I grow my bacteria in a define culture medium (C-limited) in 96-well plates. I tried TTC assay, but it doesn't seem to work well. I have to add extra glucose to intensify the reaction and the red precipitate is hard to dissolve for measurement.
Assuming you want a measurable indication of generic activity:
a. Use microelectrodes to determine O2 concentration profiles inside the biofilm.
b. Check for O2 changes in a small, closed recipient. You could add an energy source (sugar?) to estimulate activity.
c. Use a respirometer (if there's some left alive somewhere..)
How about Alamar Blue Assay? Maybe check these publications:
Pettit, R.K.; Weber, C.A.; Kean, M.J.; Hoffmann, H.; Pettit, G.R.; Tan, R.; Franks, K.S.;
Horton, M.L. Microplate alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob. Agents Chemother. 2005, 49, 2612–2617.
Mariscal, A.; Lopez-Gigosos, R.M.; Carnero-Varo, M.; Fernandez-Crehuet, J. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm. Appl. Microbiol. Biotechnol. 2009, 82, 773–783.
Cells trapped in biofilm can be easily recovered by a mild sonication treatment. Once the cells are extracted you have access to the whole panel of reagents available for metabolic activity characterization (bis-oxonol, cfda, CTC,....). The interest rely on the fact that the extracted cells can be analyzed by flow cytometry in order to get a quantification of the population heterogeneity inside biofilm (localization can be performed in a second time by confocal fluorescence microscopy)
Not sure of your sample origin, but if you can/want to do an in-situ determination you can use the fluorescein diacetate method. It has been used for in-situ determinations of activity in subsurface flow wetland systems.
McHenry, J., Werker, A., 2005. In-situ monitoring of microbial biomass in wetland mesocosms. Wat. Sci. Technol. 51, 233–241.
-> or for a recent applied example:
Ecological Engineering 37 (2011) 666–677
I don't recommend the alamar blue (resasurin method) assay. I am also in the process of optimizing some redox assays for biofilm metabolism. I'm not sure what your model is, but alamar blue being so similar to phenazines that it is often the case that it becomes expelled from the cell by RND efflux pumps and stuff. The TTC assay should work as a measure of electron transport chain activity, and for assay of dehydrogenase activity or NADH reduction I would recommend the CTC assay, which is also a tetrazolium assay. But this should be given alot of attention to, as both oxidized and reduced reagent can be toxic to cells. What concentration of reagent are using to supplement your agar? I have read that generally concentrations of 1-5nM are used. Its simple, but no trivial protocol. There is always going to be some level of troubleshooting to use these in conjunction with bacteria, and especially with biofilms, as there just isn't tons of precedence there.
I believe resazurin is a quick and simple visual method (pink to blue) for metabolic activity. I've used it before. You can see that the pink (anaerobic/no metabolic activity) levels as a comparison to mutants etc. You can observe how fast the color changes indicating the amount of metabolic activity but you have to observe the reaction immediately. After a while (15-30 mins) I think all would turn blue.
Hi.
Do TTC mean 2,3,5- triphenyltetrazolium chloride or titanium tetrachloride? Can TTC and CTC be used for fluorescence microscopy? If so, at what wavelength? Which filter should be used? I am using Nikon Eclise Ti fluorescence microscope.
Can someone provide details for biofilm sample freezing and sectioning? Have anyone tried vertical sectioning of biofilm samples, grown in microplates? I would also like to know how to calculate the diffusion co-efficients of molecules in biofilms.
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