10 replies to this topic

[biology_06er]

Hi there,

I plan to do some flow next week and an one of the steps is to resuspend the bacterial pellet to an OD600 0.5. So say I had a 1.5mL overnight culture and the following morning the OD600 was 1.7. Once I spin down, wash etc etc I read to get to a certain OD (0.5 in my case)...this is the calculation

Divide the OD you want (0.5) by the OD bacteria was at in morning (1.7)=0.5/1.7=0.29=giving you a concentration factor (cf)
Divide the original culture volume (1.5mL) by the cf
1.5/0.29=5.1mL

Ok..so I don't need 5.1mL of culture to subsequently use..say altogether I only need 2mL how do I go about this? So that in the end I have 2mL of bacteria with an OD of 0.5?

Man, I hope I make sense!
Cheers,
b_06er

[Paulgs3]

You're on the right track.  The formula you want is the old: C1V1=C2V2.

Edit for clarity:

(0.5)(2ml) = (1.7)(x)

X= 0.58ml

Take 0.58 ml of the 1.7 OD and QS to 2ml

Edited by Paulgs3, 29 June 2012 - 02:31 AM.

[pito]

Just curious: why do you need the bacteria?

In theory you could try this, but I am not a big fan of such work.

Overnight culture, resuspending, spin down, wash...

Normally when you need an OD of 0,6 there is a specific reason: cells in exponential growth , cells that are growing well and are "happy".
You are changing this by using older cells, washing them, resuspending them etc... also: that OD you measure.. not sure if this is just "cells" and not some waste made by the cells etc...

[biology_06er]

Hey Pito!

Thank you so much for your reply..you're a lifesaver!..was stressing out incase Monday came and I went into the lab not 100% sure..and can't really afford to screw it up!..so double checking..I take 588uL out, spin it down, wash, block, wash, and the final pellet I get I resuspend in 2mL?

Um can I let you know the answers to those questions after I've done the exp haha..I'm spending this weekend reading up etc..I'm trying to detect surface protein and I think 0.5 was the ideal OD someone else got when they did a similar exp.

Did you mean you're not a fan of the work I'm doing..such as washing etc? How come?

Thank you again *10000000

[pito]

biology_06er, on 29 June 2012 - 05:38 AM, said:

Hey Pito!

Thank you so much for your reply..you're a lifesaver!..was stressing out incase Monday came and I went into the lab not 100% sure..and can't really afford to screw it up!..so double checking..I take 588uL out, spin it down, wash, block, wash, and the final pellet I get I resuspend in 2mL?

Um can I let you know the answers to those questions after I've done the exp haha..I'm spending this weekend reading up etc..I'm trying to detect surface protein and I think 0.5 was the ideal OD someone else got when they did a similar exp.

Did you mean you're not a fan of the work I'm doing..such as washing etc? How come?

Thank you again *10000000

I am pretty sure that you need bacteria in exponential growth, you want a specific surface protein to be expressed. So the question: is this a protein specific for growing bacteria or not?

If you keep the bacteria and let them grow overnight , they will not be that happy and they will suffer stress and change their surface proteins etc.. So you need to know what you are trying to do.

What I do not understand: you know that the previous ones had the best results with an OD of 0,5, so why dont you do it like them? Same protocol? How many hours did they grow their bacteria culture..?Dont you have a protocol?

When working with that 588µl , you dont spin it down or wash it, you just use 588µl and you add 2ml - 588µl (final volume is 2ml).
But I dont advice you to work like this.
If you do pellet it down and wash it: you will change the OD because the OD of your sample is made not only by cells but also by other factors. (extracellular products etc).
If you dont want those: yeah, you can wash it down etc.. but you will never get that od you want, you will need to adjust it and play with it...
You are making it a lot harder for yourself then you should.

I would advise you to check your literature and look for a similar protocol.

[mdfenko]

also keep in mind that the change in absorbance may not be linear, especially in the higher range.

