We can estimate the blood glucose ,starch content of plants,like that is there any methods by which we can estimate the glucose content of bacteria?Actually I want to know about the mode of action of a plant extract which have antimicrobial activity,so by estimating the sugar content I can screen either the plant extract obstruct the carbohydrate metabolism or not.
Hi Sumana,
For most high-throughput assays, an enzyme cocktail oxidizes glucose to generate a product which reacts with a dye to generate color and fluorescence. The generated color and fluorescence is proportional to the glucose amount. These types of assays are suitable for monitoring glucose level during fermentation and glucose feeding in protein expression processes, and detect 1-10000 µM glucose samples.
I f you want to see intracellllular glucose level then fist go for sonication of cell mass then DNS test.
I SUGGEST BREAKDOWN CELL STRUCTURE BY CENTRIFUGATION/SONICATION ETC FOLLOWED BY SUGAR REAGENT TEST LIKE OSAZONE TEST AND UNDER U.V ANALYSIS STUDY ITS QUANTITY BY COMPARING WITH STANDARDS
Take dry wet /live count as toolfor bacterial number/density study
Hi Sumana,
For most high-throughput assays, an enzyme cocktail oxidizes glucose to generate a product which reacts with a dye to generate color and fluorescence. The generated color and fluorescence is proportional to the glucose amount. These types of assays are suitable for monitoring glucose level during fermentation and glucose feeding in protein expression processes, and detect 1-10000 µM glucose samples.
@Kettner what sort of enzyme cocktails are you suggesting..? One more question added to the topic, will this technique be helpful in estimating the glucose content associated with structural components as well..? Or do you suggest sonicating until the final leg where all structural components are neutralized.
I was meaning other organelles, mitochondria, microsomes.. We do have glucose concentrates in these organelles as well. So my question is - should we sonicate to the level of shredding these organelles as well to estimate the level of glucose via the enzyme cocktails which you mentioned? Or would it estimate only the level of free blood glucose.
It seems that you are concerned with the plant cell remnants in your extracts. I am concerned that the glucose signal you are looking for from the bacteria will not be sufficiently strong if you proceed otherwise. To ensure that organelles will not be involved in the reaction I suggest you use a .22 micron filter to purify your extract prior to inoculation. After a growth phase, centrifuge out the bacterial pellet, then lyse with buffer and sonication if necessary. After these steps the lysate should be sufficient to measure bacterial glucose using the enzyme cocktail described. It will be necessary to assess the background glucose concentration when computing your results.
See http://www.biovision.com/glucose-assay-kit-2902.html
I am unclear whether you are interested in free glucose (for which the enzyme assay is a good option) or free + conjugated glucose, in which case a monosaccharides analysis by GC might be a better option for you. The Complex Carbohydrate Research Center in Athens GA does such analyses on a fee for service basis and they may be able to help you in planning your experiments.
If you want to see intracellllular glucose level then fist you should "kill" all enzyme activities then go for cell rupture otherwise your intracellular sugar will be always close to zero.
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