Spc agar

C. muytjensii ATCC51329 and C. sakazakii ATCC29544 were cultured at 37 °C for 16 h using Brain Heart Infusion Broth (Eiken Chemical Co., Ltd., Tokyo, Japan). An aliquot of 5 ml of the culture was placed in a 15-ml Falcon tube (Becton Dickinson Labware, Franklin Lakes, NJ, USA) and subjected to refrigerated centrifugation at 3,000 × g for 10 min at 4 °C. The supernatant was removed, and 5 ml of physiological saline was subsequently added to the pellet to prepare a stock live cell suspension of Cronobacter. The live cell suspension was appropriately diluted with physiological saline as in the test sample. Independently, 1 ml of the aforementioned stock of live cell suspension was placed in a 1.5-ml volume microtube (Eppendorf, Hamburg, Germany), and the tube was subsequently immersed in a boiling water bath for 50 sec and immediately quenched. To generate a stock of heat-killed (dead) cell suspension, we confirmed that the bacteria did not thereafter form any colonies on a standard plate count agar medium (SPC agar: Eiken). The viable cell count of C. muytjensii or C. sakazakii live cell suspension was conducted on SPC agar medium, and measurements of turbidity were simultaneously performed at a wavelength of 600 nm using a spectrophotometer U-2800 A (Hitachi, Tokyo, Japan) to determine the relationship between the live cell count and turbidity.

To determine the bacterial counts, 3 g of the sample was obtained aseptically from the core portion of the sausages and transferred to a sterile plastic bag. Then, the sample was homogenized with 27 mL of sterile saline (0.9% salt) using a stomacher (Exnizer 400, Organo, Tokyo, Japan). Ten-fold serial dilutions were performed, and suitable dilutions (0.1 mL) were poured into the Petri dish in duplicate. The Petri dishes containing two different growth media, standard plate count (SPC) agar (Eiken, Chemical Co. Ltd., Tochigai, Japan) for the total bacterial count and MRS agar (Oxoid, Kanto Chemical Co. Ltd., Tokyo, Japan) for the total lactic acid bacterial count, were used. The dilutions were spread onto the plate using a sterilized glass rod. Subsequently, the SPC agar plate was incubated aerobically at 30 °C for 24 h, and the MRS agar plate was incubated anaerobically at 30 °C for 48 h. Finally, the number of bacteria was calculated and expressed as log CFU/g.