as far as i know you can multiply the result of cfu/ml by 3.

i am not sure but mathematically may be right, still waiting answers from other researchers.

Carole C Tranchant Thank you it seems so helpful

Shadman Sadiq I didn't get your answer, why multiple and why by 3?

Hi Ayda Khosravanihaghighi,

I am not sure to understand what you want to measure... Do you want to measure the amount of CFU of S. aureus that have attached to your 1*1*0.05cm sample? If that is the case, it seems to me that what was attached to your sample will be in the suspension from which you will do the CFU and thus the CFU/mL you get is the amount of CFU per the total surface of your sample (sum of the areas of each side of your sample). I don't know if it makes sense or if I understood your question completely wrongly.

Best,

Fabio

Fabio Pamieri Yes you are totally right so you mean CFU/ml can be reported as CFU/Cm2 cause they are all the bacteria attached to the samples beforehand.

Ayda Khosravanihaghighi, yes that is exactly what I mean. After having washed your sample in PBS or whatever solution you will use to count the CFU, it will give you the total CFU you have in the volume from which you do the counting. Then, since the bacteria would have been attached to the surface of your sample, you can report the count you had by cm2 (total surface of your sample).

Fabio Pamieri thank you.

@fabio pamieri There is one more point: we didn‘t pour all the bacteria on the nutient agar so how can we say all the counting is the same number which had been attached to the substrate beforehand. For the nutrien agar is 20micro litre per each aliquot. we put 2ml in the container containing sample and PBS but, we just took 100 micro litre to start the serial dilution and then 3 aliquot (each 20 micro liter) for each dilution.

I am not sure if I totally follow this, but my first question is why does the 'thickness' matter? Are there bacteria only on the surface of your sample or do they also exist down inside the thickness layer? If it is only a sample from the surface you would use number / cm square when you calculate out the CFU's from your serial dilution numbers.

Fabio Pamieri There is one more point: we didn‘t pour all the bacteria on the nutient agar so how can we say all the counting is the same number which had been attached to the substrate beforehand. For the nutrien agar is 20micro litre per each aliquot. we put 2ml in the container containing sample and PBS but, we just took 100 micro litre to start the serial dilution and then 3 aliquot (each 20 micro liter) for each dilution.

Robert John Wolff There has to be some bacteria on the sides of the sample as well. because the samples are quiet rough. But the number that I read from the nutrient agar is just from small amount of bacteria containing solution which the samples are in. Because after incubation of the samples in the bacteria solution in 6 well plates, I transferred each in to a container with the magnetic stirrer and 2 ml PBS and after being vortex, all the attached bacteria on the samples, would be released in the PBS, at this point we just take 20 micro litters in triplicate from this PBS+Bacteria from the samples on to the nutrient agar. So how can we say these three calculated area should be multiply by 3 and will give us the amount of the Bacteria attached on the sample beforehand?!

I am saying that there have to be much more bacteria left in the PBS+bacteria container, so this number would not be accurate for the CFU/CM2.

Ayda Khosravanihaghighi, the volume doesn't matter as you will take it into account in your calculations, and you assume you have an even suspension of your bacteria. Does it make sense? For the 20uL from the CFU counting, you get back to 100uL and then you get back to your 2mL, in which you soaked your sample, by multiplying by the inverse of the 10-fold dilutions.

I think the easiest way to calculate CFU/cm2 is by multiplying the number of discrete colonies by the dilution factor. Dilution factor is the inverse of serial dilution exponent used.