[biology_06er]

Hye there,


I am using the same protocol..I just don't get how they did the dilution factor---and I don't want to ask because a) I made it look as though I knew what they were talking about but now I'm not so sure. I can't take the 588 and top up to 2mL because I need to block before hand. I know they told me to take only a little bit of my overnight culture and spin that down as my first step...so the first time I did this experiement this is what I did:



I had a 15mL o/n culture.....the next day I measured OD600 it was 1.7. I took 1.5mL of that culture..I put in eppendorf tube, spun, down, washed, blocked, washed.
The final pellet I resuspended in 1.5mL of buffer. I needed a final sample of 1.2mL at OD600 0.5
From that 1.5mL I took 0.294uL (0.5/1.7) and topped up to 1.2mL

When I started my flow I def had cells...I was told it didn't matter if the OD was not exact but even so, I am sure what I did was wrong above! (well mainly the step in bold)



EDIT==maybe I should have taken 0.5/1.7*(1.2mL)=353uL instead?

Edited by biology_06er, 29 June 2012 - 02:28 PM.

[biology_06er]

Oh, and trust me I have looked at the lit..but they only state "bacteria was resuspended to an OD of....." that doesn't tell me HOW to do get the certain OD

[pito]

biology_06er, on 29 June 2012 - 02:19 PM, said:

Hye there,


I am using the same protocol..I just don't get how they did the dilution factor---and I don't want to ask because a) I made it look as though I knew what they were talking about but now I'm not so sure. I can't take the 588 and top up to 2mL because I need to block before hand. I know they told me to take only a little bit of my overnight culture and spin that down as my first step...so the first time I did this experiement this is what I did:



I had a 15mL o/n culture.....the next day I measured OD600 it was 1.7. I took 1.5mL of that culture..I put in eppendorf tube, spun, down, washed, blocked, washed.
The final pellet I resuspended in 1.5mL of buffer. I needed a final sample of 1.2mL at OD600 0.5
From that 1.5mL I took 0.294uL (0.5/1.7) and topped up to 1.2mL

When I started my flow I def had cells...I was told it didn't matter if the OD was not exact but even so, I am sure what I did was wrong above! (well mainly the step in bold)



EDIT==maybe I should have taken 0.5/1.7*(1.2mL)=353uL instead?

I dont get it: you talked to the people that did this before? So you can "see" them and talk to them? They work in your lab? If so: ask them!
There is no point asking it here.. We dont know all the details..they do! Ask them... If you are not sure: ask!
Ask them if you need growing cells or not?
Normally OD 0,5-0,6 is because you need cells in their exponential growth fase, but I am starting to wonder whether this is the case here since they use overnight culture too (?).



I dont know what you mean by "block before hand", you need to add the liquid before you can add the overnight culture? I dont get it.
Especially because you did this the first time... you say yourself that you "From that 1.5mL I took 0.294uL (0.5/1.7) and topped up to 1.2mL"
So I am not sure why you cant redo this till you reach 2ml.

[Papaver]

biology_06er, on 29 June 2012 - 02:19 PM, said:

EDIT==maybe I should have taken 0.5/1.7*(1.2mL)=353uL instead?

I agree with the others that it would be better to take a culture with an OD of 0.5, means cells in the exponential growth phase. So you would take your overnight culture and dilute them 1:50 or 1:100 in 5 ml LB and grow them until they reach the OD you need.

Considering your calculation...353 µl would have been right. Although I cannot image that you would get a suspension of an OD of 0.5 after the procedure of washing and so on. But if the protocol says this....??? I would, like pito have already said, ask the others. It will make it easier...also to understand the protocol.

Edited by Papaver, 30 June 2012 - 04:55 AM.

[biology_06er]

Oook..I'll ask--I was hopoing to have it sorted this weekend..so I could just go in on Monday and start...anyhow..I assume I can use an o/n culture because the protein in constitutively expressed..hence it isn't really a problem if I use the bacteria at SP.