Virulence Plasmid (pYV)-Associated Expression of Phenotypic Virulent Determinants in Pathogenic Yersinia Species: A Convenient Method for Monitoring the Presence of pYV under Culture Conditions and Its Application for Isolation/Detection of Yersinia pestis in Food

Table 3

Isolation and maintenance of pYV in Y. Pestis [30].

Day 1
(i) Frozen stock cultures were streaked onto CR-MOX and CR-BHO.
(ii) Plates were incubated at 37°C for 48 h for differentiation and isolation of pYV-bearing cells from pYV-less cells.
Day 3
(i) Using a stereomicroscope, red pinpoint colonies were examined to ensure Lcr and CR uptake. Using a sterile loop, 2-3 red pinpoint colonies were then inoculated into sterile 10 mL of BHI broth.
(ii) The broth was inoculated and incubated at 28°C for 18–24 h.
Day 4
(i) The overnight culture was divided into three portions: frozen stock cultures, working stock cultures, and cells used for PCR assay and for expression of pYV-encoded virulent phenotypic characteristics including Lcr, CR uptake, and CV binding.
(ii) Frozen stock cultures: 5 mL of overnight culture was mixed with equal portions of BHI broth and 20% glycerol and dispensed into 500 μL portions for storage at −80°C.
(iii) Working stock cultures: using a 10 μL loop, cells were streaked on CRMOX and CR-BHO. The plates were incubated for 48 h at 37°C. Plates were then stored at 2°C for future use. Plates can be stored for 15 days for CR-MOX and 30 days for CR-BHO.
(iv) PCR assay: 1 mL portion of cells was centrifuged, and DNA was prepared for PCR assay. Presence of pYV was confirmed by PCR assay targeting the virF gene in pYV.
(v) The presence of pYV was also confirmed by demonstrating expression of phenotypic virulence characteristics including colonial morphology, CV binding, Lcr, and CR binding.