Transcript الشريحة 1
NEW DEVELOPMENT IN CHROMOGENIC AND FLUOROGENIC CULTURE MEDIA Prepered by A.ElKader ElOttol Supervisor Abdelraouf A. Elmanama (PhD. Microbiology) 1 • New techniques have been developed for detection and differentiation of bacteria. • Based on utilization of chromogenic and fluorogenic substrates or detection of activities of specific enzymes. • The incorporation of such substance into a selective media can eliminate the need for subculture. 2 Detection of enzymatic activity • Three groups of fluorogenic and chromogenic reaction have been used 1. Hydrolysis of synthetic subestrate by bacterial enzymes causing considerable increase in the fluorescence. e.g Coumerin derivatives of 4methylumbelliferone ( 4-MU) . 3 2. Change in the fluoresence or absorbance of PH indicator which caused by specific enzymatic activity. e.g an increase in the PH due to urease activity. Example of indicator include 4-MU for PH increase and quinine for decrease PH. 4 3.Change in intensity of fluorescence as a result of absorbance of fluorescent dye onto some componant of the bacteria cell. e.g acridine orange(AO) binding to DNA and 8-anilino-1-naphthalene sulfonic acid (ANS) binding to protein. 5 6 Detection of Activity of Individual enzymes • Glycosidases • β-D-Glucuronidase (GUD) Catalze the hydrolysis of β-D-glucopyranosiduronic (GLR) derivatives into their corsponding aglycons and Dglucuronic acid. Can be measured by using different chromogenic and fluorogenic substrate e.g release of phenolphthalin from a phenolphthalin-mono-β-D-glucuronide complex (PHEGLR) ,PNP from p-nitrophenol- β-D-glucuronide (PNPGLR). 7 β-D-Glucuronidase (GUD) con… The most commonly used substrate is 4methylumbelliferyl- β-D-glucuronide (MUGLR) which hydrolyzed by GUD yielding (4methylumbelliferone 4-MU) , show blue fluorescence when irradiated with long wave UV light(365). 94-96% positive in E.coli. Few strain of Shigella , Salmonella and Yersinia positive. 4-MU PH dependant . 8 9 10 β-D-Galactosidase (β -GAL) . • β-D-Galactosidase (β-GAL) trivially called lactase, catalyzes the breakdown of lactose into galactose and glucose. • used mostly for enumerating the coliform group. • The activity of β-GAL was determined by using substrates as: o-nitrophenyl-β,-D-galactopyranoside (ONPG) p-nitrophenyl-β-D-galactopyranoside (PNPG) or 6bromo-2-naphthyl-β-D-galactopyranoside (BNGAL) . 11 β-D-Galactosidase con….. • The tendency of chromogenic nitrophenolic substances to diffuse through solid media was observed with both ONPG and PNPGAL. Therefore, agars containing these substrates cannot be used . • 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (XGAL) is preferred for the rapid detection of coliforms . • o-nitrophenyl-,-D-galactopyranoside (ONPG) break down and give yellow color. 12 13 14 Media for Stimultaneous Detection of E.coli and Coliforms • Commercially available media that permit rapid simultaneous detection of E.coli and coliforms in water. • EMXID agar is a diagnostic medium and provides an inexpensive means of rapid identification of Enterobacteriaceae. • It can detect β-D-galactosidase, β-D-glucuronidase, β-D-xylosidase, tryptophanedeaminase and cysteine desulfhydrase . • Oxidase and indole production can also be demonstrated. 15 COMPOSITION 16 17 18 Salmonella spp. • Rapid detection of Salmonella using fluorogenic and chromogenic media. Rambach agar • Rambach agar is composed of propylene glycol, peptone, yeast extract, sodoum deoxycholate,neutral red, and XGAL. • The formation of acid from propylene glycol causes precipitation of the neutral red in Salmonella colonies yielding a red color. • Salmonella strain show a bright red color, coliform blue(βgalactosidase activity) or violet (the formation of acid from propylene glycol and β—D-galactosidase activity) and Proteus remain colorless. • Sodium deoxycholate inhibits the growth of Gram positive. 19 The main disadvantage of Rambach agar is that it doesn’t detect S.typhi or S.paratyphi. 