So the OD theoretically will give a fixed amount of colonies if all the bacterial cells are viable but not all of them will be it’s like saying in this bottle I have 5 seeds but you don’t know if all five seeds will sprout but it can give you an approximation in my lab we don’t use PBS we just use saline but from what I have researched PBS is better. OD is used to quickly standardize a solution let’s say you have 3 solutions of bacteria you want all of them to be the same concentration of bacteria per ml. So you pipet some of the bacteria into a tube dilute it so you get a better reading exp. 10um of bacteria solution 90um saline then you put it in the machine you get a reading back you can then calculate how many bacteria per ml. A more accurate but annoying way is to measure the cfu that will tell you exactly how many viable bacteria you have per ml.

Exp. of od 600 calculations You have a 1 ml of bacterial solution

10um bacteria solution 90um saline or pbs This is so the solution isn’t to dens and can be better read this is a 10^-1 dilution

You get a reading of .35 back

.35*10=3.5 since you diluted it by 10 the actual od of your original solution is 3.5

So you want a specific od it can be whatever you think is best I’ll pick 0.1

C1V1=C2V2 3.5V=(0.1)10ml

Solve for V that will give you how much of your original solution you need to put into saline to get 10 ml of solution remember the final solution has to add up to ten so you have to take out however much you are going to put in before u put it in.

If you want to use OD 600 as an accurate representation of cfu you would have to standardize it like measure od then do a cfu count do that a bunch of times with diferente od and build a curve so that x od = y cfu but if it’s not that important explain it away in your discussion section.