The largest database of trusted experimental protocols

10 protocols using Sodium chloride

The bacterial strains and plasmids used in this study are listed in Table 1. All Salmonella strains were maintained in animal-product-free Hy-Soy (HS) medium (10 g/L soytone (Teknova, Hollister, CA, USA), 5 g/L Hy-yeast (Kerry BioScience, Beloit, WI, USA) and 5 g/L sodium chloride (AmericanBio, Natick, MA, USA)) at 37 °C. Agar (AmericanBio) was added when necessary at 15 g/L. Medium was supplemented with guanine (0.005% (w/v) final; Sigma-Aldrich, St. Louis, MO, USA) for mutants harboring ΔguaBA deletions. Antibiotic (carbenicillin (Corning, Glendale, AZ, USA) or kanamycin (50 µg/mL final; Sigma-Aldrich)) was added when necessary. For immunization studies, vaccine strains were streaked onto HS medium containing guanine and grown for 18–20 h at 37 °C. Bacterial colonies were then resuspended in sterile phosphate-buffered saline (PBS) to a concentration of 108 to 109 colony-forming units (CFU) per 10 µL for infant mice and 100 µL for adult mice.
+ Open protocol
+ Expand
An ice chip from PA M2 glycerol stock was streaked for single colony isolates on Tryptic Soy Agar (TSA) (Sigma-Aldrich, St. Louis, MO) plates and incubated at 37°C for 18 hours. A single colony was transferred to 3.0 mL of Hy-Soy Broth, containing 0.5% sodium chloride (American Bio, Canton, MA), 0.5% HY-Yeast (Kerry Bio-Science, Norwich NY), and 0.25% animal-free soytone (Teknova, Hollister, CA) and grown to stationary phase at 37°C in a shaking incubator. Next, 1% volume was inoculated into 12 mL of Hy-Soy broth and grown at 37°C in a shaking incubator until mid-log-phase, OD600 of 0.6 to 0.8. The bacteria were pelleted, washed twice with sterile phosphate-buffered saline (PBS), and resuspended to the desired concentration.
+ Open protocol
+ Expand
Unless otherwise specified, all bacterial strains were grown in Hy-Soy medium (10 g/L soytone [Teknova, Hollister, CA], 5 g/L hy-yest [Kerry Bio-Science, Norwich, NY], and 5 g/L sodium chloride [AmericanBio, Natick, MA]). When needed, agar (AmericanBio) was added at 15 g/L.
+ Open protocol
+ Expand
4

