Five oral pathogenic bacterial species were used for this study. Gram-negative strains: Fusobacterium nucleatum (ATCC 10953), Aggregatibacter actinomycetemcomitans (NCTC 9710/DSM 8324) and Porphyromonas gingivalis (ATCC 33277). Gram-positive strains: Streptococcus mutans (ATCC 25175) and Enterococcus faecalis (DSMZ 20376). The bacterial strains were cultivated for 24 h at 37 °C under appropriate (aerobic/anaerobic) conditions in nutrient solution (SCHAEDLER-Broth; Oxoid, Germany) enriched with vitamin K (Roche, Germany). In the next step, the suspensions of bacteria in the logarithmic growth phase were centrifuged (4000 rpm, 5 min/Sigma, Eppendorf, Germany), rinsed twice using PBS (phosphate buffered saline/GIBCO, Germany) and resuspended to obtain a bacterial density of 108 bacteria/mL, that equals an optical density of 0.1 at 640 nm.
Schaedler broth
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom, United States
Schaedler broth is a microbiological culture medium used for the growth and isolation of anaerobic bacteria. It provides the necessary nutrients and growth factors for the cultivation of a wide range of anaerobic microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Tryptic soy broth (tsb), by bioMérieux (1 mentions)
Horse serum, by Merck Group (1 mentions)
Glucose, by Carl Roth (1 mentions)
Vitamin k, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by Carl Roth (1 mentions)
Modified mycoplasma broth, by BD (1 mentions)
4 protocols using schaedler broth
1
Pathogenic Bacterial Species Cultivation
2
Streptococcus agalactiae and Lactobacilli Characterization
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In this study, 7 strains of Streptococcus agalactiae were used, including ATCC 27956 and 6 clinical strains, namely 1030/06, 1029/06, 2306/06, 1153/07, 2974/07 and 3301/08, representing serotypes IA, IB, III, IV, V and IX, respectively. Izabela Sitkiewicz, Translational Medicine Center, Warsaw University of Life Sciences, Warsaw, Poland kindly provided the clinical strains. Additionally, 3 Lactobacillus species were included in the analysis, specifically L. gasseri LMG13134, L. crispatus LMG 12005 and L. jensenii LMG 06414, which were purchased from BCCM/LMG. Columbia blood agar plates (GRASO, Starogard Gdanski, Poland) were used for colony-forming unit (CFU) determination. Tryptic soy broth (TSB) (Biomerieux, Craponne, France) was used for overnight planktonic culture of S. agalactiae and Schaedler broth (Oxoid, Basingstoke, UK) with a 10% addition of horse serum (Sigma Aldrich, Saint Louis, MO, USA) for Lactobacilli.
3
Bacterial Attachment and Biofilm Formation on Nanotopographies
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To analyze bacterial attachment and biofilm formation on nanotopographies,
three individual precultures were centrifuged and resuspended in phosphate-buffered
saline. They were adjusted to an optical density at 600 nm of 0.001
or 0.2 for S. aureus or A. ac, respectively. Test specimens (N = 18 per structure and strain) were incubated with bacterial suspensions
using each preculture in triplicates for 5 h at 37 °C and with
continuous shaking under aerobic (in the case of S.
aureus) or microaerophilic (5% CO2, in
the case of A. ac) conditions. After
this initial attachment, half of the specimens (N = 9 per structure and strain) were processed for microscopy as described
below.
On the other specimens, the bacterial suspension was
removed and replaced with fresh medium: tryptone soy broth supplemented
with 10% yeast extract and 50 mM glucose (Carl Roth GmbH & Co.
KG) for S. aureus or Schaedler broth
(Oxoid Limited) supplemented with 10 μg/mL vitamin K (Oxoid
Limited) for A. ac. To allow for
biofilm formation of the adhered cells, specimens were further incubated
for a total of 24 h at 37 °C under aerobic conditions and continuous
shaking in the case of S. aureus and
under static microaerophilic conditions (5% CO2) in the
case of A. ac.
three individual precultures were centrifuged and resuspended in phosphate-buffered
saline. They were adjusted to an optical density at 600 nm of 0.001
or 0.2 for S. aureus or A. ac, respectively. Test specimens (N = 18 per structure and strain) were incubated with bacterial suspensions
using each preculture in triplicates for 5 h at 37 °C and with
continuous shaking under aerobic (in the case of S.
aureus) or microaerophilic (5% CO2, in
the case of A. ac) conditions. After
this initial attachment, half of the specimens (N = 9 per structure and strain) were processed for microscopy as described
below.
On the other specimens, the bacterial suspension was
removed and replaced with fresh medium: tryptone soy broth supplemented
with 10% yeast extract and 50 mM glucose (Carl Roth GmbH & Co.
KG) for S. aureus or Schaedler broth
(Oxoid Limited) supplemented with 10 μg/mL vitamin K (Oxoid
Limited) for A. ac. To allow for
biofilm formation of the adhered cells, specimens were further incubated
for a total of 24 h at 37 °C under aerobic conditions and continuous
shaking in the case of S. aureus and
under static microaerophilic conditions (5% CO2) in the
case of A. ac.
4
Anaerobic Bacterial Culture Preparation
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Bacterial cultures of P. gingivalis, T. denticola and T. forsythia were grown in Schaedler broth (Oxoid Basingstoke, GB) supplemented with 0.25 mg·L−1 of vitamin K (and 10 mg of N-acetyl muramic acid for T. forsythia) and modified mycoplasma broth (BD, Franklin Lake, NJ, USA) supplemented with 1 mg·mL−1 of glucose, 400 mg· mL−1 of niacinamide, 150 mg·mL−1 of spermine tetrahydrochloride, 20 mg·mL−1 of Na isobutyrate enriched with 1 g·mL−1 of cysteine and 5 mg·mL−1of cocarboxylase. Forty-eight-hour broth cultures of the bacteria were centrifuged. The resulting supernatants were filtered through 400-µm membranes, and the sediments were exposed to intensive ultrasonication for 10 min.
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