For my experiment, I am trying to observe the growth of two gut microbes against in vitro agar substances which represent dietary fibre (turmeric, golden seal, and citric acid) and fats (palmitic acid, stearic acid, and yeast).
i plan to do this using a spread plate method which would have my substances mixed in agar and the bacterial culture spread on top.
i won't be using different concentrations, I will be using recommended daily intake for each substance.
my problem lies as I want to have a statistical analysis. I am already using CFU technique but I would also like to measure the optical density of my substances for bacteria growth.
How would I go from my various petri dish cultures to a spectrometer to analyze the absorbance?
would I have to perform two tests? using a petri dish for CFU and a liquid culture for optical density?
I think it is going to be tricky to accurately measure the OD of a cell lawn on a Petri dish. Probably much better to elute all the cells from the plate and then measure the OD from the buffer, or do a CFU count on them. It would certainly be much simpler if you were do do the experiment in liquid culture and monitor it that way.
You should also consider whether final OD is relevant or whether time is more relevant, you may have conditions where the cells are growing more slowly but given sufficient time will reach the same final cell number. This might also be simpler to do in liquid. But obviously you need to decide on what is the appropriate experimental conditions for your tests.
Article Estimating microbial population data from optical density
Continuing on Michael J. Benedik answer, simply add 5 ml of saline to the Petri dish, let it stand for 10 minutes, and suspend the bacteria with a cell spreader. Transfer the liquid to a tube/beaker, dilute if needed, and measure O.D. Be warned that this is likely to be a noisy experiment so do many replicates...
I would support opinion of Michael J. Benedik that it might be simpler to do in liquid, and would support Yoram Gerchman - such experiment must be done with many replications.
Generally, it would be a step down from CFU evaluation in reliability. You must study correlation between CFU and optical density for each microbe to confirm that O.D. is reliable measure of bacterial population.
If you simply repeat CFU measurements under different conditions (temperature, pH, etc.) it will be more informative study.
Michael J. Benedik Alex Ignatov Yoram Gerchman Muazzam Sheriff Maqbul
Thank you all so much for your replies, I really appreciate your time and knowledge.
I wanted to measure OD for more quantitative/statistical analysis but I think it may be easier/more reliable to count CFU against time and possibly temperature as Alex Ignatov suggested.
I support all the replies... O.D can only be accurately measured if the cells are suspended in liquid medium... And replicate as much as possible.... All the best
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