2.1. Swab sampling of egg shells
The egg shell surface of 500 eggs sampled from farms was swabbed
with a sterile cotton swab. A clean pair of gloves was used for each egg.
Prior to swabbing, the sterile swab was soaked either with buffered
peptone water (Oxoid, Australia) or nutrient broth (Oxoid, Australia).
The swab was then dipped into 4 mL buffered peptone water or nutrient
broth, vortexed and incubated for further isolation.
2.2. Shell crush methodology
For the isolation of bacteria from egg shell pores, egg shells were
processed as described by Musgrove et al. (2005). Briefly, after
swabbing, each egg shell surface was dipped into 2% tincture iodine
for 1 min to kill any bacteria on the outside of the shell and was
allowed to air dry in a biosafety cabinet. The egg was cracked open
into a sterile container. The inside of the egg shells was then washed
with sterile phosphate buffered saline to remove the adhering egg
albumen because of the antimicrobial activity of albumen. Shell and
shell membranes were transferred to a sterile bag and crushed gently.
To each bag, 50 mL of either buffered peptone water or nutrient broth
was added. The bag was incubated at 37 °C overnight for further
processing.
2.3. Egg internal contents
The egg internal contents collected in the sterile containers were
thoroughly mixed and 1 mL of egg internal content was inoculated
with 4 mL of either buffered peptone water or nutrient broth for
further processing.