# 10 millimolar (mM) to 250 micromolar - (Aug/28/2012 )

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ascacioc on Wed Aug 29 22:17:42 2012 said:

I just found the best explanation here on the forum:
http://www.protocol-...s-calculations/

This is +- what I mean.

I know that you are right in the end, but its not the best start.
The opening poster needs to understand it, not blindly fill in some formula he doesnt understand.

I'll try to add the calculations needed for this question later today or in the future days.

-pito-

Maybe you do another thread that we can pin on top of the general techniques forum for having it as a reference forever and ever and not have the same explanation repeated over and over (I am tired of typing) and then we can just refer to that thread, don't you think?

Andreea

-ascacioc-

I merged the two threads because we are talking about the same kind of calculations (I hope this is not opposed by nobody involved)

PS: Indeed I made a mistake with the 4 uL; I missed a zero. Pito is right, 10x for pointing it out.

-ascacioc-

ascacioc on Thu Aug 30 09:51:29 2012 said:

Maybe you do another thread that we can pin on top of the general techniques forum for having it as a reference forever and ever and not have the same explanation repeated over and over (I am tired of typing) and then we can just refer to that thread, don't you think?

Andreea

You are the moderator, I am not.
Do what you think is a good idea.
Perhaps its a good idea to make 1 general post with some simple examples in it and pin this somewhere so they can always check this post.

I can only tell you that I have seen hundreds of these kind of posts. People suffer a lot with elementary dilution problems. And almost everytime its the same problem: they have heard of a formula or they even know CV = CV , but have no idea how to use it or what it means.

-pito-

ascacioc on Tue Aug 28 20:04:00 2012 said:

For which final volume? Let us assume you want to have final volume 2 mL for your experiment. You pipette 2 mL = 2000 uL of saline/media whatever.. to which you add X uL of your bacteria at OD600 = 0.4; X being the solution of the below:

2.5*10^9 CFU/mL * X uL of this = (uL final solution+X uL that you add on top) * 10^6 CFU/mL (get rid of the extra zeroes)

2500x = 2000 + x
x = 2000/2499 uL (even though I wouldn't add this little amount < 1 uL) I would do some dilution series before, something like 1 to 100 in order to add 100 times the X volume.

Andreea

C1*V1 = C2* V2, Problem original stock conc. 2.5*10^9 CFU/mL required conc. 10^6 CFU/ml

2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
so, X uL = (10^6 * 2000uL)/2.5*10^9
X = 1.25 uL

I would add 1.25 uL of 2.5*10^9 CFU/mL to 1998.75 uL of saline / media to get 10^6 CFU/ml

I agree with Andreea that adding small volume to big may not be good idea why don't you go to back to stored serial dilutions and pick

X uL = (10^6 * 2000uL)/2.5*10^7
= 125 uL from 2.5*10^7 CFU/mL ( from 10^4 serial diluted sample) to 1875 uL of saline / media to get 10^6 CFU/ml

-Inbox-

prabhubct on Thu Aug 30 15:44:56 2012 said:

ascacioc on Tue Aug 28 20:04:00 2012 said:

For which final volume? Let us assume you want to have final volume 2 mL for your experiment. You pipette 2 mL = 2000 uL of saline/media whatever.. to which you add X uL of your bacteria at OD600 = 0.4; X being the solution of the below:

2.5*10^9 CFU/mL * X uL of this = (uL final solution+X uL that you add on top) * 10^6 CFU/mL (get rid of the extra zeroes)

2500x = 2000 + x
x = 2000/2499 uL (even though I wouldn't add this little amount < 1 uL) I would do some dilution series before, something like 1 to 100 in order to add 100 times the X volume.

Andreea

C1*V1 = C2* V2, Problem original stock conc. 2.5*10^9 CFU/mL required conc. 10^6 CFU/ml

2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
so, X uL = (10^6 * 2000uL)/2.5*10^9
X = 1.25 uL

I would add 1.25 uL of 2.5*10^9 CFU/mL to 1998.75 uL of saline / media to get 10^6 CFU/ml

I agree with Andreea that adding small volume to big may not be good idea why don't you go to back to stored serial dilutions and pick

X uL = (10^6 * 2000uL)/2.5*10^7
= 125 uL from 2.5*10^7 CFU/mL ( from 10^4 serial diluted sample) to 1875 uL of saline / media to get 10^6 CFU/ml

This is an excellent example of using a formula without thinking (= you use it on auto-pilot rather then reasoning, thinking the entire time) or checking it.

