Microbiology Focus. Fluorescence Assays in Microbiology. Volume 5.3, FluoroSELECT Assays Fluorogenic Media... 4

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1 Micobiology Focus Volume 5.3, 2013 Fluoescence Assays in Micobiology FluooSELECT Assays Fluoogenic Media Fluoescence assays ae inteesting tools used fo the detection of micooganisms. Fluoogenic substates and fluoescence labelling of oganisms ae possible, as ae sensitive methods of detection eithe by using a fluoescence eade, viewing the sample unde the fluoescence micoscope, o by simply viewing the sample diectly with the naked eye. Quantitative Cetified Contol Stains Image fom CDC: Mixed cultue of micooganisms stained with fluoescent antibodies (1959)

2 FluooSELECT Assays Jvo Siegist, Poduct Manage Micobiology FluooSELECT Assays FluooSELECT is a apid test based on an assay with fluoogenic substates. Simplified testing pocedues: 1. A cotton swab is used to wipe a paticula suface of the detection aea. The swab is then inseted into 0.5 ml of distilled wate fo an immediate test, o into 0.5 ml of incubation solution fo an incubation peiod anging fom 3 to 10 hous at 38.5 C, befoe measuing. Add an enzyme induce afte inseting the swab into the wate o incubation solution. The FluooSELECT Assay system is a pesumptive sceen intended fo the apid detection of E. coli and Gam-negative bacteia fom a suface swab sample using the FluooSELECT handheld fluoomete and the fluoescent assay. It can also be used as a confimation test afte using a classical o moden method. E. coli is commonly found in the lowe intestine of wam-blooded oganisms and is theefoe an indicato fo fecal contamination. Most stains ae hamless, but some seotypes can cause seious food poisoning in humans, and ae occasionally esponsible fo poduct ecalls due to food contamination. Gam-negative bacteia ae bacteia that ae defined by thei inability to etain cystal violet dye in the Gam staining potocol. This test was and still is vital fo the diffeentiation and the classification of bacteia. Compaed with Gam-positive bacteia, Gam-negative bacteia ae moe esistant to antibiotics because of thei impenetable cell wall. FluooSELECT utilizes specific fluoogenic substates which ae cleaved fom chaacteistic enzymes of E. coli o Gam-negative bacteia. Duing the typical peptide hydolysis, the specific enzymes hydolyze the fluoogenic substates and poduce a fluoescence which is ead by a potable, low cost fluoomete. The excitation wavelength is at 360 nm (UV), while the emission wavelength is at 460 nm (blue light), the intensity of which is then measued to decide if the sample is positive o negative. It is a apid test since the assay takes only 30 minutes, although it may equie some incubation time. Because the enzymes ae specific, the assay is highly specific and obust. In addition, since it is a typical fluoescence assay, it is also highly sensitive and small amounts of the cleaved fluoogen will aleady have been detected. The detection pefomance fo both tests is immediate detection of 100,000 cfu (colony foming units), while 100 cfu ae detected in 8 hous, and 1 cfu is detected within 10 hous of incubation time. 2. Then add a lysing agent to the solution and let incubate fo 5 minutes. 3. Next, a substate is added to the solution and allowed to stand fo 5 minutes. 4. Aftewads, the solution is tansfeed into a glass test tube and then inseted into the Fluoomete. The fist measuement is taken immediately, and afte 20 minutes, anothe measuement is taken. The findings can then be used to detemine a positive o negative esult. 2 sigma-aldich.com

