Growth Rates using "R studio" - Overview

Determining growth rates for many cultures or from plate reader data can be a daunting task. To automate this process, we've found that the Grofit package for R can be very useful and reliable.

Download and install "R" and "R studio"

You'll need to download and install R on your computer. Once R is installed, you can then download and install "R studio" which is easier to work with.

You can download "R" here: https://cran.r-project.org/mirrors.html
You can download "R studio" here: https://www.rstudio.com/

Growing cells in plate reader and measuring OD600

All strains tested should at least have 3 replicates. Due to small variations in temperature throughout a 96-well plate, best results will be obtained if replicates are randomly located at different wells. Set up 200µL cultures with 2µL from a pre-conditioned (same media as actual test) overnight culture. These 100-fold dilutions should be good enough for OD600 measurements that will give out reliable information for lag phase, exponential phase and stationary phase. To allow cultures to aerate properly, set the plate reader to shake (1mm radious) at least every 5 minutes; normally this would be before and after taking reads.

Converting your file with OD data to the proper format

What is most critical is the format and file type of the input data file containing the OD600 values. Most plate readers produce an Excel worksheet with the data results. With such a file, the first thing that must be done is to convert it to a comma delimited file. This is simply done by using "save as" in Excel and selecting *.csv as the file format.

Create a "times only" csv file

Grofit needs an additional file that will only have the times (e.g., 10, 20, 30 mins ...) for every time point measurement. An example of this file is given here:

Calculating Mutation Rates using Grofit R package

As a reference, the following is a short script on "R studio" that performs the downloading, installing, running and printing out results. You execute each line in the script by pressing Ctrl+Enter. For each one, you must check no errors are given.

setwd("C:/.../R_GrowthData") #sets the working directory to the folder (here named R_GrowthData) you'll be working on your computer
install.packages(c("grofit", "tidyr", "reshape2"))
library(grofit)
library(tidyr)
library(reshape2)

growthdata <- read.csv("yourdatafile.csv",sep=",", header=TRUE, check.names = FALSE)
timedata <- read.csv("timesonlyworksheet.csv", sep=",", header=TRUE, check.names = FALSE)
gro <- grofit(timedata, growthdata) #runs grofit

summary_table<-summary.gcFit(gro$gcFit) #makes a summary table
write.csv(summary_table,"FinalTable.csv") #makes a csv file of the summary table

Accepting/declining model fits:

Once Grofit is run, you will be prompted to answer yes (y) or no (n) to accept or decline the model fits generated for each individual well. Most likely, all you need to do is say yes to all of them, unless there is some obvious model fit mistake which would make some sample unreliable.

Reading summary table results:

The table summary file produced by Grofit will give you all the parameter values (mu=GrowthRates, lambda=LagPhaseTime and A=MaxAbsorbance) generated from the best model fits. Grofit uses 4 possible models; see documentation for more information.

References

1. https://www.jstatsoft.org/article/view/v033i07
2. https://cran.r-project.org/web/packages/grofit/index.html

-- Main.GabrielSuarez - 15 Dec 2016