The biofilm samples will be first dried, resuspended in 1 N NaOH, and heated at 80°C for 30 min in a water bath.
The samples will be then centrifuged at 10,000 rpm and 4°C for 15 min.
The supernatants will be collected for polysaccharide quantification.
The polysaccharide content will be measured using the anthrone method.
1. 1ml sample +1ml anthrone (0.2% in 75% H2 SO4)
2. 100 C in water bath 20mins
3. 10 mins cooling in refrigerator
4. OD at 630nm.
Do I need to centrifuge before the OD? It has lot of precipitate.
I used it before with some modifications, for OD measurement I used microplate reader, So I just take 200ul by the pipit to each well of 96 plates.
It is must be centrifuge before readering OD if it has precipitate. But this phenomenon has never happened before in my experment .
I use Somogyi-Nelson method for quantification of glucose. The procedure is almost similar for Anthrone method. Since these are spectrophotometric methods, its better to centrifuge before OD measurement, if any precipitation occurs. So that we can avoid any experimental errors.
Thank you so much for all your suggestion to improve my results.
1. In any case NaOH make any reaction with H2 SO4 to make precipitation? (Some chemistry people may be help with this)
2. Or may be Trypticase soy broth i am using for the biofilm, is this makes precipitation?
After using NaOH in biofilm, I could see turbid biofilm turns into clear solution after water bath process.
I am using well plates and microcentrifuge tube for my assay
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