3 Did you know Cat. No Desciption FluooSELECT E. coli Assay Kit Qty. FluooSELECT Gam-Negative Assay Kit Z FluooSELECT single channel fluoomete (λex 360 nm; λem 460 nm) 1 ea. The oute membane of Gam-negative bacteia contibutes to this intinsic esistance by acting as an efficient pemeability baie, because the naow poin channels limit the penetation of hydophilic solutes and the low fluidity of the lipopolysacchaide leaflet slows down the inwad diffusion of lipophilic solutes. Z Glass vials fo FluooSELECT fluoomete (O.D. L 6 mm 25 mm, volume nominal capacity 200 μl) 100 ea. [Souce: P. Plésiat, et al., Mol. Micobiol. 1992] Table 1: Poduct list of micobiology FluooSELECT kits and equipment Thee ae moe enzymatic tests available fo quantitative detection of acetate, ammonia, ascobic acid, fomaldehyde, glyceol and lactose with the FluooSELECT system. Visit sigma-aldich.com/fluooselect o efe to Table 2. Cat. No Desciption FluooSELECT Ammonia Kit FluooSELECT Assays Gam-negative bacteia ae geneally less sensitive to antibiotics? Qty FluooSELECT Fomaldehyde Kit FluooSELECT Glyceol Kit* FluooSELECT Ascobic acid Kit* FluooSELECT Acetate Assay Kit FluooSELECT Lactose Detection Assay* Table 2: Non micobiology FluooSELECT assays *Inquie on availability. Figue 1: Gam-negative bacteia cell wall Micobiology Special Catalog This is the pemiee Sigma-Aldich catalog fo micobiology, and it contains ove 900 specific micobiology poducts. The tests, media, eagents, micooganisms and base ingedients ae soted to help micobiologists find the best test, media and additives fo the desied micobiological analysis. Micobiology Special Catalog Contents: Micooganisms Standads (Vitoids ) Poficiency Testing Biological, Immunological, Molecula Biochemical Tests Micoscopical Kits and Reagents fo Specific Oganism, Special Media Application, o ISO Guideline Supplements Base Ingedients fo Media - Media by Oganisms by Applications by ISO Applications Supplements Base Ingedients & Tests - Micoscopy Reagents & Kits - Micooganisms Standads & Testing Reseve you catalog via eply cad o place you equest online.* sigma-aldich.com/micobiology-cat *only available as long as supplies last 3

4 Fluoogenic Media Jvo Siegist, Poduct Manage Micobiology Fluoogenic Media Fluoogenic media combine taditional methods with new knowledge fo beneficial esults. Taditional validated media vesus impoved media fomulations containing chomogenic and fluoogenic substates is an impotant topic in the field of micobiology. The focus behind such developments is the poduction of media that would make the detection and identification of micooganisms moe apid and moe eliable. Divese chomogenic and fluoogenic substates such as ONPG, X-Gal, X-Glu MUP o MUG ae available and ae aleady combined in today s media fomulations. Fluoogenic substates have the disadvantage of equiing a UV-lamp, but the fluoogenic media ae moe sensitive and give an ealie detectable esult. They can also be combined with a chomogenic system without intefeence. These easons give such media additional value, and togethe with the specified selectivity of the medium and the substate, new possibilities ae available to solve poblems in a smat way. Did you know about upconvesion luminescence? In nomal cases, the colo of emitted light equies a highe enegy light souce (in most cases UV light, invisible to the human eye). But thee ae also some exceptions. Now it is possible to have an upconvesion luminescence whee infaed is used as the excitation wavelength. sigma-aldich.com/upconvesion γ Enegy Ultaviolet UCP 2γ Visible Infaed Figue 3: Upconvesion Luminescence The pinciple behind fluoogenic media is simple - the taget oganisms ae chaacteized by enzyme systems that metabolize the substates to elease the fluoogen. The fluoogen can then be visually detected by diect obsevation unde UV lamp. Diect confimation of the taget oganism without futhe testing is sometimes possible. Today, it is also possible to detect and diffeentiate moe than one oganism on the same plate in combination with chomogenic substates and the help of selective media. Figue 2: Fluoogenic media containing E. coli stains and Pseudomonas putida (ight at the bottom) and Lactobacillus spp. (left). 4 sigma-aldich.com

5 4-Methylumbellifeyl galactoside + H 2 O 4-Methylumbellifeon (fluoescent at 360 nm) + Galactose Figue 4: An example of a fluoogenic eaction. In the pesence of a β-galactosidase positive oganism, the 4-methylumbellifeyl galactoside (MUG) is split and esults in the fluoophoe and in fee galactose. Advantage of fluoogenic media: Faste esults (compaed to taditional methods) Reliable visual detection (often no futhe testing equied) Additional testing possible diectly fom the media In ecent yeas, geat stides have been made in the secto of fluoescence. Thee ae dyes, labels and substates which have a stonge fluoescence and ae moe stable. Additional advancements in the knowledge of enzyme and species specificity have also occued within the past yea. These ecent gains in the development of selective agents and divese fluoescence compounds have led to an impessive ange of fluoescence tests. Fluoogenic Media Cat. No. Medium Desciption Plate Count MUG Aga Fo detemination of plate count of micooganisms in milk and othe daiy poducts and especially E. coli by fluoogenic method VRB MUG Aga Selective medium fo the detection and enumeation of colifom bacteia, in paticula E. coli. Gam-positive accompanying floa ae extensively inhibited by cystal violet and bile salts. A colochange to ed indicates lactose-positive colonies MacConkey MUG Aga Fo the isolation of Salmonella, Shigella and colifom bacteia, in paticula E. coli, fom divese mateial. Bile salts and cystal violet extensively inhibit the Gam-positive floa. The pesence of lactose and neutal ed indicate lactose-positive colonies fom which E. coli can be identified by fluoescence in the UV HiFluoo Pseudomonas Aga Base Used as a selective medium fo the isolation of Pseudomonas aeuginosa fom pus, sputum and dains etc. Pseudomonas aeuginosa beaks the fluoogenic compound to elease the fluoogen which poduces a visible fluoescence unde long wave UV light LST-MUG Both Fluoescent method fo the detection of E. coli BRILA MUG Both* Bile and billiant geen extensively inhibit the gowth of accompanying floa, in paticula Gampositive micooganisms. The pesence of E. coli esults in fluoescence in the UV E. coli O157:H7 MUG Aga* Selective aga fo the isolation and diffeentiation of enteohemohagic (EHEC) E. coli O157:H7- stains fom food and clinical mateial HiCome Rapid Colifom Both Used fo detection and confomation of E. coli and colifoms on the basis of enzyme substate eaction fom wate samples, using a combination of chomogenic and fluoogenic substate HiCome ECD Aga with MUG* Fo the detection of E. coli in wate and food samples by using a combination of chomogenic and fluoogenic substate ECD MUG Aga* The bile-salt mixtue in this E. coli Diect Aga extensively inhibits the non-obligatoy intestinal accompanying floa. Fluoescence in the UV demonstates the pesence of E. coli. M1678 MUG EC Both Used fo the detection of E. coli by a fluoogenic pocedue MUG Typtone Soya Aga Fo cultivation of fastidious and nonfastidious micooganisms by fluoogenic method. Table 3: Fluoogenic media (*not available in U.S.A.) 5

6 Vitoids The Quantitative and Qualitative Contol Stains Jvo Siegist, Poduct Manage Micobiology Vitoids The Quantitative and Qualitative Contol Stain Contol of test esults, o geneal testing pefomance, is cuently an impotant issue in micobiology. Oganizations like ISO and UKAS eithe have egulations o ecommendations to egulaly veify the accuacy of vaious test methods. One eally impotant equiement is to use cetified micooganism standads, o if not available, at least a efeence stain fom a ecognized cultue collection like ATCC, DSMZ and NCTC. The test stains should show all elevant popeties used fo the applied application. Accoding to ISO , it is possible to cultue efeence stains one time to poduce efeence stock cultues which ae then contolled fo puity and fo use in biochemical tests. They should be stoed in a feeze o in a feeze-died fom in small aliquots; howeve, defosted cultues should not be efozen. It is pefeable fo woking stain cultues to be made out of stock cultues, and they should not be subcultued again. In UKAS LAB 31 it is witten that to confom with ISO/IEC 17025:2005, it is necessay to use, whee possible, a taceable contol stain which is cetified accoding to ISO The stains should be fom a ecognized national cultue collection o fom a efeence mateial poduce accedited to ISO Guide 34. Fo detailed pefomance testing, oganizations such as ISO ecommend special potocols, an example being media testing unde ISO/TS Refeence Stain Fom an official stain collection (e.g. ATCC, NCTC, DSMZ) Defined, descibed and cataloged, at least down to the genus and species Stock Cultue Pepaed fom a efeence stain Woking Cultue Poduced fom a stock cultue and used fo contol Figue 5: Definition of ISO fo contol stains Micooganisms in Discs Sigma-Aldich s innovation, small discs called Vitoids, is a solution which suppots all of these equiements. The patented technology allows the poduction of stable and highly eliable cetified micooganisms. The discs contain a defined cetified numbe of colony foming units (CFU) fom defined bacteia, molds and yeasts fom a ecognized cultue collection like ATCC and NCTC. The discs ae easy-to-use since they can be placed diectly in wate, diluent, both o even on aga plates. Figue 6: Classical plate count The Vitoids contain highly viable bacteia, and when placed in contact with media, they dissolve apidly and stat to gow without a lag-phase. The viability of the CFU in a disc is stable fo at least one yea (fo most oganisms, moe than two yeas) when kept at -20 C. It is not a poblem if the poduct is tanspoted at ambient tempeatue o if it is kept fo a shot time in the efigeato. The discs ae poduced unde the ISO guideline 34 and ae cetified accoding ISO Each disc is packed in an individual tube with some desiccant and the tubes ae then packed in myla foil. Each package comes with a compehensive cetificate of analysis epoting the CFU and standad deviation. All of the above mentioned featues help micobiologist to have eliable esults, save a lot of time (labo, documentation) and lowe costs. Refeence micooganisms in cetified and defined colony foming units (CFU) Standads in concentations of 30 50,000 CFU pe disc Poduced acc. ISO Guide 34 Cetified acc. ISO Deliveed with detailed cetificate of analysis Refeence stains fom ATCC, NCTC, etc. Minimum one yea shelf life at -20 C (usually two yeas) No lag-phase Amazingly low standad deviation (e.g. 100 CFU+/- 4%) 6 sigma-aldich.com

