Columbia colistin nalidixic acid agar

EC-isolation was performed on Columbia colistin–nalidixic acid (CNA) agar (Oxoid GmbH) from Amies medium swabs taken at necropsy. The plates were incubated for 24 h at 37 °C and then screened for colonies of EC (small, gray, mucoid colonies with slight alpha-hemolysis). Pure subcultures from respective colonies were produced on Columbia sheep blood agar. After another 24 h of incubation, catalase and oxidase testing were performed and Gram staining of colony material complemented the microbiological examination. Besides typical colony morphology, isolates were identified as EC when they were oxidase and catalase negative, and gram positive to gram variable ovoid cocci were seen under the light microscope. Bacterial isolates that were not reliably identified by these methods were further analyzed via 16 S rRNA partial gene sequencing at Microsynth AG, Lindau, Germany [52 (link)–54 (link)].

For EC-reisolation, Amies medium swabs from different organs (heart, liver and spleen) were inoculated onto Columbia colistin-nalidixic acid (CNA) agar (Oxoid GmbH, Wesel, Germany). After incubation at 37°C for 24 hours under microaerophilic conditions, plates were screened for colonies showing a morphology typical for EC. Subcultures were produced on Columbia sheep blood agar and incubated for another 24 hours. From pure subcultures, catalase and oxidase testing as well as Gram staining was performed. Isolates were considered EC when they fulfilled the following criteria: small, gray, mucoid colonies with slight alpha-hemolysis; catalase and oxidase negative; gram positive to gram labile ovoid cocci. Isolates giving questionable results were further analyzed via 16S rRNA partial gene sequencing at Microsynth AG, Lindau, Germany [21 (link), 29 (link)–31 (link)].