Brain heart infusion (BHI), sheep blood agar (SBA), and THY were from BD Biosciences (San Jose, CA) or RPI. Kanamycin and D-glucose were purchased from Research Products International (RPI; Mt. Prospect, IL). Glycine was purchased from AMERSCO (Solon, OH). Unless otherwise specified, all reagents were purchased from Sigma Aldrich. M9 minimal medium was prepared as previously described (Miller, 2010 (link)).
Sheep blood agar
Manufactured by BD
Sourced in United States, Japan
Sheep blood agar is a microbiological culture medium used for the isolation and identification of bacteria. It consists of a standard agar base supplemented with sterile defibrinated sheep blood. The sheep blood provides essential nutrients and growth factors for a wide range of bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Macconkey agar, by BD (5 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Dent antibiotics supplement, by Thermo Fisher Scientific (3 mentions)
Bordet gengou agar, by BD (2 mentions)
Casamino acid, by Thermo Fisher Scientific (2 mentions)
Dna mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Skim milk, by BD (2 mentions)
Anaerobe blood agar plate, by BD (2 mentions)
D glucose, by Research Products International (1 mentions)
Brain heart infusion, by BD (1 mentions)
Kanamycin, by Research Products International (1 mentions)
Blood agar, by BD (1 mentions)
Todd hewitt broth, by BD (1 mentions)
Trypticase soy agar, by BD (1 mentions)
Vancomycin, by BD (1 mentions)
Kanamycin, by BD (1 mentions)
Protein assay dye, by Bio-Rad (1 mentions)
Anaerobic jar, by BD (1 mentions)
36 protocols using sheep blood agar
1
Bacterial Growth Media Preparation
2
Helicobacter pylori Infection Model in RGM1 Cells
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H. pylori (ATCC 43504) with the typical S shape, gram-negative rods, possessing the CagA and VacA were provided in a frozen state by ATCC. H. pylori ATCC 43504 strains were grown on tryptic soy agar with 5% sheep blood agar (BD Diagnostics) and Dent antibiotics supplement (Oxoid) at 37°C under microaerophilic conditions (Campy-Pak 273 System, BBL). RGM1 cells were incubated overnight in fresh serum- and antibiotic-free RPMI 274 medium and were infected with H. pylori at multiplicities of infection (MOI) of 100:1 for 48 h. Though we found no influence of WPE on H. pylori culture, we repeated the status of H. pylori colonization in group treated with WPE in the presence of H. pylori and H. pylori infection alone before RNA isolation for RNAseq analysis.
3
Isolation and Serotyping of Group B Streptococcus
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Vaginal and rectal swabs from pregnant women during delivery and swabs from external ear, nasal, throat and umbilical area of newborns were placed into Lim broth (BD Diagnostics, USA) supplemented with colistin (10 μg/ml) and nalidixic acid (15 μg/ml). The inoculated selective medium was incubated for 18–24 h, at 37 °C in CO2 enriched atmosphere, sub-cultured onto sheep blood agar (BD Diagnostics, USA) and further incubated in CO2 enriched atmosphere at 37 °C for 18–24 h. If GBS was not identified after incubating for 18–24 h, the blood agar plate was re-incubated and examined after 48 h to identify suspected colonies. All suspected GBS colonies (β-hemolytic, or non-hemolytic, gram positive cocci, catalase negative) were sub-cultured and isolated for confirmatory testing. A CAMP (Christite, Atkins, Munch, Petersen) test were considered presumptive identification of a positive GBS culture. Ambiguous CAMP test culture results were re-tested using a Strpt. B Grouping Latex (Remel, USA).
Capsular serotyping was done for all GBS isolates by slide agglutination tests by using type specific 10 antisera for serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX (Statens Serum Institute, Denmark) as previously described by Slotved et al [9 (link)].
Capsular serotyping was done for all GBS isolates by slide agglutination tests by using type specific 10 antisera for serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX (Statens Serum Institute, Denmark) as previously described by Slotved et al [9 (link)].
4
Epidemiology of Linezolid-Resistant CoNS
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Linezolid-resistant coagulase-negative staphylococci (LRCoNS) were identified through retrospective review of laboratory records between January 2007 and January 2012 for CoNS isolated from blood with a linezolid MIC of ≥8 µg/ml. Linezolid susceptibility testing was routinely performed by the clinical laboratory on CoNS isolated from patients with ≥2 blood culture sets or upon physician request for CoNS isolated from a single blood culture set. Incidence of LRCoNS in blood cultures was calculated using the total number of single-patient CoNS bloodstream isolates with susceptibility test results as the denominator. Isolates were stored at room temperature on tryptic soy agar slants (BD, Sparks, MD) with mineral oil overlay for up to 2 years. All isolates were subcultured twice on sheep blood agar (BD) prior to testing in this study. Patient medical records were reviewed to determine patient demographics and linezolid exposures. This study was approved by the UCLA Institutional Review Board with exemption of informed consent, as this was a review of existing data.
