MRSA Colonization of Necrotizing Enterocolitis Cultures

Sterile, NMB communities- or methicillin-resistant Staphylococcus aureus (MRSA)-colonized NEC cultures in 24 well transwell cups (n = 3–4 for each condition) were fixed 48 h after bacterial application in primary fixative solution (1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% CaCl₂,50 mM cacodylate buffer) and stored at 4°C. The MRSA strain USA300 isolated in our lab from a pediatric nasal sample was cultivated in Tryptic Soy broth overnight at 37°C with agitation, and 5 × 10² bacteria were added to the NEC culture. Each sample was processed by rinsing in 0.1 M cacodylate buffer, postfixed in 1% OsO₄ in 0.1 M cacodylate buffer and then dehydrated in absolute ethanol and hexamethyldisalazane. After air drying, the membrane was removed, mounted for sputter-coating with iridium in an Emitech K575 × Sputter Coater (Emitech, Houston, TX, USA) at 20 mA for 20 s and then examined as described previously (Rose et al., 2012 (link)). Imaging was completed on a Hitachi S4700 SEM (Hitachi High Technologies, Schaumburg, IL, USA) with indicated magnifications.