I am trying to use the protocol outlined by Dominenco to extract capsular polysaccharides from Klebsiella pneumoniae. The samples are then used to determine uronic acid concentration as an indirect method of measuring capsule.As of now I am using the protocol in the attached file. However, this protocol is yielding inconsistent results. I have seen some variations on the methods and am wondering which is the most precise and accurate. Variations are as follows:
-Dissolve the pellet in 200uL of 100mM HCl as opposed to DI
-Use concentrated sulfuric acid to make the sodium tetraborate solution
Also using this protocol there has been no visual difference in color once the m-hydroxydiphenyl solution is added. Is there anyone who has performed this protocol before that can tell me if there should be a visible color change or is it only detectable by a spectrophotometer?
Any advice would be greatly appreciated.
Debra - yup, these are difficult assays. Kleb is a pretty consistent capsule former, but we've noted variability as a function of phase of culture growth and how mechanically forceful the growth conditions are (presumably causing capsular shredding). My lab published several papers looking at capsular- and O-antigen Kleb mutants. Two other authors' work you should know about in this arena are Chris Whitfield at the University of Guelph and Christiane Forestier at the University d'Auvergne in Clermont-Ferrand. They're both brilliant, very experienced with the vagaries of Klebsiella extracellular carbohydrate behavior, and are both very approachable.
I've attached a handful of papers from my shop - there may be some procedural help contained within. Hopefully it can be of some assistance. Best, John
Article The Klebsiella pneumoniae O Antigen Contributes to Bacteremi...
Article Contribution of the Klebsiella pneumoniae Capsule to Bacteri...
Article Multicellularity and Antibiotic Resistance in Klebsiella pne...
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