mixing with the inoculated sand. At each sampling point, 25 g of
sample was added to 225 mL of sterile 0.1% peptone water and
stomached (Seward 400) for 1 min at 260 strokes per min. Serial
dilutions were prepared using 0.1% peptone water, and 100 ml
aliquot plated onto Salmonella-Shigella agar (Acumedia) in duplicate.
Colonies were counted after 36e48 h incubation at 35 C. The
lower detection limit was set at 1.0 log CFU/g of sample by plating
1 ml of aliquot in duplicate sets of plates. For samples containing
fewer than 1.0 log CFU/g, the presence or absence of Salmonella was
determined by pre-enriching 25 g of sample in 225 mL of lactose
broth (Acumedia) for 24 h at 35 C. The pre-enriched culture
(0.1 mL) was then transferred to 10 mL of selective enrichment,
Rappaport-Vassiliadis R10 (RV) broth (Acumedia) and incubated for
24e48 h at 35 C. The presence or absence of Salmonella was
confirmed by further plating the enriched culture on SalmonellaShigella
agar and examined for typical colony formation on the
plate (as per manufacturers specifications) that was incubated for
24e48 h at 35 C. In order to calculate the total reduction of Salmonella
counts in paste formulations, a 5-tube most probable
number (MPN) technique was carried out at day 86. The 24 hlactose
broth enriched samples were transferred (0.1, 1.0, and
10 ml) into MPN tubes filled with RV broth of appropriate concentrations.
The tubes were incubated for 24e48 h at 35 C.
Confirmation of the growth of Salmonella in the tubes was performed
by streaking a loop of the culture onto Salmonella-Shigella
agar and incubating for 24e48 h at 35 C.
A control plating of supplied chicken base paste and powder
formulations were also performed to confirm the absence of Salmonella
spp. before inoculation. Each formulation was analyzed in
triplicate by pre-enriching 25 g of sample in 225 mL of lactose broth
(Acumedia) for 24 h at 35 C. The samples were further processed
as discussed in previous paragraph to determine the presence or
absence of Salmonella spp. in all treatments.