The Gram stain is a differential staining technique used to classify bacteria into two large groups based on the physical properties of their cell walls. The Gram stain process involves staining bacteria with crystal violet dye, then treating with an iodine solution, followed by a decolorizer, and finally a counterstain. Based on their cell wall structure, bacteria will either retain the crystal violet dye (Gram positive) or lose the crystal violet dye and take on the color of the counterstain (Gram negative). The conclusion of the Gram stain provides information about the broad classification of bacterial species which can aid in diagnosis and treatment of bacterial infections.
How does the Gram stain work?
The Gram stain relies on the different properties of Gram positive and Gram negative cell walls. Gram positive bacteria have a thick peptidoglycan layer in their cell wall which retains the crystal violet dye during the decolorizing step. Gram negative bacteria have a thinner peptidoglycan layer and an outer membrane containing lipopolysaccharide which does not retain the crystal violet dye during decolorization.
The steps of the Gram staining procedure are:
Staining with Crystal Violet
A primary stain of crystal violet dye is applied to a smear of bacteria on a microscope slide. The crystal violet penetrates through porous areas of the cell walls in both Gram positive and Gram negative bacteria.
Treatment with Iodine
An iodine solution is added which acts as a mordant to fix the crystal violet dye inside the cell. The iodine forms a complex with the crystal violet.
Decolorization with Ethanol or Acetone
A decolorizing agent such as ethanol or acetone is used to wash away excess stain. In Gram negative bacteria, the crystal violet-iodine complex leaks out of the outer membrane leaving cells colorless. In Gram positive bacteria, the thick peptidoglycan layer prevents the dye from being removed.
A secondary counterstain such as safranin is applied which stains the decolorized Gram negative cells pink or red. Gram positive cells remain purple from the retention of crystal violet dye.
Gram Positive vs Gram Negative Cell Walls
The main difference between Gram positive and Gram negative bacteria is in the structure of their cell walls:
|Gram Positive||Gram Negative|
|Thick peptidoglycan layer (50-90% of cell wall)||Thin peptidoglycan layer (5-10% of cell wall)|
|Teichoic acids present||Outer membrane containing lipopolysaccharide|
|No outer membrane||Periplasmic space between inner and outer membrane|
The thick peptidoglycan layer of Gram positive bacteria retains the crystal violet dye even after decolorization. The thin peptidoglycan layer of Gram negative bacteria cannot retain the crystal violet dye after decolorization.
The Gram stain provides immediate, useful information to clinicians about unknown bacterial samples. It can narrow down the identity of a pathogen and guide treatment approaches before definitively identifying the species. Some key clinical applications include:
– Distinguishing between Gram positive and Gram negative causes of infection to guide antibiotic therapy
– Identifying presence of bacteria in normally sterile fluids like blood, spinal fluid or joint fluid which indicates infection
– Diagnosing sources of infection from samples like sputum, wounds, urine, etc. Presence of Gram positive diplococci in sputum suggests pneumonia due to Streptococcus pneumoniae.
– Monitoring response to antibiotics – if appropriate Gram positive or Gram negative bacteria persist after treatment, antibiotic resistance may be present
– Screening for particular pathogens like Group B Strep in pregnant women
Limitations of the Gram stain
While the Gram stain provides a quick overview of potential pathogens, it has some limitations:
– It cannot conclusively identify bacterial species – further biochemical testing is required
– Some bacteria may appear Gram variable, retaining or losing dye inconsistently
– The technique requires an experienced microbiologist to accurately perform staining and interpret results
– Non-bacterial pathogens like viruses and fungi will not be detected
– Viable but non-culturable bacteria may retain stain even if cells are dead
– Errors can occur due to factors like poor staining, excess decolorization, or obscured morphology
The Gram stain provides a rapid, inexpensive method to categorize bacteria as Gram positive or Gram negative based on properties of their cell wall structure. The crystal violet dye is retained in Gram positive bacteria with a thick peptidoglycan layer but not in Gram negative bacteria with a thinner peptidoglycan layer and outer membrane. The conclusion of the Gram stain gives clinicians key preliminary information about potential pathogens to help guide diagnosis and empiric treatment while definitive culture results are pending. However, supplementary testing is still required for identification of specific species and antimicrobial susceptibilities. When performed and interpreted correctly, the Gram stain remains one of the most useful tools in clinical microbiology.