R. Whittenbury developed and tested a soft agar medium to study how lactic acid bacteria utilize fermentable substrates under varying oxygen conditions. The medium, which created a gradient from aerobic (surface) to anaerobic (bottom) regions in a single culture, was evaluated using 250 strains of lactobacilli, streptococci, leuconostocs, pediococci, and aerococci. The study revealed that oxygen requirements varied considerably among these organisms and depended on specific substrates, incubation temperature, pH, salt concentration, and sterilization methods. Some bacteria required aerobic conditions for substrate utilization, others required anaerobic conditions, and many were facultative. The soft agar medium proved superior to liquid media for detecting these relationships and identifying when substrate fermentation resulted from mutation rather than normal metabolism. The work provides a practical method for characterizing oxygen requirements in lactic acid bacteria and suggests biochemical mechanisms underlying their metabolic flexibility.

Key findings

• Soft agar medium successfully distinguished aerobic, anaerobic, microaerophilic, and facultative growth patterns in lactic acid bacteria, revealing that many strains previously described as strictly anaerobic actually require or benefit from oxygen for utilizing certain substrates
• Substrate utilization in some organisms was dependent on mutation rather than inherent metabolic capability, with mutants forming isolated colonies within the culture under specific oxygen conditions
• Oxygen requirements varied significantly with environmental factors including incubation temperature, medium pH, salt concentration, substrate sterilization method, and additives like citrate and manganese sulfate
• Hydrogen peroxide formation and catalase activity played important roles in regulating anaerobic growth in some leuconostocs, with H2O2 diffusing downward from aerobic zones and inhibiting anaerobic growth initiation
• Liquid media gave inconsistent and uninformative results compared to soft agar, often failing to detect oxygen-dependent substrate utilization or mutation-based fermentation abilities