For the microbiological analysis, samples of 5 g of disinfected larvae were collected and diluted tenfold in physiological peptone solution (PPS, 0.85% NaCl, 0.01% peptone) before pulverizing the larvae in the solution by using an ethanol sterilized home type mixer (Bosch CNHR 25). Similarly, 5 g of substrate was sampled from each replicate and diluted 1:10 in PPS. Next, both larval and substrate samples were homogenized using a stomacher (BagMixer 400CC, Interscience, France) for 1 min. For each sample, the total aerobic viable count and the specific Salmonella count were determined. All plate counts were performed according to the ISO standards for microbial analyses of food and feed as compiled by Dijk et al. (2015) . For the total aerobic viable count, serial dilutions were made in PPS, plated on Plate Count Agar (PCA, Biokar Diagnostics, Beauvais, France), and incubated at 30 °C for 72 h. Salmonella was counted by plating the diluted samples on a chromogenic RAPID′ Salmonella agar (BioRad Laboratories, Belgium) and incubating the plates at 37 °C for 24 h. The cell density of all inocula was also verified by plating a serial dilution on both the RAPID’ Salmonella agar and PCA.
Plate count agar pca
Manufactured by Solabia
Sourced in France
Plate Count Agar (PCA) is a culture medium used for the enumeration of aerobic microorganisms in food, water, and other samples. It provides a solid growth surface for the cultivation and counting of viable bacterial colonies.
Automatically generated - may contain errors
Lab products found in correlation
Rapid salmonella agar, by Bio-Rad (2 mentions)
Cnhr 25, by Bosch (1 mentions)
Bagmixer 400cc, by Interscience (1 mentions)
Muller hinton broth, by Solabia (1 mentions)
Sodium hydrogen carbonate, by Merck Group (1 mentions)
Phosphate buffered saline powder, by Merck Group (1 mentions)
Sodium polyacrylate, by Merck Group (1 mentions)
Sodium carbonate decahydrate, by Thermo Fisher Scientific (1 mentions)
Blood agar plates, by Biolife (1 mentions)
Copper 2 sulfate pentahydrate, by Merck Group (1 mentions)
5 5 dimethyl 1 pyrroline n oxide dmpo, by Enzo Life Sciences (1 mentions)
Milli q plus system, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Peptone, by Merck Group (1 mentions)
Model 400 circulator, by Seward Medical (1 mentions)
Potato dextrose agar (pda), by Solabia (1 mentions)
Sodium hydroxide (naoh), by ITW Reagents (1 mentions)
Ferric chloride hexahydrate, by ITW Reagents (1 mentions)
10 protocols using plate count agar pca
1
Microbiological Analysis of Disinfected Larvae
2
Antimicrobial Activity Evaluation of NPs
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Evaluation of the compounds’ MIC was executed as previously described Silva, Costa [49 (link)]. Briefly, a 0.5 MacFarland inoculum of each microorganism was prepared and inoculated in Muller Hinton Broth (Biokar Diagnostics, Beauvais, France) with dye loaded NPs concentrations ranging from 0.1 to 7 mg/mL. Simultaneously, a negative (non-inoculated media with NPs at 0.1 mg/mL) and a positive control (inoculated media with microorganism only) were also evaluated. The MIC was determined by observing the lowest sample concentration at which no bacterial growth was visible. All assays were performed in triplicate. Determination of the MBC was performed as previously described by Costa, Silva [26 (link)]. Succinctly, MBCs were defined as the lowest sample concentration at which bacterial growth in agar plates was prevented and it was evaluated by inoculation of 100 µL aliquots of negative wells (absence of turbidity in MIC determination) in Plate Count Agar (PCA, Biokar Diagnostics, Beauvais, France), using the plate spread technique. All assays were performed in quadruplicate.
3
Listeria monocytogenes Strain Cultivation
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D-(+)-glucose, sodium polyacrylate, acetic acid 99–100% (glacial), nitric acid (≥65%), hydrogen peroxide 30% (w/w), Triton X-100, sodium tartrate dehydrate, sodium hydrogen carbonate, ascorbic acid and phosphate buffered saline powder were obtained from Sigma-Aldrich (Steinheim, Germany). Copper (II) sulfate-pentahydrate and ethanol were purchased from Merck (Darmstadt, Germany); bicinchoninc acid disodium salt and sodium carbonate decahydrate from Alfa Aesar (Haverhill, MA, USA); and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) from Enzo Life Science (Farmingdale, NY, USA). The olive oil (a mixture of refined and virgin olive oils) was purchased in a supermarket in Italy. Ultrapure water was obtained from a Milli-Q Plus system (Millipore, Bedford, MA, USA) and was always used freshly prepared.
