The best way would be to perform a growth curve of each organism and for each time point do a cell count (for colony forming units) so that you can determine cell number per OD. Ideally this should be done for each of them, although if the organisms are genetically similar it is likely the curves will be the same. You can do this ahead of time and only need to do it once. Once you have created the growth curve (it is important to use the same conditions as you would use to prepare the starter cultures for your experiment) you can measure the OD of each culture and determine what volume to add in order to prepare the proper ratios of your two cell types.

Dear Thulasidharan,

what microorganisms do you use, do you inoculate them at the same time or successively in few days? What individual (and complementary) features have these two microorganisms useful for their mixed cultivation?

This question boils down to how to quickly determine the concentration of a microorganism solution. Once you know the concentration of microorganism in the two cultures, it's a simple calculation step to find the volumes needed to make the require mixture.

The best method should be directly counting the number of microorganism in each culture using microscope and a hemocytometer. If you are counting bacteria you will need to dye them first with methylene blue or some type of dye in order to see them. If it's yeast you can count them directly without staining.

Another indirect method is to measure OD of the solution. Make several solutions of each microorganism in PBS or physiological saline (0.9% NaCl), then use those solutions to measure OD and do plate count at the same time. From that you can make a standard curve of OD vs number of microorganisms. Then when you do experiment, take an exact volume of your culture, centrifuge and resuspend the microorganism in PBS or physiological saline (depend on which one you used last time) and measure OD. Compare it to your standard curve and you can find out the concentration of microorganisms in your cultures.

Calculating the concentration of your cultures by growth curve is another indirect method which I do not recommend because it is very inaccurate and have large error. Basically you do a new culture of your microorganism. At interval (~ every 6-12 hours) you take a sample from the culture and do plate count to see how many microorganism are there. From those data you can make a growth curve of the concentration of microorganism vs time. Then make new cultures and when you do experiment, note the age of that culture when you use it (e.g. 20 h since starting that culture) and using the growth curve you can find out the concentration of microorganism in your culture at that time. Needless to say the culture you make for experiment has to be in exact condition as the culture you make for the growth curve in order for this method to be accurate. Everything from starting number of microorganisms, their condition (age, growth stage, etc.), the volume of culture, temperature, humidity, pH, nutrient concentration, etc. has to be exactly the same, which is very difficult to control, especially for the condition of the microorganisms. If you use slightly older microorganisms to make the new culture, you can find that the growth curve diverses quite a lot.