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An improved method is described for the detection of Escherichia coli by an enzyme linked immunofilter assay (ELIFA) using nitrocellulose membrane sandwiched between two 96-well plates. The incorporation of a pumping system permits a continuous flow of reagents and/or wash fluids through the membrane and provides an assay procedure capable of detecting 10(3) bacteria per well within 40 min. Quantitative bacterial detection was based on precipitated chromogen determined by scanning densitometry. The procedure represents a significant improvement in assay time and/or sensitivity over previously described ELIFA and ELISA methods for whole bacterial cells.

Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane.

The propionate (Pro), decanoate (Dec) and laurate (Lau) esters of 5-(4-hydroxy-3,5-dimethoxyphenylmethylene)-2-thioxothiazoline++ +-3-acetic acid were assessed as substrates for lipase and esterase. On hydrolysis these substrates yield an intensely red coloured phenol which could be assayed at 505 nm. The Pro ester was an effective substrate for porcine esterase and was hydrolysed at a rate 20 times greater than the Lau and Dec esters. Conversely, Pseudomonas lipase had a high activity towards the Lau and Dec esters, especially in the presence of bovine serum albumin, but little activity towards the Pro ester. The Dec and Lau were used to detect lipolytic activity in Pseudomonas strains associated with milk spoilage. For this purpose, the substrates were absorbed onto filter paper disks, which were placed over bacterial colonies growing on agar plates; activity was indicated by bright red colouration of discs within 2 h. Escherichia coli colonies hydrolysed the Pro but not the Lau or Dec esters.

Flow microcalorimetric assays for polymyxin B sulphate and neomycin sulphate have been developed using Escherichia coli as the test organism, suspended in a glucose plus buffer medium. These assays have a better reproducibility (relative standard deviations 3.2 and 2.0%, respectively), and require a shorter time (1 h including time required for preparation of the calorimeter) than do conventional microbiological assays, but are not as sensitive. It is suggested that a screening programme might produce a small group of more suitable (i.e. more sensitive) test organisms, which could be used to develop rapid and reproducible flow microcalorimetric assays for a wide range of antibiotics by the procedure described. The effect of combinations of polymyxin B sulphate, neomycin sulphate and zinc bacitracin (the antibiotic components of the commercial preparation Trisep, ICI, Macclesfield, UK) on the power output of cells suspended in glucose plus buffer medium is also reported. In defined combinations, the effects of neomycin sulphate appeared to be exerted before those of polymyxin B sulphate.