3 replies to this topic

[PVB]

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

[bob1]

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.

[PVB]

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.

[Phil Geis]

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.