Ft LVS (American Type Culture Collection 29,684) and Ft ΔclpB [obtained from Conlan et al. (2010) (link)] were grown to mid-log phase in modified Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, MI), harvested, and frozen in aliquots in broth alone at−80°C (Baker et al., 1985 (link); Fortier et al., 1991 (link)). Ft strain SchuS4 (BEI Resources, Manassas, VA, NR-10492) was sub-cultured in modified cysteine partial hydrolysate (MCPH) broth to produce a sub-master stock and a working stock, which were frozen in aliquots with 20% glycerol at −80°C. Of note, Ft SchuS4 NR-10492 has been demonstrated to be identical and with similar level of virulence compared to Ft SchuS4 NR-28534 (Lovchik et al., 2021 (link)), which was used previously to evaluate vaccine efficacy in NHsd rats (De Pascalis et al., 2022 (link)). Bacteria were periodically thawed for quality control by quantification of viability on MH agar plates or chocolate agar plates (Hardy Diagnostics, Santa Maria, CA); in addition, quantification was also evaluated at the time of vaccination or challenge procedures. Although different media were used at each site, within each laboratory’s work with the three rat strains, bacteria were cultured using the same media.
Chocolate agar plates
Manufactured by Hardy Diagnostics
Sourced in United States
Chocolate agar plates are a type of microbiological culture medium used for the isolation and identification of certain bacterial species. They are made by adding defibrinated blood to a standard agar base, which is then heated to create a chocolate-brown colored medium. This medium supports the growth of fastidious organisms, including Neisseria and Haemophilus species.
Automatically generated - may contain errors
Lab products found in correlation
Hemin, by Merck Group (2 mentions)
Modified mueller hinton broth, by BD (1 mentions)
Millipore filter, by Merck Group (1 mentions)
Gswp02500, by Merck Group (1 mentions)
Nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Bacto brain heart infusion, by BD (1 mentions)
Miseq dna sequencer, by Illumina (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Brain heart infusion broth, by Merck Group (1 mentions)
Pierce lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Pierce high capacity endotoxin removal column, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Brain heart infusion media, by Merck Group (1 mentions)
7 protocols using chocolate agar plates
1
Preparation and Characterization of Francisella Strains
2
Nontypeable Haemophilus Influenzae Biofilm Protocol
3
Cultivation and Characterization of Francisella Strains
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F. tularensis LVS (ATCC 29684) and LVS-R (originally obtained from Dr. Francis Nano, University of Victoria, Canada) [20 (link)], were grown to mid-log phase in supplemented Mueller-Hinton broth, and working infection stocks were quality controlled as previously described (Difco Laboratories, Detroit, MI) [9 ]. Heat killed LVS (HK-LVS) was prepared immediately prior to use by treating LVS at 60°C for 60 minutes; killing was confirmed by plating onto chocolate agar plates (Hardy Diagnostics; Santa Maria, CA) or enriched Mueller-Hinton plates [21 (link)]. F. tularensis strain SchuS4 was originally derived from Master Cell Bank, NR-28534 (BEI Resources, Manassas, VA). The Master Cell Bank was sub-cultured in Modified Cysteine Partial Hydrolysate (MCPH) broth once to produce a sub-master stock and a second time to produce a working stock, which as frozen in aliquots with 20% glycerol at -80°C.
