Inoculation of pork
Raw, 90 % lean pork was purchased from a local store. The
samples to be tested were prepared as follows: The sample
was divided into different parts (10 g for each) and sterilized
with radiation. Prior to inoculation, the absence of live
pathogens was assessed according to ISO standard microbiological
methods for enumeration of microorganisms
[25] and detection of Salmonella spp. [26], L. monocytogenes
[27], E. coli O157:H7 [28], S. aureus [29] and
Y. enterocolitca [30].
Sterile petri dishes containing pork samples were inoculated
with bacteria at numbers ranging from 109 to 100,
for inoculation of each species alone, or 5 9 109 to 5 9 100 for inoculation of the five species together. A
100-lL aliquot of each dilution was inoculated onto the
surface of the pork sample and stored at 4 C for four
hours. Controls consisted of uninoculated samples treated
identically to the inoculated ones.
When each of the pork samples transferred into a sterile
250-mL Erlenmeyer flask, Ninety milliliters of TSB was
added to obtain the proportion 1:10 (sample weight/total
weight). To suspend the bacteria, the bottles were shaken
for 10 min at 200 rpm. The same selective media and
protocol described above were used for enumeration of
bacteria.
The samples were incubated for overnight at 30 ± 1 C.
After overnight culture, a 1.5-mL portion of rinse fluid also
was removed from each bottle and placed in a sterile 2-mL
microcentrifuge tube for DNA extraction. DNA of the
pellet was extracted using the TIANamp Bacteria DNA Kit
according to the manufacturer’s instruction, and the PCR
products were checked as above.
Results
Specificity of
Inoculation of pork
Raw, 90 % lean pork was purchased from a local store. The
samples to be tested were prepared as follows: The sample
was divided into different parts (10 g for each) and sterilized
with radiation. Prior to inoculation, the absence of live
pathogens was assessed according to ISO standard microbiological
methods for enumeration of microorganisms
[25] and detection of Salmonella spp. [26], L. monocytogenes
[27], E. coli O157:H7 [28], S. aureus [29] and
Y. enterocolitca [30].
Sterile petri dishes containing pork samples were inoculated
with bacteria at numbers ranging from 109 to 100,
for inoculation of each species alone, or 5 9 109 to 5 9 100 for inoculation of the five species together. A
100-lL aliquot of each dilution was inoculated onto the
surface of the pork sample and stored at 4 C for four
hours. Controls consisted of uninoculated samples treated
identically to the inoculated ones.
When each of the pork samples transferred into a sterile
250-mL Erlenmeyer flask, Ninety milliliters of TSB was
added to obtain the proportion 1:10 (sample weight/total
weight). To suspend the bacteria, the bottles were shaken
for 10 min at 200 rpm. The same selective media and
protocol described above were used for enumeration of
bacteria.
The samples were incubated for overnight at 30 ± 1 C.
After overnight culture, a 1.5-mL portion of rinse fluid also
was removed from each bottle and placed in a sterile 2-mL
microcentrifuge tube for DNA extraction. DNA of the
pellet was extracted using the TIANamp Bacteria DNA Kit
according to the manufacturer’s instruction, and the PCR
products were checked as above.
Results
Specificity of
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