20 21 Esterases and Lipases • • Esterases hydrolyze molecules with shorter chain organic acids, whereas lipases are capable of acting on derivatives of longer-chain acids. For detecting these enzymes, they used fluorescein derivatives butyryl, hexanoyl, heptanoyl, nonanoyl, palmitoyl, andoleyl esters of 4-MU. • A plate assay was further designed to detect bacterial lipases in a medium containing trioleylglycerol and the fluorescent dye rhodamine B. • Substrate hydrolysis causes the formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation. • Butyrate esterase has been found in cultures of Branhamella catarrhalis, absent from other members of the family Neisseriaceae negative. 22 • Sprit blue dye icorperated to olive oil in media which give the agar an opaque blue appearance. • When olive oil hydrolyzed a clear zone a round the growth will appear 23 24 MUCAP • MUCAP is confirmation test for Salmonella species based on rapid detection of caprylate esterase using 4-methylumbelliferyl-caprylate. • In the presence of C8 estrase the substrate is cleaved with the release of 4-methylumbelliferone (4-MU), which produced strong blue fluorescence when excited by UV light source. • One drop of of MUCAP add to each colony tested on columbia agar and observed under UV light for (1-5 min). 25 DNases • Tests commonly used for DNase activity based on hydrolysis of natural DNA. • detection of hydrolysis of DNA by flooding the incubated plate with 1 N HCI was modified. • Modifications of this, involving tolidine blue and methyl green, have advantages because they do not require the addition of reagents after plates are incubated. • Methyl green dye and polymerized DNA form a complex that gives the agar a blue green color . • Production of the enzyme will hydrolyze the DNA, unbound the methyl green , give clear area around the colony. 26 27 Peptidases and Proteases • Pyroglutamyl aminopeptidase (PYRase). Substrates used for detection of PYRase activity include Lpyrrolidonyl-β-naphthylamide( PYR) , L-pyroglutamyl-pnitroanilide , and L-pyroglutamyl- 7-amido-4-methylcoumarin. all of the Enterococcus faecalis strains, 90% of the E. faecium strains, and 96% of the Streptococcus bovis, and Group A Streptococcus positive. Rapid method for detection of PYRase by using impregnated paper strips with PYR and after incubation add of pdimethylaminocinaldehyde reagent. Formation of deep red color indicate positive test. 28 Gram differentiation • Enzyme substrate used to distinguish Gram positive from Gram negative bacteria. • Based on L-alanine-aminopeptidase activity of the Gram negative bacteria that act on the substrate L-alanine-7-amido-4-methylcoumarin (AAMC). • Give blue long wave UV light. • Anther substrate used the fluorgenic protein specific dye, 8-anilino-1-naphathalene sulphonic acid . 29 Summary Enzyme Action Methods Result GUD Catalze the hydrolysis of(GLR) derivatives into their corsponding aglycons and Dglucuronic acid. MUGLR) which hydrolyzed by GUD yielding (4-MU) blue fluorescence when irradiated with long wave UV light(365). β -GAL catalyzes the breakdown of lactose into galactose and glucose. o-nitrophenyl-β,-Dgalactopyranoside (ONPG) (ONPG) break down and give yellow color. Esterases and Lipases Esterases hydrolyze molecules with trioleylglycerol and shorter chain the fluorescent dye organic acids, whereas lipases are rhodamine B . capable of acting on derivatives of longer-chain acids orange fluorescent halos around bacterial colonies visible upon UV irradiation. 30 caprylate esterase caprylate esterase using 4-methylumbelliferylcaprylate 4methylumbelliferone (4-MU), strong blue fluorescence with UV. DNases hydrolysis of natural DNA. tolidine blue and methyl green hydrolyze the DNA, unbound the methyl green , give clear area around the colony. PYR hydrolysis impregnated paper strips with PYR and after incubation add of pdimethylaminocinald ehyde reagent. deep red color indicate positive test. PYRase 31 Discussion and Conclusions • Advantages Can eliminate the need for subculture and further biochemical test to establish the identity of certain microorganisms. • Disadvantages Expensive. Some compound are unstable and some are water insoluble. Media used for primary isolation may inhibit the synthesis of enzymes of interest. 32 33