Right after microwave and conventional treatments, sample aliquots were diluted in duplicate with sterile 1%-peptone water (Scharlab Chemie S.A., Barcelona, Spain), and 0.1 mL of diluent spread-plated onto Tryptic Soy Agar (TSA, Scharlab S.A., Barcelona, Spain). The plates were then incubated at 37 °C for 24 h for E. coli and 48 h for L. monocytogenes. Afterwards, the counting step was performed. The reduction of viable cell count was expressed as the decimal logarithm of the quotient of the treated ( ) and untreated samples ( ).
Tryptic soy agar tsa
Manufactured by Scharlab
Sourced in Spain
Tryptic Soy Agar (TSA) is a general-purpose microbiological growth medium formulated with tryptone, soy peptone, and agar. It provides nutrients for the cultivation of a wide range of microorganisms, including bacteria and fungi.
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Lab products found in correlation
Tryptic soy broth (tsb), by Scharlab (3 mentions)
Stomacher 400 circulator, by Seward Medical (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Lb broth, by Scharlab (1 mentions)
Chitosan, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Buffered peptone water (bpw), by Scharlab (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Sterile swab, by Copan (1 mentions)
Sabouraud dextrose agar, by Scharlab (1 mentions)
Luria bertani agar lba, by Merck Group (1 mentions)
Malt extract agar, by Thermo Fisher Scientific (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Listeria monocytogenes, by American Type Culture Collection (1 mentions)
Salmonella enterica serovar typhimurium, by American Type Culture Collection (1 mentions)
Escherichia coli atcc 8739, by American Type Culture Collection (1 mentions)
12 protocols using tryptic soy agar tsa
1
Microbial Cell Viability Assay
2
Salmonella Growth in Plant Infusions
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Both treated and untreated S. Typhimurium samples were inoculated in tubes with cauliflower and mandarin infusions (1, 5, and 10% (w/v)) and incubated at 10 and 37 °C. During the incubations, the S. Typhimurium population was determined by plate count in Tryptic Soy Agar (TSA) (Scharlab Chemie, Barcelona, Spain) at regular time intervals after serial dilution with 0.1% (w/v) buffered peptone water The initial counts in the samples without PEF treatment were 10 7 cfu/mL and in the samples with previous PEF treatment were 10 3 cfu/mL. The plates were incubated at 37 °C for 24 hours. All analysis was done in triplicate.
3
Maintenance and Cultivation of Bacterial Strains
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Pseudomonas aeruginosa PAO1 (ATCC 15692) and Staphylococcus aureus SA31 (ATCC 29213) were maintained in initial cryo-stock cultures at −80°C, and they resuscitated in Luria-Bertani (LB) agar (Scharlab, S.L., Spain) and Tryptic Soy Agar (TSA) (Scharlab, S.L., Spain) plates, respectively. Isolated colonies were grown overnight in LB broth and Tryptic Soy Broth (TSB) (Scharlab, S.L., Spain), respectively, at 37°C with shaking at 200 rpm. Unless otherwise specified, all reagents were purchased from Sigma, Spain.
4
Microbial Population Enumeration in Dough
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Approximately 10 g of dough sample were decimally diluted in sterile peptone water (10 g/L Bacto-peptone (Costantino SpA), pH 6.8) and homogenized in a Stomacher 400 Circulator (Seward, Worthing, UK) for 5 min at 260 rpm. The appropriate dilutions were plated onto MRSm agar (MRSm broth added with agar 15 g/L) for the determination of L. sanfranciscensis population, as well as onto DSM agar (DSM broth added with agar 15 g/L) for Z. mobilis. Plates were incubated at 30 °C for 3 d in anaerobic conditions. Aerobic bacterial count (ABC) was determined by pour plating in Tryptic Soy Agar (TSA, Scharlab, Barcelona, Spain) after incubation at 30 °C for 48–72 h. The enumeration of yeasts and moulds were carried out in Yeast Glucose Chloramphenicol Agar (YGC-Scharlab, Barcelona, Spain) plates after incubating at 25 °C for 3–5 day.
5
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).
6
Quantification of Bacillus cereus in Food
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Sterile swabs (Copan, Brescia, Italy) were used to swab surfaces of about 10 × 10 cm2. After sampling, swabs were placed in sterile tubes with transport medium, kept on ice during transport to the laboratory, and analyzed within 24 h from collection. The presumptive presence of B. cereus was assessed by striking the swabs onto PEMBA (Scharlab, Barcelona, Spain), followed by incubation at 37 °C for 48 h. From each positive sample, a presumptive B. cereus colony was sub-cultured onto tryptic soy agar (TSA, Scharlab, Barcelona, Spain) at 37 °C for 24 h. Samples of cheese curd and PDO Taleggio were also submitted to the enumeration of B. cereus. Briefly, 10 g of sample was ten-fold diluted in chilled sterile diluent solution (0.85% NaCl and 0.1% peptone) and homogenized for 60 s in a Stomacher 400 (Seward Medical, London, UK). Then, appropriate ten-fold dilutions of the homogenates were made in chilled saline, and spread onto PEMBA plates. Plates were incubated at 30 °C for 48 h (limit of quantification equal to 102 CFU/g).
