The enriched bacterial cultures (100 μl) from the samples were transferred and spread on PALCAM agar (Merck, Germany). The PALCAM agar plates were incubated at 37°C for 48 hr. Listeria spp. colonies appeared to be greyish-green or black in color and surrounded by black halo on PALCAM agar [26 ]. These colonies were observed, counted, and recorded.
Palcam agar
Manufactured by Merck Group
Sourced in Germany
PALCAM agar is a selective and differential culture medium used for the isolation and identification of Listeria monocytogenes in food and environmental samples. It contains ingredients that inhibit the growth of most other bacteria, allowing Listeria species to be easily detected.
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Lab products found in correlation
Tryptic soy broth (tsb), by Merck Group (2 mentions)
Mrs agar, by Merck Group (1 mentions)
Listeria enrichment broth, by Merck Group (1 mentions)
Tryptone soy agar, by Merck Group (1 mentions)
Oxford agar, by Merck Group (1 mentions)
Buffered listeria enrichment broth, by Merck Group (1 mentions)
Xylose lysine deoxycholate agar xld agar, by Merck Group (1 mentions)
Buffered peptone water (bpw), by Merck Group (1 mentions)
Bacteriological agar, by Avantor (1 mentions)
Phosphate buffered saline (pbs), by Avantor (1 mentions)
Tsa ye, by Merck Group (1 mentions)
Macconkey agar, by Merck Group (1 mentions)
Plate count agar, by Lab M (1 mentions)
12 protocols using palcam agar
1
Listeria spp. Isolation from Samples
2
Probiotic Inhibition of Foodborne Pathogens
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The ability of the selected Lactobacillus spp. strains to inhibit the growth of pathogenic bacteria, namely S. Typhimurium, S. Enteritidis, S. Choleraeusis, and L. monocytogenes, was evaluated in co-cultures with the plate count method.
Monocultures of each probiotic or pathogenic strain were propagated in the MRS liquid medium or nutrient broth, respectively, with 2% (w/v) of the prebiotic substance, or glucose for comparison. The strains were cultivated at 37 °C for 24 h without oxygen limitation. Afterward, the bacterial monocultures grown on specific prebiotic were mixed in the amount of 1 mL of cultures of all probiotic strains with each pathogen separately. An initial number of bacteria was assessed by preparing serial decimal dilutions of each mixture, which were then used in the pour plate technique with MRS agar, Salmonella Shigella agar (SS agar; Merck Millipore), and PALCAM agar (Merck Millipore) for probiotics, Salmonella spp., and L. monocytogenes, respectively. Next, the co-cultures of probiotics and pathogens were incubated at 37 °C without oxygen limitation.
The changes in the bacterial count were evaluated after 4, 8, 12, and 24 h as it was previously done, in order to determine the initial number of bacteria. The plates were incubated at 37 °C for 24 h and the results are presented in CFU/mL. Each co-culture was conducted in three repetitions.
Monocultures of each probiotic or pathogenic strain were propagated in the MRS liquid medium or nutrient broth, respectively, with 2% (w/v) of the prebiotic substance, or glucose for comparison. The strains were cultivated at 37 °C for 24 h without oxygen limitation. Afterward, the bacterial monocultures grown on specific prebiotic were mixed in the amount of 1 mL of cultures of all probiotic strains with each pathogen separately. An initial number of bacteria was assessed by preparing serial decimal dilutions of each mixture, which were then used in the pour plate technique with MRS agar, Salmonella Shigella agar (SS agar; Merck Millipore), and PALCAM agar (Merck Millipore) for probiotics, Salmonella spp., and L. monocytogenes, respectively. Next, the co-cultures of probiotics and pathogens were incubated at 37 °C without oxygen limitation.
The changes in the bacterial count were evaluated after 4, 8, 12, and 24 h as it was previously done, in order to determine the initial number of bacteria. The plates were incubated at 37 °C for 24 h and the results are presented in CFU/mL. Each co-culture was conducted in three repetitions.
3
Listeria monocytogenes Detection Protocol
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A similar volume of samples was primarily homogenized with 225 ml of Listeria enrichment broth (Merck, Germany). Incubation was carried out for 24 h at 37°C. Afterward, a milliliter of samples was transferred to Frazer broth (9 ml) (Merck, Germany). Incubation was carried out for 24 h at 37°C. PALCAM agar (Merck, Germany) and Oxford agar (Merck, Germany) media were used for other enrichment. Incubation was done for 48 h at 35°C. Three black colonies with black sunken were cultured on Tryptone Soy agar (Merck, Germany) containing yeast extract (0.6%). Incubation was carried out for 24 h at 37°C. Listeria monocytogenes was identified by biochemical tests [11 ].
