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Prism version 4.0b

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Prism version 4.0b is a data analysis and graphing software. It provides tools for data visualization, curve fitting, and statistical analysis.

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Lab products found in correlation

F96 microwell plate, by Thermo Fisher Scientific (1 mentions) Faststart universal sybr green master mix rox, by Roche (1 mentions) Analar normapur, by Avantor (1 mentions) 2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)

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Prism 4.0b Prism version 4.0 GraphPad Prism version Prism 4

4 protocols using prism version 4.0b

1

Pneumococcal Growth Curves Comparison

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Pneumococcal growth curves were performed in BHI broth comparing the growth of TIGR4, TIGR4 P2 and Xen35. 1x106 CFU of S. pneumoniae were resuspended in 200μl BHI in a 96 well plate. Absorbance (600nm) readings were taken every 20 minutes for 10 hours using a a FLUOstar OPTIMA (BMG) plate reader. Triplicate readings were taken and assays performed in triplicate. Graphical presentation was performed in Prism version 4.0b (GraphPad Software).
Herbert J.A., Mitchell A.M., Ritchie R., Ma J., Ross-Hutchinson K, & Mitchell T.J. (2018). Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence. PLoS ONE, 13(1), e0189426.
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2

Pneumococcal Growth Dynamics Assay

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1x106 CFU of S. pneumoniae were added in 20μl BHI to a black F96 MicroWellTM plate (Nunc, Fisher Scientific, UK) containing 180μl BHI, each strain was tested in triplicate. Luminescence was measured on a FLUOstar OPTIMA (BMG) plate reader taking readings every 20 minutes for 10 hours. Graphical presentation was performed in Prism version 4.0b (GraphPad Software), each data point representing the mean of the triplicate luminescence reading minus background.
Herbert J.A., Mitchell A.M., Ritchie R., Ma J., Ross-Hutchinson K, & Mitchell T.J. (2018). Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence. PLoS ONE, 13(1), e0189426.
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3

RT-PCR Analysis of Gene Expression

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cDNA synthesis and RT-PCR was performed as described in [16 (link)]. The primers used for RT-PCR analysis are listed in S5 Table. Real-time PCR was performed using FastStart Universal SYBR green master mix (ROX) (Roche) as per the manufacturers instructions. gyrA was used as an internal control to normalise for cDNA synthesis variations. Analysis was performed in Opticom Monitor™ version 3.1. Background was subtracted using No-Reverse Transcriptase controls and replicates grouped together with at least two replicates used for analysis. Data was analysed using the 2-ΔΔCT method [18 (link)], graphical data representation was performed in Prism version 4.0b (GraphPad Software), each bar representing the sample replica means ± standard deviation.
Herbert J.A., Mitchell A.M., Ritchie R., Ma J., Ross-Hutchinson K, & Mitchell T.J. (2018). Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence. PLoS ONE, 13(1), e0189426.
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4

Hydrogen Peroxide Production Assay

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Hydrogen peroxide assay was performed to assess the ability of the different strains to produce hydrogen peroxide. S.pneumoniae cultures were grown to OD600nm 0.6 in BHI, 180μl of culture were added in triplicate to a 96 well flat bottom plate. To this 20μl of 3mg/ml ABTS (2,2 azinobis (3-ethylbenzthiazolinesulfonic acid) diammonium salt, Sigma-Aldrich, UK), 0.2mg/ml HRP (Peroxidase from horseradish Type II, Sigma-Aldrich, UK) in a 0.1M sodium phosphate buffer pH7.0 was added. The assay was left to develop for 30 minutes and 24 hours at 37°C followed by centrifugation at 1500g for 3 minutes. 100μl from each well was removed and placed into a fresh 96 well plate. Absorbance was measured using a spectrophotometer at 540nm (FLUOstar OPTIMA, BMG). Hydrogen peroxide standards were used as a positive control by making serial dilutions of Hydrogen peroxide 30% w/v AnalaR NORMAPUR (VWR, UK) in BHI. Graphical presentation of hydrogen peroxide assay data was performed in Prism version 4.0b (GraphPad Software). Data presented was the absorbance at each time point from each strain tested. No statistical analysis was performed.
Herbert J.A., Mitchell A.M., Ritchie R., Ma J., Ross-Hutchinson K, & Mitchell T.J. (2018). Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence. PLoS ONE, 13(1), e0189426.
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