Viable cells of Lactiplantibacillus plantarum were estimated by serial dilution from 10−1 to 10−8 g/L with buffered peptone water (Scharlab, Barcelona, Spain), seeding on MRS agar (Scharlab, Barcelona, Spain) and incubation at 37 °C for 24 h. First dilution (10−1 g/L) was obtained by mixing 3 g of solid sample with 27 mL of sterile buffered peptone water in a stomacher bag and homogenizing for 2 min. Finally, colonies present on the plates were counted.
Buffered peptone water (bpw)
Manufactured by Scharlab
Sourced in Spain
The BPW is a laboratory equipment designed for general-purpose use. It is a compact, portable device that can be used for a variety of applications in the laboratory setting. The core function of the BPW is to provide a reliable and consistent power source for various laboratory instruments and equipment.
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Lab products found in correlation
Formic acid, by ITW Reagents (2 mentions)
Acetonitrile, by ITW Reagents (2 mentions)
Milli q advantage a10 ultrapure water purification system, by Merck Group (2 mentions)
Methanol, by ITW Reagents (2 mentions)
Hesperidin, by Merck Group (2 mentions)
Nutrient agar plates, by Scharlab (2 mentions)
Xylose lysine deoxycholate xld, by Liofilchem (2 mentions)
Api 20e, by bioMérieux (2 mentions)
Potato dextrose agar (pda), by Condalab (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Ketoconazole, by Merck Group (1 mentions)
Triphenyl tetrazolium chloride (ttc), by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Potato dextrose broth (pdb), by Scharlab (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Mrs agar, by Scharlab (1 mentions)
Heracell 150, by Thermo Fisher Scientific (1 mentions)
Plate count agar, by Scharlab (1 mentions)
Rose bengal agar, by Scharlab (1 mentions)
Ethylenediaminetetraacetic acid disodium salt 2 hydrate edta, by ITW Reagents (1 mentions)
17 protocols using buffered peptone water (bpw)
1
Enumeration of Lactiplantibacillus plantarum
2
Quantification of Hesperidin and Cyanidin
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Hesperidin was obtained from Merck (Darmstadt, Germany), and cyanidin 3-O-glucoside was purchased from TransMIT (Geiben, Germany). Acetonitrile, formic acid, methanol, and ethylenediaminetetraacetic acid disodium salt 2-hydrate (EDTA), were obtained from Panreac (Barcelona, Spain). Buffered peptone water, Plate Count Agar (PCA), Rose Bengal Agar (RBA), Brilliant Green Bile 2% Broth (BGBB) and Man Rogosa Sharpe Agar (MRSA) were purchased from Scharlab (Barcelona, Spain). L-ascorbic acid (AA) and dehydroascorbic acid (DHAA) were acquired from Acros Oganics (Thermo Fisher Scientific Inc., Madrid, Spain) and Sigma-Aldrich (St. Louis, MO, USA), respectively. All solutions were prepared with ultrapure water from a Milli-Q Advantage A10 ultrapure water purification system (Millipore, Burlington, MA, USA).
3
Microbiological Culture Media Assays
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Microbiological culture media used in this study consisted of potato dextrose broth (PDB) and buffered peptone water (BPW) (purchased from Scharlab (Barcelona, Spain)); tryptic soy broth (TSB) and tryptic soy agar (TSA) (from Labkem (Barcelona, Spain)); potato dextrose agar (PDA) (from Condalab (Madrid, Spain)); reinforced clostridial medium (RCM) (from Thermo Scientific (Madrid, Spain)). Antibiotics, such as kanamycin, gentamycin, erythromycin, ketoconazole, and triphenyl tetrazolium-chloride (TTC) (from Sigma-Aldrich (Madrid, Spain)), were also used on these assays.
4
Enumeration of Gut Microbiota
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The cecal contents were diluted in 9 mL (1 gm/1,000) Buffered Peptone Water (Scharlau, Spain), then the tube was 10-fold serially diluted. From each tube, 0.1 mL was transferred to each different selective media. The Lactobacillus spp. was enumerated on the MRS agar. The media was incubated at 37°C with 5% CO2 using anaerobic incubator (Thermo fisher Heracell150, Golden Valley, MN) for 72 h. Escherichia coli and Salmonella spp. were enumerated using MacConkey agar and Salmonella Shigella agar at 37°C for 24 h, respectively. All data were expressed in colony-forming units (cfu ) (Log10 CFU/g of tissue).
5
Simultaneous Analysis of Anthocyanin and Flavonoid
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Cyanidin 3-O-glucoside was obtained from TransMIT (Geiben, Germany) and Hesperidin from Sigma-Aldrich (St. Louis, MO, USA). Brilliant Green Bile 2% Broth (BGBB), Plate Count Agar (PCA), buffered peptone water, Man Rogosa Sharpe Agar (MRSA), and Rose Bengal Agar (RBA) were acquired from Scharlab (Barcelona, Spain). Methanol, acetonitrile, formic acid, and ethylenediaminetetraacetic acid disodium salt 2-hydrate (EDTA), from Panreac (Barcelona, Spain). Dehydroascorbic and L-ascorbic acids (DHAA and AA, correspondingly) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Acros Oganics (Thermo Fisher Scientific Inc., Madrid, Spain), respectively. All solutions were prepared with ultrapure water from a Milli-Q Advantage A10 ultrapure water purification system (Millipore, Burlington, MA, USA).
