IMPROVED METHODS FOR THE DETECTION OF BETA-GALACTOSIDASE ACTIVITY IN COLONIES OF ESCHERICHIA-COLI USING A NEW CHROMOGENIC SUBSTRATE - VBZTM-GAL (2-(2-(4-(BETA-D-GALACTOPYRANOSYLOXY)-3-METHOXYPHENYL)-VINYL)-3-METHYLBENZOTHIAZOLIUM TOLUENE-4-SULFONATE)

The use of a new substrate 2-(2-(4-(beta-D-galactopyranosyloxy)-3-methoxyphenyl)-vinyl)-3-methylbenzothiazolium toluene-4-sulphonate (VB-zTM-gal) is described for the detection of beta-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-mu-m) 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-degrees-C for several days, and this identified the expression of beta-galactosidase activity. This was found to be more specific than methods using triphenyltetrazolium or Eosin Methylene Blue media, and more economical than methods using X-gal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside). VBzTM-gal should have applications in gene cloning technology and in the detection of coliform organisms in polluted water.