We are trying to measure the reduction in glucose concentration in a bacterial culture as the OD increases. For this, I grew my cells at a starting OD of 0.11 and let them grow to an OD of 1.2. I took 1 ml aliquots at regular intervals, spun the cells down and froze the supernate. Next, I tried to measure the glucose concentration in the supernate with a glucose oxidase kit from Sigma Aldrich (http://www.sigmaaldrich.com/catalog/product/sigma/gago20?lang=en®ion=US) . The problem I am facing is that the kit does not have a good dynamic range. So I am not able to rely on the results that I am getting. Can anyone suggest a good kit with a broader range that I can use with bacterial cultures? Thank you!
I do use DNS method for glucose estimation in culture medium. This DNS method is for estimation of reducing sugars, not specifically for glucose estiamtion
Hi there,
What do you mean by "the kit does not have a good dynamic range"? This coupled assay is the one I use during practical courses without any problem to measure glucose depletion in yeast media (from 10g/L to 0)...
Thank you for your answers. I will look up DNS assay.
Dominique, what I mean is that the standard curve of this kit ranges from 20 to 80 ug/ml only. In terms of absorption, the standard range goes from 0.1 to 0.35. That is too small for my system. I need an assay that has a bigger range which allows me to measure depletion more accurately.
Hi again,
I still don't see where the problem is as just for any other spectrophotometric determination the reading has to be made within the linearity domain (here it's 0 to 80µg of glucose in sample and the response is 0 to 0.35, by the way you should be able to trace a straight line from standard samples data).So wathever the width of this linearity domain, it doesn't change the basic rule. And finally including a necessary dilution step of concentrated samples prior to determination isn't a problem. For instance I guess that to follow your bacterial culture growth you have to dilute the sample at some point before reading the OD at 600nm(depending on the spec but roughly when undiluted OD is beyond 0.5-0.6) ... So if you do it for cell growth monitoring, why not doing it for any other determination?
Of course, we dilute our samples to fit them in the linear range. We dilute them 25, 50, 75 and 100-fold. While 25-fold diluted samples are out of the standard curve, 50 and 75-fold are within the curve. The problem is that both 50 and 75-fold diluted samples give similar numbers for all time points (around 40 ug/ml). Hence it is indicating that there is no reduction in glucose concentration when OD changes from 0.11 to 1.2. This is completely against existing literature and we want to make sure that the results we are seeing are real results and not artifacts due to the kit's limitations. I am sorry I can't divulge more details on a public forum but I hope this helps.
It sounds very weird... What diluent are you using? What is the starting concentration of glucose? Does it correspond to the amount detected?
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials
- Polymers