Hi:

Your calculation is right.  Don't be afraid; Go ahead sir.

Correct! Have fun counting ;) To achieve more accurate values you should plate different dilutions. (in triplicates optimally) It also helps you to avoid plates with no colonies or no single colonies at all. 

Hi! Check the links given below you get useful information.

Colony-forming unit

https://en.wikipedia.org/wiki/Colony-forming_unit

BAM: Aerobic Plate Count

http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063346.htm

COUNTING BACTERIA

http://wiki.hackuarium.ch/images/0/09/Bacterial_counts.pdf

earlier somebody asked the same question check the following link

https://www.researchgate.net/post/How_can_I_calculate_colony_forming_unit_cfu_for_bacteria

In theory, if you dropped the 30uL onto an agar medium and it was from a pure culture and the agar used was conducive to the growth of your culture.  If you used a spreaders pl refer to J Appl Microbiol. 2012 Aug;113(2):339-50. doi: 10.1111/j.1365-2672.2012.05327.x. Epub 2012 May 25.

Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU.

Thomas P1, Sekhar AC, Mujawar MM.

Thank you to all for your answers

Hi:

Your calculation is right.  Don't be afraid; Go ahead sir.

Dear Paul Rios,

You are absolutely correct. I think you should increase the dilution factor for better and accurate result.

Hi Paul, I am just adding some more hints and tips to make it enlightened further and more meaningful for the scenario of CFU counting. CFU countable range is 30-300 colonies and this range indeed is offering a statistical accuracy too. If you get more than 300 colonies then obviously it’s difficult to count so you need to increase your serial dilution range and colonies less than 30 you are in an urgent need to decrease your dilutions no doubt and this low number may also give statistical errors in calculations later on.

CFU formula can be simplified as "number of colonies x volume factor (not the volume) x dilution factor (not the dilution itself)".  Also you can use this alternative formula to count CFU; “number of colonies / volume plated (in ml) X dilution factor (DF)”.

So here let’s say if I have 50 colonies after inoculating 75uL culture from a 10-2 dilution so CFU / ml by the first formula would be; 50 x (1000/75) x 102 (DF)= 666.6 x 102= 6.6 x 104 CFU / ml.  The same case by using second formula would be as; 50/0.075 ml (which is 75 uL) x 102= 66666.6 x 102= 6.6 x 104 CFU / ml.

We your prepared culture broth is in your hands, you can have the idea of serial dilutions you may need by viewing at its turbidity or even looking at photometric values. Usually a turbid broth has cells above 107 in general. And for CFU rule of thumb is; 1 O.D. = 1 x 109 CFU / ml for bacteria. So at least, you can have some ideas by reviewing these values before start.

So if you have a certain CFU value for your culture and you want a certain number of colonies only from a proper dilution range which you have no idea so far, then you need to calculate the TDF (total dilution factor) by using this formula;

TDF = number of colonies required / ml of culture plated and then divide this whole value with the CFU / ml which you have, so ultimately the required dilution of your culture, which is needed to get this particular count will be in your hands. So whatever amount of culture you want to plate, just convert it in mL first (if it is in uL) and feed in the above formula.

Also in CFU calculations the volumetric size of inoculating loop matters (in the case if you are using loops for plate inoculation) like are you using a 10 uL loop or 1 uL loop? For a certain culture, colonial ratio should be the same ideally and logically of getting the same strength of colonies by using either of the loops. For example if a 10 uL loop is giving you 80 colonies so from the same culture using a 1 uL loop, the colonies are 80/10 (simple unitary method) which is 8. So, actually 80 = 8 from the same culture.

Dear Paul Rios,

No need to be worried. You are 100% correct

It is correct, if any clarification i have attach the dilution work sheet method.

Yes Sir, you are right.