AUTOMATED MICROBIOLOGICAL ASSAY FOR QUANTITATION OF NIACIN PERFORMED IN CULTURE MICROPLATES READ BY DIGITAL IMAGE-PROCESSING

An automated microbiological assay for the quantitation of niacin is d escribed. The assay, involving the use of Lactobacillus plantarum, was performed in culture microplates. The performance of the assay in bot h 48-well and 96-well culture microplates was investigated. Pipetting was carried out by a computer-controlled laboratory robot. Growth resp onse was read by video digital-image processing (VDIP) and by ELISA re ader. The effect of shaking the microplates prior to reading was also investigated. The use of a niacin-depleted culture as inoculum shows l ower blanks and an increased growth rate at low concentrations of niac in. The incubation time is reduced from 48 to 24 h by using the cultur e of L. plantarum directly after thawing of the cryopreserved working culture. The 48-well plate assay showed better performance than the as say performed in 96-well culture plates. The coefficient of variation (CV) was improved in the 48-well plate assay when compared with the co nventional microbiological assay. The relative intra-assay variation r anged from 3.7 to 8.5% in the 48-well plate assay and from 16.7 to 25. 8% in the 96-well plate assay for samples of foods and feeds. The reco very of added standard in the 48-we11 and in the 96-well culture-plate assay was 101% +/- 3% (n = 6), and 105% +/- 12% (n = 6), respectively . The results of the 48-well plate assay correlated well with the resu lts obtained by the conventional microbiological assay (r = 0.985, n = 40). The detection limit for the 48-well assay was found to be 0.5 ng niacin per well. Avoiding shaking the 48-well plates prior to reading of growth response increased the precision significantly. The techniq ue used in this assay reduces reagent costs and time spent on pipettin g and evaluating results when compared with the conventional microbiol ogical assay performed in 5-ml tubes. The microplate assay is more com pact and easier to perform. Digital image processing is introduced as a measurement of turbidity.