Once pure cultures were obtained, colonies of Salmonella were added to Mueller Hinton (MH) broth (BD Diagnostics, Sparks, MD). Based on quantitative Salmonella culture completed previously to determine the correlation between the optical density and CFUs of Salmonella, a target optical density (OD600) of 0.09 would approximately correlate to 1 ×108 CFU/mL; this was the goal of the inoculum. Once the inoculum was prepared, serial dilutions of the inoculum were plated to BA to determine the actual concentration of Salmonella, and the remaining inoculum was stored at 4°C for <2 h until administered to the piglets.
Mueller hinton broth
Manufactured by BD
Sourced in United States
Mueller–Hinton (MH) broth is a general-purpose culture medium used for the growth and susceptibility testing of a wide range of bacteria. It provides the necessary nutrients and growth factors to support the proliferation of various bacterial species.
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Lab products found in correlation
Ampicillin, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Levofloxacin, by Merck Group (1 mentions)
Bhi medium, by Thermo Fisher Scientific (1 mentions)
Iron 2 chloride tetrahydrate, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Potassium sorbate, by Merck Group (1 mentions)
Hydroxypropyl methylcellulose, by Merck Group (1 mentions)
Benzene 1 3 5 tricarboxylic acid h3btc, by Merck Group (1 mentions)
κ carrageenan, by Merck Group (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Dna isolation kit, by Tiangen Biotech (1 mentions)
Gel dna purification kits, by Tiangen Biotech (1 mentions)
9 protocols using mueller hinton broth
1
Salmonella Inoculum Preparation
2
TGII Antibiotic Efficacy Evaluation
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TGII was provided and identified by Lih-Geeng Chen (NCU, Jiayi, Taiwan). The bio-activity of anti-bacterial infection obtained from TGII was granted to the United States patent on 10 March 2015 (Patent No. US8,975,234 B2). The abstract and the detail claim can be seen in the open patent document. All the conventional antibiotics were purchased from Sigma-Aldrich (St Louis, MO, USA), namely, oxacillin, erythromycin, ampicillin, kanamycin, levofloxacin, vancomycin, and doxycycline, as well as BOCILLIN FL used to detect the penicillin-binding capacity of PBP2a. Mueller–Hinton (MH) broth for bacteria dilution and enrichment culture was purchased from BD Company (San Diego, CA, USA). The PBP2a latex agglutination assay kit was purchased from Denka Seiken Co., Ltd. (Tokyo, Japan).
3
Antimicrobial Susceptibility Testing
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A standard assay (CLSI, 2019) was conducted in a 96-well microplate (Bio-kont, China). The tested bacterial colonies on each blood agar plate were aseptically transferred into Mueller-Hinton (MH) broth (BD Biosciences, America), and a homogenous suspension with a density equivalent to a 0.5 McFarland's standard was prepared. Then the bacterial suspension was diluted with MH broth at a ratio of 1:200.
One hundred microliters of prepared bacterial solution were inoculated into wells with sequentially diluted antimicrobials and incubated at 35°C with 180 rpm for 16 h. Aztreonam, cefepime, imipenem, ticarcillin/clavulanic acid, piperacillin/tazobactam, amikacin, tobramycin, minocycline, levofloxacin and trimethoprim/sulfamethoxazole were used in this study. The concentrations of the ten antimicrobials were based on the breakpoints of other Non-Enterobacteriaceae listed in guidelines M100 (CLSI, 2019). MIC value and the results of antimicrobial susceptibility were interpreted based on guidelines M100 (CLSI, 2019).
One hundred microliters of prepared bacterial solution were inoculated into wells with sequentially diluted antimicrobials and incubated at 35°C with 180 rpm for 16 h. Aztreonam, cefepime, imipenem, ticarcillin/clavulanic acid, piperacillin/tazobactam, amikacin, tobramycin, minocycline, levofloxacin and trimethoprim/sulfamethoxazole were used in this study. The concentrations of the ten antimicrobials were based on the breakpoints of other Non-Enterobacteriaceae listed in guidelines M100 (CLSI, 2019). MIC value and the results of antimicrobial susceptibility were interpreted based on guidelines M100 (CLSI, 2019).
4
Evaluation of Enterococcus and Staphylococcus Strains
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E. casseliflavus strains KB1733 and JCM 8723T were evaluated in this study. Enterococcus faecalis JCM 7783 (which is identical to E. faecalis ATCC 29212) and Staphylococcus aureus JCM 2874 (which is identical to S. aureus ATCC 29213) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan, and were used as control strains. The four strains used in this study were stored in glycerol stock vials at −80 °C until use.
Mueller–Hinton (MH) broth (Becton Dickinson, Franklin Lakes, NJ, USA, product number: 275730) was used for the inoculation and passaging of each strain in culture. Each strain was inoculated with 1% (v/v) of a thawing glycerol stock in 10 mL MH liquid medium and then incubated for 20 h at 37 °C. After incubation, 100 µL of the culture was inoculated into 10 mL of fresh MH liquid medium and then incubated for 24 h at 37 °C. The culture medium was diluted to McFarland 1 (OD600 = 0.257) in fresh MH liquid medium, and the minimum inhibitory concentration (MIC) and hemolytic activity were determined.
