Letters in Applied Microbiology 1998, 27, 349–351 Isolation of Aeromonas salmonicida in association with purple-pigmented bacteria in sediment from a Scottish loch D.A. Austin, P.A.W. Robertson, D.K. Wallace, H. Daskalov and B. Austin Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh, UK 1844/98: received 28 August 1998 and accepted 1 September 1998 D.A. AUSTIN, P.A.W. ROBERTSON, D.K. WALLACE, H. DASKALOV AND B. AUSTIN. 1998. Aeromonas salmonicida was recovered in close association with an unidentified purple-pigmented organism, which was isolated from sediment in a Scottish loch during November (1997) and February (1998). However, there has not been any evidence of A. salmonicida infections, specifically furunculosis, associated with the fish in this loch. INTRODUCTION Aeromonas salmonicida, the causal agent of furunculosis in salmonids, has been regarded as obligate to fish, not being culturable from surface waters (Popoff 1984). However, this definition does not explain the spread of furunculosis between fish populations. Certainly, there is evidence of a non-cul- turable phase of the pathogen in the aquatic environment (e.g. Morgan et al. 1993; Effendi and Austin 1995). Also, the organism has been found to survive and multiply in protozoa, namely Tetrahymena pyriformis (King and Shotts 1988). More recently, during a microbiological study of freshwater sedi- ment, it became apparent that cells of A. salmonicida were emeshed in seemingly pure cultures of an unidentified pur- ple-pigmented organism. Consequently, the aim of this study was to determine whether A. salmonicida was indeed present as co-cultures with other bacteria in freshwater sediment. MATERIALS AND METHODS Sampling site The experimental site was a Scottish loch of 7606 m 2 in area and an average depth of 1·5–2·0 m. The loch, which is fed by two streams, contains low populations of brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), eel (Anguilla anguilla), carp (Cyprinus carpio) and stickleback (Gasterosteus spp.), and supports a population of ducks and swans. Accord- ing to local records, there has not been any evidence of disease associated with the fish during the preceding 25 years. Correspondence to: Professor B. Austin, Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK (e-mail: b.austin@hw.ac.uk). © 1998 The Society for Applied Microbiology Collection and examination of samples Samples (approximately 1 kg) of sediment were collected in beakers on three occasions from a site 0·3 m from shore (the water depth was 0·3 m), during November (1997) and February (1998), when the water temperature was 4–5 °C. Within 15 min of collection, three random 1 g subsamples of sediment were mixed by vortexing for 5 min in 9·0 ml volumes of 0·85% (w/v) sterile (121 °C, 15 min −1 ) saline, and serial dilutions prepared to 10 −7 . Volumes (0·1 ml) of the dilutions were spread over triplicate plates of tryptone soya agar (TSA; Oxoid) and Coomassie brilliant blue agar (CBB; Udey 1982) with incubation at 25 °C for 7 d. Plate counts were recorded and purple-pigmented colonies from TSA purified by streak- ing and re-streaking on fresh media. Also, deep blue-pig- mented colonies from CBB, considered to resemble A. salmonicida (Markwardt et al. 1989), were subcultured to fresh media. Identification of bacterial cultures Purple-pigmented cultures were compared with the diag- nostic tables of Sneath (1984a,b). Sediment bacteria con- sidered to resemble A. salmonicida were compared with reference cultures, including the type strain of A. salmonicida subsp. salmonicida (American Type Culture Collection 14174, Rockville, MD, USA). The Aeromonas isolates were exam- ined for the phenotypic traits described by Austin et al. (1989, 1998) and compared with the characteristics in Popoff (1984). All cultures were examined by the API 20NE rapid identi- fication system (bioMe ´rieux, Basingstoke, UK), with incu- bation at 25 °C for 48 h. Whole cell agglutination of the sediment isolates of A. salmonicida was carried out with mono- specific antiserum to A. salmonicida (titre  1:-8192; Austin et al. 1998) produced in a New Zealand white rabbit (Schill