Also: in the second part of your post I dont see what you are trying to do.
You use 2.5*10^7cfu/ml ? but the conc is 2.5*10^9 cfu/ml in his stock ... not sure where you got this factor 100 difference. I know you can use the less diluted ones, but thats not the issue here and its confusing. In this you are changing the "stock" solution with the "diluted (100 times)" solution.
+you make the same mathematical mistake as in the first calculation.

-pito-

ascacioc on Tue Aug 28 19:49:31 2012 said:

Your calculation is correct only if you plated 100 uL of your dilution.

Andreea

pito on Thu Aug 30 09:42:30 2012 said:

I know this seems complicated and a bit stupid since if you simple use C1V1 = C2V2 , you can do it a lot faster ==>
250 x 10^6 X V1(?) = 10^6 x 10 ml
Meaning X = 0.04 ml or 40µl

Here I suppose that 100 µl of culture taken from 10^6 dilutions to get

(250colonies/100 µl)* 10^6 = 2.5 * 10^9 colonies/ml
i.e 2.5 * 10^9 CFU/ml

From Pito's post ""You use 2.5*10^7cfu/ml ? but the conc is 2.5*10^9 cfu/ml in his stock ... not sure where you got this factor 100 difference. I know you can use the less diluted ones, but thats not the issue here and its confusing. In this you are changing the "stock" solution with the "diluted (100 times)" solution."

Here I suppose changing stock to less diluted one will be good idea. I am not changing "stock" solution with the "diluted (100 times)" solution but to concentrated 100 times. you have 10^1 to 10^9 diluted series use 10^4 instead of 10^6 one to get 10^6 CFU/ml

-Inbox-

prabhubct on Thu Aug 30 17:48:00 2012 said:

ascacioc on Tue Aug 28 19:49:31 2012 said:

Your calculation is correct only if you plated 100 uL of your dilution.

Andreea

pito on Thu Aug 30 09:42:30 2012 said:

I know this seems complicated and a bit stupid since if you simple use C1V1 = C2V2 , you can do it a lot faster ==>
250 x 10^6 X V1(?) = 10^6 x 10 ml
Meaning X = 0.04 ml or 40µl

Here I suppose that 100 µl of culture taken from 10^6 dilutions to get

(250colonies/100 µl)* 10^6 = 2.5 * 10^9 colonies/ml
i.e 2.5 * 10^9 CFU/ml

From Pito's post ""You use 2.5*10^7cfu/ml ? but the conc is 2.5*10^9 cfu/ml in his stock ... not sure where you got this factor 100 difference. I know you can use the less diluted ones, but thats not the issue here and its confusing. In this you are changing the "stock" solution with the "diluted (100 times)" solution."

Here I suppose changing stock to less diluted one will be good idea. I am not changing "stock" solution with the "diluted (100 times)" solution but to concentrated 100 times. you have 10^1 to 10^9 diluted series use 10^4 instead of 10^6 one to get 10^6 CFU/ml

You are mixing things!

You first state that you would take 1.25 uL from a stock solution that is, as you wrote it, original stock conc. 2.5*10^9 CFU/mL

Then you state 125 uL from 2.5*10^7 CFU/mL (which is correct) however you also state

Here I suppose changing stock to less diluted one will be good idea. I am not changing "stock" solution with the "diluted (100 times)" solution but to concentrated 100 times. you have 10^1 to 10^9 diluted series use 10^4 instead of 10^6 one to get 10^6 CFU/ml

going from 10^9 to 10^7 is not going from less concentrated to more concentrated....

Read the bleu text: changing stock to less diluted? Stock is always the "less diluted" one!
The stock is what you dilute.

Also: a problem here is that we speak about 10^9 and 10^7 and dilutions etc...
While its clear that you might be speaking about 10^-9 and 10^-7 because you mention the 10^4 diluted series.. thus it should be 10^-4 you are mentioning....

I think you are mixing things up because you are not using the "-" sign in your dilution series!

==>
2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
so, X uL = (10^6 * 2000uL)/2.5*10^9
X = 1.25 uL

This is NOT correct.

2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
==>
X uL = 10^6 * 2000uL/2.5*10^9 CFU/mL =/= 1.25 !

the numerator is smaller then the denominator, so how can you end up with 1.25? You see this immediatly by just looking at the numbers (thinking).

You should directly see that the numerator has 10^6 and the denominator 10^9 , so 1000 times bigger while the numerator has only 2000 left to multiply and the denomintor 2,5.....

-pito-

Thanks Pito for correcting math error,

2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
so, X uL = (10^6 * 2000uL)/2.5*10^9
= 0.8 uL
add 0.8 uL of 2.5*10^9 CFU/mL to 1999.2 uL of saline / media to get 10^6 CFU/ml

@ Pito going from 10^9 to 10^7 is not going from less concentrated to more concentrated....

