Rappaport

For quantitative bacteriology, 1 g of pig feces was combined with 5 ml PBS, vortexed, and 0.1 ml directly plated to XLT-4 medium (Beckton, Dickinson and Co., Sparks, MD, USA) containing 30 μg/ml of nalidixic acid. For tissue samples, 1 g of each tissue was combined with 2 ml of PBS in a whirlpak bag, pounded with a mallet, and homogenized in a Stomacher (Seward, Westbury, NY, USA) for 1 min. One hundred microliters of the resulting solution was aliquoted onto XLT-4 medium containing nalidixic acid. One hundred microliters of a 10-fold dilution of each fecal and tissue sample was also plated, and additional dilutions were performed when CFU reached >300/plate. Following 48 h of incubation at 37°C, black colonies were enumerated and a single colony from each plate was confirmed to be Salmonella by serogroup antiserum agglutination (Beckton, Dickinson and Co., Sparks, MD, USA). The total number of CFU for each quantitative tissue or fecal sample was calculated per gram of sample by obtaining the number of Salmonella per plate and multiplying by the dilution factor.
Qualitative bacteriology of Salmonella was performed as follows: 1 g (fecal) or 0.1 ml (homogenized tissue) samples were inoculated in 10 ml tetrathionate broth (TET; VWR, Rutherford, NJ, USA) for 48 h of growth at 37°C. Following incubation, 0.1 ml of each culture was transferred to 10 ml Rappaport–Vassiliadis medium (RV; Difco) and incubated at 37°C for 18–20 h. Cultures were streaked on XLT-4 medium containing nalidixic acid. Colonies suspicious for Salmonella were confirmed by serogroup antiserum agglutination.
Statistical analysis for Salmonella shedding in feces (CFU/g) was Log10-transformed and analyzed using a mixed linear model for repeated measures (Proc Mixed in SAS for Windows, version 9.2; SAS Institute Inc., Cary, NC, USA). Covariance structures within pigs across time were tested and modeled using the REPEATED statement to determine the optimal covariance structure. Linear combinations of the least-squares mean estimates were used in a priori contrasts after testing for a significant (P < 0.05) treatment group effect. Comparisons were made between each group at each time point, using a 5% level of significance (P < 0.05) to assess statistical differences. The endpoint data for bacterial colonization (CFU/g) of tissues collected at necropsy were Log10-transformed and analyzed by analysis of variance using a general linear model for unbalanced data. A 5% level of significance (P < 0.05) was used to assess statistical differences.

In each experiment, 30 chicken carcasses prior to inside outside wash step were collected from a processing plant in large sterile bags. Chickens were weighed and the deep muscle breast temperature was recorded within 20 min of arrival in the laboratory. Individual carcasses were subsequently placed in a whirl pack bag (ThermoFisher Scientific, Scoresby, Australia) and washed with massaging for 2 min in 200 mL buffered peptone water (BPW; ThermoScientific, Oxoid, Scoresby, Australia). Two mL of the BPW wash were spread plated onto five modified charcoal-cefoperazone-deoxycholate agar (mCCDA) (ThermoScientific, Scoresby, Australia) plates (400 µL per plate) and incubated at 42 °C with 10% CO₂ for 48 hours to assess direct Campylobacter spp. counts. From the initial 200 mL BPW wash, 40 mL was incubated overnight at 37 °C. Then, 100 µL of the incubated BPW was transferred into 10 mL Rappaport Vassiliadis soya peptone broth (RVS, ThermoScientific, Oxoid, Scoresby, Australia) and incubated overnight at 42 °C for selective growth of Salmonella enterica serovars. A loop-full of the RVS broth was streaked on to xylose lysine deoxycholate agar (XLD; ThermoScientific, Oxoid, Australia) plates. Suspected Salmonella colonies were subcultured onto Brilliance Salmonella agar (ThermoScientific Oxoid, Scoresby Australia) for confirmation. To quantify the total viable counts (TVC) in the carcass wash, the carcass wash was diluted 10-fold and plated on nutrient agar. The limit of detection for TVC was 0.25 colony forming units (CFU)/cm² of chicken carcass. The plates were incubated overnight at 37 °C and colonies were recorded in colony forming units (CFU). For Campylobacter spp. the limit of detection was 10 CFU/mL of rinsate.
Following the initial BPW wash, 5 carcasses each were placed into six different treatment groups, water and sanitizer wash each at 5 °C, 15 °C, and 22 °C (Table 1). Carcasses were placed into large containers filled with diluted sanitizer and agitated continuously for the entire treatment period (Table 1). Carcasses were then removed and placed in a sterile bag and rinsed as with BPW. The miniaturized Most Probable Number (MPN) method described by [20 (link)], was used to determine the Salmonella load in culture-positive samples. Campylobacter and Salmonella counts as well as the TVC were interpreted per square centimeter of carcass as per the Australian standard [21 ]. Briefly, surface area of a whole chicken carcass in square centimeters was calculated by the following formula, 0.87 m + 635 (m = total mass in grams) and the microorganisms per square centimeter of surface area from the rinse fluid was calculated using the following formula,

