Results 
High Throughput Assay for Nematode Killing by P. aeruginosa It was reported earlier that relatively high-throughput screening procedures performed on plates could be used to identify virulence factors in various pathogens such as Staphylococcus aureus [32], P. aeruginosa [8] or Serratia marcescens [33].
Here, we developed a high throughput screening method, in which C. elegans killing is assessed in a liquid assay and which allows a quick selection of attenuated candidates within large transposon libraries of P. aeruginosa.
We prepared a bacterial culture medium that both allowed the worms to thrive feeding on the non-pathogenic E. coli OP50, and bacterial isolates being tested to have normal (see Materials and Methods).
We filled 96 well plates with the medium and inoculated the bacterial strains to be tested.
The virulence of each isolate was assessed in a second microtitre plate, into which a specific population of nematodes had been deposited.
Evaluation of virulence was based upon the number of live worms recovered after 24 h exposure to bacteria.
Under our assay conditions, more than 90% of the worms grown on E. coli were still alive after 24 h.
In contrast, more than 50% of the worms were killed within 24 h when cultured with any one of three isolates of P. aeruginosa, PA14, PAO1 and TB (data not shown).
In order to confirm that this assay would allow the recovery of P. aeruginosa mutants with an attenuated virulence, we tested previously characterized mutants.
We used a gacA mutant from the PA14 transposon library (http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) [34], which is affected in a gene encoding a response regulator involved in virulence [8].
C. elegans infection using this mutant in the liquid assay resulted in only 40% killing at 24 h, whereas 95% killing was observed with the parental PA14 strain (data not shown).
In order to screen a large number of bacterial isolates more rapidly, the assay was automated using the COPAS (Complex Object Parametric Analyzer and Sorter) Biosort system (Union Biometrica) (http://www.unionbio.com/).
The sorter is able to dispatch systematically a fixed number of worms of a precise developmental stage, into each well of a microtitre plate.
