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washing pellets; recovery of isolates in glycerol


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#1 Dima

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Posted 13 June 2014 - 11:07 AM

Hello ! Could you pls help me know more on this?

1- after 24 hrs incubation of colonies in a broth, the culture is centrifuged, supernatant discarded, pellets washed and resuspendedbin broth. What is the purpose of centrifugion, washing? To get rid of dead cells?? Or nutrients outproducts?

2- i have e.coli isolates in glycerol.what s the best way to recover? I have rapid e.coli agar from biorad. Shall i take a loopful from the surface and suspend in brain heart infusion broth? And later Shall i streak the colonies on the agar or follow pour plate techniques??

Thanks loads in advance for the help!

#2 Adriana Reis

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Posted 01 August 2014 - 03:39 AM

well you centrifugate to separate the cells from the broth. since I dont know your work I have no clue what the main purpose

do you change into a different broth?

 

E coli in glycerol? just make a suspension in nutrient broth and make mcconkey and streak the colonies 



#3 Dima

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Posted 03 August 2014 - 02:24 AM

Thanks a lot Adriana..!

as for my question re. Why they centrifuge an overnight broth cultte and wash pellets to later resuspend it in broth Instead of directly using the overnight culture stems from studies i read when they follow this procedure for antimicrobial tests. or if the culture is freeze dried , such steps overnight broth-centrifugation-resuspension were described before streaking .

One more question if possible: would different volumes of broth used for an overnight culture generate a difference in cfu/ml?

#4 phage434

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Posted 03 August 2014 - 06:40 AM

This could make sense if you are using ampicillin resistant bacteria. They excrete beta lactamase into the medium, so non-plasmid containg cells can overgrow (satellite colonies on a plate). The spin down would remove this and allow for more selective growth conditions on recovery. I doubt it makes much difference.



#5 Dima

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Posted 12 August 2014 - 06:42 AM

Thank you loads for always helping.

 

sorry for this basic question: Would the volume of broth that i use to subculture colonies produce different cell densities? 5 ml or 10 ml?  i may need to try it and get the answer by plating but i thought to ask experts before doing all those tedious  plating for all the organisms i am working with. I started my experiments by subculturing in 10 ml BHI and i am considering to reduce the volume-resources issue :( - but I am afraid i may end up with different cell densities after overnight incubation.

 

Thx!



#6 pito

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Posted 12 August 2014 - 07:26 AM

Thank you loads for always helping.

 

sorry for this basic question: Would the volume of broth that i use to subculture colonies produce different cell densities? 5 ml or 10 ml?  i may need to try it and get the answer by plating but i thought to ask experts before doing all those tedious  plating for all the organisms i am working with. I started my experiments by subculturing in 10 ml BHI and i am considering to reduce the volume-resources issue sad.png - but I am afraid i may end up with different cell densities after overnight incubation.

 

Thx!

yes, but its not that easy to tell you what to expect!

 

But you add 1 "cfu" to your start culture or?
 


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


#7 Dima

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Posted 18 August 2014 - 09:31 AM

so better to stick to what I started with to avoid changes in results...

 

normally i take one colony from agar plate (freshly streaked-overnight culture) and i dispense in 10 ml broth.



#8 pito

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Posted 18 August 2014 - 09:38 AM

so better to stick to what I started with to avoid changes in results...

 

normally i take one colony from agar plate (freshly streaked-overnight culture) and i dispense in 10 ml broth.

 

So you use  10ml for an overnight incubation or?

 


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


#9 Dima

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Posted 18 August 2014 - 10:17 AM

yes, i use 10 ml, and i was thinking to continue my experiment while using 5 ml instead.



#10 pito

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Posted 18 August 2014 - 10:26 AM

Why 10ml?


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


#11 Dima

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Posted 18 August 2014 - 10:41 AM

it started by using 10 ml based on our lab technician initial "say" and i noticed in some papers they used such volumes maybe to prepare enough inoculum, and somehow i did the same. But then with time i realized i don't need 10 ml! 5 ml would have been sufficient.



#12 pito

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Posted 18 August 2014 - 10:47 AM

But whats the goal? Just to inoculate a larger volume the next day?

You might simple use 3 ml, and take 2-3 tubes...

If the liquid is too high, it will not reach the needed OD..

I am not sure what you are doing in the end, so perhaps the 10ml will do the trick too.


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


#13 Dima

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Posted 18 August 2014 - 10:52 AM

yes that's it to have a larger volume...you are right , i could have done the way you are saying.  this is why i was questioning if i change to lower volume will that change my expected cfu/ml at a specific OD.

 

I like what i am being through in this PhD, few unneeded procedures i am doing, yet i am learning.

 

Thx always!!



#14 pito

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Posted 18 August 2014 - 10:55 AM

I would just use 3ml than rather than 10.
5ml is still ok.. but 3ml is what most people do.


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


#15 Dima

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Posted 18 August 2014 - 11:11 AM

yes..i will !






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