Cryo-preservation of Lactobacillus leichmannii for vitamin B12 microbiological assay

An optimised method for preparing cryo-preserved cultures of Lactobacillus leichmannii for vitamin B12 microbiological assay is described. Cultures are mixed with an equal volume of 800 ml/l sterile glycerol before freezing. The percentage recovery of viable cells after thawing is highest when cultures are enclosed in polystyrene insulation and cooled to -70 degrees C. Cells cryo-preserved in the stationary phase of growth have a higher percentage recovery after thawing, but the same numbers of log phase cells give a larger growth response to cyanocobalamin under assay conditions. Cryo-preserved cultures are stable for many months, giving consistency of standard curve shape. Results obtained for control sera are also more consistent over time, with a range of CV values from 2.58% to 4.82%, as compared to a range of 4.63% to 6.11% for control sera in assays using conventionally prepared fresh cultures. The use of large inocula also allows for assay completion after overnight incubation.