Help with dilution

[marinerush]

So I'm currently growing 5 bacterial cell cultures. I start at the dilution of 10^-1 by adding 300 microliters of a media and 33 microliters of the bacterium. So when we do these dilutions fewer cells are developed. Is it because we are starting with fewer cells or is it because we are using less spent media?

[ewmon]

So when we do these dilutions fewer cells are developed.

"Fewer" as compared to what?

[marinerush]

"Fewer" as compared to what?

 

So I start different dilutions. I would do 10^-1, 10^-2, 10^-3 and so on. If you could imagine like 10 wells. I would transfer 33 microliters from the 10^-1 well to the next one, making it a 10^-2. So as I go further into dilution of the bacterium, the spent media is also decreasing.

 

I start growth and end growth at the same time for all of these dilutions, but for some reason I see more growth after 24 hrs in the 10^-1 dilution than the 10^-5 dilution of bacterium.

[CharonY]

Adding 33 µl of bacterial culture to 300 µl of medium is not a 1:10 dilution but it is rather slightly off. If you continue the dilution, errors will propagate.

For the rest of the question, you will have to be careful with the words. The way you phrased it, it is e.g. not clear vs you refer to growth rate, or total cell count (which are quite different things). If you refer to the latter, it should be rather obvious, you start with less, you end with less.

[marinerush]

Adding 33 µl of bacterial culture to 300 µl of medium is not a 1:10 dilution but it is rather slightly off. If you continue the dilution, errors will propagate.

For the rest of the question, you will have to be careful with the words. The way you phrased it, it is e.g. not clear vs you refer to growth rate, or total cell count (which are quite different things). If you refer to the latter, it should be rather obvious, you start with less, you end with less.

 

 

Isn't 33 microliters of bacterium in 300 microliters of medium a 1/10 dilution? --> 33/333 = 1/10?

 

Either way, say we measure the growth rate (cell count) over a period of time for each dilution factor (eg. 10^-1, 10^-2, 10^-3, etc)

Obviously the 10^-3 dilution will have a lower cell count at the end of the period compared to the 10^-1 dilution.

 

What I'm asking is what could be the reason for this?

1. Is it because of the actual initial dilution factor of the bacterium?

2. Or is it because of the dilution factor of the spent media?

 

And ultimately, what would be the best way to figure out which reason it is?

[CharonY]

Ah yes you are correct I read it as in 300 µl final volume, for some reasons. The growth rate is not the cell count, but rather the change of cells over time. It is usually expressed as generation time (i.e. time/number of generations). Using the cell number at the beginning and at the end of the interval, you can calculate the value. That value is dependent on growth conditions and relatively independent of initial cell count. However, if you are talking about about the total cell count, it refers to the number of cells you end with. In your description it is still not clear to which value you are actually measuring.

 

That being said, you come back to the dilution of the media. What kind of impact do you think it has. Or in if you hypothesize, should the spent medium increase or decrease growth of the bacterium. And how?

 

 

[ewmon]

So I start different dilutions. I would do 10^-1, 10^-2, 10^-3 and so on. If you could imagine like 10 wells. I would transfer 33 microliters from the 10^-1 well to the next one, making it a 10^-2. So as I go further into dilution of the bacterium, the spent media is also decreasing.

 

I start growth and end growth at the same time for all of these dilutions, but for some reason I see more growth after 24 hrs in the 10^-1 dilution than the 10^-5 dilution of bacterium.

I'm letting CharonY handle this, someone who, as far as I can tell, knows more about this than me.

 

FYI, you're describing "serial dilutions" of the bacterium (and spent medium?). What do you mean by "spent medium"? Do you mean that you don't otherwise add any medium to the wells? That the wells get all their medium, along with their bacterium, through the serial dilutions?

[CharonY]

Well, actually my reading comprehension seems to be crap. 333/33 is of course not 1:10. The correct dilution would have been 33/330, 33.3/333 or 30/300. Also admittedly the error is relatively low (but one should still avoid it, pipetting errors are going to be bad enough). *sigh* Posting without sleep (PWS) is not good...

[Mark Ian]

Haha, I do the final volume mistake allot too.

 

I start growth and end growth at the same time for all of these dilutions, but for some reason I see more growth after 24 hrs in the 10^-1 dilution than the 10^-5 dilution of bacterium.

 

As I'm sure you are aware of, the reason you are diluting the stuff is so that you can compare it more easily to other strains. If you were to take only one colony and compare it with another at similar concentrations, you risk of growing too many or to little bacteria for comparison- plus its more convincing.

 

I see why this can be a little confusing: I would think the exponential growth of bacteria would, given enough time, overpower the small starting number of cells used. Try typing in numbers into your calculator:

 

n^t; while 'n' would be the amount of starting bacteria and 't' would be the time that has past.

 

This would sorta mimic exponential growth. For example if you compare growths with 2 cells and 5 time units: 2^5=32 AND with 4 cells and 10 time units: 4^5= 1024 there will always be a significant difference between outcome, given the same time past, and starting amount. You might find the Wikipedia article about Bacterial Growth helpful to understand the concept, especially the different phases; complicated stuff.