Gradient plate assay

Rexroat MA, Oberly TJ, Bewsey BJ, et al. The gradient plate assay A modified Ames assay used as a prescreen for the identification of bacterial mutagens. Mutat Res. 1995 341(3) 185-192. 

Fig. 5. Average in vitro gram-negative minimum inhibitory concentration (in p.g/ml) versus -TSE. The MICs are from gradient plate assay for the 7-(thiopheneacetyl)cepha-losporins with the 3-R groups as shown.

Figure 5. Existence of a Crcomponent in P. funiculosum cellulose, shown by isoelectric focusing. The column had a volume of 110 ml. and the pH gradient was 3-6. The Crcomponent was measured by a zone-clearance technique in a plate assay involving solubilization of cellulose suspended in agar...

Commercial tube diffusion or multi-plate assays are by nature simple to use and do not require the use of expensive instrumental equipment. Over the years some improvements have been realized. For example, the incubation step is typically performed using a water bath. However, the use of water baths has some disadvantages. Temperature gradients can occur within the unit (unless the water is adequately... 

Bevan et al. [97] have developed a solubility screen for. small amounts of compounds (10 pi of a 10 mM DMSO solution) on a 96 well plate format by applying reversed-phase HPLC with a fast gradient elution. The DMSO solutions of the compounds are dispensed into a 96 well microtitre plate format at a known concentration (typically 10 mM). Duplicate plates are prepared each containing 10 pi of a 10 mM DMSO solution. For the so-called standard plate the DMSO solution is diluted with known amounts of solvent possibly dissolving the compounds (methanol or DMSO). The wells on the so-called sample plate are diluted with the same known amounts of aqueous buffer used in the enzyme-assay screens. The compounds having poor aqueous solubility will eventually precipitate out in the wells of the sample plates. The precipitation and the equilibrium formation between the saturated solution and the solid can be promoted by applying sonication. Before HPLC analysis of the wells of the standard... 

MARA is an innovative bioassay devised for the evaluation of toxicity of chemicals and environmental samples. The assay utilizes a taxonomically diverse array of ten bacterial species (prokaryotes) and a yeast (eukaryote). The assay is performed in a 96 well micro titre plate and involves exposure of the microorganisms provided in a freeze-dried state. The toxicity of the test sample using a concentration gradient is determined with the employment of the redox dye tetrazolium red (TZR). The dye is transformed from a soluble colourless state to a red insoluble form upon reduction. The dye is a growth indicator and detects enzyme systems by acting as an electron acceptor. 

Metabolite/residue analysis. Milk, urine and plasma samples were first analyzed by a microbiological cylinder/plate procedure against M.luteus which has a limit of detection of 0.02 ppm. A sub-sample of the milk was prepared for this assay by a centrifugation step followed by a pH adjustment to 8.5. In addition, an HPLC/RAM analysis was conducted after treating another sub-sample with FTSH (10% formic acid, 30% trifluoroacetic acid, 2% sodium chloride, 2N hydrochloric acid) followed by centrifugation to precipitate the proteins. The supernatant was basified and concentrated by C-18 solid phase extraction (SPE) techniques. The HPLC conditions were Column - 20 cm x 4.8 mm C-8 Mobile-phase -linear gradient at 5%/minute from 90 10 O.IM pH 7 phosphate buffer methanol to 20 80 Detectors - UV operated at 214 nm and a radioactivity flow detector operated in the DPM mode. 

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