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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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