4
Listeria Isolation and Identification Protocol
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Initially, the specimens were inoculated in Buffered Listeria Enrichment Broth (BLEB, Merck, Germany) and were incubated at 4°C for 2 weeks to 1 month. The inoculum was then plated on PALCAM agar (Merck, Germany), Oxford agar (Difco, USA) and CHROM agar Listeria (Paris, France) plates. After 48 h of incubation at 37°C, colonies morphologically resembling Listeria were submitted for confirmatory examinations using Gram staining, catalase and oxidase tests, motility and sugar fermentation tests (xylose, rhamnose, mannitol, α-methyl D-mannopyranoside), hemolysis on 5% sheep blood agar and CAMP test (8 (link), 9 (link)). In CAMP test, the L. monocytogenes isolates were streaked perpendicular to Staphylococcus aureus on 5% sheep blood agar plates and zones of hemolysis were investigated, after 24–48 h of incubation at 35°C (10 (link)).
5
Enumeration of Salmonella and Listeria
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For the determination of the remaining (surviving) inoculum, an appropriate volume of maximum recovery diluent (MRD; Merck, Darmstadt, Germany) was added into each bag (in a 1:10 ratio) [13 (link)]. The bags were shaken and the serial decimal dilutions were prepared. From each dilution, 100 μL was spread into the appropriate medium: xylose-lysine-deoxycholate agar (XLD agar; Merck, Darmstadt, Germany) and PALCAM agar (Merck, Darmstadt, Germany) for S. enterica and L. monocytogenes, respectively. The plates were then incubated at 37 °C for 24–48 h, typical colonies for each bacterium were counted and the results were expressed as log cfu/mL.
6
Ozone Treatment for Microbial Reduction
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Treatments with ozone were performed as previously described using artificially inoculated peel paste. Approximately 35.0 g of sample was immersed during 1 min in 100 mL of bacterial suspension, and excess was discarded. For enumeration, 225 mL of sterile peptone water (BPW, Merck, Darmstadt, Germany) was added to approximately 25.0 g of peel paste into stomacher bags and pummelled for 2 min at normal speed. L. innocua was determined following the spread plate method on Palcam agar (Merck, Darmstadt, Germany) at an incubation temperature of 37 °C for 48 h. Microbial counts were achieved in duplicate and expressed as cfu/mL.
7
Survival of Listeria monocytogenes in Chicken Offal
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The survival determination study was carried out as described in our previous study with modification (Tang et al., 2014) . Three grams of chicken liver, gizzard and heart were placed in universal bottles meant for different holding time (0, 1, 3, and 6 h). A total of 20 µl of inoculum was spiked onto the chicken offal and incubated at 28 o C. L. monocytogenes enumeration was performed using a spread plate method on PALCAM agar (Merck, Germany). The experiments were done in triplicates.
• Data analysis Data collected during the experiment was analyzed using SPSS 17.0 software. The data were analyzed using oneway ANOVA. The significance level was set at p < 0.05.
• Data analysis Data collected during the experiment was analyzed using SPSS 17.0 software. The data were analyzed using oneway ANOVA. The significance level was set at p < 0.05.
8
Listeria Survival on Chicken Offal
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L. monocytogenes growth on chicken liver, gizzard and heart were determined to simulate its survival patterns in cases of crosscontamination due to poor food handling. The level of L. monocytogenes on chicken offal will affect the amount being transfer to the cooked chicken offal via food contact surfaces (Goh et al., 2014) .
• Preparation of L. monocytogenes inoculum L. monocytogenes ATCC 7944 was revived in 10 ml of Tryptic Soy Broth (TSB) (Merck, Germany) and incubated at 37 o C for 24 h. The revived culture was streaked on PALCAM agar (Merck, Germany) and incubated at 37 o C for 24 h to obtain a purified culture. Pure L. monocytogenes culture was grown in TSB (Merck) in a condition described earlier before the culture was harvested for inoculum preparation. The inoculum of L. monocytogenes was determined using spectrophometer at 600 nm wavelength to give OD 0.99 which corresponds to 9.20 log CFU/ml.
