bacteriostatic assay... troubleshooting!!! - (Nov/03/2012 )
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.
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.
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.
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.