Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species

While pulsed field gel electrophoresis has become an important tool for gen otyping of bacteria, one of its drawbacks is that standard methods are rath er time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field ge l electrophoresis of Clostridium botulinum could be applied to other Clostr idia species. For this, the protocol was modified and used to prepare the D NA of 34 strains of 25 different Clostridia species. In contrast to a stand ard procedure, which takes at least 5 days from DNA extraction to completio n of the electrophoresis, this protocol yielded results within 2 days. In o rder to directly compare the results of the short protocol with those of th e standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding th e bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. Thi s was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were re peated four times with fresh TE. After a 2-h restriction with SmaI, electro phoresis was carried out overnight. (C) 1999 Federation of European Microbi ological Societies. Published by Elsevier Science B.V. All rights reserved.