From the Guidelines
The Gram stain is a crucial diagnostic tool that should be performed on all patients being evaluated for suspected meningitis, as it permits a rapid and accurate identification of the causative bacterium in 60%–90% of patients with community-acquired bacterial meningitis, with a specificity of 97% 1.
Importance of Gram Stain
The Gram stain is a fundamental laboratory technique used to classify bacteria into two major groups: Gram-positive and Gram-negative. This staining method involves applying crystal violet dye to bacteria, followed by iodine as a mordant, decolorization with alcohol or acetone, and counterstaining with safranin.
Clinical Applications
Gram-positive bacteria retain the crystal violet dye and appear purple under microscopic examination because they have thick peptidoglycan cell walls that trap the crystal violet-iodine complex. Gram-negative bacteria, with their thinner peptidoglycan layers and outer lipid membrane, lose the primary stain during decolorization and take up the safranin counterstain, appearing pink or red.
Diagnostic Value
The Gram stain is essential for rapid preliminary identification of bacterial infections, guiding initial antibiotic therapy before culture results are available. It's particularly valuable in clinical settings for diagnosing infections like pneumonia, meningitis, and urinary tract infections, allowing healthcare providers to distinguish between bacterial types and select appropriate antimicrobial treatments.
Key Considerations
- The likelihood of visualizing the bacterium on Gram stain correlates with the CSF concentration of bacteria, with concentrations of 10^5 CFU/mL leading to positive microscopy results in 97% of cases 1.
- The probability of visualizing bacteria on a Gram stain can be increased up to 100-fold by using cytospin techniques 1.
- The yield of CSF Gram stain may be ~20% lower for patients who have received prior antimicrobial therapy 1.
From the Research
Gram Stain Overview
- The Gram stain is a method used to distinguish between Gram-positive and Gram-negative bacteria based on differential staining with a crystal violet-iodine complex and a safranin counterstain 2, 3.
- The cell walls of Gram-positive organisms retain the crystal violet-iodine complex after treatment with alcohol and appear purple, whereas Gram-negative organisms decolorize following such treatment and appear pink 2, 3.
Gram Stain Mechanism
- The Gram stain differentiates bacteria into two fundamental varieties of cells based on the chemical and structural makeup of their cell walls 4.
- Gram-positive bacteria have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers, while Gram-negative bacteria have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane 4.
Applications of Gram Stain
- The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures 2, 3, 5.
- The Gram stain can also be used to separate bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods 5.
Gram Stain and Antibiotic Resistance
- The Gram stain can be used to identify bacteria that are resistant to certain antibiotics, such as cefuroxime-resistant Gram-negative bacilli 6.
- Cefepime, a new, parenteral, fourth-generation antibiotic, has been shown to have activity against Gram-positive cocci and cefuroxime-resistant Gram-negative bacilli, although its activity against certain Gram-negative bacilli is less than that of other antibiotics 6.