washing pellets; recovery of isolates in glycerol - (Jun/13/2014 )

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!

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 

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?

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.

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!

Dima on Tue Aug 12 14:42:55 2014 said:

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!

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

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

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.

Dima on Mon Aug 18 17:31:14 2014 said:

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?

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

Why 10ml?