6
3D Printed PCL Scaffolds for Antimicrobial Evaluation
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Polycaprolactone (PCL) 3D printing filament (molecular weight: 50,000 g/mol) was purchased from 3D4makers (Haarlem, The Netherlands). Chitosan (#448869, 75–85% deacetylated, low molecular weight), acetic acid, and NaOH were obtained from Sigma-Aldrich (Saint Louis, MO, USA) and used as received. Vancomycin hydrochloride was purchased from Lab. Reig Jofre S.A. (Barcelona, Spain).
Staphylococcus aureus (CECT 239) and Staphylococcus epidermidis (CECT 231) strains were purchased from the Spanish Type Culture Collection (CECT) (Valencia, Spain). Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and buffered peptone water were obtained from Scharlau (Barcelona, Spain).
Staphylococcus aureus (CECT 239) and Staphylococcus epidermidis (CECT 231) strains were purchased from the Spanish Type Culture Collection (CECT) (Valencia, Spain). Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), and buffered peptone water were obtained from Scharlau (Barcelona, Spain).
7
Microbial Enumeration and Pathogen Detection
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Microbial enumeration was performed on the 20 collected samples. A 10-fold dilution was prepared by homogenizing 10 g of each sample in 90-mL sterile buffered peptone water (Scharlau). Successive 10-fold dilutions were then prepared and plated on nutritive and selective media and incubated with respect to necessary conditions (Table 1). Two values for de Man, Rogosa, and Sharpe agar, and M17 lactose agar were identified (Velasco et al., 2000) (link) and removed from the analysis. The results were expressed as log cfu/g of sample. In addition, samples were tested in an external laboratory (Microbiological Quality Control Laboratory, Saint Joseph University USJ, Beyrouth) for the presence of the following food pathogens: Brucella melitensis (NF U47-105 ISO), Escherichia coli (RAPID' E.coli 2 Agar -NF validation according to ISO 16140, AOAC-RI approved N° 050601), Staphylococcus aureus (ISO 6888_1-1999) followed by coagulase and DNase confirmation tests, Listeria monocytogenes (ISO 11290-1/A1-2004), and Salmonella spp. (ISO 6579-2002) . In addition, coliform bacteria at 37°C and thermotolerant coliforms at 44°C (ISO 4832-1991) were enumerated.
8
Preparation of Cauliflower and Mandarin By-Product Infusions
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Cauliflower and mandarin by-products were provided from agro-industrial primary production of TRASA S.L. and INDULLEIDA S.A., respectively. Both by-products were processed according to Brandi, Amagliani, Schiavano, De Santi and Sisti (2006) . In brief, they were washed in sterile water, dried, triturated, and homogenized with a laboratory grinder (Janke & Kunkel, IKA-Labortechnik) to obtain a powder with a particle size of 40 µm.
A 10% (w/v) infusion of cauliflower or mandarin by-product was obtained by boiling the powder in 0.1% (w/v) buffered peptone water (Scharlab, S.A., Barcelona, Spain) for 30 min. Then the infusions were centrifuged at 4 °C, at 2450 g for 15 min for the cauliflower by-product infusion and at 3000 rpm for 5 min in the case of the mandarin by-product infusion. Finally, both infusions were filtered through filters (Whatman) with a pore size of 11 and 2.5 μm and then sterilized by filtering through a PVDF syringe filter with a pore size of 0.45 μm.
A 10% (w/v) infusion of cauliflower or mandarin by-product was obtained by boiling the powder in 0.1% (w/v) buffered peptone water (Scharlab, S.A., Barcelona, Spain) for 30 min. Then the infusions were centrifuged at 4 °C, at 2450 g for 15 min for the cauliflower by-product infusion and at 3000 rpm for 5 min in the case of the mandarin by-product infusion. Finally, both infusions were filtered through filters (Whatman) with a pore size of 11 and 2.5 μm and then sterilized by filtering through a PVDF syringe filter with a pore size of 0.45 μm.
9
Microbial Analysis of Salad Samples
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For microbiological analysis purposes, salad samples were prepared according to ISO 6887-2:2003 [32 ]. Enterobacteriaceae colony counts were carried out according to ISO 21528-2:2017 [33 ], and aerobic mesophilic colony enumeration was performed conforming to ISO 4833-1:2013 [34 ]. L. monocytogenes detection and enumeration were performed according to ISO 11290-1 [35 ] and 2:2017 [36 ], respectively. Salad extracts were kept for further molecular assessment purposes and comprised the initial suspension of ISO 11290-2—the salad’s test portion and the diluent, i.e., buffered peptone water (BPW; Scharlab, S.L., Barcelona, Spain). All countings were expressed as log colony-forming units per gram of salad (cfu/g).
10
Staphylococcus Isolation and Identification
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A pre-enrichment in buffered peptone water (BPW; Scharlau, Barcelona, Spain), at a ratio of 1:10 v/v, of the sample swabs collected were carried out, followed by an incubation at 37 ± 1 °C for 24 h. After that, the suspension was streaked onto the non-specific agar Columbia CNA agar with 5% sheep blood, Improved II (BD, Becton Dickinson, Madrid, Spain), and incubated at 37 ± 1 °C for 24–48 h. The plates were examined at 24 and 48 h, and the suspected colonies, matching the typical morphology of Staphylococcus spp. in blood agar and the positive result of the catalase test, were identified using a MALDI-TOF MS Biotyper System (Bruker Daltonics, Madrid, Spain) at the Microbiology Service of the Consorcio Hospital General Universitario de Valencia.
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