Escherichia coli inhibition methods

Several methods for performing the test have been described, the guideline describes the diffusion and suspension test. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain. Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) pairs are recommended. The test should initially be performed over a broad range of concentrations. 

Selected entries from Methods in Enzymology [vol, page(s)] Equilibrium isotope exchange study of kinetic mechanism, 249, 466 site-directed mutagenesis of Escherichia coli enzyme, 249, 93 positional isotope exchange studies, 249, 423 product inhibition studies of three substrates three products reactions, 249, 207-208. 

The first step in identification of herpesvirus inhibitors that act via this mechanism is the evaluation of compounds for their ability to inhibit the protease enzyme. A number of assay formats have been used to examine the effect of herpesvirus protease inhibitors. This chapter describes four of these assays, and their relative merits and problems are discussed. In particular, the conditions for HCMV protease are described, with reference to modifications for other herpesvirus proteases where possible in the notes. A source of purified recombinant enzyme will be assumed, because a number of studies have described the use of Escherichia coli to express active herpesvirus proteases from a number of viruses, along with suitable purification methods (4-6). 

A sensitive and simple chemiluminescent (CL) method for measuring antioxidant activity was developed. The determination of TEAC (Trolox equivalent antioxidant capacity) is based on the inhibition of CL intensity of luminol by an antioxidant. Antimicrobial activity was tested as a minimal inhibitory concentration (MIC) by broth microdilution method on gram-positive Enterococcus faecalis. Staphylococcus aureus) and gram-negative Pseudomonas aeruginosa, Escherichia coli) bacterial strains. 

The strain of Actinamadura spiralis isolated from a soil sample produced new antibiotics, pyralomicins (169-175). Pyralomicins inhibited the growth of Micrococcus luteus and Escherichia coli at the concentration of 0.2-25 (xg/ml by agar dilution method. Pyralomicin la (169) and pyralomicin 2a (173) did not show any acute toxicity in mice at 100 mg/kg ip [205]. 

To measure the antibacterial activities of amphiphilic cationic polynorbornene derivatives, they developed a slightly different method based on optical density (OD). In this method, bacteria were grown in suspensions (Escherichia coli D31 and Bacillus subtilis ATCC 8037 ) of Miieller-Hinton broth (MHB) overnight at 37 °C, diluted with fresh MHB to an OD of 0.1 at 600 nm and further diluted by a factor of 10. This suspension was mixed with different concentrations of freshly prepared polymer solutions in rm-buffered saline (pH 6.5-7.0), by serial dilution in a 96-well plate, and incubated for 6 h at 37 °C. The OD 600 nm was measured for bacteria suspensions that were incubated in the presence of a polymer solution or fm-buffered saline only. After 6 h, the MIC for 90% inhibition of growth was determined. 

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