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

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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

-chandch-

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

-ascacioc-

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

-chandch-

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

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)

-chandch-

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

-ascacioc-

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

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

-chandch-

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

-ascacioc-

ascacioc on Tue Aug 28 20:31:52 2012 said:

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

-chandch-

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.

-ascacioc-

ascacioc on Tue Aug 28 20:47:04 2012 said:

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

-chandch-
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