Isolation and verification of anatoxin-a producing clones of Anabaena flos-aquae (Lyngb.) de Breb. from a eutrophic lake

We present a technique to isolate and confirm anatoxin-a producing clones (single trichome-isolates) of Anabaena flos-aquae (Lyngb.) de Breb. from blooms of this cyanobacterium. A single trichome is isolated from a field sample and grown in ASM medium. Single trichomes are then isolated from this culture and grown in ASM medium to produce single clone cultures. Mouse bioassay, and thin-layer chromatography (TLC) using purified anatoxin-a as reference is then used to confirm the anatoxin-a producing clones. Using this methodology, Anabaena flos-aquae samples collected during July 1991 from Hebgen Lake, Montana, were found to contain only 8.7% anatoxin-a producing clones. This minor proportion of anatoxin-a producing clones apparently accounts for the anatoxin-a produced by the entire population of A. flos-aquae. Our technique is simple and reproducible. A selected clone of A. flos-aquae that produces anatoxin-a and one that does not produce anatoxin-a were deposited in the UTEX culture collection, University of Texas at Austin.