4
Biofilm Formation of Non-Typeable Haemophilus influenzae
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50 µl from frozen stock cultures of non-typeable Haemophilus influenzae strains PittAA, PittEE, PittGG, PittII [42] (link), [43] (link), 2019 [44] (link) and 9274 [45] (link) were spread onto chocolate agar plates (Hardy Diagnostics, Santa Maria, CA, USA) and incubated at 37°C in an atmosphere of 5% CO2 with 95% relative humidity. After 24-hour incubation, multiple colonies were used to inoculate BHI broth (Bacto Brain Heart Infusion; BD Diagnostics, Sparks, MD, USA) supplemented with hemin (Sigma-Aldrich Inc., St. Louis, MO, USA) at 10 µg/ml and nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich Inc., St. Louis, MO, USA) at 2 µg/ml (sBHI) [46] . The bacterial suspension was grown to stationary phase (1×109 CFU/ml) at 37°C, 5% CO2, with 95% relative humidity. Biofilms in this study were prepared by diluting 1∶200 bacterial suspensions at stationary phase with fresh sBHI, aliquoting into culture vessels appropriate for subsequent applications, and incubating at 37°C with 5% CO2 and 95% relative humidity for 24 hours. All NTHi strains were isolated from chronic otitis media patients, with the exception of 2019, which was isolated from a patient with chronic bronchitis.
5
Whole-Genome Sequencing of L. monocytogenes
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All 31 L. monocytogenes strains were passaged twice from − 80 °C frozen stocks on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA) at 37 °C. A single colony of each isolate was then inoculated in 1.5 mL of Brain-Heart Infusion (BHI) broth and grown overnight without shaking at 37 °C. Genomic DNA was extracted from the cultures using MO BIO microbial DNA isolation kits (MO BIO Laboratories, Carlsbad, CA) according to the manufacturer’s instructions. The extracted DNAs were quantified and checked for purity using 260/280 absorbance readings on a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). Individual libraries were constructed for each of the strain DNA preparations using Illumina Nextera XT DNA sample preparation kits with appropriate indices tags according to the manufacturer’s instructions (Illumina Inc., San Diego, CA). The libraries were pooled together and run on an Illumina MiSeq DNA sequencer (Illumina Inc., San Diego, CA). The genome of each strain was sequenced to a minimal depth of 10X coverage.
6
NTHi DNA Extraction and Endotoxin Removal
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NTHi glycerol stock (strain R2846/12, a gift from Stephen Barenkamp at Saint Louis University School of Medicine, St Louis, MO, USA) was grown on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA, USA) in a 37°C incubator. A single colony was selected and cultured in brain heart infusion broth (Sigma, St Louis, MO, USA) for 24 h at 37°C. Bacteria were centrifuged three times at 15 294×g for 5 min and resuspended in PBS to wash the bacteria. A QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract DNA from bacteria according to the manufacturer's instructions. Briefly, bacterial pellet was resuspended in 200 µL of PBS containing 20 µL of proteinase K to extract DNA. To generate endotoxin-free bacterial DNA, a Pierce High-Capacity Endotoxin Removal column (Thermo Fisher Scientific, Waltham, MA, USA) was used to remove any potential endotoxin from NTHi-derived DNA as per manufacturer's instructions. A Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific) was used to confirm endotoxin removal from NTHi-derived DNA showing <0.096 EU·mL−1 (0.0096 ng·mL−1) of endotoxin contamination in NTHi DNA preparation, which is well below the endotoxin contamination level (0.5 EU·mL−1 or 0.05 ng·mL−1) set by the US Food and Drug Administration (FDA) for research.
7
NTHi Strain 1479 Protein Extraction
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NTHi strain 1479 (a gift from Dr Brahm Segal, University of Buffalo) was grown overnight on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA) and then used to inoculate 20 ml of brain–heart infusion media (Sigma-Aldrich) containing 10 μg ml−1 NAD and 10 μg ml−1 Hemin (both from Sigma-Aldrich). The culture was incubated for 4 h at 36 °C with constant shaking and then used to inoculate an additional 200 ml of liquid media. After another 4 h of growth, bacteria were pelleted, boiled for 1 h, sonicated twice for 1 min each and filtered through a 0.22-μM polyethylsulfone filter (EMD Millipore, Darmstadt, Germany) using 1 ml of added PBS for each pellet generated from 50 ml of liquid culture. Protein concentration was adjusted to 1.5 mg ml−1 in PBS using Bradford assay (Pierce, Rockford, IL).
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