@eljonco and @Stoyan_Pavlov raise very good points. It also illustrates why analysing agar plates like this may not necessarily be the best practice for accurate quantification. This especially applies if your agar plates are over confluent (i.e., multi layered). I think that is perfectly fine to use the extent of area covered by the bacteria as a measure of bacterial growth for initial assessment.
A better experimental design could have been to make a standard curve of some sorts and see if there is any linearity:
- Perform serial dilutions
- Make pour plates with each serial dilution
- Perform colony counts using the normal technique (counting colonies from the plate where colonies are distinguishable) and establishing what the bacterial count/mL is for the original sample (background
- Perform ImageJ analysis on the agar plates and get area fraction.
- Plot area fraction against CFU/mL for each serial dilution, and check for correlation.
You may have to do this for 24 hour and 48 hour as well. This will establish
- if there is any linearity?
- and if so, which dilution ranges have the best linearity with CFU/mL
Again, am sure there are some issues with this method, but making sure the plates are not overconfluent is a good way to negate the problem with multilayered growth. Again, it depends on bacterial strain I think…
As this is a assignment, I’m not sure how much more you are supposed to do, but use the points raised by the others as criticisms of your method. To account for some modicum of error, you could express your treated plates as percentage of control…
I think doing this in liquid media and measuring OD is the best way for accurate quantification.