Hale and Bisset compared staining reactions of bacterial cell walls using various mordants and dyes on Gram-positive bacteria, Gram-negative bacteria, and Azotobacter chroococcum. They tested 20 different mordanting compounds and direct dyes including Alcian blue and Janus green on representative bacterial strains. Results showed that Gram-positive bacteria stained much more readily than Gram-negative organisms. Notably, A. chroococcum stained exceptionally well despite being Gram-negative, behaving similarly to Gram-positive bacteria in staining properties. Calcium hydroxide and Janus green provided particularly clear visualization of A. chroococcum's morphology, revealing Bacillus-like cellular structures including septations and spore coats. Different bacterial genera showed unpredictable and varied responses to specific mordants, indicating that cell wall chemical composition differs significantly between Gram-positive and Gram-negative bacteria. The authors suggest that A. chroococcum's unusual staining behavior supports earlier observations of its relationship to the Bacillaceae family, despite its typical Gram-negative reaction.

Key findings

• Gram-positive bacteria stained much more readily with mordants than Gram-negative bacteria, while A. chroococcum stained exceptionally well despite being Gram-negative
• Azotobacter chroococcum showed Bacillus-like morphological features when stained with calcium hydroxide and Janus green, including visible cell septations and spore-like structures
• Different bacterial genera responded unpredictably and variably to specific mordanting compounds, indicating distinct chemical differences in cell wall composition between bacterial groups
• Alcian blue and Janus green proved effective direct dyes for cell wall staining without prior mordanting
• The staining properties of A. chroococcum support evidence of its relationship to Bacillaceae despite its typical Gram-negative staining reaction