<I>Salmonella Enteritidis</I>

1022 Journal of Food Protection, Vol. 60, No.9, 1997, Pages 1022-1028 Copyright ©, International Association of Milk, Food and Environmental Sanitarians Visualization of Eggshell Membranes and Their Interaction with Salmonella enteritidis Using Confocal Scanning Laser Microscopy J. W. WONG LIONG,l J. F. FRANK,l* and S. BAILEy2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1022/1665105/0362-028x-60_9_1022.pdf by guest on 02 October 2021 tCenter for Food Safety and Quality Enhancement, Department of Food Science and Technology, University of Georgia, Athens, GA 30602-2106; and 2Poultry Microbiological Safety Research, United States Department of Agriculture, Russell Research Center, Athens, GA 30605, USA (MS# 96-255: Received 27 September 1996/Accepted 24 December 1996) ABSTRACT systems, CSLM has been used to visualize in real time the formation of yogurt structure and the responsible microorgan- Confocal scanning laser microscopy (CSLM) was used to isms (11). Specimens for CSLM do not need to be dehy- visualize eggshell membrane and observe its interaction with drated or fixed like samples that are to be examined by Salmonella enteritidis. Two- and three-dimensional images of electron microscopy. Special fluorescent dyes can be used to fluorescein isothiocyanate (FITC)-stained egg membranes were visualize specific features of a specimen. CSLM's analytical obtained for observation of structure. Outer membrane fibers 1 to 7 power derives from its ability to exclude light not in the 11m in thickness could be seen emerging from the calcified layers of focal plane so as to produce images with higher resolution the eggshell. Inner membrane fibers 0.1 to 3 flm in thickness were interlaced with the outer membrane. The limiting membrane, when than normal light microscopy (6, 7). The data, in digital stained with FITC, appeared as particles that filled spaces between form, can be collected and used to produce two-dimensional, the inner membrane fibers several microns outward from the level three-dimensional, and volume renderings and temporal at which the inner membrane fibers first appeared. The outer image sequences (3). membrane layer, approximately 50 to 70 11m thick, and the inner Contamination of eggs by Salmonella spp. can occur by membrane, approximately 15 to 26 11m thick, consisted of several numerous routes. An understanding of the mechanisms of discontinuous layers that were discernible as shifts in fiber position contamination can provide a basis for developing and or orientation and changes in fiber size. Large egg membranes, introducing controls to prevent or reduce this contamination. which had been detached from the eggshell, were submerged in a Introduction of Salmonella spp. can occur by transovarian 109-CFU/ml suspension of S. enteritidis over a 24-hour period to infection of the egg or contact with a contaminated environ- observe cell penetration. Cells were able to penetrate 28 flm into the membrane after 24 hours. Under moist conditions, Salmonella cells ment (19). Little can be done to rid an egg of microorgan- did not appear to attach to the fibers but floated easily in between isms which have infected it during its formation, but by them. Under dry conditions, Salmonella cells adhered to the understanding the natural barriers that protect the egg from membrane fibers. CSLM could be a useful tool in examining the environmental contamination, preventive measures might be effects of current storage and handling practices on eggshells and developed. membranes. The egg has a number of protective mechanisms to prevent the successful invasion of microorganisms (14). Key words: Eggshell membrane structure, Salmonella enteritidis, There are chemical as well as physical barriers to prevent the confocal scanning laser microscopy entrance of microorganisms and reduce their viability (1). Salmonella cells are known to survive the chemical barriers Confocal scanning laser microscopy (CSLM) allows in whole egg (1). The physical barriers, including the cuticle, access to spatial and topographical information with mini- the shell, and the shell membranes, limit contamination from mal sample manipulation (3) and offers a rapid means of particulates and microorganisms (1,2,20). collecting data regarding the interaction of microorganisms The objective of this study was to characterize the with their environment. With samples stained using the membranes of a hen's egg with confocal scanning laser fluorescent dye pyronin Y, CSLM has been used to examine microscopy and visualize the interaction of S. enteritidis the attachment of Salmonella cells to chicken skin and with the membranes. obtain information about skin topography (12). In food MATERIALS AND METHODS Cultures * Author for correspondence. Tel: 706-542-0994; Fax: 706-542-7472; S. enteritidis Rochester was obtained from the USDA, Russell E-mail: [email protected]. Research Center, Athens, GA. The culture was stored on slants of SALMONELLA