Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

Background: A now cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of gen omic DNA, stained with an intercalating dye, are passed through an ultrasen sitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. Methods: The isolation of bacterial genomic DNA and digestion by restrictio n enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution wa s used to move the DNA fragments from the agarose plug into a solution cont aining polyamines to protect the DNA from shear-induced breakage. The DNA w as stained with the bisintercalating dye thiazole orange homodimer and intr oduced into our ultrasensitive flow cytometer. A histogram of the fluoresce nce intensities (fingerprint) was constructed. Results: Gram-positive Bacillus globigii and Gramnegative bacteria Escheric hia coli and Erwinia herbicola were distinguished by the fingerprint patter n of restriction fragments of their genomic DNA. DNA sizes determined by FC M are in good agreement with pulsed-field gel electrophoresis (PFGE) analys is. Flow cytometry requires only picogram quantities of purified DNA and ta kes less than 10 min for data collection and analysis. When the total sampl e preparation time is included, the analysis times for PFGE and ECM are sim ilar (approximate to 3 days). Conclusions: FCM is an attractive technique for the identification of bacte rial species. It is more sensitive and potentially much faster than PFGE. ( C) 1999 Wiley-Liss, Inc.