Gut microbiota obtained from an IgA-deficient patient was incubated with purified mAbs (final concentration, 1 µg/ml) in a 96-well V-bottom plate (107 bacteria/well, 10 wells/mAb) for 30 min at 4°C. After washing with 1X-PBS (10 min, 4,000 ×g, 4°C), cells were stained with goat anti-human IgA-FITC (1/200) or isotype control antibody (Jackson ImmunoResearch). For sorting of in vivo IgA1- or IgA2-bound commensal bacteria, purified fecal microbiota from five healthy donors were thawed, washed, and directly stained with mouse anti-human IgA1-FITC or mouse anti-human IgA2 Alexa Fluor 647 (Southern Biotech) in a 96-well V-bottom plate (107 bacteria/well, five wells/subclass) for 20 min at 4°C. After washing, sorting was performed using a microbiota-dedicated single laser S3 cell sorter (Bio-Rad). Of note, while flow cytometry analysis is performed on fixed samples, combined flow sorting and DNA sequencing is performed on live bacteria in order to allow unbiased and optimal DNA extraction. Sorted bacteria (9.105) were collected in 1X-PBS at 4°C, centrifuged (8,000 ×g, 10 min, 4°C), and stored at −80°C until DNA extraction. Purity for both fractions was systematically verified after sorting. To check the absence of contaminants in flow cytometer fluid lines, sheath fluid was regularly incubated in Brain-Heart Infusion Broth (BioMérieux) at 37°C for 7 d.
Brain heart infusion broth
Manufactured by bioMérieux
Sourced in France
Brain heart infusion broth is a culture medium used for the growth and isolation of a wide range of microorganisms, including bacteria and fungi. It provides essential nutrients and growth factors to support the proliferation of various microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Single laser s3 cell sorter, by Bio-Rad (2 mentions)
Mannitol salt agar, by bioMérieux (2 mentions)
Isotype control antibody, by Jackson ImmunoResearch (1 mentions)
Mouse anti human iga2 alexa fluor 647, by Southern Biotech (1 mentions)
Goat anti human iga fitc, by Jackson ImmunoResearch (1 mentions)
Maldi tof mass spectrometry, by Bruker (1 mentions)
Chromid esbl, by bioMérieux (1 mentions)
Mcfarland standard, by bioMérieux (1 mentions)
Genbox anaer, by bioMérieux (1 mentions)
Defibrinated sheep blood, by Liofilchem (1 mentions)
Brain heart infusion broth, by Merck Group (1 mentions)
Sheep blood, by Avantor (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Maldi tof, by Bruker (1 mentions)
Sabouraud dextrose broth (sdb), by Thermo Fisher Scientific (1 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by bioMérieux (1 mentions)
11 protocols using brain heart infusion broth
1
IgA-bound Gut Microbiota Sorting
2
Detecting ESBL-Producing Enterobacteriaceae from Boot Swabs
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Sterisox® boot swabs were incubated 20 ± 4 h at room temperature with 100 ml of physiological water and 900 µL of Brain-Heart Infusion broth (BioMérieux SA, Marcy l’Etoile, France). Ten µL of the enriched suspension was directly streaked onto selective chromogenic agar plates (ChromID-ESBL, Biomérieux, Marcy l’Etoile, France) and incubated overnight at 37 °C under aerobic condition. Presumptive ESBL-producers were sub-cultured individually on Drigalski lactose agar and bacterial species identification performed using MALDI-TOF mass spectrometry (Bruker Daltonics, Breme, Germany). All Enterobacteriacae isolates identified, one or more by positive farms, were considered ESBL-E if confirmed by the combination disc test according to the European Committee on Antimicrobial Susceptibility Testing guidelines [19 ]. Thus, Muller Hinton agar with cefotaxime, ceftazidime, cefixime and cefepime disks with and without clavulanic acid allowed testing. The result was considered positive if the inhibition zone diameter was ≥5 mm larger with clavulanic acid than without for at least on cephalosporin tested.
If ESBL-E were identified, antibiograms were performed on isolates with ertapenem (ETP), nalidixic acid (NA), ofloxacin (OFL), gentamicin (GEN), Amikacin (AMK), trimethoprim/sulfamethoxazole (SXT) and tetracycline (TCN) tested.
If ESBL-E were identified, antibiograms were performed on isolates with ertapenem (ETP), nalidixic acid (NA), ofloxacin (OFL), gentamicin (GEN), Amikacin (AMK), trimethoprim/sulfamethoxazole (SXT) and tetracycline (TCN) tested.
3
Microbiota-Antibody Interaction Profiling
4
Streptococcus mutans Interaction with Odontoblasts
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The Streptococcus mutans strain (CIP 103220) was purchased from Institut Pasteur (Paris, France). Bacteria were cultured in Brain Heart Infusion broth (Biomérieux, Marcy-L'Etoile, France) at 37°C for 24 h. After centrifugation at 2500 rpm for 5 min, the supernatant was discarded and the bacterial pellet resuspended in water for determination of the bacteria number with McFarland Standard (Biomérieux). Samples containing 108 bacteria were then taken and centrifuged at 15,000 rpm for 5 min. Bacterial pellets were resuspended in 100 μL odontoblast-like cell culture supernatants, then maintained at 37°C for 15, 30, 60, or 90 min, the later time being just before the end of the bacteria growth phase as determined by our preliminary experiments (not shown). Bacteria-containing samples were then plated onto Columbia agar supplemented with 5% defibrinated horse blood. Bacterial cultures were performed in anaerobic conditions (GENbox anaer, Biomérieux) for 5 days at 37°C, then S. mutans colonies were counted.
