Post on 13-Jan-2016
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NEW DEVELOPMENT IN CHROMOGENIC AND FLUOROGENIC CULTURE MEDIA
Prepered by A.ElKader ElOttolSupervisor Abdelraouf A. Elmanama
(PhD. Microbiology)
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• 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.
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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) .
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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.
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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.
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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 D-glucuronic 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).
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β-D-Glucuronidase (GUD) con…
The most commonly used substrate is 4-methylumbelliferyl- 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 .
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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 6-bromo-2-naphthyl-β-D-galactopyranoside (BNGAL) .
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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 (X-GAL) is preferred for the rapid detection of coliforms .
• o-nitrophenyl-,-D-galactopyranoside (ONPG) break down and give yellow color.
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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.
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COMPOSITION
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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(β-galactosidase 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.
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The main disadvantage of Rambach agar is that it doesn’t detect S.typhi or S.paratyphi.
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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.
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• 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
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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).
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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.
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Peptidases and Proteases
• Pyroglutamyl aminopeptidase (PYRase).
Substrates used for detection of PYRase activity include L-pyrrolidonyl-β-naphthylamide( PYR) , L-pyroglutamyl-p-nitroanilide , 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 p-dimethylaminocinaldehyde reagent.
Formation of deep red color indicate positive test.
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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 .
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SummaryEnzyme Action Methods Result
GUD
Catalze the hydrolysis of(GLR) derivatives into their corsponding aglycons and D-glucuronic 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,-D-galactopyranoside (ONPG)
(ONPG) break down and give yellow color.
Esterases and Lipases
Esterases hydrolyze molecules with shorter chain organic acids, whereas lipases are capable of acting on derivatives of longer-chain acids
trioleylglycerol and the fluorescent dye rhodamine B .
orange fluorescent halos around bacterial colonies visible upon UV irradiation.
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caprylate esterase
caprylate esterase using 4-methylumbelliferyl-caprylate
4-methylumbelliferone (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.
PYRase
PYR hydrolysis impregnated paper strips with PYR and after incubation add of p-dimethylaminocinaldehyde reagent.
deep red color indicate positive test.
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Discussion and Conclusions
• AdvantagesCan eliminate the need for subculture and
further biochemical test to establish the identity of certain microorganisms.
• DisadvantagesExpensive.Some compound are unstable and some are
water insoluble.Media used for primary isolation may inhibit the
synthesis of enzymes of interest.
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