Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2005
Publication Date: August 7, 2005
Citation: McLaughlin, M.R. 2005. Enhanced phage plaque assays in H2S+ salmonella. [Abstract]. In: Proceedings 16th Evergreen International Phage Biology Meeting, August 7-12, 2005, Olympa, Washington.
Ferric ammonium citrate (FAC) and sodium thiosulfate (ST) have traditionally been used in selective media to aid identification of Salmonella. The chemistry supporting this application is well known. Sulfate-reducing strains of Salmonella convert thiosulfate to sulfite and hydrogen sulfide (H2S) gas. The presence of H2S is detected by its reaction with ferric ions from the ferric ammonium citrate to produce an insoluble black precipitate, which marks the H2S+ bacterial colony. The FACST was used here to enhance the plaque assay of a Salmonella bacteriophage in trypticase soy agar. Bacteriophage PR05-43, isolated from an anaerobic swine effluent lagoon, was used in a traditional double agar layer plaque assay with FACST and Salmonella enterica subsp. enterica (ex. Kauffmann and Edwards) Le Minor and Popoff serovar Enteritidis (ATCC 13076). The phage, FACST and Salmonella were added to the soft agar overlay. Plates were inverted and incubated in the dark at 35C. The result was a dark stained bacterial lawn with distinctly contrasting clear bacteriophage plaques. The precipitate reaction, however, requires acid pH and carbohydrate as a source of H+ ions. If oxidative exhaustion of carbohydrate occurs, such as after prolonged or highly concentrated bacterial growth, ammonia is produced, raising the pH, reversing the reaction and clearing the black precipitate. To avoid this potential problem, FACST-enhanced plaque assays with H2S+ Salmonella should be read within 6-12 hr. This simple enhancement is useful for traditional visual assessment of plaque assays and may be exploited for digital automation.