Brain heart infusion broth (BHI), listeria selective agarb (LSM), and blood agar plates were produced by Biolife, while plate count agar (PCA) was produced by Biokar diagnostics. The reference strain for experimental trials was the American Type Culture Collection (ATCC 19117), which came from a Listeria monocytogenes (serotype 4d) third line culture to prevent the risk of phenotypic alteration.
Brain heart infusion broth (BHI), listeria selective agarb (LSM), and blood agar plates were produced by Biolife, while plate count agar (PCA) was produced by Biokar diagnostics. The reference strain for experimental trials was the American Type Culture Collection (ATCC 19117), which came from a Listeria monocytogenes (serotype 4d) third line culture to prevent the risk of phenotypic alteration.
4
Antimicrobial Efficacy Evaluation
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A 1000 μg mL−1 extract solution in TSB was inoculated with an overnight inoculum of each of the pathogenic microorganisms and incubated at 37 °C for 24 h. At the 0, 6, 12 and 24 h mark, the total viable cells were determined using decimal dilutions and plated in Plate Count Agar (PCA, Biokar Diagnostics, Beauvais, France) [16 (link),17 (link)]. The PCA plates were then incubated at 37 °C for 24 h. Each condition was assessed in three independent assays, each considering triplicate of analysis and plating in duplicate.
5
Microbial analysis of food samples
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After one night of defrosting at 4 °C, only the larvae underwent a grinding step of 10 s three times (Blender Nutriboost 23180-56, Russell Hobbs, Courbevoie, France). The mesophilic, psychrotrophic, and endospore aerobic bacteria were quantified with a fraction of 25 g of sample diluted to the tenth with buffered peptone water (VWR) in stomacher plastic bag. The samples were stomached at 25.5 stroke/s for 2 min with a lab paddle blender (Masticator®, IUL, Barcelona, Spain).
After a phase of revival of 1 h at room temperature, serial dilutions were performed in buffered peptone water to 10−8. The total mesophilic flora and bacterial endospore counts were determined with the pour-plate method on Plate Count Agar (PCA, Biokar diagnostics) or PCA with starch 2 g/L at 30 °C for 72h after a heat treatment at 80 °C for 10 min. The spread plate technique was used to count the psychrotrophic bacteria on PCA at 6.5 °C for 10 days. The major food pathogens were investigated and the enterobacteria, lactic bacteria, and yeasts/molds were quantified according to the standards used in the LAB Eurofins (Table 2 ).
After a phase of revival of 1 h at room temperature, serial dilutions were performed in buffered peptone water to 10−8. The total mesophilic flora and bacterial endospore counts were determined with the pour-plate method on Plate Count Agar (PCA, Biokar diagnostics) or PCA with starch 2 g/L at 30 °C for 72h after a heat treatment at 80 °C for 10 min. The spread plate technique was used to count the psychrotrophic bacteria on PCA at 6.5 °C for 10 days. The major food pathogens were investigated and the enterobacteria, lactic bacteria, and yeasts/molds were quantified according to the standards used in the LAB Eurofins (
6
Microbial Safety and Sensory Evaluation of Fermented Milk
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Before sensory analysis, additional fermented milk samples were used for the determination of total microbial and Enterobacteriaceae counts, through plating of sequential dilutions in Plate Count Agar (PCA; Biokar, Allonne, France) and Violet Red Bile Glucose Agar (VRBGA; Biokar) plates, and incubation for 24 h at 30 °C or 37 °C in aerobiosis, respectively.
Once microbiological safety was guaranteed, fermented milk samples were subjected to sensory characterization by a senior trained panel (n = 8). Samples were assessed in terms of appearance, aroma, texture, mouthfeel, flavor and after-taste.
Once microbiological safety was guaranteed, fermented milk samples were subjected to sensory characterization by a senior trained panel (n = 8). Samples were assessed in terms of appearance, aroma, texture, mouthfeel, flavor and after-taste.
