I am working on osmotic stress in a member of family alpha-proteobacteriaceae. My organism keeps doubling time constant but there is a visible effect on end O.D. I used 10% PEG as osmotic stress inducer. Has anyone tested PEG to be a problematic for O.D measurement at 600nm? Secondly, if PEG is fine then why cell keep its doubling time constant? I also measured the substrate consumption and it is much faster in PEG media but membrane bound enzyme activity for that substrate is half than normal. These two things are quite confusing.
Nageena,
Osmotic stress has multiple effects on bacteria. One physical impact of osmotic stress conditions is that size and shape of the bacterial cells change. When measuring OD @ 600 nm for bacteria you are not observing much absorbance since not many molecules in a bacteria or in the media absorb at that long wavelength but you are measuring defraction of long wavelength light by the relatively large particles in the solution, the bacterial cells. Defraction is correlated not only with the number of particles in the solution but also the size and shape of the particles. Thus under high osmotic stress conditions, when the individual cells become smaller and more spherical their defraction decreases lowering the OD. This means that the same OD value at high and low osmotic stress conditions does not correlate to the same CFUs. The best way to correlate the CFUs under different osmotic stress conditions is to count the number of colonies per unit volume of culture by plating the culture on the appropriate agar plates. You'll probably have to do multiple dilutions.
Also, what average MW PEG are you using? PEG will not only have an effect on osmotic effect on a solution but also on a hydrodynamic effect. Since PEG is a large linear molecule that can interact with water it can form matrices in a solution and alter the activity of water (water's effective concentration not only its actual concentration). That is why for most osmotic stress studies small molecule osmolytes are used such as glycerol, ethylene glycol, sucrose, or salt are used.
Christopher Barbieri,
Thank you very much for reply. I am using PEG6000 in my media. But if it interacts with water and reduce watrer effective concentration, will it not provide stress of water to my organisms??. I also have checked the substrate consumption rate in my organism (Cell free extract and membranes also). I found same activity in cell free extract but lower activity in membranes in comparison to control sample. But the product formation rate is higher in stressed sample. Can i say that more substrate is oxidized by periplasmic or cytosolic enzymes??
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology