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10 millimolar (mM) to 250 micromolar

Bacteria CFU

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#1 chandch

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Posted 28 August 2012 - 11:34 AM

Hi
I am trying to calculate colony forming units calculation. I cultured bacteria overnight (20ml) and did O.D reading at 0.4 and did serial dilution (10*1 to 10*9) and I got 250 colonies at 10*6 dilution. So I got 2.5 × 10*9 cfu/ml (original, if I am right). But I need 10*6 or 10*7 CFU/ml (for my experiment). How can I reduce it down to it. Please give example calculation.

Thank you

#2 ascacioc

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Posted 28 August 2012 - 11:49 AM

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

2.5*10^9 CFU/mL * X uL of this = (uL final solution+X uL that you add on top) * 10^6 CFU/mL. Solve equation for X inserting the amount needed for your experiment in uL. It is a simple dilution from stock solution problem (c1*V1=c2*V2)

Andreea

#3 chandch

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Posted 28 August 2012 - 11:55 AM

Am little bit confused...Could you please do sample calculation ( From this step I am quite confusing from the last few days)

#4 chandch

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Posted 28 August 2012 - 11:58 AM

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

2.5*10^9 CFU/mL * X uL of this = (uL final solution+X uL that you add on top) * 10^6 CFU/mL. Solve equation for X inserting the amount needed for your experiment in uL. It is a simple dilution from stock solution problem (c1*V1=c2*V2)

Andreea


Do I need to dilute it down? Am little bit confused...Could you please do sample calculation ( From this step I am quite confusing from the last few days). In most of the papers people say use 10*6 cfu/ml ( I think plating is initial step to check the cfu at a desired O.D)

#5 ascacioc

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Posted 28 August 2012 - 12:04 PM

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

#6 chandch

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Posted 28 August 2012 - 12:29 PM

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


For example I need 10 ml

So 2.5*10^9 *** 10000 = ( What is uL final solution+X uL that you add on top) * 10^6 CFU/ml

Please reply...Today I will learn how to check conc

#7 ascacioc

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Posted 28 August 2012 - 12:31 PM

No: 2.5*10^9 *X = ( 10000+X) * 10^6 CFU/ml

#8 chandch

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Posted 28 August 2012 - 12:43 PM

No: 2.5*10^9 *X = ( 10000+X) * 10^6 CFU/ml


Thank you

So

2.5 *10^9 *X = (10000+X)*10^6 CFU/ml

2500 X = 10000+X

x = 10000/2499 uL = 4 ul

Am I right

#9 ascacioc

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Posted 28 August 2012 - 12:47 PM

yup; but again: i wouldn't pipette 4 uL but I would make 1 mL culture at 0.4 diluted to 9 ml media, thorough mix and I would add 40 uL of this to 10 mL.

#10 chandch

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Posted 28 August 2012 - 12:49 PM

yup; but again: i wouldn't pipette 4 uL but I would make 1 mL culture at 0.4 diluted to 9 ml media, thorough mix and I would add 40 uL of this to 10 mL.


Thank you. Now I understand. I will practice some more examples, so that I will get good idea on this calculations

#11 cell_bee

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Posted 28 August 2012 - 03:03 PM

Hello,
I am a very new person to cell culture and would need some help with the calculations...
1. I am testing few inhibitors on mouse cellline. I have a chemical compound whose MW is 243. I need to dissolve it in DMSO and get the concentration to 10mM. Then I need to dilute it to 200uM and serial dilute this to 20uM-10um-5um-2.5um and so on...
How do I do this and when serial diluting it, do I have to use the growth media or do I use DMSO? I do not want the concentration of DMSO to go more than 1%. Please help

2. I have BMP4 whose concentration is 10ug/ml. I need 10ul of this, whose concentration is 250pM. How do I calculate?

#12 bob1

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Posted 28 August 2012 - 03:13 PM

First off - there are many many threads on here about calculations - have a look through some of them, they will tell you how to calculate the answers you want.

You may need to dilute in DMSO, but probably you can dilute in the medium of choice. Note that DMSO is toxic to cells at concentrations above 0.1-0.5% (cell line dependent), so you will have to work out how much you need to add to the cells to keep the DMSO below this concentration range. You should also have what is known as a vehicle only control -this is pure DMSO diluted in your medium to the same concentration as for your treatments, just to make sure that any effect you see isn't just from the DMSO.

#13 cell_bee

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Posted 29 August 2012 - 09:51 AM

Thanks for the reply. I am kinda lost, could you please link me to the thread as there are tons of post and its hard for me to find a particular thread realted to my question.

Thanks again!

#14 ascacioc

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Posted 29 August 2012 - 10:49 AM

useful formulae:
concentration (in M)= mass (in grams)/MW (in grams/mol)/volume (in L); since you know your concentration, volume and MW --> mass of substance in grams = volume (in L) * MW (in g/mol) * concentration (in M). This is your stock solution (10 mM), or the most concentrated from which you can take a volume V1 to add to V2 of media to get 200 uM final conc
c1*V1=c2*(V2+V1); where c1 is 10 mM = 10000 uM; c2 is 200 uM and V2 is the final volume on top of which you add your drug (~12 mL for a small flask of cells)
solve for V1 = c2 * V2 /(c1-c2). since you plug in V2 in mL, you will get V1 also in mL. (for V2 = 12 mL you get ~0.245 mL = 245 uL). For 20 uM you need to add 10 times less --> 24.5 uL or you dilute your stock solution 10 times (1 part stock of 10 mM and 9 parts media) (down to 1 mM) and still add 245 uL.

With the two formulae I gave you, you can also answer the second question.

Link to similar discussion with drug soluble in DMSO:
http://www.protocol-...-added-to-dmem/
http://www.protocol-...__hl__micronagu

Andreea

#15 pito

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Posted 29 August 2012 - 01:08 PM

I would really like to know where your formula: 2.5*10^9 CFU/mL * X uL of this = (uL final solution+X uL that you add on top) * 10^6 CFU/mL) comes from!
In symbols: C1 x V1 = (V2 + V1) x C2 or C1 x V1 = V2 x C2 + V1 x C2


Also: plz recheck it, it does not add up.

Many mistakes have been made here.

He wants a final (fixed) end volume, this is not the case here.

I noticed you are a huge fan of c1*V1=c2*(V2+V1), but plz dont use the formula when its not appropriate.

Edited by pito, 29 August 2012 - 01:18 PM.

If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.





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