Revised pyocin typing method for Pseudomonas aeruginosa

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Abstract
In the Gillies and Govan method of pyocin typing for Pseudomonas aeruginosa a cross-streaking technique was used, and 105 main types and 25 subtypes were identified by the patterns of inhibition observed on 13 indicator strains. Disadvantages of the technique included the need to remove test strain growth before application of the indicator strains, the 48-h period needed to obtain a result, and the inability to reliably type mucoid P. aeruginosa. Recent studies have enabled us to overcome these disadvantages and significantly improve the speed and application of pyocin typing. Our revised technique utilizes the same 13 indicator strains which are already used internationally. Test strains were rapidly applied to the surface of agar plates with a multiple inoculator. After incubation for 6 h and exposure to chloroform, the indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, the pyocin type was recognized by inhibition of particular indicator strains. Additionally, the activity of particulate (R and F) and nonparticulate (S) pyocins could be distinguished on the basis of inhibition zone size, which thus allowed further discrimination. The revised technique allows typing within 24 h, increases the number of identifiable types, and can be used to type mucoid strains.