The cefazolin minimal inhibitory concentrations (MICs) were determined by a broth microdilution method using cation-adjusted Mueller-Hinton II broth (Becton, Dickinson and Company, Sparks, MD), according to Clinical and Laboratory Standards Institute (CLSI) guidelines, except for the inoculum size of the strains [21 ]. MICs of high inoculum (HI, ~5 x 107 CFU/ml) were compared to the standard inoculum (SI, ~5 × 105 CFU/ml) to identify the stains with the CIE. The MIC value of each isolate was measured by two independent researchers. S. aureus strain TX 0117 (a high-level producer of type A β-lactamase), S. aureus ATCC 29213 (known to produce small amounts of type A β-lactamase), and S. aureus ATCC 25923 (a β-lactamase negative strain) were used as controls [1 (link)22 (link)]. The CIE was defined as an increase in MICs to ≥16 μg/ml at high inoculums from the susceptible range of MIC at standard inoculum [23 (link)]. The MICs of vancomycin and linezolid were measured by the broth microdilution method and E-test (bioMérieux, Marcy-l'Étoile, France). The E-test was performed according to the manufacturer’s protocol. The data regarding susceptibility to agents other than cefazolin, vancomycin, and linezolid were collected through a review of medical record of microbiological data.
Mueller hinton 2 broth
Manufactured by BD
Sourced in United States
Mueller-Hinton II broth is a microbiological culture medium used for the growth and isolation of various bacteria. It is a standardized medium used in antimicrobial susceptibility testing to determine the sensitivity of bacterial isolates to different antimicrobial agents.
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Lab products found in correlation
Thymol, by Fujifilm (2 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (2 mentions)
Albumin dextrose catalase, by BD (2 mentions)
Oleic acid albumin dextrose catalase oadc, by BD (2 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (2 mentions)
Etest, by bioMérieux (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Rifampin, by Merck Group (1 mentions)
Special grade, by Fujifilm (1 mentions)
Tetrahydrofuran, by Thermo Fisher Scientific (1 mentions)
Trypticase soy broth (tsb), by BD (1 mentions)
Gold chloride, by Thermo Fisher Scientific (1 mentions)
Sodium citrate dihydrate, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Thermo Fisher Scientific (1 mentions)
2 2 azobis 2 methylpropionitrile, by Merck Group (1 mentions)
N isopropylacrylamide nipaam, by Merck Group (1 mentions)
Vancomycin, by Cayman Chemical (1 mentions)
Atcc 43300, by American Type Culture Collection (1 mentions)
Atcc 33591, by American Type Culture Collection (1 mentions)
28 protocols using mueller hinton 2 broth
1
Evaluating Cefazolin Susceptibility in Staphylococcus aureus
2
Bacterial Growth Monitoring Protocol
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Bacterial growth was monitored by measuring the turbidity (that is, the optical density at 600 nm [OD600]) using an Infinite M200 PRO multimode microplate reader (Tecan, Kawasaki, Japan). Strains (eight clones per strain) were grown in 0.5 ml of tryptic soy broth (Becton Dickinson) overnight at 37°C, and 1 × 105 CFU/ml bacteria were cultured in 0.1 ml Mueller-Hinton II broth (Becton Dickinson) in a 96-well plate at 37°C with shaking at 140 rpm for 16 h. Bacterial growth curves were created based on measurements made every 10 min for 16 h at least in triplicate.
3
Antimicrobial Susceptibility of S. aureus
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The antimicrobial susceptibility profiles of the S. aureus isolates were determined using the broth microdilution method according to the Clinical and Laboratory Standard Institute (CLSI) guidelines [38 ]. For the broth microdilution method, serial two-fold dilutions were carried out in cation-adjusted Mueller–Hinton II broth (Becton Dickinson, Sparks, MD, USA) in microtiter plates according to standard criteria [38 ]. The MIC was determined using the broth microdilution method, and each spot was inoculated with 106 CFU. After incubation at 37 °C for 16–20 h, the MIC value was considered when the bacteria did not grow at the minimum antibiotic concentration. The reference strain from the American Type Culture Collection 29,213 was used for quality control. The MIC for rifampin (Sigma, Darmstadt, Germany) was determined using the broth microdilution method, following standard criteria [38 ]. Based on the CLSI guidelines, the isolates were classified as RIF susceptible (MIC ≤ 1 mg/L) or RIF resistant (MIC ≤ 4 mg/L).
