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Maintaining E. Coli (ATCC 25922)


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#1 pinkrainbow

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Posted 16 January 2014 - 11:30 AM

I have just obtained a stock of E. Coli, which I am using to test novel compounds for antibacterial activity. I am not too familiar with microbiology techniques, so there are some questions I'd like to clear up.

 

I plan to first streak the bacterial stock on a normal agar plate, and let the plate incubate at 37 degrees overnight. Then I will pick a colony to grow in an overnight liquid culture of Mueller Hinton broth, monitor the OD values and use the liquid solution to perform the bacterial assay.

 

Several questions I have:

1) Should I supplement the agar plate with any antibiotics, as to prevent the growth of other bacteria other than E. Coli? If so, what antibiotic should I use?

2) How should I maintain the agar plates so that I won't need to re-order anymore strains in the future? 

 

Thanks all! 


Edited by pinkrainbow, 16 January 2014 - 11:57 AM.


#2 bob1

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Posted 16 January 2014 - 11:45 AM

Hmmm - I see 29522 as Aggregatibacter actinomycetocomitans, not E. coliPerhaps you have the number reference wrong, or have got the wrong bacterium!

 

Anyway, for E. coli, your process seems fine.  You shouldn't supplement the plate or broth with any antibiotic unless the strain needs it to maintain a certain characteristic.  This should be mentioned in the documentation for the strain and in the genotype.  You will need to use a standardized OD for the bacteria to ensure that the number of cells is roughly the same in each treatment. 

 

Your best bet for long term storage of E coli is to make a glycerol stock (or several stocks), this essentially will keep the bacteria forever.  The way to do this is streak a plate, pick a single colony and grow in liquid broth overnight.  The next morning, mix in glycerol to 15-20% final concentration (dilute glycerol to 50% in water and autoclave to make it easier to pipette), then freeze in the -80.  Once frozen they will keep for many many years.  To re-use, simply take a loop and scratch the surface of the still frozen stock (return it to freezer immediately) then streak on a plate.



#3 pinkrainbow

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Posted 16 January 2014 - 11:59 AM

You're right, I meant 25922! 

Thanks for your help. 

I have some further questions:

 

1) What kind of tubes do you suggest I make the glycerol stocks in? 

2) We have a plate reader in our lab, but not a spectrophotometer. Is it acceptable to use the plate reader to measure the OD values? or is it best to use a large volume in a cuvette with the spectrophometer? 


Edited by pinkrainbow, 16 January 2014 - 12:38 PM.


#4 bob1

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Posted 16 January 2014 - 12:42 PM

Glycerol stocks I would make in 2-5 ml screw cap tubes.  Cryovials are good for this sort of thing.

 

Plate readers should be fine so long as you use a big enough volume in the plate.  I would recommend 200 ul per well of a 96 well plate.



#5 pinkrainbow

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Posted 17 January 2014 - 10:25 AM

Since I am growing a pure culture, I have concerns about contamination... the incubator and shaker I use are shared with other labs. Are there any special precautions I should take to prevent the growth of other strains? 



#6 phage434

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Posted 17 January 2014 - 11:28 AM

No, just normal sterile technique. This is why you streak stored samples out for single colonies when you are using the strain. And also one of the reasons why you should prepare multiple samples.






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