Bacteria bioluminescent determination

A feasibility study on the potential use of a luminescent bacteria bioassay in the toxicity screening oftrichothecenes is presented. The toxic effects of four 12,13-epoxytrichothecenes to bioluminescent bacteria were determined for the first time using the Microtox " Toxicity Analyzer (TM Trademark of Microbics Corporation, Carlsbad, California). The influence of test temperature and exposure time on the toxic effect oftrichothecenes to the luminescent bacteria are discussed. The toxicities measured are compared with toxic effects determined using other bioassays. Potential relationships between the toxicities found and variations in the chemical structure of these compounds are considered. 

Bioluminescence can be used for spedfic detection of separated bioactive compounds on layers (BioTLC) [46]. After development and drying the mobile phase by evaporation, the layer is coated with microorganisms by immersion of the plate. Single bioactive substances in multicomponent samples are located as zones of differing luminescence. The choice of the luminescent cells determines the specificity of detection. A specific example is the use of the marine bacterium Vibrio fischeri with the BioTLC format. The bioluminescence of the bacteria cells on the layer is reduced by toxic substances, which are detected as dark zones on a fluorescent background. BioTLC kits are available from ChromaDex, Inc. (Santa Ana, CA). 

These bacteria were cultivated in each product for 24 h at 37 °C and each cultured broth sample was serially diluted into the corresponding aseptic products. The total colony count was determined with Plate Count Agar (MERCK). Bioluminescent Assay... 

Cellular ATP Determination The intensity of the bioluminescence catalyzed by luciferase was used to estimate the ATP content in bacterial cells. Bacterial samples were treated with 50% (v/v) of B-PER obtained from Pierce Biotechnology, Rockford, Il.(bacterial protein extraction reagent), a cell lysis reagent specific to bacteria. The released cellular ATP was determined from the luminescence emitted from luciferase catalyzed oxidation of luciferin. A Berthold FB12 luminometer was used to record the light output. 

A promising flash method was proposed by Lappalainen etal. (1999), applying a new measurement technique. Bioluminescence is determined immediately after Vibrio fischeri is exposed to the soil slurry and again after an incubation period of less than one minute. The first value serves as reference, assuming that the bacteria are not yet affected by the toxicants in the sample. The second value, expressed relative to the first value, indicates the effect of the toxicant. This procedure provides an alternative for coloured and turbid samples. 

Quantitative criteria of expression efficiency of bioluminescence were copy number of lux-operon (level of a replication) in a cell, duration of the latent period of induction of bioluminescence (level of a transcription), the maximal intensity of luminescence (level of a translation). The following quantitative criteria and coefficients are used fi - number of copies of /wx-operons in a cell (for natural marine bacteria - 1 operon/chromosome, for transgenic luminous bacteria the parameter varies depending on strain). Number of copies of plasmid was determined on electrophoregrams with program Scion Image f2 = 1/t - coefficient (s 1), which reflects influence of the latent period in dynamics of bioluminescence on expression... 

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