IN EGGSHELL MEMBRANES 1023 trypticase soy agar (TSA, Difco, Detroit, MI) at 4°C and trans- matching shell were placed in a sterile Petri dish and submerged in ferred on a monthly basis. FITC stain. After staining for one hour, the shell pieces and membranes were rinsed for 30 seconds with sterile distilled water. Eggs Shell pieces and membrane were individually taped onto slides. Large size grade A brown-shelled eggs were used in all An MRC-600 confocal scanning laser microscope (Bio-Rad microstructure experiments. Eggs for S. enteritidis interaction Inc. Hemel Hempstead, England) with 60X oil immersion objec- experiments were obtained from the USDA, Russell Research tive (numerical aperture 1.4, Nikon, Japan) and an Ar/Kr laser Center, Athens, GA. These eggs originated from a 65-week-old operating at 488 nm was used to visualize the FITC-stained fibers. flock of Peterson roosters and Avian Farms hens. Images (768 by 512 pixels) were digitally captured as data sets of 256-level gray scale values. Stacks of 10 images taken at 0.5-l1m intervals were merged. Fiber diameters were measured at the Stain, buffer, and fixative solution preparations thickest portion of the fiber image to make sure that the entire cross A 40-g/liter stock solution of osmium tetroxide (Sigma section was accounted for. The diameters of three hundred fibers Chemical Company, St. Louis, MO) was prepared by dissolving 1 g each were measured in the inner and outer membrane layers. of osmium tetroxide crystals in 25 ml of distilled water. A stock solution of 0.2 M cacodylate buffer (Sigma Chemical Company, Limiting, inner, and outer membrane layer thicknesses were Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/9/1022/1665105/0362-028x-60_9_1022.pdf by guest on 02 October 2021 St. Louis, MO) was prepared by dissolving 10.7 g of sodium determined by stepping with the stage motor through the sample until the corresponding fibers changed in diameter or disappeared. cacodylate in 250 ml of distilled water. The pH was adjusted to 7.2 Interstitial spaces were measured between fibers in the inner and with 0.1 N HC!. The 10-g/liter OS04 fixative was prepared by mixing 25 ml of the 40-g/liter OS04 stock solution with 75 mlofthe outer membrane layers. 0.1 M cacodylate buffer. Stock solution of fluorescein isothiocyanate was prepared by Thermal differential contamination of shell and outer membranes dissolving 30 mg of fluorescein isothiocyanate (FITC) crystals by S. enteritidis (Sigma Chemical Company) in 100 ml of 0.5 M sodium bicarbon- Approximately, 0.1 ml of stock S. enteritidis was transferred ate. The working solution was made by diluting the stock solution to 10 ml of Trypticase soy broth (TSB, Difco Laboratories, Detroit, lOO-fold in 0.5 M sodium bicarbonate. MI) and incubated in 10 ml at 34°C for 24 h. The entire contents Stock solution of pyron in Y (Sigma Chemical Company) was were transferred to 300 ml of nutrient broth in a 500-ml beaker. The prepared by dissolving I g of pyronin Y crystals in 100 ml of sterile inoculated broth and a sterile control were incubated at 34°C for 24 distilled water. The working solution was made by diluting the h. The final cell concentration of the cell suspension was 105 stock solution IDO-fold in sterile distilled water. CPU/m!. The cell suspension (in broth) was cooled to a temperature of Preparation of egg membranes for scanning electron microscopy 21°C. Dry eggs equilibrated to 40°C were submerged in the cell Membranes were detached from the shell using flat Teflon- suspension for approximately one minute, removed, and stored in coated forceps. The membranes were placed on aluminum stubs, 100-ml beakers containing 10 ml of sterile distilled water. The and carefully anchored using an aluminum retaining ring. While beakers containing the eggs were incubated for 24 h at 34°C. The the stubs were being prepared, cork was used to hold the stubs eggs were then cracked open, the yolk and albumen removed, and upright in a petri dish containing sterile water to maintain a moist membranes detached from the shell pieces. The remaining outer environment. Membranes were vapor fixed with osmium tetroxide membrane on the shell and the cuticle on the outer surface of the to minimize actual manipulation of the membrane surfaces and to shell were stained by flooding with O.I-g/liter pyronin Y solution minimize dehydration. Once the samples were anchored, the water for 10 min. The pieces were then rinsed in 100 m1 distilled water for was drawn off and replaced with a lO-gIliter OS04 solution. The 30 s and mounted on a slide. dishes were covered, and the samples were allowed to vapor fix for The confocal scanning laser microscope with 60X oil immer- 30 minutes. The samples were removed from the Petri dishes sion objective and Ar/Kr laser operating at 568 nm was used to containing the OS04 and sequentially dehydrated by submersion in visualize the pyronin Y-stained bacteria.

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