Cultivation of Pseudomonas aeruginosa M2 for Experimentation

An inoculum of P. aeruginosa M2 (O5/FlaB), originally isolated from the gastrointestinal tract of a CF-1 mouse by Ian Alan Holder (37 (link)), was grown overnight to stationary phase from glycerol stocks in 2.0 ml of Hy-Soy broth, containing 0.5% sodium chloride (American Bio, Canton, MA), 0.5% HY-Yeast (Kerry Bio-Science, Norwich NY), and 0.25% animal-free soytone (Teknova, Hollister, CA). One-hundred twenty microliters of overnight stationary P. aeruginosa M2 was transferred into 12 ml of fresh medium and grown to mid-log phase (optical density at 600 nm [OD600] of 0.6 to 0.8). Bacteria were pelleted, washed, and resuspended (to an OD600 of 0.3, corresponding to ∼1 × 108 CFU/ml) in sterile PBS. Final dilutions were made to achieve the desired concentrations and verified by colony count on TSA (tryptic soy agar; Sigma-Aldrich, St. Louis, MO) plates.
+ Open protocol
+ Expand
The bacterial strains used in this study are listed in Table 1. Plasmids pKD13, pKD46, and pCP20 were used to delete genes from specific chromosomal loci. All Salmonella strains were maintained in animal-product-free Hy-Soy (HS) medium (10 g/L Soytone (Teknova, Hollister, CA), 5 g/L Hy-yest (Kerry BioScience, Beloit, WI, USA) and 5 g/L sodium chloride (American Bio, Natick, MA, USA)) at 37 °C. When needed, agar (American Bio) was added at 15 g/L. Medium was supplemented with guanine (0.005% (w/v) final concentration; Sigma–Aldrich, St. Louis, MO, USA) for mutants harboring ΔguaBA deletions. Carbenicillin (Corning, Glendale, AZ, USA) or kanamycin (Sigma–Aldrich) at a final concentration of 50 µg/mL were added when necessary.
+ Open protocol
+ Expand
Bacterial strains used in this study are shown in Table 1. All bacterial strains were maintained in animal-product-free Hy-Soy (HS) medium (10 g/L Soytone [Teknova, Hollister, CA], 5 g/L Hy-yest [Kerry Bio-Science, Beloit, WI] and 5 g/L sodium chloride [American Bio, Natick, MA]) at 37°C. When needed, agar (Sigma–Aldrich, St. Louis, MO) was added at 15 g/L. For CVD 1926, medium was supplemented with guanine (0.005% weight/volume [w/v] final concentration; Sigma–Aldrich). For S. Typhimurium SL1344, medium was supplemented with 50 μg/mL streptomycin sulfate (Research Products International, Mt. Prospect, IL).
+ Open protocol
+ Expand
Isolates used for whole-genome sequencing are listed in Table S1. Strains and plasmids used in this study are listed in Table S6 in the supplemental material. S. Paratyphi B, S. Paratyphi A, and S. Typhi strains were originally isolated from adults who presented with enteric fever symptoms in Santiago, Chile, in the 1980s. S. Paratyphi B strains were typed as Java or sensu stricto based on their ability to ferment d-tartrate. (Java strains are able to ferment d-tartrate, whereas sensu stricto strains do not.) We also confirmed the presence or absence of an intact d-tartrate fermentation gene by PCR as previously described (35 (link)). Bacteria were grown in animal-product-free Hy-Soy (HS) medium (1% [wt/vol] soytone [Teknova, Hollister, CA], 0.5% [wt/vol] Hy-Yest [Kerry Bioscience, Beloit, WI], 0.5% [wt/vol] NaCl [American Bio, Natick, MA]) at 37°C. Bacterial strains carrying ΔguaBA mutations were grown on media supplemented with 0.005% (wt/vol) guanine.
+ Open protocol
+ Expand
Invasive Salmonella Typhimurium ST313 (D65, Q55, S11) and Salmonella Typhimurium ST19 (I77, I41, S52) have previously been isolated from the blood of infants in Mali, West Africa [14 (link),15 (link),16 (link)]. These clinical strains were maintained on animal product-free Hy-Soy (HS) agar (0.5% Hy-yest [Kerry Biosciences, Beloit, WI], 1% Soytone [TEKNova, Hollister, CA], 0.5% NaCl [American Bio, Natick, MA]).
+ Open protocol
+ Expand
(i) Bacterial motility assay. Bacteria were grown overnight in HS medium supplemented with 20 µg/ml chloramphenicol to maintain complementation plasmids and 0.005% Guanine to supplement ΔguaBA mutants as required. Cultures were stab inoculated onto motility agar (1% [wt/vol] tryptone [Fisher Scientific, Hampton, NH], 0.5% [wt/vol] NaCl [American Bio], 0.4% [wt/vol] bacteriological agar [American Bio]) and incubated at 37°C for 18 h. The diameter of the zone of motility was then measured.
(ii) Guanine auxotrophy. Bacterial lawns were created on chemically defined medium (47 (link)), with or without the addition of 0.005% (wt/vol) Guanine (Sigma-Aldrich, St. Louis, MO). Plates were assessed for growth after incubation at 37°C for 18 h.
+ Open protocol
+ Expand
Bacterial strains used in this study consisted of both prototypic and clinical strains and are listed in Table 4. Bacteria were grown under aerobic conditions at 37°C. S. enterica, E. coli, P. aeruginosa, K. pneumoniae, and C. freundii strains were grown on Hy-Soy medium (0.5% wt/vol Hy-yest [Kerry Biosciences, Beloit, WI], 1% wt/vol Soytone [TEKNova, Hollister, CA], and 0.5% wt/vol NaCl [American Bio, Natick, MA]). E. cloacae and E. faecalis were grown on Bacto Tryptic Soy Agar (BD, Sparks, MD). S. aureus, S. pneumoniae, and N. meningitidis were grown on sheep’s blood agar plates (Tryptic soy agar, 5% vol/vol sheep blood [Waltz Farm, Smithsburg, MD]), with N. meningitidis incubated with 5% CO2. Genomic DNA was isolated using the Bacterial Genomic DNA isolation kit (Norgen Biotek Corp., Thorold, Canada). A. baumannii genomic DNA (gDNA) was a gift from D. Rasko at the Institute for Genome Sciences, University of Maryland, Baltimore. Bile spiking experiments were performed with S. Typhi strains Ty2 and ISP1820.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!