This 1951 study investigated factors affecting vitamin B12 assay using Lactobacillus lactis Dorner ATCC 8000 via a cup-plate test method. The researchers identified critical conditions for reliable B12 detection: both oxygen and reducing agents (ascorbic acid or thiolacetic acid at 0.1%) were essential, while excess oxygen prevented growth. Agar thickness between 1-2.5 mm optimized zone formation. The organism stored sufficient B12 when cultured in B12-rich media, rendering it unsuitable for inocula; this was resolved by growing cells in B12-deficient medium after vitamin depletion. The authors developed a standardized inoculum preparation method involving initial growth in B12-rich medium followed by transfer to deficient medium for 5-8 hours. Under anaerobic or highly reducing conditions, B12 was not required, suggesting either synthesis or replacement by alternative factors. The strain remained stable under standard maintenance conditions with no detected variants. Deoxyribosides above 5 µg/ml and antibiotics interfered with assay accuracy. These findings established practical conditions for reproducible B12 microbiological assays.

Key findings

• Vitamin B12 assay requires both oxygen and reducing agents (ascorbic acid or thiolacetic acid); excess oxygen alone prevents growth
• Proper inoculum preparation is critical: cells must be depleted of stored B12 by growth in deficient medium before use in plates
• Agar thickness between 1-2.5 mm is optimal; thicker agar allows unrestricted growth independent of B12, while thinner agar shows poor response
• Under anaerobic conditions, B12 is not essential, suggesting synthesis or replacement by alternative growth factors
• The L. lactis Dorner strain remained stable under maintenance conditions; no variants were detected despite previous reports of strain instability