S. pyogenes 10782 zymogen SpeB clone (residues 28–398) was overexpressed and purified as a C-terminal His6-tag fusion from E. coli BL21DE3 (Strategene) in a pET23b vector (Novagen), as previously described.21 (link), 42 (link) Caspase-3 was expressed and purified as previously described.43 Papain was purchased from MP Biomedicals, reconstituted, and purified by size-exclusion chromatography. Human sputum leucocyte elastase was purchased from Elastin Products Company, Inc. and human complement C3 and C3b were purchased from Complement Technologies. Streptococcus pyogenes serotype M1 (strain 5448), WT or ΔspeB, was grown in Todd-Hewitt medium (HiMedia Laboratories) supplemented with 0.2% yeast extract (Difco) (THY media) or in C medium which consists of 0.5% Proteose Peptone #3 (Hardy Diagnostics), 1.5% yeast extract, 10 mM K2HPO4, 0.4 mM MgSO4, 17 mM NaCl, adjusted to pH 7.5.44 (link)
Todd hewitt medium
Manufactured by HiMedia
Sourced in Brazil, India
Todd-Hewitt medium is a microbiological culture medium used for the isolation and cultivation of Streptococcus species. It provides the necessary nutrients and growth factors required for the optimal growth of these bacteria.
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Lab products found in correlation
3 protocols using todd hewitt medium
1
Purification of Streptococcus pyogenes Zymogen SpeB
2
Vaginal and Anorectal Bacterial Sampling
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Clinical specimens from the vaginal distal third were collected by introducing sterile swabs labeled 1, 2, and 3, without using a speculum. Anorectal specimens were collected by introducing sterile swabs labeled 4, 5, and 6 in the anorectal region.
Vaginal swab 1 and anorectal swab 4 specimens were immediately plated on 5% desfibrinated sheep blood agar-SBA (Himedia, Curitiba, Paraná, Brazil) and incubated at 35–37°C for 18–24 h.
Vaginal swab 2 and anorectal swab 5 were each submerged in 2ml of HPTH medium [12 (link)], and 100 μl of sterile defibrinated sheep blood (Laborclin, Pinhais, Paraná, Brazil) was added, followed by incubation at 35–37°C for 18–24 h. Afterward, subculturing was performed on SBA, with incubation at 35–37°C for 24–48 h.
Vaginal swab 3 and anorectal swab 6 were submerged in Todd-Hewitt medium (Himedia, Curitiba, Paraná, Brazil) supplemented with 8 μg/ml gentamicin (Inlab, São Paulo, Brazil) and 15 μg/ml nalidixic acid (Inlab, São Paulo, Brazil) according to the manufacturer's instructions and incubated at 35–37°C for 18–24 h. Afterward, an aliquot of each culture was plated on SBA (Himedia, Curitiba, Paraná, Brazil) and incubated at 35–37°C for 24–48 h.
Vaginal swab 1 and anorectal swab 4 specimens were immediately plated on 5% desfibrinated sheep blood agar-SBA (Himedia, Curitiba, Paraná, Brazil) and incubated at 35–37°C for 18–24 h.
Vaginal swab 2 and anorectal swab 5 were each submerged in 2ml of HPTH medium [12 (link)], and 100 μl of sterile defibrinated sheep blood (Laborclin, Pinhais, Paraná, Brazil) was added, followed by incubation at 35–37°C for 18–24 h. Afterward, subculturing was performed on SBA, with incubation at 35–37°C for 24–48 h.
Vaginal swab 3 and anorectal swab 6 were submerged in Todd-Hewitt medium (Himedia, Curitiba, Paraná, Brazil) supplemented with 8 μg/ml gentamicin (Inlab, São Paulo, Brazil) and 15 μg/ml nalidixic acid (Inlab, São Paulo, Brazil) according to the manufacturer's instructions and incubated at 35–37°C for 18–24 h. Afterward, an aliquot of each culture was plated on SBA (Himedia, Curitiba, Paraná, Brazil) and incubated at 35–37°C for 24–48 h.
3
Isolation and Growth Kinetics of Cariogenic Streptococcus
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Samples were collected from plaque surface of patients with dental caries using sterile toothpick and suspended in 1 ml phosphate buffer saline (PBS) buffer. 100 μl of the sample was spread on the mitis salivarius agar supplemented with 0.5 IU/mL bacitracin (MSB agar) (Sigma, USA) and incubated at microaerophilic condition at 37°C for 48 hours. Streptococcus strains were routinely grown in Todd-Hewitt medium (Hi-Media, India) supplemented with 0.2% yeast extract (THY) at 37°C. For the monitoring of growth, overnight cultures were diluted into fresh medium (1:100), grown to late exponential phase and absorbance was taken at 630 nm at various time intervals. Doubling time was calculated based on two OD values taken from the logarithmic phase of the growth by using the formula, r = ln [OD2/OD1]/(T2-T1) and represents the average value of at least two measurements. We have included first 40 strains (SN1 to SN40) for simplicity.
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