What is the basis for classifying bacteria as gram-positive or gram-negative?

Classification of Gram-Positive and Gram-Negative Bacteria

Bacteria are fundamentally classified based on their cell wall composition: gram-positive bacteria possess a thick peptidoglycan layer fused to the cytoplasmic membrane, while gram-negative bacteria have a thin peptidoglycan layer sandwiched between an inner cytoplasmic membrane and an outer hydrophobic lipopolysaccharide capsule containing porins. 1, 2

Structural Basis of Classification

The classification system relies on distinct architectural differences in bacterial cell walls that determine their response to Gram staining 1, 2:

Gram-Positive Cell Wall Structure

• Thick peptidoglycan layer (almost entirely composing the cell wall) fused directly to the outside of the cytoplasmic membrane 1
• Peptidoglycan consists of oligosaccharide chains cross-linked by short peptides that maintain cell-wall integrity 1
• Contains secondary polymers embedded within the thick peptidoglycan matrix 3
• Penicillin-binding proteins (PBPs) are located in the cytoplasmic membrane 1

Gram-Negative Cell Wall Structure

• Thin peptidoglycan layer located between two membranes 1, 2
• Outer hydrophobic lipopolysaccharide capsule surrounding a lipoprotein-phospholipid membrane 1
• Small channels called porins embedded in the outer membrane 1
• Periplasmic space between the outer and inner membranes serves as an important site for drug-inactivating enzymes such as β-lactamases 1

The Gram Staining Mechanism

The American Academy of Otolaryngology-Head and Neck Surgery recommends Gram staining as the cornerstone of bacterial classification 2:

• Gram-positive bacteria retain crystal violet stain (appear purple) due to their thick, relatively impermeable peptidoglycan wall that resists decolorization 3
• Gram-negative bacteria are decolorized and stain red with carbol fuchsin or safranin because their thin peptidoglycan layer and outer membrane can be disrupted during decolorization 3

Clinical Application and Diagnostic Accuracy

When evaluating respiratory samples for bacterial identification 1:

• Gram staining focuses on presence or absence of gram-negative or gram-positive bacteria, followed by semi-quantitative assessment (rare, moderate, predominant) and morphological shape evaluation 1
• For Staphylococcus aureus specifically, the presence of gram-positive cocci in grape-like clusters has 68% sensitivity and 95% specificity 1
• Quality control requires samples with <25 squamous epithelial cells per high-power field to ensure adequate specimen quality 1

Important Caveats

Not all bacteria follow traditional classification patterns 4:

• Some deeply branching Bacillota species stain gram-negative despite having monoderm (single membrane) cell wall structure 4
• Bacteria with intermediate wall structures may stain variably, even within the same species 3, 5
• The Archaea domain has such variable wall structure that Gram staining is not a useful differentiating tool 3

Modern Molecular Classification

The American Journal of Respiratory and Critical Care Medicine advises against relying solely on phenotypic methods in the era of antimicrobial resistance 2:

• 16S rRNA sequencing for broad taxonomic classification and species-specific identification 2
• Single nucleotide variant (SNV) analysis with ≥10× sequencing coverage for precise strain-level differentiation 2
• Molecular methods provide superior accuracy and stability for clinical decision-making compared to traditional staining alone 2