Visualization of Eggshell Membranes and Their Interaction with Salmonella enteritidis Using Confocal Scanning Laser Microscopy

Abstract

Confocal scanning laser microscopy (CSLM) was used to visualize eggshell membrane and observe its interaction with Salmonella enteritidis. Two- and three-dimensional images of fluorescein isothiocyanate (FITC)-stained egg membranes were obtained for observation of structure. Outer membrane fibers 1 to 7 μm in thickness could be seen emerging from the calcified layers of the eggshell. Inner membrane fibers 0.1 to 3 μm in thickness were interlaced with the outer membrane. The limiting membrane, when stained with FITC, appeared as particles that filled spaces between the inner membrane fibers several microns outward from the level at which the inner membrane fibers first appeared. The outer membrane layer, approximately 50 to 70 μm thick, and the inner membrane, approximately 15 to 26 μm thick, consisted of several discontinuous layers that were discernible as shifts in fiber position or orientation and changes in fiber size. Large egg membranes, which had been detached from the eggshell, were submerged in a 10⁹-CFU/ml suspension of S. enteritidis over a 24-hour period to observe cell penetration. Cells were able to penetrate 28 μm into the membrane after 24 hours. Under moist conditions, Salmonella cells did not appear to attach to the fibers but floated easily in between them. Under dry conditions, Salmonella cells adhered to the membrane fibers. CSLM could be a useful tool in examining the effects of current storage and handling practices on eggshells and membranes.