• Preparation of L. monocytogenes inoculum L. monocytogenes ATCC 7944 was revived in 10 ml of Tryptic Soy Broth (TSB) (Merck, Germany) and incubated at 37 o C for 24 h. The revived culture was streaked on PALCAM agar (Merck, Germany) and incubated at 37 o C for 24 h to obtain a purified culture. Pure L. monocytogenes culture was grown in TSB (Merck) in a condition described earlier before the culture was harvested for inoculum preparation. The inoculum of L. monocytogenes was determined using spectrophometer at 600 nm wavelength to give OD 0.99 which corresponds to 9.20 log CFU/ml.
9
Listeria monocytogenes Detection and Enumeration
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Listeria monocytogenes detection (enrichment protocol) and enumeration were performed according to the ISO 11290-1:2017 and ISO 11290-2:2017 standards, respectively (ISO, 2017). The selective agar medium used was PALCAM agar (Merck, Darmstadt, Germany) plus selective supplement (Merck).
Pediococcus acidilactici was serially diluted in sterile ¼ Ringer's solution and plated in duplicate for enumeration by the drop count technique (Miles, Misra, & Irwin, 1938) (link) on MRS agar and incubated at 37 ºC for 48 h, aerobically.
Phage Listex™ P100 titre (PFU mL -1 ) was determined by the double-layer method (plaque assay) as previously described by (Kropinski, Mazzocco, Waddell, Lingohr, & Johnson, 2009) (link) with modifications in the media and diluent. TSAYE was selected as the solid media (underlay) and TSBYE, containing 7g L -1 of bacteriological agar (VWR Chemicals), was used as molten soft agar (overlay). The diluent used in the experiments was PBS (0.1 M, pH 7.4, VWR Chemicals) and the detection limit of the enumeration technique was 10 PFU mL -1 .
Pediococcus acidilactici was serially diluted in sterile ¼ Ringer's solution and plated in duplicate for enumeration by the drop count technique (Miles, Misra, & Irwin, 1938) (link) on MRS agar and incubated at 37 ºC for 48 h, aerobically.
Phage Listex™ P100 titre (PFU mL -1 ) was determined by the double-layer method (plaque assay) as previously described by (Kropinski, Mazzocco, Waddell, Lingohr, & Johnson, 2009) (link) with modifications in the media and diluent. TSAYE was selected as the solid media (underlay) and TSBYE, containing 7g L -1 of bacteriological agar (VWR Chemicals), was used as molten soft agar (overlay). The diluent used in the experiments was PBS (0.1 M, pH 7.4, VWR Chemicals) and the detection limit of the enumeration technique was 10 PFU mL -1 .
10
Enumeration of Listeria monocytogenes in Ground Beef
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Serial dilutions from the cooled TSBYE tubes were prepared using MRD (Maximum Recovery Diluent; 1 g/l peptone, 8.5 g/l NaCl) and 0.1 ml portions from appropriate dilutions were spread on Tryptic Soy Agar supplemented with 0.6% yeast extract (TSAYE, Merck, Darmstadt, Germany) and plates were incubated at 30°C for 24-48 h. After incubation colonies were counted manually and the results were expressed as log cfu/ml of TSBYE.
A ten gram ground beef sample was transferred to stomacher bag added with 90 ml MRD and homogenized for 2 min. Serial dilutions were prepared using MRD and 0.1 ml portions from dilutions were spread plated on TSAYE. Plates were kept at 25°C for 3 hours. Then, 10 ml PALCAM Agar (Merck, Darmstadt, Germany) added with selective supplement at 45°C was poured on TSAYE (Miller et al., 2010) (link). Plates were incubated at 30°C for 48 h. After incubation the typical grey-green colonies with black zone were counted as L. monocytogenes and the results were expressed as log cfu per gram of ground beef (log cfu/g).
A ten gram ground beef sample was transferred to stomacher bag added with 90 ml MRD and homogenized for 2 min. Serial dilutions were prepared using MRD and 0.1 ml portions from dilutions were spread plated on TSAYE. Plates were kept at 25°C for 3 hours. Then, 10 ml PALCAM Agar (Merck, Darmstadt, Germany) added with selective supplement at 45°C was poured on TSAYE (Miller et al., 2010) (link). Plates were incubated at 30°C for 48 h. After incubation the typical grey-green colonies with black zone were counted as L. monocytogenes and the results were expressed as log cfu per gram of ground beef (log cfu/g).
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