Enumeration of E. coli were carried out using the pour plate technique on Tryptone Soy Agar (Oxoid), at pH 7.3. The Tryptone Soy Agar was overlaid with Violet Red Bile Agar (Oxoid), at pH 7.4. Incubation was at 37°C for 24 h for total coliforms and at 44°C for 24 h for E. coli. Dark red to purple colonies suspected to be coliforms were confirmed in Brilliant Green Bile Broth (Oxoid), pH 7.4, and incubated at 37°C for 24 h (NMKL No. 44) [15] . Colonies suspected to be E. coli colonies were confirmed in EC Broth (Oxoid), pH 6.9. Positive tubes showing gas formation were sub-cultured into Tryptone Water (Oxoid), pH 7.5, and incubated at 44°C for 24 h. The Indole test was then performed for E. coli according to the NMKL No. 125 method [16] .
Tryptone soy agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Tryptone Soy Agar is a general-purpose microbiological growth medium. It is used for the cultivation of a wide range of microorganisms, including bacteria and fungi. The medium provides nutrients and growth factors necessary for the proliferation of various microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Sabouraud dextrose agar, by Thermo Fisher Scientific (3 mentions)
Macconkey agar, by Thermo Fisher Scientific (3 mentions)
Nutrient agar, by Thermo Fisher Scientific (2 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (2 mentions)
Blood agar, by Thermo Fisher Scientific (2 mentions)
Tryptone soya broth (tsb), by Thermo Fisher Scientific (2 mentions)
Tryptone soy broth (tsb), by Thermo Fisher Scientific (2 mentions)
Miglyol 812, by Merck Group (1 mentions)
Peg 8 caprylic capric glycerides, by Gattefosse (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Transcutol p, by Gattefosse (1 mentions)
Salmonella shigella agar, by Thermo Fisher Scientific (1 mentions)
Difcotm, by BD (1 mentions)
Biometra tone 96g thermal cycler, by Analytik Jena (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Peptone water, by Thermo Fisher Scientific (1 mentions)
Triple sugar iron agar, by HiMedia (1 mentions)
Citrate agar, by Thermo Fisher Scientific (1 mentions)
29 protocols using tryptone soy agar
1
Quantification of E. coli and Coliforms
2
Bacterial Strain Isolation and Identification
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A total of three to five typical colonies were selected from each quail meat sample and culture medium. Strains were purified on tryptone soy agar (Oxoid, Hampshire, UK). The purified strains were kept at −80 °C. Bacterial identification was conducted using the MALDI-TOF Biotyper technology (Bruker, Daltonik, Bremen, Germany).
3
Isolation and Identification of MRSA, Salmonella, and Hemolytic E. coli
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Five positive MRSA colonies (if present) were subcultured on Tryptone Soy Agar (Oxoid, CM0131) and DNA was extracted according to the method of Stranden et al. [21 (link)]. A multiplex PCR, as described by Maes et al. [22 (link)], was performed for MRSA and a CC398 specific PCR, as described by Stegger et al. [23 (link)], for MRSA ST398 confirmation.
Positive Salmonella colonies on Xylose Lysine Deoxycholate agar medium (Oxoid, CM0469) were subcultured on Nutrient Agar (Oxoid, CM0003). After incubation, PCR confirmation on cel lysates was performed as described by Aabo et al. [24 (link)].
From the third down-time and production round, five positive E. coli colonies (when possible) were subcultured on Columbia base Blood Agar (Oxoid, CM0331) with 5 % sheep blood and incubated for 24 h at 37 °C for analysis of haemolytic E. coli. If a plate was negative after 24 h, it was incubated for a further 24 h. To calculate the enumerations of haemolytic E. coli, the ratio of the number of positive haemolytic E. coli colonies on the 5 selected colonies was multiplied by the mean E. coli enumeration of that sample.
Positive Salmonella colonies on Xylose Lysine Deoxycholate agar medium (Oxoid, CM0469) were subcultured on Nutrient Agar (Oxoid, CM0003). After incubation, PCR confirmation on cel lysates was performed as described by Aabo et al. [24 (link)].
From the third down-time and production round, five positive E. coli colonies (when possible) were subcultured on Columbia base Blood Agar (Oxoid, CM0331) with 5 % sheep blood and incubated for 24 h at 37 °C for analysis of haemolytic E. coli. If a plate was negative after 24 h, it was incubated for a further 24 h. To calculate the enumerations of haemolytic E. coli, the ratio of the number of positive haemolytic E. coli colonies on the 5 selected colonies was multiplied by the mean E. coli enumeration of that sample.
