Transcript Serotyping Salmonella spp. from Environmental Waterways: A Visual Protocol Timothy M.

Serotyping Salmonella spp. from Environmental Waterways: A Visual Protocol
Timothy M. Smith and David W. Buckalew
Department of Biological and Environmental Sciences
Longwood University
Farmville, VA 23909
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Water Samples are collected from 3 different locations: Appomattox River (APP2), Sayler’s
Creek (SAY5), and Green Creek (GRE16).
Upon returning to the laboratory, samples are processed via membrane filtration.
After the filter is incubated in BGB for 24 hours, the presumptive Salmonella spp. colonies are
enumerated and representative samples aseptically transferred to TSI slants.
Courtesy of Oxoid™ website
Each isolate was prepared for multiple rounds of MPCR to determine O, H1, and H2 antigen
allelic formulae. Control primers were used to further confirm Salmonella spp. identity and
for experimental integrity.
Genomic DNA was extracted from all 32 isolates using Qiagen’s DNeasy Blood and Tissue kit
according to manufacturer’s instructions.
An example of (+) agglutination from this experiment
After 48 hrs. incubation, TSI results are recorded and the Rapid Salmonella Antibody Beads™
test is performed to serologically confirm Salmonella spp. Gram-staining was then done on
the isolates testing positive for Salmonella spp.
Upon completing a PCR cycle, the samples were prepared for pulsed-field gel electrophoresis
on a 1.5% agarose gel. Completed gels were visualized and photographed on a UV
transilluminator. After an isolate’s antigenic formulae was determined, it was correlated with
the World Health Organization’s 2007 “Antigenic Formulae of the Salmonella Serovars”
publication for serotyping analysis.
LONGWOOD UNIVERSITY
Department of Biological and Environmental Sciences