From each turkey meat sample and culture media five colonies of the highest dilution that generated growth were randomly selected and isolated. The morphology of suspected colonies was taken into consideration when specific media were used. Isolates were purified in Tryptone Soy agar (Scharlau) and Brain Heart Infusion broth (Scharlau). The purified isolates were maintained at −80 °C. Bacterial identification was carried out by a MALDI-TOF biotyper (Bruker, Daltonik, Bremen, Germany).
Brain heart infusion broth
Manufactured by Scharlab
Sourced in Spain
Brain Heart Infusion broth is a microbiological growth medium used for the cultivation of a wide variety of aerobic and anaerobic bacteria. It provides the necessary nutrients and growth factors for the cultivation of fastidious microorganisms.
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5 protocols using brain heart infusion broth
1
Isolation and Identification of Bacterial Colonies from Turkey Meat
2
Quantifying Vaginal Bacterial Populations
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Each vaginal swab was homogenized in 1 mL of brain heart infusion broth (Scharlab, Barcelona, Spain) and vortexed for 1 min at maximum speed to suspend attached bacteria. Then, decimal dilutions in phosphate-buffered saline (PBS) were plated in 13.5 cm diameter Petri dishes containing the following media: Man Rogosa and Sharpe (MRS) agar (Scharlab, Barcelona, Spain) for the selective growth of lactic acid bacteria (LAB) and Columbia blood agar (BA; Dismalab, Valdemorrillo, Madrid, Spain) as a general bacterial growth medium. Plates were incubated for 48 h at 37 °C microaerobically and aerobically, respectively. The number of colony forming units (CFU/mL) was counted.
3
Multispecies Biofilm Formation Assay
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Biofilm development was performed in sterile polystyrene 24-well tissue culture plates (Greiner Bio-one, Frickenhausen, Germany).
In order to determine the effect of the oral products to be studied on bacterial viability, a multispecies biofilm formation was designed, where the substrates were incubated with poly-L-lysine solution 0.01% (Sigma-Aldrich, Darmstadt, Germany) for 1 h at 37 °C to promote and stabilize the biofilm.
The growth kinetics were evaluated by generating growth curves. The bacterial concentration was adjusted by spectrophotometry (optical density (OD) at 600 nm) in order to obtain a bacterial suspension containing 105 CFU/mL (colony-forming units/mL) for S. mutans and 106 CFU/mL each for F. nucleatum, P. gingivalis, and P. intermedia.
The culture medium used for the growth of S. mutans was brain–heart infusion broth (Scharlab, Barcelona, Spain) under CO2 conditions at 37 °C for 24–48 h.
F. nucleatum, P. intermedia, and P. gingivalis were grown on fastidious anaerobe broth (EO labs, Bonnybridge, UK) under anaerobic conditions at 37 °C for 48–72 h for F. nucleatum and 72–96 h for P. intermedia and P. gingivalis.
In order to determine the effect of the oral products to be studied on bacterial viability, a multispecies biofilm formation was designed, where the substrates were incubated with poly-L-lysine solution 0.01% (Sigma-Aldrich, Darmstadt, Germany) for 1 h at 37 °C to promote and stabilize the biofilm.
The growth kinetics were evaluated by generating growth curves. The bacterial concentration was adjusted by spectrophotometry (optical density (OD) at 600 nm) in order to obtain a bacterial suspension containing 105 CFU/mL (colony-forming units/mL) for S. mutans and 106 CFU/mL each for F. nucleatum, P. gingivalis, and P. intermedia.
The culture medium used for the growth of S. mutans was brain–heart infusion broth (Scharlab, Barcelona, Spain) under CO2 conditions at 37 °C for 24–48 h.
F. nucleatum, P. intermedia, and P. gingivalis were grown on fastidious anaerobe broth (EO labs, Bonnybridge, UK) under anaerobic conditions at 37 °C for 48–72 h for F. nucleatum and 72–96 h for P. intermedia and P. gingivalis.
4
Bacterial Isolation and Identification Protocol
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From each sample and culture media five colonies of the highest dilution that yielded growth were randomly selected and isolated. The morphology of suspected colonies was taken into account when specific media were used. Isolates were purified on Tryptone Soy Agar (Scharlau, Barcelona, Spain) and Brain Heart Infusion broth (Scharlau, Barcelona, Spain). The purified isolates were kept at −80 °C. Bacterial identification was performed by a Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass-Spectrometry (MALDI-TOF MS) Biotyper (Bruker, Billerica, MA, USA).
5
Campylobacter Detection in Broiler Carcasses
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The sampling was performed from November 2018 to June 2019. At the slaughterhouse level, samples of broiler carcasses, neck skin, and cecal content were collected from diverse flocks on different working days. For neck skin, sampling was performed according to the recommendations described in the regulation (EU) 2017/1495 of August 23, 2017. Campylobacter enumeration and isolation were performed according to ISO 10272-1 and 10272-2 (2017) . Fecal samples were obtained from cecum extracted from broiler carcasses as Fraqueza et al. (2016) described. Fecal samples were streaked with a loop on mCCD agar (Campylobacter selective agar, Neogen) supplemented with Cefoperazone and Amphotericin (Neogen). All plates were incubated at 42°C for 48 h under microaerobic conditions: 6% O2; 7.1% CO2; 7.1% H2 (Anoxomat, Advanced Instruments). All isolates presenting typical colony morphology were submitted to the Gram staining procedure and oxidase, catalase, and Hippurate test, as described in ISO 10272-1 (2017) . Campylobacter isolates were stored in cryotubes with Brain Heart Infusion broth (Scharlau, Spain) and 15% glycerol at −80°C. For DNA extraction, the culture from each isolate was consistently stored in Eppendorf tubes with TE 1X buffer (10 mmol−1 Tris-HCl, 1 mmol−1 EDTA, pH = 8). A total of 143 C. jejuni and C. coli isolates comprised this collection.
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