What is the purpose of a Gram stain?

From the Guidelines

Gram stain examination is a crucial diagnostic tool that should be performed in all patients being evaluated for suspected meningitis, as it permits a rapid, accurate identification of the causative bacterium in 60%–90% of patients with community-acquired bacterial meningitis, and it has 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 bacterial cells, followed by iodine, decolorization with alcohol or acetone, and counterstaining with safranin.

Diagnostic Accuracy

Gram-positive bacteria retain the crystal violet dye and appear purple under microscopic examination, while Gram-negative bacteria lose the primary stain during decolorization and take up the counterstain, appearing pink or red. The difference in staining results from structural variations in bacterial cell walls - Gram-positive bacteria have thick peptidoglycan layers that trap the crystal violet-iodine complex, while Gram-negative bacteria have thinner peptidoglycan layers and an outer membrane that allows the dye to wash out.

Clinical Application

This simple, rapid test provides crucial preliminary information for diagnosing bacterial infections and guiding initial antibiotic therapy, as certain antibiotics are more effective against specific bacterial types. According to a recent study, Gram staining morphology suggestive of Staphylococcus in respiratory samples of patients with VAP holds great potential for predicting VAP caused by S. aureus 1.

Key Points

• The likelihood of visualizing the bacterium on Gram stain correlates with the CSF concentration of bacteria 1.
• The probability of visualizing bacteria on a Gram stain can be increased up to 100-fold by using cytospin techniques 1.
• Gram stain examination has a specificity of 97% for the diagnosis of bacterial meningitis 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 differential staining technique used to identify and classify bacteria into two main groups: Gram-positive and Gram-negative 2.
• This technique is based on the chemical and structural makeup of the cell walls of bacteria, with Gram-positives having a thick, relatively impermeable wall and Gram-negatives having a thin peptidoglycan layer plus an overlying lipid-protein bilayer 2.

Gram Stain Procedure and Applications

• The Gram stain procedure involves a series of steps, including staining with crystal violet, decolorization, and counterstaining with carbol fuchsin or safranin 3.
• The Gram stain is useful for assessing bacterial contamination of tissue culture samples, examining the Gram stain status and morphological features of bacteria, and identifying bacteria in clinical samples 3.
• The Accelerate Pheno™ system can be used in combination with Gram stain to rapidly identify and perform antimicrobial susceptibility testing of Gram-positive and Gram-negative bacteria from positive blood cultures 4.

Gram-Positive and Gram-Negative Bacteria

• Gram-positive bacteria have a thick peptidoglycan layer and retain the initial crystal violet stain, appearing purple under a microscope 2.
• Gram-negative bacteria have a thin peptidoglycan layer and an overlying lipid-protein bilayer, and are decolorized by the Gram stain, appearing red or pink under a microscope 2.
• Examples of Gram-positive bacteria include Staphylococcus aureus and Enterococcus, while examples of Gram-negative bacteria include Escherichia coli and Klebsiella 5.
• Antibiotic susceptibility profiles vary between Gram-positive and Gram-negative bacteria, with some antibiotics being more effective against one group or the other 5, 6.