NRMRL approach
Samples of raw sludge from a large egg processing facility
were collected and held under refrigeration (4–1C) for no
more than 1 day prior to processing. Samples of raw sludge
from a municipal wastewater treatment facility were collected
and held under refrigeration (4–1C) for no more than 2-h
prior to processing. Lime treatment of samples was by direct
addition of calcium hydroxide (hydrated lime) to the municipal sludge in a stirred beaker. Controls included
laboratory grown cultures of wild type Salmonella and E. coli.
Undiluted samples were transferred to a sterile blending cup
and blended for 2 min at 18 000 rev min)1
; 10 ml of this
suspension was used to inoculate fermentation tubes containing
10 ml of double strength BPW. Thirty grams of
sample were blended for 2 min at 18 000 rev min)1 with
270 ml of sterile phosphate-buffered dilution water to
produce a 1 : 10 (w : v) suspension. Ten milliliter portions
of this suspension were transferred to fermentation tubes
containing 10 ml of double strength BPW. Decimal dilutions
of the suspension were used to inoculate tubes containing
single strength BPW through 10)3 ml. Inoculated tubes were
incubated for 24 h at 35C. Following incubation 0Æ1 ml of
the BPW was inoculated into tubes containing 10 ml of RV
broth. These were incubated for 24 h at 41Æ5C. The DNA
hybridization assays were performed according to the test kit
instructions (Gene-Trak) using 0Æ5 ml of each RV culture
for a given assay. To confirm assay results, an inoculating
loop of each RV culture was streaked on to xylose lysine
Tergitol 4 (XLT4) agar plates following 24 and 48 h of
incubation at 35C. Black or black-centered colonies appearing
on XLT4 plates were considered presumptive positive
for Salmonella spp. and were biochemically confirmed based
upon growth characteristics exhibited on triple sugar iron
agar (TSIA) slants and LIA slant cultures after 24 h at 35C.
Representative atypical colonies (yellow, red and white)
when observed, were also transferred to TSIA slants.
Following incubation, individual cultures were assayed for
agglutination with Salmonella poly O antisera. Cultures
yielding typical biochemical responses and agglutination
were scored as positive for Salmonella spp. Total solid
analysis of the biosolids was by method 2540G as described
by APHA et al. (1992).
RES