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Transcript
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