Download الشريحة 1

NEW DEVELOPMENT IN CHROMOGENIC AND
FLUOROGENIC CULTURE MEDIA
Prepered by A.ElKader ElOttol
Supervisor 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
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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 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).
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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 .
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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 6bromo-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 (XGAL) 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(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.
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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
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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.
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A plate assay was further designed to detect bacterial lipases in a
medium containing trioleylglycerol and the fluorescent dye rhodamine
B.
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Substrate hydrolysis causes the formation of orange fluorescent halos
around bacterial colonies visible upon UV irradiation.
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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 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.
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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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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.
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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
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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.
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