I whole-heartedly agree with Richard. I have used the Syto/PI bacterial kit since its creation, and I am quite pleased with its overall utility. I would add that in some instances, one can see binding of both dyes to single cells: a sort of indeterminate metabolic state. Furthermore, I strongly suspect that many cells which stain "dead" can in fact be cultured. Bacteria, especially in their natural habitats (i.e., NOT in pure cultures grown on laboratory media), do not fit well into pigeonholes of live vs dead. >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 Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 308 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396
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