2.3.1: Gram Stain

The Gram stain is one of the most widely employed staining techniques in microbiology. The Gram stain was developed by Han’s Christian Gram in 1884. The technique separates bacterial cells into two types of cells based on structural differences in their cell walls. Those cells that are referred to as gram positive cells have thick peptidoglycan cell walls, while those that are known as gram negative cells have thin peptidoglycan cell walls with outer membranes. These structural differences in the cell walls of these two types of cells results in their having different susceptibilities to various antimicrobial drugs. Thus, knowing the Gram stain result aids in the selection appropriate treatments for bacterial infections.

Bacterial Cell Walls - A. Gram-positive cell wall structure. B. Gram-negative cell wall structure

Credit: Susan Decker

The Gram stain is a differential stain and thus will employ more than one stain. The first stain (primary stain) employed in the Gram stain procedure is crystal violet, a basic stain, that will stain both Gram-positive and Gram-negative cells. After the primary stain is rinsed from the smear the mordant, Gram’s iodine, is added to the smear. Mordants help to fix the dyes into the samples. Thus, Gram’s iodine binds to crystal violet forming an insoluble complex that is trapped within the cell. The next reagent used in the procedure is Gram’s decolorizer. Decolorizers act as the differential step of a differential staining process. Decolorizers work based on differences in the cell to remove the primary stain from one cell type/structure and allowing it to remain inside another cell type/structure. The decolorizer used in the Gram stain procedure, Gram’s decolorizer, may be alcohol and/or acetone. The decolorizer used in this lab is a 50/50 mixture of ethanol and acetone. This reagent reacts with the two cell walls very differently. In Gram-positive cell walls the peptidoglycan molecules become more tightly connected making it more difficult for the crystal-violet iodine complexes to leave the cell during the decolorization step. Gram-negative cells on the other hand have very little peptidoglycan. The decolorizer interacts with the outer membrane and cell membrane to cause them to become more porous because it dissolves some components of these two structures. As a result, the crystal violet iodine complex is washed out of the cell, leaving it colorless. You should be aware that it is important to use the decolorizer cautiously, as it is possible to remove the crystal violet from Gram-positive cells if too much decolorizer is utilized, thereby resulting in a false Gram-negative result. At this point in the procedure Gram-positive cells appear violet/purple, while Gram- negative cells are colorless.

Differential staining procedures require a counter-stain be added after the differential step so that the cells that had the dye stripped from them can be observed. The counter-stain in this procedure is safranin, a red/pink dye. Safranin enters both cells. In Gram-positive cells the safranin is overshadowed by the darker crystal violet that is already present in the cell. Gram-negative cells however do not have any dye to compete with the red/pink safranin Thus at the end of the procedure Gram-positive cells will appear violet/ purple while Gram-negative cells appear light red/pink. The procedure for the Gram stain is provided in this exercise. It is vital that the stains/reagents be used in the correct order to obtain accurate results.

There are several factors that you need to be aware of before beginning the procedure, that may affect the outcome of your Gram stain. The first of these is the age of the sample. As bacterial cells age their cell walls become weak. This is problematic more so with Gram-positive cells because as they age, they would fail to be capable of retaining the crystal violet-iodine complex and therefore may falsely appear Gram- negative. In order to avoid this problem, it is important to use cultures that are less than 24 hours old, preferably cultures that are 16-18 hours old.

Another factor that may affect the results of the Gram stain is the thickness of the smear. If the smear is too thick the crystal violet may become trapped in the smear and not wash out during the decolorizing step. This would cause Gram-negative cells to falsely appear Gram-positive. Therefore, it is important that the smears to be stained are prepared correctly.

---