7
Microbial Inactivation in By-Product Infusions
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Both by-product infusions were inoculated with 10 8 cfu/mL of S. Typhimurium and incubated at 10 and 37 °C until its inactivation. The microbial inactivation curves were obtained by removal of aliquots at regular time intervals and plate count in Tryptic Soy Agar (TSA, Scharlab Chemie, Barcelona, Spain) after serial dilution with 0.1% (w/v) buffered peptone water. The plates were incubated at 37 °C for 24 hours. All analysis was done in triplicate.
8
Cultivation of Biofilm-Producing Microbial Strains
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The rhamnolipid-producer strain Pseudomonas aeruginosa 89, a clinical isolate from a patient with cystic fibrosis, was cultured from frozen stocks onto Tryptic Soy Agar (TSA, Scharlab, Barcelona, ES) at 37°C for 18–20 h. The lipopeptide-producer strain Bacillus subtilis AC7, from the inside of stems of Robinia pseudoacacia, was cultured from frozen stocks onto Luria Bertani agar (LBA, Sigma–Aldrich, St. Louis, MO, United States) at 28°C for 18–20 h. The sophorolipid-producer strain Candida bombicola ATCC 22214, obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), was cultured from frozen stocks onto Sabouraud dextrose agar (SDA, Scharlab, Barcelona, ES) at 25°C for 18–20 h. All the biofilm-producer strains used in this study were obtained from the ATCC (Manassas, VA, United States). C. albicans ATCC 10231, S. aureus ATCC 25923, S. aureus ATCC 6538, and S. epidermidis ATCC 35984 were cultured from frozen stocks onto SDA and TSA plates, respectively, and incubated overnight at 37°C.
9
Cauliflower Extract Antimicrobial Effect
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The initial population of S. enterica var Typhimurium (10 7 CFU/mL) was exposed to three consecutive antimicrobial treatments. Each treatment consisted in exposure to 5 % (w/v) cauliflower by-product infusion at 37 °C for 4 h with continuous shaking (200 rpm) (sub-lethal treatment) (Sanz-Puig et al., 2015a) . Afterwards, the sample was centrifuged to recover the microbial cells. Recovered cells were grown in TSB culture overnight to achieve stationary phase (10 9 CFU/mL), inoculated in cauliflower by-product extract (10 7 CFU/mL) and treated again using the same conditions as described above. Before and after each treatment, the antimicrobial effect against the S. enterica var Typhimurium population was evaluated by plate count in tryptic soy agar (TSA) (Scharlab Chemie, Barcelona, Spain). The entire experiment was carried out in triplicate with three different infusion batches.
S. Typhimurium treated once and S. enterica var Typhimurium treated three times were included in the C. elegans infection study, because they were the most different populations with regard to their resistance to the cauliflower antimicrobial exposure.
S. Typhimurium treated once and S. enterica var Typhimurium treated three times were included in the C. elegans infection study, because they were the most different populations with regard to their resistance to the cauliflower antimicrobial exposure.
10
Microbial Enumeration in Bakery Dough
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At appropriate leavening intervals, 5 g of dough was diluted in 45 mL sterile peptone water (10 g/L Bacto-peptone in distilled H2O, pH 6.8) and homogenized in a Stomacher (Seward, Worthing West Sussex, UK) for 3–4 min. After appropriate decimal dilutions, suspensions were plated in proper media: Z. mobilis onto DSM agar, incubated at 30 °C for 3 days under anaerobic conditions; and baker’s yeast (S. cerevisiae) onto MEA (Malt Extract Agar, Oxoid, Basinstoke, UK), then incubated at 30 °C for 48 h. Total mesophilic aerobic bacterial count (TBC) was determined by pour plating on Tryptic Soy Agar (TSA, Scharlab, Barcelona, Spain) after incubation at 30 °C for 24–48 h. When S. cerevisiae was used, TSA was added with 0.1% (v/v) cycloheximide (Sigma-Aldrich) to avoid TBC overestimation due to yeast growth. Yeasts and molds were determined by plating on Yeast Glucose Chloramphenicol Agar (YGC-Scharlab, Barcelona, Spain) and incubated at 25 °C for 3–5 days. Counts were reported as logarithms of the number of colony-forming units (Log CFU/g of dough) with means and standard deviation values of two technological replicates.
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