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.