4
Formulation and Characterization of Topical Antimicrobial Agents
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Thymol (TH), Tween 20 (TW), poloxamer 188 (P), caprylic/capric triglyceride (Miglyol 812) (CCTG), benzalkonium chloride (BZ), glycerin (GLY), propylene glycol (PG), hydroxypropyl methylcellulose (HPMC), and poloxamer 407 (Pluronic®F127) (PP) were acquired from Sigma-Aldrich (Madrid, Spain). Glyceryl behenate (GBH) (Compritol CG 888 ATO), PEG-8 Caprylic/Capric Glycerides (PCCG), and Transcutol P® (Diethylene glycol monoethyl ether) were provided by Gattefossé (Cedex, France). Carbomer® 934 (Polyacrylic acid, CB) was purchased from Fagron Iberica (Barcelona, Spain). Double-distilled water was filtered through a Millipore filter (Molsheim, France). All other reagents used were of analytical grade.
DMEM (Dulbecco’s Modified Eagle’s medium) was purchased from ThermoFisher, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) from Sigma-Aldrich (St. Louis, MO, USA) and DMSO (99% dimethyl sulfoxide) from Sigma-Aldrich (Barcelona, Spain).
Mueller–Hinton broth (MHB), Brain Heart Infusion (BHI), Clostridium reinforced medium (CRM), Tryptone Soy Agar (TSA), and Sabouraud Dextrose Agar (SDA) were purchased from Oxoid (Basingstoke, UK). Beren (cosmetic diluent) was acquired from Scharlab (UK).
DMEM (Dulbecco’s Modified Eagle’s medium) was purchased from ThermoFisher, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) from Sigma-Aldrich (St. Louis, MO, USA) and DMSO (99% dimethyl sulfoxide) from Sigma-Aldrich (Barcelona, Spain).
Mueller–Hinton broth (MHB), Brain Heart Infusion (BHI), Clostridium reinforced medium (CRM), Tryptone Soy Agar (TSA), and Sabouraud Dextrose Agar (SDA) were purchased from Oxoid (Basingstoke, UK). Beren (cosmetic diluent) was acquired from Scharlab (UK).
5
Salmonella Isolation from Blood and Stool
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Blood cultures were collected and processed as previously described [4 (link),15 (link)]. About 1 g of stool was suspended in 10 ml selenite broth (Oxoid, Basingstoke, UK and BD DifcoTM, Becton, Dickinson and Company, Franklin Lakes, NJ, U.S.), and incubated at 36°C for 18–24 hours. Next, 10 μl of selenite broth was inoculated on two plates of Salmonella-Shigella (SS) agar (Oxoid) which were incubated at 36°C for 18–24 hours. In case of absence of growth on the SS plate, the plate was re-incubated for another 24 hours. Two colonies suspected of Salmonella per SS plate were inoculated on a Kligler Iron Agar (KIA) tube (Oxoid) and incubated at 36°C for 18–24 hours. KIA tubes suggestive of Salmonella species were further identified by their biochemical characteristics [2 (link),16 (link)]. Salmonella spp. isolates from blood and stool were stored in tubes with Tryptone Soy Agar (Oxoid) and shipped to INRB for serotyping and ITM for confirmation and molecular testing. Salmonella spp. isolates were serotyped using commercial antisera (Sifin, Berlin, Germany; and VisionTM, Pro-Lab Diagnostics, Richmond Hill, Canada) according to the Kaufman-White scheme [17 ].
6
Identification of Semen Microbiome
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All semen samples were cultured on tryptone soy agar (Oxoid, UK) with 5% sheep blood and MacConkey agar (Oxoid, UK) incubated at 37 °C for 18-24 h. All different colonies were identified using standard biochemical tests followed by 16S rRNA sequencing and stored in Brain Heart Infusion (BHI) (Oxoid, UK) with 20% glycerol at -80 °C. Genomic DNA of all isolates was performed using G-spinTM genomic DNA extraction kit (iNtRON, Republic of Korea) and amplified 16S rRNA by PCR with a BiometraTOne96G thermal cycler (AnalytikJena, Germany) using UFUL (5’- GCCTAACACATGCAAGTCGA-3’) and 800R (5’-TACCAGGGTATCTAATCC-3’) primers. The PCR was performed with the following protocol: initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 45 sec, with a final extension step at 72 °C for 5 min. The PCR products were purified by MEGAquick-spinTM Plus Total Fragment DNA purification kit (iNtRON, Republic of Korea) and sequenced with an Applied Biosystems 3730XL DNA Analyzer (Bionics, Republic of Korea). Each 16S rRNA sequences was blasted against the NCBI nucleotide database (https://blast.ncbi.nlm.nih.gov ) to identify all isolates.
