The data from any experiment are no better than the design and execution of that experiment. This is particularly true when the data are obtained automatically from a machine such as a plate reader. Some key elements of growth experiment design are discussed below.
Pay attention to the environmental conditions
Most plate readers can control the temperature and the shaking of the plates during the experiment.
Set a temperature slightly above ambient room temperature. NEVER just run the experiment at room temperature. Room temperature can vary with the season, time of day, and even the number of occupants of the lab. Always control the temperature.
Shaking serves to aerate the culture and to maintain cells in uniform suspension. Set a shaking speed that is high enough to provide good aeration but low enough to ensure no splashing over between wells.
Always use the same volume in every well
The volume must be consistent from well to well. The OD reading depends critically on the volume. The same culture density will give a higher reading with a higher volume. This requires care and attention during the process of filling the wells.
- Avoid very low volumes because slight evaporation affects low volumes more than it does high volumes.
- Avoid very high volumes to avoid contamination between wells during shaking. Variation in volume from well to well will result in unreliable and uninterpretable results.
Know the background OD
The growth rate calculations require correcting readings for background. Background is the OD attributable to the uninoculated culture medium. You can either determine the mean background from several uninoculated wells before the experiment, or you can leave one well uninoculated so that GrowthRates can use that well to automatically correct for background.
Inoculate the cultures at an appropriate initial OD
If the initial OD is too low, too close to the background OD, the accuracy of reading the OD attributable to the cells will be poor. The initial OD must be at least 0.01 above the background OD. Lower ODs result in considerable scatter.
Read ODs at appropriate time intervals
Ideally the culture density should be determined between 3 and 12 times during each doubling. If reading intervals are too long there will not be a sufficient number of points during the exponential growth phase to get a reliable estimate of growth rate.
Surprisingly, reading too frequently can also be a problem. If reading intervals are so low that there is very little growth, i.e. change in culture density, between readings the effect is much the same as having too low an initial density: lots of scatter and unreliable estimates of growth rate.
The program EditReadingIntervals solves the problem of having set the reading interval too low and does not require repeating the experiment. EditReadingIntervals simply rewrites the input file retaining, for instance, only every third reading. The effect is exactly as though you read 1/3 as frequently.
When growth rates vary a lot among wells it can be difficult to pick an appropriate reading interval. You should always set the reading interval to be appropriate for the fastest growing culture. You can then use EditReadingIntervals to rewrite the input file to a reading interval suitable for the slowest growing cultures.