The techniques used to isolate and identify Salmonella were recommended by the International Organization for Standardization (ISO-6579, 2002) and the World Health Organization [27 ]. Global foodborne infections network (formerly WHO global Salmonella Surveillance) [27 ]. In a nonselective liquid medium (buffered peptone water (BPW) (Oxoid CM509, Basingstoke, England), a 10 ml milk sample was mixed with 90 ml of pre-enrichment, and the sample mixture was thoroughly shaken before being incubated at 37°C for 24 hours. Following incubation, the culture was mixed, and a portion (0.1 ml) was transferred to a tube containing 10 ml of selective enrichment liquid medium (Rappaport Vassiliadis (RV)) broth and incubated at 41.5°C for 24 hours. A 10 µl of loop full inoculum from selective enrichment media was streaked onto Xylose Lysine Deoxycholate (XLD) (Oxoid CM0469, Basingstoke, England) agar and Salmonella Shigella (SS) agar plates prepared on petri-dishes and incubated at 37 ± 1°C for 24 ± 3 hours. After proper incubation, the plates were examined for the presence of typical Salmonella colonies. The typical colonies of Salmonella grown on Xylose Lysine Deoxycholate agar medium produce black centers with distinct red colonies due to the color change of phenol red in medium and colorless transparent colonies on Salmonella Shigella agar. The presumptive Salmonella colonies on the XLD (Oxoid CM0469, Basingstoke, England) and SS agar medium were transferred onto the surface of predried nutrient agar plates in a manner that allow isolated colonies to develop and incubated at 37°C for 24 hours in further confirmation with biochemical tests. Thus, all suspected Salmonella colonies were picked from the nutrient agar and inoculated into the biochemical test including Triple Sugar Iron (TSI) agar (Oxoid CM0277, Basingstoke, England) for the TSI test, Simmons's Citrate agar (Oxoid CM53, Basingstoke, England) for the citrate utilization test, Tryptone Soya Broth (Becton Dickinson, USA) for the indole test and Methyl red-Voges Proskauer (MR-VP) (Micromaster Thane, India) for methyl red and Voges Proskauer test and incubated for 24 or 48 hours at 37°C. Colonies producing an alkaline (red) slant, with acid (yellow) but on TSI with blackening or hydrogen sulfide production, negative for Tryptophan utilization on indole test (yellow-brown ring), positive for Methyl red (produce red color on the surface of medium), negative for Voges–Proskauer (yellow color), and positive for Citrate utilization (deep blue slant) were consider to be Salmonella positive [28 (link), 29 ].

A 25 g of dried beef meat powder was mixed with 225 mL of buffered peptone water (Oxoid, UK) with a nonselective enrichment followed by serial dilutions using 9 mL of 1% buffered peptone water and normal saline water. Total coliform and fecal coliform counts were conducted using pour plate method, whereby 1 mL of each serial dilution was pipetted into a Petri dish and then mixed with a 5 mL of tryptone soya agar; the agar with 10-15 mL of violet-red bile lactose agar (VRBLA) (Oxoid, UK) was covered. For the total coliform count, the plates were incubated at a temperature of 37.0°C and for fecal coliform incubated at 44.0°C for the duration of 24 ± 2 hours. For the presumptive coliform and fecal coliform, approximately 1 mm diameter size colonies with pink color were counted. Confirmatory tests were done by taking five representative colonies and inoculating to brilliant green bile broth and Escherichia coli (E. coli) broth, respectively, for coliform and fecal coliform count. E. coli count was conducted after fecal coliform count was made using E. coli broth and nutrient broth according to [35 (link)].
Aerobic mesophilic count was determined using a pour plate method, kept at 30°C for 48 hours. Sample of buffered peptone water (BPW); then, serial dilutions were made; plates were put at 30°C for 72 hours [36 ]. Colonies were counted in each of the Petri dishes with a count between 30 and 300 colony-forming units (cfu). Salmonella was detected according to the method described by [37 (link)]. Samples were nonselectively enriched in buffered peptone water (BPW). After 24-hour growth at 36±1⁰c, selectively enriched using selenite cysteine broth and rappaport vassiliadis enrichment broths, then incubated at 35±1⁰c. After purification and biochemical identification, qualitative results were presented as present and absent. Mold and yeast counts were performed by spread plate techniques; 0.1 mL of the respective diluted sample was added to prepare rose bengal agar supplemented with chloramphenicol. Samples were spread using a spreader after being incubated at 22°C for 5-7 days according to the method described for total mold and yeast counts (cfu/g) [38 ]. Microbial load of MP was checked after completing the process of slicing, air-drying, and making the dried meat into powder form. The same process was followed for a standard diet, except slicing which does not need standard feed. Final microbial analysis was done to estimate the degree of microbial contamination at the final product after many traditional meat powder preparation processes as similar steps as food preparation at household level aimed to sprinkle the MP in complementary foods of children in the future.