For the all the bacterial assays, each strain was cultured in Cation-adjusted Mueller Hinton broth (CAMHB; Bacto Laboratories 212322) at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh CAMHB and incubated at 37 °C for 1.5–3 h. The resultant mid-log phase cultures were diluted with CAMHB (CFU mL–1 measured by OD600), then added to each well of the compound-containing plates (384-well non-binding surface (NBS) plates; Corning CLS3640), giving a cell density of 5 × 105 CFU mL–1 and a total volume of 50 μL. Plates were covered and incubated at 37 °C for 18 h without shaking. Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD600), using media only as negative control and bacteria without inhibitors as positive control. MIC values were determined as the lowest concentration at which the growth was inhibited at ≥80%. Colistin sulfate (Sigma C4461) and vancomycin HCl (Sigma 861987) were used as internal controls on each plate for Gram-negative and Gram-positive bacteria, respectively.
Cls3640
Manufactured by Corning
The CLS3640 is a piece of lab equipment manufactured by Corning. It is designed for general laboratory use, but its core function is not specified in the given information. A more detailed and unbiased description cannot be provided without making assumptions or interpretations.
Automatically generated - may contain errors
5 protocols using cls3640
1
Antimicrobial Susceptibility Assay Protocol
2
Fungal Growth Inhibition Assay
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For the fungal assays, both fungi (yeast) strains were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD; Becton Dickinson 242720) agar at 30 °C. A yeast suspension of 1 × 106 to 5 × 106 CFU mL–1 (as determined by OD530) was prepared from five colonies from the agar plates, and subsequently diluted with Yeast Nitrogen Base media (YNB; Becton Dickinson 233520), and added to each well of the compound-containing plates (384-well plates, NBS; Corning CLS3640) giving a final cell density of 2.5 × 103 CFU mL–1 and a total volume of 50 μL. Plates were covered and incubated at 35 °C for 36 h without shaking. Growth inhibition of C. albicans was determined by measuring absorbance at 630 nm (OD630), while the growth inhibition of C. neoformans was determined by measuring the difference in absorbance between 600 and 570 nm (OD600–570), after the addition of resazurin (0.001% final concentration; Sigma R7017) and incubation at 35 °C for 2 h, using media-only as negative control and fungi without inhibitors as positive control. MIC values were determined as the lowest concentration at which the growth was inhibited at ≥80%. Fluconazole (Sigma F8929) was used as internal control on each plate for both strains.
3
Fungal Growth Inhibition Assay
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For the fungal assays, yeast strains C. neoformans var. grubii H99 ATCC 208821 and C. albicans ATCC 90028 were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD; Becton Dickinson 242720) agar at 30 °C. A yeast suspension of 1 × 106 to 5 × 106 CFU/mL (as determined by OD530) was prepared from five colonies form the agar plates, and subsequently diluted with Yeast Nitrogen Base media (YNB; Becton Dickinson 233520), and added to each well of the compound containing plates (384-well plates, NBS; Corning CLS3640) giving a final cell density of 2.5 × 103 CFU/mL and a total volume of 50 μL. Plates were covered and incubated at 35 °C for 36 h without shaking. Growth inhibition of C. albicans was determined measuring absorbance at 630 nm (OD630), while the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD600-570), after the addition of resazurin (0.001% final concentration; Sigma R7017) and incubation at 35 °C for 2 h, using media only as negative control and fungi without inhibitors as positive control. MIC values were determined as the lowest concentration at which the growth was inhibited at ≥ 80% (equivalent to no visible growth by eye). Fluconazole (Sigma Aldrich, F8929) was used as a positive inhibitor control on each plate for both strains.
4
Antimicrobial Susceptibility Assay for Bacterial Strains
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For the bacterial assays, each bacterial strain was cultured in Cation-Adjusted Mueller Hinton Broth (CAMHB; Bacto Laboratories 212322) at 37 °C overnight. Bacterial strains tested were methicillin resistant S. aureus (methicillin-resistant Staphylococcus aureus - MRSA) ATCC 43300, E. coli ATCC 25922, K. pneumoniae K6/ESBL ATCC 700603, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606. A sample of each culture was then diluted 40-fold in fresh CAMHB and incubated at 37 °C for 1.5–3 h. The resultant mid-log phase cultures were diluted with CAMHB (CFU/mL measured by OD600), then added to each well of the compound-containing plates (384-well non-binding surface (NBS) plates; Corning CLS3640), giving a final cell density of 5 × 105 CFU/mL and a total volume of 50 μL. Plates were covered and incubated at 37 °C for 18 h without shaking. Inhibition of bacterial growth was determined by measuring absorbance at 600 nm (OD600), using media only as negative control and bacteria without inhibitors as positive control. MIC values were determined as the lowest concentration at which the growth was inhibited by ≥ 80% (equivalent to no visible growth by eye). Colistin sulfate (Sigma Aldrich, Castle Hill, Australia C4461) and vancomycin HCl (Sigma Aldrich, 861987) were used as positive inhibitor controls on each plate for Gram-negative and Gram-positive bacteria, respectively.
5
Bacterial Growth Inhibition Assay
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For the all the bacterial assays,
each bacterial strain was cultured in cation-adjusted Mueller Hinton
broth (CAMHB; Bacto Laboratories 212322) at 37 °C overnight.
A sample of each culture was then diluted 40-fold in fresh CAMHB and
incubated at 37 °C for 1.5–3 h. The resultant mid-log
phase cultures were diluted with CAMHB (CFU/mL measured by OD600) and then added to each well of the compound-containing
plates (384-well non-binding surface (NBS) plates; Corning CLS3640),
giving a cell density of 5 × 105 CFU/mL and a total
volume of 50 μL. Plates were covered and incubated at 37 °C
for 18 h without shaking. Inhibition of bacterial growth was determined
by measuring the absorbance at 600 nm (OD600) using media
only as negative control and bacteria without inhibitors as positive
control. MIC values were determined as the lowest concentration at
which the growth was inhibited by ≥80% (equivalent to no visible
growth by the eye). Colistin sulfate (Sigma C4461) and vancomycin
HCl (Sigma 861987) were used as internal controls on each plate for
Gram-negative and Gram-positive bacteria, respectively. All compounds
were tested as two technical replicates in two independent biological
assays, n = 4 final data.
each bacterial strain was cultured in cation-adjusted Mueller Hinton
broth (CAMHB; Bacto Laboratories 212322) at 37 °C overnight.
A sample of each culture was then diluted 40-fold in fresh CAMHB and
incubated at 37 °C for 1.5–3 h. The resultant mid-log
phase cultures were diluted with CAMHB (CFU/mL measured by OD600) and then added to each well of the compound-containing
plates (384-well non-binding surface (NBS) plates; Corning CLS3640),
giving a cell density of 5 × 105 CFU/mL and a total
volume of 50 μL. Plates were covered and incubated at 37 °C
for 18 h without shaking. Inhibition of bacterial growth was determined
by measuring the absorbance at 600 nm (OD600) using media
only as negative control and bacteria without inhibitors as positive
control. MIC values were determined as the lowest concentration at
which the growth was inhibited by ≥80% (equivalent to no visible
growth by the eye). Colistin sulfate (Sigma C4461) and vancomycin
HCl (Sigma 861987) were used as internal controls on each plate for
Gram-negative and Gram-positive bacteria, respectively. All compounds
were tested as two technical replicates in two independent biological
assays, n = 4 final data.
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