Mueller–Hinton (MH) broth (Becton Dickinson, Franklin Lakes, NJ, USA, product number: 275730) was used for the inoculation and passaging of each strain in culture. Each strain was inoculated with 1% (v/v) of a thawing glycerol stock in 10 mL MH liquid medium and then incubated for 20 h at 37 °C. After incubation, 100 µL of the culture was inoculated into 10 mL of fresh MH liquid medium and then incubated for 24 h at 37 °C. The culture medium was diluted to McFarland 1 (OD600 = 0.257) in fresh MH liquid medium, and the minimum inhibitory concentration (MIC) and hemolytic activity were determined.
5
Antibiotic Susceptibility Testing Protocol
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Clindamycin hydrochlorid (European Pharmacopeia Reference Standard) was purchased by Council of Europe EDQM (Strasbourg, France), the brain heart infusion (BHI) medium by Oxoid (München, Germany), Mueller Hinton (MH) broth, cation adjusted by Becton, Dickinson (Heidelberg, Germany) and Muller Hinton-agar by Roth GmbH (Karlsruhe, Germany).
6
Synthesis of Antimicrobial Hydrogel Composite
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Kappa-carrageenan (Kc, 0.3% aqueous solution viscosity at 25 °C: 5–25 cP), hydroxypropyl methylcellulose (HPMC, 2% aqueous solution viscosity at 20 °C: 40–60 cP), glycerol (≥99.5%), benzene-1,3,5-tricarboxylic acid (H3BTC, 95%), iron(II) chloride tetrahydrate (≥99.0%), sodium hydroxide (≥98%), and potassium sorbate (KS, 99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Tea tree essential oil (TTO) was purchased from Siberina LLC (Kirov, Russia). Mueller–Hinton (MH) broth and agar solid medium (Becton Dickinson and Company) were purchased from Hem Ltd. (Moscow, Russia)
7
Bacterial Strain Cultivation for Research
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The major bacterial strains and plasmids used in this study are listed in Table 1 . E. coli strains were grown in Luria-Bertani (LB) broth or Mueller Hinton (MH) broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with shaking (250 rpm) or on agar at 37 or 32 °C overnight. When needed, culture media were supplemented with ampicillin (100 μg/mL, Fisher Scientific, Pittsburgh, PA, USA), kanamycin (30 μg/mL, Fisher Scientific, Pittsburgh, PA, USA) and/or colistin sulfate (2 μg/mL, ACROS, Geel, Belgium).
8
Biofilm formation of Serratia marcescens
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S. marcescens SR 41-8000 [24 (link)] was routinely grown at 37°C with a constant agitation at 200 rpm overnight in Luria-Bertani (LB) broth containing (per L) 10 g tryptone, 5 g yeast extract, and 5 g NaCl. To study the biofilm formation, bacteria were grown in 3 different media, LB broth, minimal broth Davis (MBD) containing (per L) 7 g K2HPO4, 2 g KH2PO4, 0.5 g Na3C6H5O7, 0.1 g MgSO4, and 1 g (NH4)2SO4 supplemented with 0.5% glucose, and Mueller-Hinton (MH) broth (Becton Dickinson, USA), under the same conditions for 7 days at 30°C.
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S. marcescens SR 41-8000 [24 (link)] was routinely grown at 37°C with a constant agitation at 200 rpm overnight in Luria-Bertani (LB) broth containing (per L) 10 g tryptone, 5 g yeast extract, and 5 g NaCl. To study the biofilm formation, bacteria were grown in 3 different media, LB broth, minimal broth Davis (MBD) containing (per L) 7 g K2HPO4, 2 g KH2PO4, 0.5 g Na3C6H5O7, 0.1 g MgSO4, and 1 g (NH4)2SO4 supplemented with 0.5% glucose, and Mueller-Hinton (MH) broth (Becton Dickinson, USA), under the same conditions for 7 days at 30°C.
9
Bacterial Culture Media and Reagents
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Mueller Hinton (M-H) broth and de Man, Rogosa, and Sharpe (MRS) broth were purchased from Becton Dickenson (United States). Biochemical reagents (i.e. arabinose, pyruvate, tellurite, arginine, glucose, inulin, lactose, mannitol, maltose, melibiose, raffinose, ribose, sucrose, sorbitol, sorbose, trehalose, and xylose) and antimicrobial agents (i.e. ampicillin, chloramphenicol, ciprofloxacin, clarithromycin, erythromycin, gentamicin, nitrofurantoin, norfloxacin, tetracycline and vancomycin) were obtained from Hangzhou Microbe Reagent Co., Ltd. (Hangzhou, China). Primers were synthesized by the Tsingke Biotechnology Limited Company (Wuhan, China). Taq DNA Polymerase and deoxynucleotide triphosphates (dNTPs) were purchased from Takara (China). Defibrinated rabbit blood was purchased from Zhengzhou Kowloon Biological Products Co., Ltd. (Zhengzhou, China). DNA isolation kits and gel DNA purification kits were purchased from Tiangen Biotech (Beijing, China).
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