If you are willing to use a different definition of bacterial viability --- membrane permeability --- then I strongly suggest trying the BacLight Viability kits instead of CFDA, SE, a reagent whose use is frought with all kinds of potential problems when used as a "viability" assay, particularly in bacteria. CFDA, SE is a good probe for looking at cell replication but not particularly good when used for "viability" due, in part, to the problems that you have observed. The BacLight kits http://www.probes.com/servlets/product?region=USA&item=7012 utilize a green-fluorescent SYTO dye for the live population and red-fluorescent propidium iodide for the dead population. All one has to do is add the dyes and observe the fluorescence (or analyze by flow cytometry or in a fluorometer) a few minutes later. No wash step is required. The bacteria can be collected on a fliter before staining, if desired. Here are some pictures: http://www.probes.com/servlets/photo?fileid=g000508 http://www.probes.com/servlets/photo?fileid=g000004 http://www.probes.com/servlets/photo?fileid=g000011 http://www.probes.com/servlets/photo?fileid=g000652 http://www.probes.com/servlets/photo?fileid=g000509 and about 31 references: http://www.probes.com/servlets/bib?item=7007 This assay works in a wide variety of bacteria http://www.probes.com/handbook/tables/1502.html You will also not have to remember ANY of the "five things to remember" in the note below with this type of assay: > There are five things to remember when using CFDA-se or similar with bacteria: > > pH 8 is a very good way to cleave the dye very rapidly, thus you should use > buffers below pH 7.3 > Ensure that no free esterases are present in your system > Ensure you switch of the pumps that get the dye out of the bugs before it has a > chance to bind inside the cell > Unless you use di-chloro CFDA-SE the intracellular pH can seriously effect your > fluorescence readout. > > Don't say you measure 'viability'! Esterase activity only indicates metabolic > activity, not necessarily the ability of the cells to show reproductive growth > ! You can also use CCFDA-SE to stain any type of vesicle you load with esterase > which will turn green but is definitively not viable. Assuming from your email > address that you work for a water company, this is particularly problematic if > you want to use it in the context of UV treated water where you will create > DNA, not membrane damage, thus leaving metabolism intact to some extend whilst > preventing reproductive growth. > > regards > Gerhard > > -----Original Message----- > From: Daniel Hoefel [SMTP:daniel.hoefel@sawater.sa.gov.au] > Sent: Thursday, June 07, 2001 1:58 AM > To: Cytometry Mailing List > Subject: CFDA/SE for microbial analysis > > Dear List, > > I would like to pose a question regarding the use of 5(6)-carboxy fluorescein > diacetate, > succinimidyl ester (5(6)CFDA/SE). This question does not relate at all to the > current > CFSE discussion on the list. > > I was planning to use CFDA/SE to enumerate viable bacteria from water samples by > flow cytometry, where viability is obviously based on the presence of active > internal > esterases that cleave the molecule into its fluorescent carboxy fluorescein > derivative. > I have made a stock solution of CFDA/SE (25mM) in high quality, fresh, anhydrous > DMSO (stock solution stored in a desiccator). I have been trying to optimise > the > staining conditions by testing different buffers and varying pH (buffers > include PB, > PBS, MilliQ water and also seeing if the addition of EDTA facilitates > permeabilisation > of the cells for better staining). > > However, after the addition of CFDA/SE (both 50 and 100uM final concentration) > to the bacterial suspensions at 35oC, I noticed that after about half an hour > the > solutions started to turn a bright yellow/green colour. I thought that this > was due > to the bacteria taking up the molecule and cleaving it internally, and as a > result > some of the fluorescent product was leaking out of the cells into the > supernatant. > I examined the suspensions by epifluorescent microscopy (blue light excitation) > and I was nearly blinded by the intensity of the background (ie. the > supernatant was > fluorescent, tentatively indicating that the yellow/green colour of the > suspensions > was due to the cleaved fluorescent product of CFDA/SE). > > Having seen this, I set up a series of negative controls, ie. all the different > buffers > without the addition of the bacteria. I then added CFDA/SE at the same > concentrations as > before to the negative controls. To my surprise, after half an hour at 35oC > incubation, > the negative controls started to turn the yellow/green colour. To further > investigate > this, I set up the negative controls once again, this time in a real-time PCR > machine > set to detect fluorescein fluorescence. After the addition of CFDA/SE to the > negative > controls, the real-time PCR maching started to detect fluorescence in the > phosphate > buffer [pH 8.0] negative controls within 30 seconds. After 2 mins, all of the > buffers > were producing a fluorescent product and within 5 mins the PB solutions had > saturated > the fluorescent detector. The fluorescent intensity and rate of fluorescent > product > production was proportional to pH of the buffers where the buffers at a pH of > 8.0 > produced the fluorescent product faster than buffers at 7.5 and 7.0. > > Therefore, it seems clear to me that the addition of CFDA/SE to the aqueous > buffers is > causing the molecule to break down into its fluorescent derivative. The > manufacturer > states that if CFDA/SE is stored in the presence of even minimal water, it will > break > down over time (therefore CFDA/SE is obviously not stable in an aqueous > medium). And even > when stored appropriatley, CFDA/SE is said to only be good for 6 months but > this isn't > an issue with my work because I am using new CFDA/SE and storing it in > anhydrous DMSO. > Therefore, it appears to me that the molecule is breaking down upon the > addition of > it to an aqueous solution such as the buffers. If this is the case, obviously > I can't > use CFDA/SE for bacterial viability studies. > > I have read many papers that use CFDA/SE for bacterial viability studies and I > am > basically following the same protocol as they report to be successful. I was > therefore > wondering if anyone has seen this occur before or does anyone have any idea as > to why > this is happening or what infact is happening. > > Have I just got a 'dud' batch of CFDA/SE from the manufacturer? > > Any comments or suggestions would be appreciated. > > Daniel Hoefel
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