Read the bleu text: changing stock to less diluted? Stock is always the "less diluted" one!
The stock is what you dilute.

I think we have serial dilution stock of 10^1 , 10^2.......10^9.
and 10^7 must be more concentrated original stock than 10^9 because its serially diluted.

@ pito : 0.8 uL from a stock solution that is, as you wrote it, original stock conc. 2.5*10^9 CFU/mL

Then you state 80 uL from 2.5*10^7 CFU/mL
I Mean to take 80 uL from original stock of 10^4 as you are having 2.5*10^7 CFU/mL in 10^4 stock.

This means 10^6 diltion stock is of 2.5*10^9 CFU/mL.
10^5 diltion stock is of 2.5*10^8 CFU/mL.
10^4 diltion stock is of 2.5*10^7 CFU/mL.
10^4 diltion stock is of 2.5*10^6 CFU/mL.
10^3 diltion stock is of 2.5*10^5 CFU/mL.

-Inbox-

prabhubct on Thu Aug 30 19:09:00 2012 said:

Thanks Pito for correcting math error,

2.5*10^9 CFU/mL * X uL = 10^6 * 2000uL
so, X uL = (10^6 * 2000uL)/2.5*10^9
= 0.8 uL
add 0.8 uL of 2.5*10^9 CFU/mL to 1999.2 uL of saline / media to get 10^6 CFU/ml

@ Pito going from 10^9 to 10^7 is not going from less concentrated to more concentrated....

Read the bleu text: changing stock to less diluted? Stock is always the "less diluted" one!
The stock is what you dilute.

I think we have serial dilution stock of 10^1 , 10^2.......10^9.
and 10^7 must be more concentrated original stock than 10^9 because its serially diluted.

@ pito : 0.8 uL from a stock solution that is, as you wrote it, original stock conc. 2.5*10^9 CFU/mL

Then you state 80 uL from 2.5*10^7 CFU/mL
I Mean to take 80 uL from original stock of 10^4 as you are having 2.5*10^7 CFU/mL in 10^4 stock.

This means 10^6 diltion stock is of 2.5*10^9 CFU/mL.
10^5 diltion stock is of 2.5*10^8 CFU/mL.
10^4 diltion stock is of 2.5*10^7 CFU/mL.
10^4 diltion stock is of 2.5*10^6 CFU/mL.
10^3 diltion stock is of 2.5*10^5 CFU/mL.

I see what you mean...

But this is exactly my point from the start...

You are working "the other way around" (and still mixing things up, your explenation is contradictory, see below)

Normally people work like this:

Stock -> 10^-1 -> 10^-2 -> 10^-3 -> and so on...

The opening poster was indeed doing this, however rather then writing down 10^-9 he wrote down 10^9 , which caused you to make this mistake.

Remember what he said in his opening post:

I got 250 colonies at 10*6 dilution. So I got 2.5 × 10*9 cfu/ml (original, if I am right

He goes from 250 colonies at 10^6 to 2.5 x 10^9 colonies per ml in the original one (plating out 0,1ml).
this is only possible if the 10^6 actually means 10^-6 !

See what I mean?

Your explenation is "correct" (the reasoning is) but its not right in the end....(+mixing things up)

Altough, I do find it weird you write this:

I think we have serial dilution stock of 10^1 , 10^2.......10^9.
and 10^7 must be more concentrated original stock than 10^9 because its serially diluted.

10^7 is more concetrated then 10^9? 10^7 is a lower number then 10^9.
Unless you mean that 10^7 means you diluted it 10^7 times and 10^9 means diluting 10^9 times... But I have never seen people work like this...
dilutions are always given in negative powers.

Also: if you link what you stated (the quouted part) with what you write afterwars:

This means 10^6 diltion stock is of 2.5*10^9 CFU/mL.
10^5 diltion stock is of 2.5*10^8 CFU/mL.
10^4 diltion stock is of 2.5*10^7 CFU/mL.
10^4 diltion stock is of 2.5*10^6 CFU/mL.
10^3 diltion stock is of 2.5*10^5 CFU/mL.

Then something went wrong again.

you are mixing things up over and over.
===> in the bold reasoning you state that a 10^5 dilution is less diluted then a 10^4 dilution, meaning 10^5 is more concentrated (more CFUs) then 10^4, but at the same time (see quoted part) you state that "10^7 must be more concentrated original stock than 10^9 because its serially diluted"

So what is it now?
Either "and 10^7 must be more concentrated original stock than 10^9 because its serially diluted." is correct or the last part (the bold part) of your post is the correct one.
But they cant be both right.

Things are becoming more and more complicated because the same mistakes are made over and over.

-pito-
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