7 The Vitoids ae packed in micovials and ae lens shaped. They ae coloed and can easily be seen on the top of the filte plug. Below the filte plug is a desiccant which should be yellow. Clea (opaque) desiccant indicates the silica gel capacity has been depleted and is no longe effective. The tubes ae packed in a esealable aluminium foil bag. Scew Cap Vitoid Disc Filte Plug Desiccant Figue 7: Vitoids in the vial Pepaation Rehydate the disc with a common phosphate buffe, o place the disc onto a solid o into a liquid medium. The ehydation pocess takes appoximately 10 minutes. On solid media, the disc foms a doplet that can be spead with a loop. Liquid media may simply be shaken to dissolve the disc. The discs can be ehydated in as little as 100 μl of wate (pefeably a buffe) o added into lage volumes, e.g. 100 ml, fo geneal wate testing methods (MF, MTF, Quanti-Tay, etc). It is also possible to add the disc to the media fo pou plate techniques. Befoe Rehydation 0.5 ml Afte Rehydation 0.5 ml Figue 8: A Vitoid just put into buffe (left) and afte 10 minutes it is completely dissolved (ight). Befoe Rehydation plate Afte Rehydation plate Vitoids Test Stains Oigin Stain # CFU Cat. No. Aspegillus basiliensis ATCC RQC15003 Bacillus subtilis ATCC ,000 RQC02258 Bacillus subtilis ATCC RQC16003 Candida albicans ATCC RQC14003 Citobacte feundii ATCC RQC02105 Clostidium pefingens NCTC RQC02351 Clostidium pefingens NCTC RQC02355 Clostidium pefingens NCTC RQC20106 Clostidium spoogenes ATCC RQC19003 Enteobacte aeogenes ATCC RQC01652 Enteobacte aeogenes ATCC RQC01655 Enteobacte aeogenes ATCC ,000 RQC01657 Enteobacte aeogenes ATCC ,000 RQC01658 Enteococcus cloacae ATCC RQC21102 Enteococcus faecalis ATCC RQC01772 Enteococcus faecalis ATCC RQC01774 Enteococcus faecalis ATCC RQC01775 Enteococcus faecalis ATCC ,000 RQC01777 Escheichia coli ATCC RQC01702 Escheichia coli ATCC RQC01705 Escheichia coli ATCC ,000 RQC01707 Escheichia coli ATCC ,000 RQC01708 Escheichia coli ATCC RQC11003 Heteotophic Oganisms 100 RQC02504 Legionella bozemanii NCTC ,000 RQC02908 Legionella pneumophila NCTC ,000 RQC02008 (seogoup 1) Listeia monocytogenes ATCC RQC01901 Pseudomonas aeuginosa ATCC RQC02202 Pseudomonas aeuginosa ATCC RQC02204 Pseudomonas aeuginosa ATCC RQC12002 Pseudomonas aeuginosa ATCC RQC12005 Pseudomonas aeuginosa ATCC ,000 RQC12007 Salmonella enteica subsp. NCTC RQC18003 Enteica seova Abony Salmonella enteica subsp. ATCC RQC17002 Enteica seova Typhimuium Salmonella goldcoast NCTC RQC02301 Staphylococcus aueus ATCC RQC13002 susp. Aueus Staphylococcus aueus ATCC RQC13005 susp. Aueus Vitoids Blank 0 RQC0001 Vitoids The Quantitative and Qualitative Contol Stain Figue 9: A single Vitoids disc on an aga plate. Afte about ten minutes on a plate, it is ehydated automatically and foms a doplet (no wate addition is needed). The dop can be spead with a loop. Table 4: Vitoids potfolio ATCC Licensed Deivative Many of RTC s Vitoids micobiological standads ae offeed as Quality Contol micobiology poducts unde the Ameican Type Cultue Collection (ATCC ) Licensed Deivative pogam. Look fo the ATCC Licensed Deivative emblem fo poducts deived fom ATCC cultues. The ATCC Licensed Deivative emblem signifies ATCC-deived poducts ae endosed by ATCC. Poducts displaying the emblem ae the only ones fo which the quality of the ATCC ingedient can be popely assued. ATCC does not endose poducts including ATCC ingedients fom companies that ae not membes of the LDP pogam. The ATCC Licensed Deivative emblem, the ATCC Licensed Deivative wod mak and the ATCC catalog maks ae tademaks of ATCC. RTC is licensed to use these tademaks and to sell poducts deived fom ATCC cultues. 7

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