Microbiology question

[peachy3214]

Ok, so im super embarrassed to ask this, but math is not my strong point. I have the following problem that i can't do. I figure if anyone will know how to do it will be you guys!


For the following scheme, indicate the total dilution on the plate and the number of colonies on each plate.

Initial Sample= 4.2 x10^7 CFU/ml--->1 ml is put into 99 ml------>10ml is put into 90ml---->1 ml is put into 9 ml----> .5 ml is put into 4.5 ml.

.1 ml is then taken from each dilution and plated. I need to know what the total dilution is on the each plate after each step and the number of colonies after each step on each plate.
If you could explain it that would be great, because i want to know how to do it.

Thanks!

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[chii chan]

Not sure if this will make sense..... but.....

You need to calculate the IDFs for each step (individual dilution factors) which is amount taken (1mL, for example), over the total amount (1mL + 99mL = 100mL). So IDF2 would be 10,L/(10mL + 90mL), IDF 3 is 1ml/(1mL + 9mL), IDF4 is .5mL/(.5mL + 4.5mL).

From these individual IDFs, you find the TDF (total dilution factor), which is IDF1 x IDF2 x IDF3 x IDF4....

CFU/stock is calculated by [CFU counted/amount plated]/TDF. So in this case, you know CFU/stock, amount plated, and should know TDF by now. Just solve for CFU counted.

I think you multiply IDFs to get TDF... or maybe it was add? I just had it last semester, so I SHOULD remember it, but I don't. Pretty sure it's multiply since its usually, e.g., 10^-1 * 10^-1 * 10^-2 etc. I KNOW for sure that the CFU/stock solution equation is correct.

Hope it helped! 🙂


Edit: Yes. You do multiply it. (It doesn't make sense to add them.)

Didn't notice you said CFU/plate for EACH step. So TDF of stock to first dilution is just IDF1 (1mL/100mL). TDF of the second step is IDF1 x IDF 2 [(1/100) x (10/100)]. etc, etc. You have to use that equation 4 times to find the number of colonies, because each time you make a new dilution, you have a new TDF. However, the other numbers (CFU/mL and amount plated (0.1mL)) remain the same.

This and non-serial dilutions were the thing I did awesome at. Got 120% on all my quizzes thanks to extra credit. But of course, it had to be broken down this far for me to get it.

PM me if you have other math questions. I actually liked the math stuff. 🙂

 

[billw]

(4.2x10^7) x (1/100) x (10/100) x (1/10) x (0.5/5) x 0.1

(For the final plate. You should be able to work backwards based on the example.)

 

[that70sfan11]

Okay here we go... I'm no expert, but I have a test on this in a few days so if anything it may help me.

Formula - D2 = (V1xD1)/V2

D2 is what you are wanting to find, the current dilution factor
D1 is the dilution of what you put in the tube (if its a pure sample, the dilution is 1)
V1 is the volume of D1
V2 is the volume of the sample (aka V1) and the volume of the diluent

So for the 2nd tube, V1 = 1 ml, V2 = 1 ml + 99 ml (the diluent) = 100 ml
D1 = well, I think it should be 1, since the original sample hasn't been diluted

this would be 1 x 1/100 = .01 = D2

so now for the next plate, D2 becomes your D1 10 ml = V1 V2 = 10 + 90 ml... so now for this next polate

D2 = (10 x .01)/ 100 = .001

and so on....

to get the # of cells on each plate
OCD (original cell density) = CFU/(volume plated) x (dilution factor)

this might be completely wrong... my questions aren't quite set up like that, so I'm trying my best. But if that doesn't work, no offense will be taken! I'm hoping you have correct answers to compare your answers to...

 

[PrimalMU]

Don't feel embarrassed. I could never get the hang of those calculations either. Neither could the rest of my class.

 

[GellaBella]

Ok, so im super embarrassed to ask this, but math is not my strong point. I have the following problem that i can't do. I figure if anyone will know how to do it will be you guys!


For the following scheme, indicate the total dilution on the plate and the number of colonies on each plate.

Initial Sample= 4.2 x10^7 CFU/ml--->1 ml is put into 99 ml------>10ml is put into 90ml---->1 ml is put into 9 ml----> .5 ml is put into 4.5 ml.

.1 ml is then taken from each dilution and plated. I need to know what the total dilution is on the each plate after each step and the number of colonies after each step on each plate.
If you could explain it that would be great, because i want to know how to do it.

Thanks!

Ok yay micro! This is right up my alley! Here we go!

Since you are taking 0.1 mL from each dilution and plating it, that is 1/10th of an mL. So, now if you plate 0.1 mL of the initial sample (at 4.2 x 10^7 CFU/mL) since there are already 4.2x10^7 CFU/ml and you are plating 0.1 mL of that (which is 1/10th of an mL) you would get 4.2x10^6 cells on the plate containing the initial sample.

Now, you take 1 mL of 4.2x10^7 cells/mL and put it into 99mL of fresh medium. Thats a 1:100 dilution. So you would now have 4.2x10^5 cells/mL. You take a 0.1 mL sample and plate it (which is 1/10th of an mL) and you would get 4.2 x10^4 cells on your plate.

You then take 10 mLs of your 4.2x10^5 cells/mL sample and dilute it into 90 mLs of fresh medium. So thats a 1:10 dilution which would mean you are now at 4.2 x 10^4 cells/mL. Take 0.1 mL of that and you would get 4.2 x10^3 cells on your plate.

1 mL of 4.2 x 10^4 cells/mL diluted in 9 mLs of medium is again a 1:10 dilution so you'd have 4.2 x10^3 cells/mL. Plating 0.1 mLs of that would give you 4.2 x 10^2 (or 420) cells on your plate.

Finally you take 0.5 mLs of your 4.2x10^3 cells/mL culture and dilute it in 4.5 mLs of medium which is a 1:5 dilution. You now have 8.4x10^2 cells/mL (840 cells). If you take a 0.1 mL sample of this culture and plate it you will end up with 84 cells on your plate.

I hope this helps!! Let me know if anything is confusing!

 

[peachy3214]

Thanks guys! that was really helpful! your awesome!

just one more thing.....For my total dilutions my results would be.....
for dilution 1= 10^-3
D2=10^-4
D3=10^-5

and then D4 confuses me......i got
D4=5^-5?
haha am I doing this right?

 

[peachy3214]

Finally you take 0.5 mLs of your 4.2x10^3 cells/mL culture and dilute it in 4.5 mLs of medium which is a 1:5 dilution.

Did you mean a .5:5 dilution?

 

[GellaBella]

yes you are correct. It is 0.5:5 mLs which is actually a 1:10 dilution (I'm sorry!)

so for that last one you had 4.2 x 10^ 3 cells/mL, dilute it 1:10 again by adding 0.5 mLs to 4.5 mLs fresh medium and you'll have 4.2 x 10^ 2 cells/mL (420 cells/mL). If you take 0.1 mLs of that you would be plating 42 cells on your plate.

Sorry about that again! For some reason I was reading it incorrectly.
🙄