5
Enterotoxin Detection in Bacillus cereus
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The production of the enterotoxins Nhe and Hbl was tested using two immunological tests, the BCET-RPLA Toxin detection kit (Oxoïd) and Tecra kit (BDE VIA, 3M-Tecra), respectively, after culture in brain heart infusion broth (Biomérieux) for 6 hours at 30 °C with stirring [18 (link)].
6
Antimicrobial Activity Evaluation of Chlorquinaldol
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The bacteriostatic and bactericidal properties of chlorquinaldol and its spectrum of activity were evaluated in comparison with gentamicin and fusidic acid.
The antimicrobial activity was assessed by determining the MIC and the MBC-values against the microbial strains described above. The MIC was determined by broth microdilution method, in accordance with EUCAST standard1 . Briefly, a suspension in growth medium (Brain heart infusion broth, Biomérieux, Marci l'Etoile, France) was prepared for each bacterial strain with an optical density equal to 0.5 McFarland (1.5 × 108 CFU/ml). For P. acnes broth was supplemented with 3% of defibrinated sheep blood (Liofilchem, Roseto degli Abruzzi, Italy). After obtaining a concentration of 105 CFU/ml using appropriate dilutions, each suspension was inoculated in a 96-wells microtiter plate containing a serial 2-fold dilution of chlorquinaldol, fusidic acid, or gentamicin. MIC-values, corresponding to the lowest concentration exhibiting no visible bacterial growth, were read after 24 h of incubation at proper conditions (except for P. acnes that required 48 h). The MBC was determined by plating 10 μl from each well showing no turbidity onto agar plates. After incubation at proper conditions, MBC was read as the lowest concentration able to kill 99.9% of the initial inoculum.
The antimicrobial activity was assessed by determining the MIC and the MBC-values against the microbial strains described above. The MIC was determined by broth microdilution method, in accordance with EUCAST standard
7
Bacterial Isolation and DNA Extraction
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Isolates from the external sources were delivered in brain heart infusion broth (bioMérieux, Nürtingen, Germany) and subcloned for 24 h at 37°C on Columbia agar with 5% sheep blood (VWR International, Darmstadt, Germany). Single colonies were picked and propagated in brain heart infusion broth (Sigma-Aldrich, Munich, Germany) for 24 h at 37°C for DNA extraction. Liquid culture (1 mL) was processed using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to manufacturer instructions.
8
S. aureus ATCC 25923 Bacterial Culture
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S. aureus strain ATCC 25923 was used in this study as described in our recent study [24] . Briefly, bacteria were cultured at 37°C overnight onto Mannitol Salt Agar (BioMerieux, France) and incubated into Brain Heart Infusion Broth (BioMerieux) at 37°C for 16 hours. The bacterial suspension was suspended in PBS to obtain a 0.5 McFarland turbidity (equal to about 1×108 CFU/mL), then serially diluted with sterile saline solution and counts were performed to check for bacterial inoculum used for the experiments.
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S. aureus strain ATCC 25923 was used in this study as described in our recent study [24] . Briefly, bacteria were cultured at 37°C overnight onto Mannitol Salt Agar (BioMerieux, France) and incubated into Brain Heart Infusion Broth (BioMerieux) at 37°C for 16 hours. The bacterial suspension was suspended in PBS to obtain a 0.5 McFarland turbidity (equal to about 1×108 CFU/mL), then serially diluted with sterile saline solution and counts were performed to check for bacterial inoculum used for the experiments.
9
Isolation and Identification of S. aureus
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After enrichment of the samples in Brain Heart Infusion Broth (bioMérieux, Mercy l’Etoile, France) at 37 °C for 24 h, we performed an isolation on mannitol salt agar (bioMérieux, Mercy l’Etoile, France) at 37 °C for 48 h. The identification of isolated strains as S. aureus was also based on the DNASE test (Bio-Rad, Marnes-la-Coquette, France). This was then confirmed by matrix-assisted laser desorption ionisation/time of flight (MALDI-TOF) (Bruker, Bremen, Daltonics, Germany) [14 (link)].
10
Microbial Preparation for Antimicrobial Testing
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The following microbes were used in the experiment: methicillin-resistant Staphylococcus aureus (MRSA) (clinical strain), beta-lactamase-producing Escherichia coli (clinical strain), Candida auris (DSM 21092), Candida albicans fluconazole-resistant (ATCC 10231), Trichophyton rubrum, and Trichophyton mentagrophytes (ATCC 9533). MRSA and E. coli (ESBL+) were cultured in a brain heart infusion broth (bioMerieux, Marcy-l’Étoile, France) for approximately 20 h at a constant temperature of 35 ± 1 °C under aerobic conditions. C. albicans and C. auris were cultivated for approximately 24 h at 35 ± 1 °C in the Sabouraud dextrose broth (Oxoid, Hampshire, UK) and Emmons’ modification of Sabouraud’s broth (BD), respectively. After the incubation period, the bacteria and fungi were centrifuged and harvested (3000 rpm for 15 min) and then re-suspended in 10 mM phosphate-buffered saline (PBS, pH = 7.0). T. rubrum and T. mentagrophytes in Sabouraud dextrose agar (Oxoid, Hampshire, UK) at 35 ± 1 °C until adequate sporulation appeared (ca. three weeks). After incubation, cultures were covered with sterile 0.9% NaCl solution supplemented with 0.1% Tween 80, carefully rubbed with a sterile cotton swab, and transferred to a sterile flask. Suspensions were homogenized and filtered. In the last step, each strain was adjusted at a concentration of ca. 107 CFU/mL.
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