you can’t really see the outcome in the Gram-positive cells, because the CV-iodine dye forms a deep purple color that isn’t affected by additional pink staining. Excess counterstain is washed off, and the final result is that Gram-positive cells are visualized as a dark purple, while Gram-negative cells appear a light pink.
This image shows what Gram-negative cells look like (Yersinia enterocolitica):
The next image shows what Gram-positive cells (Clostridium perfringens) look like:
We at Next Step hope this helps you consolidate your knowledge of this PCAT-relevant laboratory technique, and wish you the best of luck in your PCAT prep!