Improved methods for the detection of β-galactosidase activity in colonies of Escherichia coli using a new chromogenic substrate: VBzTM-gal (2-(2-(4-(β-d-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate)

Summary

The use of a new substrate (2-(2-(4-(β-d-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluence-4-sulphonate (VB-zTM-gal) is described for the detection of β-galactosidase activity in colonies of wild type and mutant strains of Escherichia coli. On enzymic hydrolysis this substrate, which is soluble in water, released a chromophore which is red at pH 7 and bound to cellulose and nitrocellulose. The best procedure for the detection of activity was to grow colonies on standard nitrocellulose membranes (pore size 0.45 μm) laid onto an agar plate and to float the membranes over a solution of the substrate. Coloured colonies developed within 3 min, which were stable at 4°C for several days, and this identified the expression of β-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium of Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms is polluted water.

β-Galactosidase, Escherichia coli, Colonies of bacteria, X-gal, Chromogenic substrate