Bacteroides fragilis type strain E-022248T (= DSM 2151 = ATCC 25285) from the VTT Culture Collection (VTT Technical Research Center of Finland) was grown under anaerobic conditions for 2 days at 37°C on Brucella agar with hemin and vitamin K (Sigma Aldrich, St. Louis, MO, United States) along with 5% defibrinated sheep blood (Bio Karjalohja Oy, Finland). Escherichia coli K12-derived TOP10 (Invitrogen, United States) was aerobically cultivated overnight in Luria–Bertani broth (Becton Dickinson, United States).
Defibrinated sheep blood
Manufactured by Merck Group
Sourced in United States
Defibrinated sheep blood is a laboratory product used as a culture medium for the growth and study of microorganisms. It is obtained by defibrinating sheep blood, which removes the fibrin and prevents clotting. This product can be used in various microbiological and diagnostic applications.
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Lab products found in correlation
Luria bertani broth, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Oxoid sr 147 supplement, by Thermo Fisher Scientific (1 mentions)
Brucella agar plates, by BD (1 mentions)
Glycerol, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Menadione, by Merck Group (1 mentions)
Hemin, by Merck Group (1 mentions)
Bhi agar, by Merck Group (1 mentions)
Bhi broth, by Merck Group (1 mentions)
Campylobacter selective supplement, by Merck Group (1 mentions)
Brucella agar, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Polysorbate 80, by Merck Group (1 mentions)
Sodium phosphate dibasic dehydrate, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Malt extract agar, by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar tsa, by Thermo Fisher Scientific (1 mentions)
Maximum recovery diluent, by Merck Group (1 mentions)
7 protocols using defibrinated sheep blood
1
Anaerobic Culturing of Bacteroides fragilis
2
Helicobacter pylori Isolation and Identification
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Two biopsy samples were achieved from each patient from the antrum and the body of the stomach. The H. pylori isolates were cultured at 37 °C on Brucella agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) containing 5% defibrinated sheep blood (Hanil Komed, Seongnam, Korea) under microaerobic conditions (5% O2, 10% CO2, 85% N2) for 4 days. Organisms were identified as H. pylori by colony morphology, rapid urease test, H. pylori-selective media (Oxoid™ SR 147 supplement (Thermo Fisher Scientific, Waltham, MA) and 5% defibrinated sheep blood), and PCR to detect ureA. All stock cultures were stored at −70 °C in Brucella broth supplemented with 15% glycerol (Sigma Chemical Co., St Louis, MO, USA) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA). These preparations were thawed and subcultured for further experiments.
3
Culturing Aggregatibacter actinomycetemcomitans
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A. actinomycetemcomitans ATCC 33384 strain, purchased from Institute of Microbiology, ETH Zurich, Switzerland, was cultured in microaerophilic conditions (<20% O2) for 48 hours at 37°C in a culture medium that was prepared by using brain heart infusion (BHI) agar (Merck KGaA, Darmstadt, Germany) to which the following compounds were added: 5% defibrinated sheep blood (Sigma-Aldrich Co., Ltd., Dorset, United Kingdom), 5 g/L of yeast extract (Merck KGaA, Darmstadt, Germany), 5 mg/L of hemin (Sigma-Aldrich Co., Ltd., Dorset, United Kingdom), and 1 mg/L of menadione (Sigma-Aldrich Co., Ltd., Dorset, United Kingdom). The strain was then inoculated into freshly prepared tubes containing BHI broth (Merck KGaA, Darmstadt, Germany), and the cell density was adjusted to 1.5×108 cells/ml as verified by a spectrophotometer (Eppendorf BioPhotometer, Hamburg, Germany) to measure the optical density at 600 nm (OD600) and to count the colonies.
