ENHANCED CONTRAST OF BACTERIOPHAGE PLAQUES IN SALMONELLA WITH FERRIC AMMONIUM CITRATE AND SODIUM THIOSULFATE (FACST) AND TETRAZOLIUM RED (TZR)

Authors: McLaughlin, Michael; El Balaa, Mohamad

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2005
Publication Date: 3/1/2006
Citation: McLaughlin, M.R., Balaa, M.F. 2006. Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR). Journal of Microbiological Methods. 65:318-323.

Interpretive Summary: Bacteriophages, or phages, are viruses that attack bacteria. Phage attack often causes the bacteria to produce new phages at the expense of shutting down its own functions. The end result is rupture of the bacteria releasing multiple copies of new phages, which launch new attacks continuing the cycle. Scientists use this natural process in tests called plaque assays. When a pure bacterial culture is spread evenly over the surface of a nutritive agar of a culture dish, the bacteria grow uniformly forming a lawn. Phage attack results in clear spots or holes, called plaques, in the cloudy bacterial lawn. The plaques can be classified and counted to provide precise information on the identity and characteristics of the bacteria. Plaque assays are widely used by medical, veterinary and food safety personnel, to help identify and track bacterial infections. In research reported here, Agricultural Research Service scientists at Mississippi State, MS tested combinations of chemicals added to the agar of plaque assays. They used chemical reactions to detect hydrogen-sulfide gas (the rotten egg odor produced during growth of many bacteria). The result was a dark black stain in otherwise cream colored bacterial lawns of most strains of Salmonella. Using this chemical combination in plaque assays with Salmonella resulted in clear plaques within black lawns. This made describing and counting plaques much easier and paved the way for digital automation of the assay. The hydrogen sulfide-based plaque assay compared favorably with a traditional method using red dye to enhance contrast of plaques. The enhanced plaque assay will be useful in studies to find and apply specific phages which attack Salmonella.

Technical Abstract: Visualization of bacteriophage plaques was enhanced by addition of ferric ammonium chloride and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar top layer of double agar layer (DAL) plaque assays. Adding either of these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement was demonstrated with a hydrogen sulfide producing (H2S+) strain of Salmonella using a bacteriophage (Podoviridae) isolated from swine lagoon effluent. Growth of Salmonella produced dark brown-black color in FACST-amended top agar and bright pinkish-red color in TZR-amended top agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts of the Podoviridae isolate used in the study. The FACST should be useful in bacteriophage plaque assays with H2S+ strains of Salmonella and other H2S+ bacteria.