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Crystal violet

colometric assay

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#1 Dima

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Posted 21 August 2013 - 10:52 AM

Hello dear members,

 

I read : Staining adherent cells with crystal violet is simple and easily done. It should be noted that some bound cells may be lost due to the staining and washing steps.

A disadvantage of this method is that it does not allow quantitation of cell number in reference to a standard curve. Changes in OD measure qualitative variation in adhesion rather than the number of bound cells.

 

My question: I thought CV assay quantify the adhered cells by measuring the solubilized crystal violet following the application of ethanol or acetic acid.

 

I did not understand the underlined : changes in OD measure qualitative variation ....

 

COuld you pls share your experience?? I just started to use this method.

 

N.B. will be posting another question in a different topic.

 

Regards

 

Dima



#2 bob1

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Posted 21 August 2013 - 12:44 PM

I think in this context that the OD referred to is due to the attached cells binding CV rather than CV that has been released.  Released CV could well be quantitative, but bound to cells it is dependent on the density and adherence pattern of the cells (e.g. compare pictures of MCF7 with say HeLa for different types of growth)  rather than the number attached, hence the qualitative.



#3 Dima

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Posted 23 August 2013 - 01:18 PM

Thanks loads! somehow I understood that the OD of the CV  assay test is dependent on quantity of adhered cells thus its value is interpreted in equivalence to cfu/ml (approx). i.e. 0.10 OD value may be equivalent to an approximate value in cfu/ml . Was too wrong then ? there is no equivalence or such interpretation in cfu/ml rather we interpret OD results qualitatively in relation to different conditions that affected the high or low values?



#4 bob1

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Posted 23 August 2013 - 07:23 PM

There will be an equivalence somewhere, but for attached cells and measurement of OD, given how a spec works, the light path is in a limited area, so any holes in the monolayer or unexpected gaps would alter the reading quite dramatically.



#5 Dima

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Posted 24 August 2013 - 11:34 AM

Thank you again for post! yes I got the point ...always great to post on this forum :)







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