How should I choose a microplate reader?
For the purposes of high-throughput growth rate experiments you do not need a particularly sophisticated plate reader, a single-mode plate reader is quite sufficient. The primary requirements are:
- It must be an absorbance plate reader.
- Temperature control. You should be able to set a temperature at which the plate will be maintained. It is essential that you control temperature. Growth experiments should never be done at “room temperature” because room temperature can vary with the time of day, season, which equipment is running and even the number of occupants of the lab.
- Shaking. The plate reader should be able to shake the plate between readings in order to provide sufficient aeration of the culture and in order to keep the cells in suspension. You should be able to control the shaking speed.
- Wavelength control. You should be able to control the wavelength at which the spectrophotometer reads the OD. Most plate readers use filters for that purpose. Be sure your reader includes a filter for 600 nanometers, or that one is available.
- You must be able to set the reading interval.
- The reader should write an output file either as a tab-delimited text file or as an Excel file.
Used microplate readers are available on eBay at prices ranging from under $1000 to over $50,000 or from used scientific equipment dealers such as LabEx https://www.labx.com/microplate-readers or BioDirect https://www.biodirectusa.com/microplate-readers/ at similar prices. As with automobiles, one must be cautious when buying used equipment, but excessive cost should not be a barrier to most labs when it comes to doing high-throughput microbial growth experiments.
What does “IOError: [Errno 2] No such file or directory: ‘BioTek'” mean?
If , for instance, you specified the input file as “Biotek”, and there is no file named Biotek in the current working directory you will get Errno 2. Either you forgot to navigate to the folder containing that file (see Command-line Programs.pdf), or you mistyped the file name. Perhaps the File was named “Biotek Exp1.txt”. File names may not contain any spaces. After the -i option the command line sees the next word as the name of the input file. You will need to change the name of that file to ” BiotekExp1″ or to ” Biotek_Exp1″ and use that name on the command line.
On which platforms has GrowthRates been tested?
Macintosh OS X version 10.14.5, Ubuntu Linux 16.04, and Windows 10. There is no reason to expect problems with different versions of those operating systems, but if problems appear on later versions please let us know at email@example.com.
GrowthRates doesn’t read my data file or wells file correctly and it gives me weird error messages. What can I do?
If you think your data file is in standard format, be sure the format conforms exactly to the format shown in Figure 3 of the GrowthRates User Guide and that there is no extraneous information before or after the data.
If you chose a formatter from the menu (Figure 4 of the GrowthRates User Guide) the resulting .in file should be fine, but if in doubt compare it with Figure 3. Some plate readers label the wells as 101, 102, etc. instead of A1, A2, etc. That is not a problem.
Is there no blank line or more than one blank line at the end of the input file? You need exactly one blank line.
How can I easily calculate the well number from the well ID?
Suppose that I want to use well C6 as my blank. How can I determine the well number to enter with the -w option on the command line?
The well ID consists of two parts, the letter and the number.
The index of the letter is its position in the alphabet minus 1, so the index of C is 2.
If you are using a 96 well plate, which has 12 wells per row, the well number is: (index x 12) + number. Thus for C6 the equation is (2 x 12) + 6 = 30.
For well G11 the well number is (6×12) + 11 = 83.
The table below shows the index for each letter used in microtitre plates.
384 well plates have 24 wells per row, so for 384 well plates well number =(index x24) + number
Well C6 = 54. Well M20 = 308.
What if you have more than one blank well?
GrowthRates uses one blank well to correct for background OD, but what if your experimental design uses more than one blank well? Suppose that well A1 through D11 have cells growing in mineral salts medium and well D12 is the mineral salts blank, and that wells E1 through H11 have cells growing in broth medium with H12 being the broth blank. Which to use as the blank?
The answer is to do the GrowthRates analysis twice. The first time use the mineral salts blank. If the input file in myfile.txt the command line for the first analysis would be GrowthRates -i myfile.txt -w 48. Well 48 is D12. The output files will be myfile.report and myfile.summary. So that the second analysis does not overwrite the first change those output file names to something else, such as myfileA.report and myfileA.summary.
Then run GrowthRates again with the command line GrowthRates -i myfile.txt -w 96. Well 96 is H12. Rename the resulting output files to myfileB.report and myfileB.summary.
Use the “A” output file for the growth rates in mineral salts medium and the “B” reports for the growth rates in broth.
This approach can be extended to as many different blanks as are used, doing one GrowthRates analysis for each blank.