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bacteriostatic assay... troubleshooting!!!


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#1 PVB

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Posted 03 November 2012 - 02:14 AM

Hi all!!!


A monoclonal antibody generated against outer membrane proteins of my microbe has bacteriostatic activity. To evaluate this activity statistically, I am standardizing a bacteriostatic assay in microtitre plate (basically, recording the absorbance on hourly basis for 8 continuous hours). At early stage of standardization, things worked out very well. But I'm facing a wierd problem now. Positive control (organism in enrichment broth) which used to give a typical bacterial growth curve, is showing a drastic decrease in absorbance @ the first hour of incubation itself, and no change in absorbance in further hours. Tried everything to get rid of the problem - used fresh broth, tried different enrichment broths, fresh culture... no improvement. Not finding out the root of the problem. Kindly help.


Regards,
PVB

#2 bob1

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Posted 03 November 2012 - 01:15 PM

Are you re-using plates for this? If so, make sure that they are very very well washed - traces of detergent can cause this sort of effect. The same applies to glassware used for making up the medium and anything else used in the experiments.

#3 PVB

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Posted 04 November 2012 - 10:41 PM

Thanks for the reply.

I am using only fresh plates. I have changed the glassware to prepare the medium and buffers too. They did not help me.

#4 Phil Geis

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Posted 06 November 2012 - 07:55 AM

You can answer this youself. Think as a scientist - isolate and test impact of potential root causes/ variables - media, glassware, plates, culture, incubator space, etc.




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