4
Culturing and Standardizing P. multocida
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A few colonies of freshly grown P. multocida CVCC 411 from Tryptic Soy agar (Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., Guangzhou, China) plates supplemented with 5 % sheep blood (Puboxin Biotechnology Co., Ltd., Beijing, China) were cultured in Mueller–Hinton II broth (Becton Dickinson, Sparks, MD, USA) with shaking at 200 rpm at 37 °C for 10 h (final cell count approximately 109 CFU/mL). Bacteria were collected by centrifugation at 3,000 g for 10 min and re-suspended in 0.9 % NaCl to obtain a final suspension containing 1010 CFU/mL. This suspension was then diluted 100 times to obtain a bacterial concentration at 108 CFU/mL.
5
Analysis of n-Alkanes by GC
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Acetone for gas chromatography was purchased from KISHIDA CHEMICAL Co., Ltd, Japan. Dimethyl sulfoxide (DMSO) and thymol (special grade) were purchased from FUJIFILM Wako Pure Chemical Corporation, Japan. A series of n-alkane standards (C9 to C40) was purchased from GL Sciences Inc., Tokyo, Japan. Mueller-Hinton II broth was purchased from Becton, Dickinson and Company, USA. Staphylococcus aureus (NBRC 12732) for antibacterial activity tests were from the National Institute of Technology and Evaluation, Biological Resource Center (NBRC), Japan.
6
Synthesis and Characterization of Gold Nanoparticles
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Gold chloride
(99%), sodium citrate dihydrate, tetrahydrofuran (THF), Ce6, ethanol,
and phosphate buffer saline (PBS, 10× solution) were purchased
from Fisher Scientific. Trypticase soy broth (TSB) and Mueller Hinton
II broth were purchased from Becton Dickinson. S. aureus (ATCC BAA-44) and MCF-7 cell line (ATCC HTB-22) were purchased from
the American Type Culture Collection (ATCC, USA). N-Isopropylacrylamide (NIPAAm, 99%), 2,2′-azobis(2-methylpropionitrile)
(99%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
were purchased from Sigma-Aldrich. Sodium borohydride was purchased
from Fluka. All chemicals were used as received.
(99%), sodium citrate dihydrate, tetrahydrofuran (THF), Ce6, ethanol,
and phosphate buffer saline (PBS, 10× solution) were purchased
from Fisher Scientific. Trypticase soy broth (TSB) and Mueller Hinton
II broth were purchased from Becton Dickinson. S. aureus (ATCC BAA-44) and MCF-7 cell line (ATCC HTB-22) were purchased from
the American Type Culture Collection (ATCC, USA). N-Isopropylacrylamide (NIPAAm, 99%), 2,2′-azobis(2-methylpropionitrile)
(99%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
were purchased from Sigma-Aldrich. Sodium borohydride was purchased
from Fluka. All chemicals were used as received.
7
Minimum Inhibitory Concentration Determination
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ATCC 43300 and ATCC 33591 were purchased from the American Type Culture Collection and were stored at −80 °C in a freezer by using the Microbank system (IWAKI & CO., Ltd., Tokyo, Japan). For each experiment, a bacterial strain was cultured overnight on mannitol salt agar plates to confirm the purity and viability of the microbe.
The minimal inhibitory concentration (MIC) of synthetics were determined by microdilution method using Mueller–Hinton broth according to Clinical and Laboratory Standards Institute (CLSI) guidelines [33 ,34 ]. Briefly, after diluting the suspension of bacteria equivalent to 1 × 106 colony-forming units (CFU)/mL with Mueller–Hinton II broth (Becton, Dickinson and Company, MD, USA) with 17.5 mg/L of calcium, the dilution (50 μL) was applied into 96-well plate, which included synthetics or vancomycin (Cayman Chemical Company, MI, USA) (50 μL) at concentrations of 0.5–256 μg/mL. Final inoculum concentration was approximately 5 × 105 CFU/mL. Synthetics concentration was 0.25–128 μg/mL. After incubation at 37 °C for 16–20 h, MICs were determined.
The minimal inhibitory concentration (MIC) of synthetics were determined by microdilution method using Mueller–Hinton broth according to Clinical and Laboratory Standards Institute (CLSI) guidelines [33 ,34 ]. Briefly, after diluting the suspension of bacteria equivalent to 1 × 106 colony-forming units (CFU)/mL with Mueller–Hinton II broth (Becton, Dickinson and Company, MD, USA) with 17.5 mg/L of calcium, the dilution (50 μL) was applied into 96-well plate, which included synthetics or vancomycin (Cayman Chemical Company, MI, USA) (50 μL) at concentrations of 0.5–256 μg/mL. Final inoculum concentration was approximately 5 × 105 CFU/mL. Synthetics concentration was 0.25–128 μg/mL. After incubation at 37 °C for 16–20 h, MICs were determined.