7
Quantifying Salmonella and Aerobic Counts
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For each sample, the total aerobic viable count and the specific Salmonella count were determined. All plate counts were performed according to the ISO standards for microbial analyses of food and feed as compiled by Dijk et al. (2015) . For the total aerobic viable count, serial dilutions were made in PPS, plated on Plate Count Agar (PCA, Biokar Diagnostics, Beauvais, France), and incubated at 30°C for 72 hours. Salmonella was counted by plating the diluted samples on a chromogenic RAPID′ Salmonella agar (BioRad Laboratories, Belgium) and incubating the plates at 37°C for 24 hours. The cell density of all inocula was also verified by plating a serial dilution on both the RAPID' Salmonella agar and PCA.
8
Microbial Enumeration in Food Samples
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The samples (10 mL) were homogenized in a stomacher (Model 400 Circulator, Seward, Norfolk, England) with peptone (Sigma, Sintra, Portugal) water (90 mL), serially diluted and plated on Plate Count Agar (PCA, Biokar Diagnostics, Solabia, France) and incubated at 30 C during 24 h. Total yeasts and molds were also determined for the raw samples using Potato Dextrose Agar -PDA (Biokar Diagnostics, Solabia, France). The plates were incubated for 2 days at 30 C. In both cases, colonies were enumerated and total viable cells (cfu/g) determined.
9
Strawberry safety and quality analysis
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Strawberries (Fragaria × ananassa) were purchased from local providers the same day of the experiment, and stored at 4 °C until it was carried out. Strawberries with no visible damages and similar in size were selected, and peduncle was removed.
Triptone soy broth (TSB), triptone soy agar (TSA), Palcam base agar and Palcam selective supplement for Listeria spp., yeast extract, plate count agar (PCA), dichloran rose bengale chloramphenicol agar (DRBC), sodium chloride and peptone were obtained from Biokar Diagnostics (Allonne, France). Dey-Engley broth was obtained from Honeywell Fluka (Madrid, Spain).
2,4,6-tris (2-pyridyl) -s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and sodium carbonate were acquired from Sigma-Aldrich (Steinheim, Germany). Methanol, chlorhidric acid (37%), sodium acetate, sodium hydroxide, ferric chloride hexahydrate and Folin Ciocalteau's reagent were procured from Panreac (Llinars del Vallès, Spain).
Triptone soy broth (TSB), triptone soy agar (TSA), Palcam base agar and Palcam selective supplement for Listeria spp., yeast extract, plate count agar (PCA), dichloran rose bengale chloramphenicol agar (DRBC), sodium chloride and peptone were obtained from Biokar Diagnostics (Allonne, France). Dey-Engley broth was obtained from Honeywell Fluka (Madrid, Spain).
2,4,6-tris (2-pyridyl) -s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and sodium carbonate were acquired from Sigma-Aldrich (Steinheim, Germany). Methanol, chlorhidric acid (37%), sodium acetate, sodium hydroxide, ferric chloride hexahydrate and Folin Ciocalteau's reagent were procured from Panreac (Llinars del Vallès, Spain).
10
Enumeration of Microbial Populations in Rose Flowers
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Microbial population was first determined in a sample of freshly picked rose flowers. Three replicates of 15 g fresh rose C. flowers were blended in 100 mL sterile demineralized water using a Stomacher® 400 blender (Seward, Worthing, UK) (2 × 60 s), and 100 μL of the appropriate decimal dilutions were spread onto Plate Count Agar (PCA) (Biokar Diagnostics) and Yeast Glucose Chloramphenicol agar (YGC) (Biokar Diagnostics), to enumerate, respectively, aerobic mesophilic bacteria and yeasts and molds. Counts of colony-forming units (CFU) of aerobic mesophilic bacteria in hydrosols were performed by spreading 100 μL of the appropriate decimal dilutions of hydrosols in sterile demineralized water onto 10 fold-diluted PCA, complemented with agar (Biokar Diagnostics) to get a final 12 g/L agar concentration (DPCA), unless otherwise specified. Previous tests in our laboratory consistently showed a better recovery on DPCA than on PCA (data not shown). All Petri dishes were incubated at 30 °C for two days before colony counts were recorded. Counts of microbial cells in hydrosols were performed using a Malassez counting chamber and an Olympus BX50 phase-contrast microscope at ×400 magnification.
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