Method of the Month: Gram Staining
Our featured method this month is Gram staining, a common procedure in microbiology used to differentiate bacteria based on their cell wall composition. This technique was developed in 1884 by Danish physician Hans Christian Gram, who wanted a simpler way to identify lung pathogens. His technique continues to play an important role in medical microbiology today.
The principle behind Gram staining is that bacterial cell walls come in two basic types. Gram-positive bacteria have thick cell walls with copious amounts of peptidoglycan and lipoteichoic acid. In contrast, Gram-negative bacteria have thin cell walls consisting of a single layer of peptidoglycan covered by a porous outer membrane of lipopolysaccharides. Both types can absorb dye, but thin cell walls will lose the dye after being washed with a solvent. For a thorough review of the differences in structure between bacterial cell wall types, see this article: The Bacterial Cell Envelope — PMC (nih.gov)
To prepare for Gram staining, an inoculation loop is used to transfer a small quantity of cultured bacteria from a petri dish into a drop of water on a microscope slide. Next, the slide is briefly waved over an open flame to evaporate the water and “fix” the bacteria to the glass (this prevents loss of the bacteria when the slide is rinsed).
The bacteria can then undergo the Gram staining process, which consists of four steps: primary staining with crystal-violet dye, iodine wash, ethanol wash, and counterstaining.
During the primary staining step, the bacteria on the slide are immersed in crystal violet dye (a.k.a. gentian violet) for 30–60 seconds. This alkaline dye is absorbed into the peptidoglycan of bacterial cell walls, staining them dark purple-blue.
After the primary staining, the slide is briefly rinsed with water to get rid of excess dye. Then, a mild iodine solution is added. The iodine bonds to the crystal violet dye in the bacterial cell walls, making it harder to remove from the cell wall. After 30–60 seconds, the excess iodine is rinsed away with water.
During the ethanol wash, the slide is flooded with a concentrated mixture of acetone and ethanol, causing all of the crystal violet dye to leach out of the thin cell walls. After a 2–3 seconds, the slide is rinsed with water to prevent removal of the dye from the thick walled cells.
The final step in the procedure is the counterstain. The cells are immersed in safranin, a bright red dye that enters the peptidoglycan of the thin-walled cells. After 30–60 seconds, the counterstain is washed off.
The color of the Gram-stained cells when viewed under a microscope determines whether they are classified as Gram-positive (thick-walled) or Gram-negative (thin-walled).
Gram-positive cells appear blue or purple because they retain the primary crystal violet stain. Several examples of Gram-positive bacteria include Clostridium botulinum, Staphylococcus aureus, and Streptococcus pneumoniae. Gram-negative cells appear pink or red because they lose the primary stain and absorb the counterstain instead. Examples of Gram-negative cells include Escherichia coli, Haemophilus influenzae, and Mycobacterium tuberculosis.
Bibliography:
Silhavy, Thomas J., et al. “The Bacterial Cell Envelope.” Cold Spring Harbor Perspectives in Biology vol. 2, no. 5, 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC2857177/#:~:text=Gram-negative%20bacteria%20are%20surrounded,found%20in%20the%20Gram-negatives.
Smith, Ann C., and Marisse A. Hussey. “Gram Stain Protocols.” American Society for Microbiology, 30 September 2005, https://asm.org/getattachment/5c95a063-326b-4b2f-98ce-001de9a5ece3/gram-stain-protocol-2886.pdf.
Tripathi, Nishant, and Amit Sapra. “Gram Staining.” NIH StatPearls, 8 August 2022, https://www.ncbi.nlm.nih.gov/books/NBK562156/#:~:text=The%20Gram%20staining%20is%20one,to%20identify%20organisms%20causing%20pneumonia.
If you are interested in reading more research on this topic, you can find similar research in Applied and Environmental Microbiology.
Grove City College students can find any of these journals by simply searching the journal name in Discover on the Henry Buhl Library’s homepage. And don’t forget — if you’d like to find more related resources, the library maintains a list of A-Z Databases with an entire tab dedicated to biology!
