For E. coli isolation, rectal swabs were spread onto selective agar BROLAC (Thermo Fisher Scientific, Waltham, MA, USA). For Pasteurellaceae isolation, nasopharyngeal swabs were spread onto Pasteurella selective agar (Thermo Scientific Oxoid, Reinach, Switzerland). The plates were incubated at 37°C for 24 h, and one single colony was picked for species identification with MALDI-TOF (Microflex LT, Bruker Daltonics GmbH, Bremen, Germany). Colonies identified as E. coli, P. multocida and M. haemolytica were purified on trypticase soy agar plates containing 5% sheep blood (TSA-SB; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and incubated at 37°C for 24 h, followed by reconfirmation of species identification using MALDI-TOF. Isolates were then cryopreserved until further analyses in 30% glycerol stocks at –80°C.
Tsa sb
Manufactured by BD
Sourced in United States
The TSA-SB is a laboratory equipment designed for sample preparation and analysis. It is a centrifuge that separates components of a liquid sample based on their density differences. The TSA-SB can be used to isolate and concentrate specific analytes or particles within a sample.
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5 protocols using tsa sb
1
Isolation and Identification of Bacterial Pathogens
2
Characterization of M. caseolyticus Strains
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The origin and characteristics of the M. caseolyticus strains used in this study are listed in Table 1 . They were obtained from the diagnostic unit of the Institute of Veterinary Bacteriology at the University of Bern. The samples were taken by veterinarians for diagnostic purposes therefore not requiring ethical approval or a permit for animal experimentation according to the current Swiss legislation (Federal Animal Protection Law, 455 (https://www.admin.ch/opc/de/classified-compilation/20022103/index.html ). Strains were routinely cultivated on trypticase soy agar plates containing 5% sheep blood (TSA-SB) (Becton, Dickinson and company, Franklin Lakes, NJ, USA) at 37 °C. Species identification was performed using matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) (microflex LT, Bruker Daltonics, Bremen, Germany). The laboratory strains E. coli DH5α and S. aureus RN422048 (link) were used for cloning and transformation experiments. They were cultivated in Luria-Bertani (LB) broth with shaking or on LB agar plates at 37 °C under aerobic conditions. The recombinant DH5α and RN4220 strains containing the S. aureus-E. coli shuttle vector pTSSCm33 (link) or derived constructs were selected and routinely grown using 10 mg/l tetracycline in the growth medium.
3
Isolation and Identification of E. coli from Clinical Samples
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The swabs were spread directly onto Enterobacterales-selective BROLAC agar (Thermo Fisher Scientific, Waltham, USA), and the plates were incubated at 37°C for 24 hours. Three lactose-fermenting colonies exhibiting a dissimilar morphology were selected per plate to take the within-sample strain diversity into account. If no difference in morphology was observed, the colonies were chosen randomly. Species identification was assigned using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Microflex LT, Bruker Daltonics GmbH, Bremen, Germany). If one of the selected colonies was not identified as E. coli (in less than 10 instances), an additional colony was selected in order to obtain a total of three E. coli per sample. Isolates identified as E. coli were regrown in pure culture on trypticase soy agar containing 5% sheep blood (TSA-SB; Becton, Dickinson and Company, NJ, USA) and stored in 30% glycerol at −80°C.
4
Preparation and Characterization of S. pneumoniae Strains
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All S. pneumoniae strains used in this study are listed in Table S1 in the supplemental material. All bacterial stocks were prepared as follows. Cells harvested from overnight culture on Trypticase soy agar supplemented with 5% sheep blood (TSA-SB [Becton Dickinson, Sparks, MD]) were used to inoculate Todd-Hewitt broth supplemented with 5% yeast extract (THY [Becton Dickinson]) at an optical density at 620 nm (OD620) of 0.05. These cultures were further diluted 2.5 × 10−1, incubated at 37°C until they reached OD620 of 0.2 to 0.25, supplemented with 10% glycerol, aliquoted to 1 ml, and stored frozen at −70°C. Prior to use, cell stocks were thawed on ice and washed by centrifugation at 12,000 × g for 2 min, and the pellet was resuspended in 1 ml of ice-cold phosphate-buffered saline (PBS). The wash step was repeated, with the exception that cells were suspended in a predetermined volume of PBS to adjust the CFU concentration to that targeted in a particular experiment. All strains were individually tested for their ability to colonize C57BL/6 mice for at least 5 days, and only mouse-passaged isolates were used in experiments that followed (31 (link), 47 (link)).
5
Screening for 3GCs-R Enterobacteriaceae
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The inoculated Tryptone Soy Broth (Oxoid) medium was transferred into 5 mL of MacConkey Broth (Oxoid) containing 8 mg/L of ceftazidime (Sigma-Aldrich, Merck, St. Louis, MO, USA) and incubated at 37 °C for 24 h under agitation. Then, a loopful (10 μL) of the mixture was plated onto MacConkey Agar (BioCen, BioCubaFarma, Bejucal, Cuba) supplemented with 8 mg/L of ceftazidime (Sigma-Aldrich) for the screening of 3GCs-R Enterobacteriaceae and reincubated overnight. Lactose-positive (pink colonies) were selected and streaked three times on selective MacConkey Agar plates to obtain pure culture. Single pink colonies from each selective plate were streaked onto Tryptone Soy Agar plates containing 5% sheep blood (TSA-SB; Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37 °C. The colonies were identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS, Microflex LT; Bruker Daltonics, Billerica, MA, USA) and frozen at −80 °C in glycerol stocks.
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