4
Isolation and Identification of H. pylori
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Two biopsy specimens were taken, the first was sent to the pathology department for histopathological examination, the second was shipped within 2–4 hours to the laboratory in Eppendorf tubes containing thioglycollate broth medium in a cold-box [14 (link)]. Bacterial culture was conducted briefly, as follows: the biopsy specimen was gently vortexed, then the homogenate was inoculated into Brucella agar (Merck, Darmstadt, Germany) plates supplemented with 5% defibrinated sheep blood (Bahar-Azma, Tehran, Iran), 10% fetal bovine serum (Sigma, St Louis, MO, USA), Campylobacter selective supplement (Merck), and 5 mg/L of amphotericin B (Merck) [14 (link)]. Incubation period was 7–14 days, and the plates were checked for suspected colonies after 5 days. Microaerophilic conditions (10% CO2, 5% O2 and 85% N2) were used during the incubation period to provide optimum growth conditions for H. pylori. Following the incubation period, the H. pylori cultures were investigated using Gram-staining, translucent colonies, as determined with the naked eye, and three common biochemical tests (catalase, oxidase and urease) [14 (link)]. The confirmed isolates were chosen for second bacterial culture to achieve a single colony (to avoid mixed infections). The selected isolates were the subject of susceptibility tests and PCR.
5
Antimicrobial Formulation for Wound Care
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Chlorhexidine digluconate (20%,) polyvinylpyrrolidone (PVP)–iodine complex, sodium phosphate dibasic dehydrate, citric acid, isopropyl, polysorbate 80, catalase, hydrogen peroxide, bovine serum albumin, and iodine were obtained from Sigma Aldrich (USA). Malt extract agar (MEA) and tryptic soy agar (TSA) were purchased from Oxoid (UK). Maximum recovery diluent, lecithin, and defibrinated sheep blood were obtained from Merck, Alfa Aesar, and Thermo Fisher Scientific, respectively.
6
Cutibacterium acnes Infection of HaCaT Cells
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A sequenced strain of Cutibacterium acnes (C. acnes), sampled from facial acne (ATCC® 6919TM, LGC standards s.r.l., Milano, Italy), was grown by solid culture and used to infect HaCaT cells. The bacteria were stored at −80 °C, at the optical density (O.D.) of 5, in cryovials containing reinforced clostridial medium 30% (Oxoid, Hampshire, UK), glycerol 20%, and defibrinated sheep blood 50% (TCS Biosciences Ltd., Oxoid, Hampshire, UK). The solid culture was achieved using agar-blood Petri dishes, containing RCM, agar 1% (Merck Life Science, Milan, Italy), and defibrinated sheep blood 5%. Petri dishes were inoculated with 100 μL of the store aliquots of bacteria, sealed in plastic bags containing an anaerobic atmosphere generated by CO2 GasPakTM EZ (Becton Dickinson, Sparks, MD, 21152 USA), and then placed in a cell incubator (37 °C) for 48 h before HaCaT cell infection. At the time of infection, bacteria were collected and suspended in PBS 1X solution, counted by the optical method at 600 nm in 1 mL cuvette (Biochrom Libra S22 UV/VIS spectrophotometer), and directly diluted in the medium of cultured HaCaT cells, with a final O.D. of 0.1.
7
Rapid Urease and Culture Detection of H. pylori
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One of the biopsy samples was routinely tested by the gastroenterologist by Rapid Urease Test; and if positive a second sample was obtained and placed in 200 μl sterile thioglycolate (Merck, Germany) broth and then immediately shipped to the diagnostic laboratory for routine culture. Upon arrival in the microbiology lab this sample was immediately grinded and 100 μl of the resultant homogenate was inoculated on a Colombia agar (Merck, Germany) plate supplemented with 7% defibrinated sheep blood (Jihad Daneshgahi, Tehran, Iran), 10% Fetal Calf Serum (FCS) and antibiotics (DENT, Supplement, Oxoid) [15 (link)]. Plates were incubated at 37°C, in 10% CO2 conditions provided by incubator (Binder, USA) and high humidity until typical H. pylori colonies appeared or for a maximum of 7 days if no suspect colonies were observed. Colony shape, morphology in microscopic examination, routine biochemical tests such as urease, catalase and oxidase tests were performed for identification of H. pylori strains.
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