7
Heat Stress Survival Assay
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For heat treatment, stationary phase cultures were harvested by centrifugation (6,000 × g, 5 min) and resuspended in an identical volume of 0.85% KCl. Subsequently, a PCR tube containing 50 µL of the resuspended culture was heat treated at the indicated temperature and time in a Biometra T3000 Thermocycler (Biometra, Göttingen, Germany). Unstressed control cultures were simultaneously kept at room temperature for the duration of the treatment. Survival was determined by aseptically retrieving the treated cultures from the PCR tubes, serially diluting heat stressed and unstressed cultures in 0.85% KCl and spotting 5 µL drops onto Tryptone Soy Agar (TSA; Oxoid) as previously described (35 (link)). After 24 h of incubation at 37°C, the CFU/mL was determined by counting colonies in spots containing between 5 and 50 colonies. The limit of quantification corresponds to 200 CFU/mL. Finally, survival was expressed as percentage (%) of the viable cells after the treatment with respect to the initial population (set at 100%).
8
Microbial Identification and Antimicrobial Testing
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Culture media used were Peptone water, Nutrient Agar, Blood Agar, MacConkey Agar, Plate Count Agar and Sabouraud Dextrose Agar (all from Oxoid Limited, Thermo Fisher Scientific Inc., UK). These culture media were prepared as per the manufacturer's instruction leaflet and under standard sterile conditions in order to prevent external contaminations. Other media such as the Tryptone Soy Agar (TSA) (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was used for storing pure isolates for further evaluation in the study. Mannitol Salt Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was used for the identification of Staphyloccocus aureus. Shigella-Salmonella Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) were used for the identification of Shigella. Triple sugar iron Agar (HiMedia Laboratories Pvt. Ltd., India) and Citrate Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) were also used in the biochemical assay of the various microbes found. Müller Hinton Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was also used in the culture and sensitivity testing. All these were prepared as directed by the manufacturer's leaflet.
9
Bacterial Culture Protocol for ATCC Strains
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The bacterial strains Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were inoculated on Tryptone Soy Agar (Oxoid, Basingstoke, UK) and taken on Tryptone Soy Broth (Oxoid, Basingstoke, UK). The culture of bacteria was incubated at 37 °C until bacterial colonies reached ~108 CFU/mL or 1.0 McFarland Standard.
10
Identification of Staphylococcus Species and MRSP/MRSS/MRSA
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The samples, which were collected using Venturi Transystem® Culture Swab
Transport System (Copan Diagnostics Inc., Murrieta, CA, USA), were cultured on mannitol
salt agar (Oxoid, Hampshire, UK) under aerobic conditions at 35°C for 48 hr. All colonies
with different colors and morphologies were selected and streak cultured on tryptone soy
agar (Oxoid) under aerobic conditions at 35°C for 24 hr. Following, the isolates were
tested using Gram staining, degradation of mannitol, and production of coagulase (PS
LATEX; Eiken Chemical, Tokyo, Japan) [28 (link)]. CoPS
species were determined using the multiplex PCR method developed by Sasaki et
al. [26 (link)]. Strains that could not be
identified using PCR were determined using 16S rRNA gene sequencing [9 (link)]. MRSP, MRSS, and MRSA were identified based on the presence of
mecA [13 (link)].
Transport System (Copan Diagnostics Inc., Murrieta, CA, USA), were cultured on mannitol
salt agar (Oxoid, Hampshire, UK) under aerobic conditions at 35°C for 48 hr. All colonies
with different colors and morphologies were selected and streak cultured on tryptone soy
agar (Oxoid) under aerobic conditions at 35°C for 24 hr. Following, the isolates were
tested using Gram staining, degradation of mannitol, and production of coagulase (PS
LATEX; Eiken Chemical, Tokyo, Japan) [28 (link)]. CoPS
species were determined using the multiplex PCR method developed by Sasaki et
al. [26 (link)]. Strains that could not be
identified using PCR were determined using 16S rRNA gene sequencing [9 (link)]. MRSP, MRSS, and MRSA were identified based on the presence of
mecA [13 (link)].
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