We are trying to pick up an effect on lysozyme by measuring the absorbance of florescent labeled M. luteus cells at OD600. We are using this as a substrate because we are comparing the results we observed using this substrate in an enz chek kit. We are getting very wavy lines(see attached) in the reading. Anyone have any suggestions as to why?
The wavy lines could be due to increased light scattering caused by cell aggregation. To check this, you can try resuspending the cells thoroughly before the measurement and then measuring at several time intervals. If aggregation is occurring, you might see a progressive change in the signal during the early time points as the cells begin to clump together. Oversaturation of the signal at high concentrations can also cause distortions, such as flattened or erratic absorbance readings that do not correspond linearly with expected changes in the sample. If this seems to be the case, you may try diluting the sample to reduce these effects.
The waviness of these lines may just be due to the limited number of pixels available in the display. If you plot the data in a graphing program, such as Excel, are the graphs also wavy?
Hi Sally,
There could be a number of explanations, but it's difficult to tell with the image you've shared. As Adam suggested, a plot in a graphing program will help. Does that decrease represent a 1% change or a 50% change?
One suggestion I have without seeing the data more clearly is to make sure the instrument is warmed up. The manufacturer generally recommends a time (or sometimes doesn't!), but for any time-dependent measurement, I find getting it up to a steady temperature and having a lamp that is warmed up helps give cleaner results.
Similar to what Tomislav suggested, the cells may be settling over the course of your measurement. I have no idea what the timescale is from your image, so that may or may not be relevant. The optics of your plate reader may not be measuring the light scatter (OD600 of cells) equally at all depths, so you may be seeing them settle away from an ideal depth. If it has a shaking/mixing function, you might be able to mix just before each measurement and see an improvement. Just make sure it doesn't shake so vigorously that you have splashed liquid on the ceiling of your plate chamber. Been there, it gets gross!
Thanks for all the suggestions! I think aggregation of the cells is the most likely reason as we hsve seen this under the microscope also.
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