5
Antibacterial Efficacy of Glycerol Monolaurate
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Bacteria were grown in Todd Hewitt broth (Difco Laboratories, Detroit, MI) overnight at 37°C in a standard incubator with aeration (shaking at 225 revolutions/min). Initial innocula were estimated by measuring absorbance at 600 nm wavelength and were verified by plate counts. For GML-treated planktonic cultures, bacteria were inoculated in 3 ml of media or 10% non-aqueous delivery vehicle solubilized in media in glass tubes to give an inoculum of 1 x 106 CFU/ml. GML or ethanol vehicle control was then introduced to cultures. Cultures were shaken at 37°C for 24 h, at which point they were diluted for plate counts.
To assess effect of GML Gel on bacteria, 0.3 ml volumes of approximately 2 x 108 CFU were added to 2.7 ml of 5% GML Gel and were mixed vigorously with sterile swabs. At desired time points, a swab of culture, containing approximately 0.1 ml volume of Gel was taken and spread on trypticase soy agar with 5% sheep blood agar (BD Biosciences, San Jose, CA).
To assess effect of GML Gel on bacteria, 0.3 ml volumes of approximately 2 x 108 CFU were added to 2.7 ml of 5% GML Gel and were mixed vigorously with sterile swabs. At desired time points, a swab of culture, containing approximately 0.1 ml volume of Gel was taken and spread on trypticase soy agar with 5% sheep blood agar (BD Biosciences, San Jose, CA).
6
S. aureus USA300 LAC Culturing
7
Culturing Gut Microbiota from IBD Patients
8
H. pylori Protein Expression Analysis
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RPMI-1640 medium, fetal bovine serum, penicillin (FBS), streptomycin were products of GIBCO BRL (Grand Island, NY) and materials for culturing H. pylori were sheep blood agar, Gaspak and anaerobic jars (BD Biosciences, Sparks, MD). Primary antibody against actin was purchased from Sigma-Aldrich Co. (St. Louis, MO), antibodies for lamin B from Santa Cruz Biotechnology (Santa Cruz, CA), other antibodies for p-STAT3Tyr705, total STAT3 from Cell Signaling Technology (Beverly, MA), horseradish peroxidase (HRP)-conjugated secondary antibody from Pierce Biotechnology (Rockford, IL). DL-dithiothreitol (DTT), TRIzol, 4',6-diamidino-2-phenylindole (DAPI) from Invitrogen (Carlsbad, CA), and polyvinylidene difluoride (PVDF) membranes were supplied from Gelman laboratory (Ann Arbor, MI). The ECL chemiluminescent detection kit was purchased from LPS solution (Daejon, South Korea) and protein assay dye (Bradford) reagent was supplied by Bio-Rad Laboratories (Hercules, CA), bicinchonic acid (BCA) protein assay reagent was obtained from Pierce Biotechnology.
9
In vitro H. pylori Infection Assay
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H. pylori (ATCC 43504) with the typical S shape, gram-negative rods, possessing the CagA and VacA were provided in a frozen state by ATCC. H. pylori ATCC 43504 strains were grown on tryptic soy agar with 5% sheep blood agar (BD Diagnostics) and Dent antibiotics supplement (Oxoid) at 37°C under microaerophilic conditions (Campy-Pak 273 System, BBL). RGM1 cells were incubated overnight in fresh serum- and antibiotic-free RPMI 274 medium and were infected with H. pylori at multiplicities of infection (MOI) of 50:1.
10
Bordetella species isolation and DNA extraction
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B. pertussis (ATCC 9797) was obtained from American Type Culture Collection (ATCC, Rockville, MD), and B. holmesii and B. parapertussis were clinical isolates obtained from the Wisconsin Department of Health Services, which were de-identified. B. pertussis was grown on Bordet-Gengou agar (BD, Sparks, MD, USA) supplemented with 10% casamino acid (Fisher Scientific, USA). B. holmesii and B. parapertussis were grown in 5% sheep blood agar (BD, Sparks, MD, USA). The microorganisms were incubated in an aerobic environment with sufficient humidity at 35 °C for 3–4 days. Genomic DNA from B. pertussis, B. holmesii and B. parapertussis was extracted and purified by using the Qiagen DNA Mini kit (Catalog No. 51304, Valencia, CA, USA) following the protocol from the manufacturer. For each organism, bacterial colonies were transferred into a centrifuge tube containing 5 mL of sterile saline adjusting the turbidity to 0.5 McFarland standard (ProLab Diagnostics, Round Rock, TX, USA) to achieve a cell density of ~ 1.5 × 108. Cells were harvested by centrifugation in a Beckman centrifuge, using a C0650 rotor with adapters for 15 mL tubes, at 7500 rpm (5000 × g) for 10 min. Bacterial cell pellets were collected to proceed with the solid-phase extraction of DNA as indicated by the Qiagen protocol. Eluted DNA samples were measured by using Nanodrop (Nanodrop 1000, Thermo Scientific, MA, USA).
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