8
Radial Diffusion Assay for Staphylococcus
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Preparation of target strain, Staphylococcus carnosus: Stock of target strain was incubated at 37 °C overnight in anaerobic conditions (AnaeroGen 2.5 L, Thermo Scientific) on to LB agar plate then resuspended in 0.9% NaCl, 20% glycerol to a McFarland turbidity standard of 1 (OD600 ~0.870). Aliquots of 150 µl are frozen at −80 °C. The colony-forming unit per mL (CFU/mL) was determined. The RDA media (5.5 g Mueller-Hinton II Broth, Beckton Dickinson; 7.5 g Agarose High resolution, Sigma–Aldrich, 500 ml MilliQ water) was autoclaved (121 °C; 15 min) and cooled down to ~42 °C. S. carnosus was added 30 ml medium to a final CFU/mL of 5x10e5 . Plates were prepared using Omnitray with NUNC-TSP lid (both Thermo Scientific) and kept at 4 °C for at least 20 min to solidify. The lid was discarded, and the wells were loaded with 10 µL sample. The RDA plate was incubated at 37 °C overnight. The plate was coloured with 1.5 mM Thiazolyl Blue Tetrazolium Bromide (MTT) (Sigma–Aldrich).
9
Atomic Force Microscopy of E. coli Cells
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E. coli K12 was grown in Mueller–Hinton II broth (Becton-Dickinson, Sparks, MD, USA) to mid-log phase, then centrifuged (9391× g for 10 min), re-suspended in HEPES buffer (10 mM) and adjusted to an optical density corresponding to108 CFU/mL. 2,4-DAPG was then added to media containing bacterial cells at final concentrations corresponding to MBC. During incubation, the samples were taken at various time of treatment. Bacterial cells were washed with distilled water, applied to a freshly cleaved mica and dried at 25 °C.
Atomic force microscopy was carried out using “Integra NT-MDT” (NT-MDT, Moscow, Russia) microscope in tapping mode. Microscopy investigation was performed using the cantilever NSG01 (Tipsnano, Tallinn, Estonia) with spring constant ~5.1 N/m. Preliminarily, wide scanning of about 50 × 50 μm2 was conducted to produce a reference map of the sample surface so as to help localize the bacterial cells. The scan size was then set with sampling of 512 by 512 points and scan rate of 0.5 Hz. Topographic, amplitude (MAG) and phase images were acquired simultaneously (AFM-images obtained in MAG-mode showed inFigures S1 and S2 ).
Atomic force microscopy was carried out using “Integra NT-MDT” (NT-MDT, Moscow, Russia) microscope in tapping mode. Microscopy investigation was performed using the cantilever NSG01 (Tipsnano, Tallinn, Estonia) with spring constant ~5.1 N/m. Preliminarily, wide scanning of about 50 × 50 μm2 was conducted to produce a reference map of the sample surface so as to help localize the bacterial cells. The scan size was then set with sampling of 512 by 512 points and scan rate of 0.5 Hz. Topographic, amplitude (MAG) and phase images were acquired simultaneously (AFM-images obtained in MAG-mode showed in
10
Determination of Minimal Bactericidal Concentration
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Determination of minimal bactericidal concentration was performed using cation-adjusted Mueller-Hinton II broth (MHB; Becton-Dickinson, Sparks, MD, USA). Bacteria at 106 CFU/mL were incubated in 96-well microtiter plates (cat. no. 0030730011, Eppendorf, Hamburg, Germany) containing growth media and various concentrations of 2,4-DAPG in series of two-fold dilutions. The dynamics of bacterial growth were assessed by reading and plotting the absorbance data at 620 nm obtained by the spectrophotometer Multiscan GO (Thermo Scientific, Waltham, MA, USA). Wells without visible bacterial growth were plated on MHB-agar and incubated. Antimicrobial activity was expressed by the minimal bactericidal concentration (MBC), and minimal inhibitory concentrations (MIC) which was defined as the lowest antibiotic dose at which no visible growth in medium and growth on agar-plate were detected, respectively.
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