- 1. Dr.T.V.Rao MDDr.T.V.Rao MD2/20/2014
2. Dr.T.V.Rao MD2/20/2014 3. Commonlypresent in large intestine
Non sporing , Non Acid fast, Gram bacilli. A complex family of
organisms, Some are non pathogenic A few are highly Pathogenic,
Some commensals turn out to be pathogenic. as in UTI after
catheterization. Dr.T.V.Rao MD2/20/2014 4. Are facultative
anaerobes If motile, motility is by peritrichous flagella Many are
normal inhabitants of theintestinal tract of man and other animals
Some are enteric pathogens and others are urinary or respiratory
tract pathogens Differentiation is based on biochemical reactions
and differences in antigenic structure 5. Most grow well on a
variety of lab mediaincluding a lot of selective and differential
media originally developed for the selective isolation of enteric
pathogens. Most of this media is selective by incorporation of dyes
and bile salts that inhibit G+ organisms and may suppress the
growth of nonpathogenic species of Enterobacteriaceae. Many are
differential on the basis of whether or not the organisms ferment
lactose and/or produce H2S. 6. All EnterobacteriaceaeGram-negative
rods Ferment glucose with acid production Reduce nitrates into
nitrites Oxidase negative Facultativeanaerobic Motile except
Shigella and Klebsiella Non-capsulated except Klebsiella
Non-fastidious Grow on bile containing media (MacConkey agar)
Dr.T.V.Rao MD 2/20/2014 7. All EnterobacteriaceaeGram-negative rods
Ferment glucose with acid production Reduce nitrates into nitrites
Oxidase negative Facultativeanaerobic Motile except Shigella and
Klebsiella Non-capsulated except Klebsiella Non-fastidious Grow on
bile containing media (MacConkey agar) Dr.T.V.Rao MD 2/20/2014 8.
Gramnegative bacilli or coccobacilli Non-spore forming Colony
morphology on BAP or CA of little value, as they look the same,
except for Klebsiella Selective and differential media are used for
initial colony evaluation (ex. MacConkey, HE, XLD agars) 9. Some
Enterobacteriaceae are true pathogensSalmonella spp. Shigella spp.
Yersinia spp. Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)
Mostmembers of the Enterobacteriaceae are opportunistic or cause
secondary infections of wounds, the urinary and respiratory tracts,
and the circulatory system e.g. E. coli. Enterobacteriaceae divided
into TWO main groups according to action on LACTOSE Lactose
Fermenters (LF) E. coli, Citrobacter, Klebsiella, Enterobacter
Lactose Non-Fermenters (LNF) Salmonella, Shigella, Proteus,
Yersinia Dr.T.V.Rao MD2/20/2014 10. EnterobacteriaceaeLactose
fermenters E. coli, Citrobacter, Klebsiella,
EnterobacterNon-lactose fermenter Salmonell, Shigella Proteus,
YersiniaThere are several selective and differential media used to
isolate distinguishes between LF & LNF The most important media
are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella
Shigella (SS) agar Dr.T.V.Rao MD In addition to Triple Sugar
2/20/2014 Iron (TSI) agar 11. Oxidase Test
NegativePositiveEnterobacteriaceaePseudomonasMacConkeys agar &
TSI O/F test: O+/F Nitrate test: +ve furtherreduction to N2Pink
colonies on MacConkey colorless colonies on MacConkey & acidic
butt and slant on TSI & acidic butt alkaline slant onTSI Growth
on cetrimide agar: Lactose non-fermenter Pale colonies with green
pigmentationLactose fermenter IMViC test & EMB IMViC ++ - &
black colonies with metalic shines on EMBE.coliNo H2S production
(no blacking in TSI) IMViC - - ++ShigellaUrease
production+veMotilityNot motileH2S production (blacking in TSI)-ve
SS agarMotilecolorless colonies with black centers Proteus
Dr.T.V.Rao MD 2/20/2014 Salmonella 12. MacConkey agar is selective
& differential medium for Enterobacteriaceae MacConkey Agar
ContainsBile saltsCrystal violetInhibit growth of G+ve
bacteriaLactoseCause of differentialCause of selectivity Lactose
feremnters Pink coloniesDr.T.V.Rao MDNeutral redpH indicator
Acidic: PinkLactose non feremnters colorless colonies 2/20/2014 13.
EnterobacteriaceaeLactose Non-FermentersLactose FermentersNo
acidAcid Neutral redColorless coloniesPink coloniesEscherichia coli
Klebsiella spp Enterobacter spp Citrobacter sppDr.T.V.Rao
MDSalmonella spp Schigella spp Proteus spp 2/20/2014 Yersinina spp
14. Method: MacConkey agar is inoculated with tested organism using
streak plate technique Incubate the plate in incubator at 37 C/24
hrs Results: LF organism appears as pink colonies (e.g. E. coli)
NLF organism appears as colorless colonies (e.g. Shigella) Flame
& Cool 213 4 5Flame & CoolFlame & CoolDr.T.V.Rao
MD2/20/2014 15. Colorless colonies Uninoculated plateLactose non
feremters Salmonella, Shigella, Proteus Dr.T.V.Rao MDPink colonies
Lactose feremters E. coli, Citrobacter Klebsiella,
Enterobacter2/20/2014 16. Growth of Enterobacteriaceae on SS
agarBoth are lactose fermenters Both Salmonella sp. & Proteus
product H2S Pseudomonas colonies are nearly colorless 17. Coli-type
colonies are very dark, almost black e.g. E. coli 18.
PrincipleAcidic pathwayGlucoseOrAcety methyl carbinol
(ACETOIN)Mixed acids pH less than 4.4Barrits A Barrit;s BMethyl Red
indicatorRed colorNeutral pathwayVP positive MR positive Klebsiella
Dr.T.V.Rao MD 2/20/2014 E. coliPink color 19. Indole,Methyl Red,
VogesProsakaur, Citrate (IMViC) Tests: The following four tests
comprise aseries of important determinations that are collectively
called the IMViC series of reactions The IMViC series of reactions
allows for the differentiation of the various members of
Enterobacteriaceae. Dr.T.V.Rao MD2/20/2014 20. Principle Certain
microorganisms can metabolize tryptophan by tryptophanase The
enzymatic degradation leads to the formation of pyruvic acid,
indole and ammonia The presence of indole is detected by addition
of Kovac's reagent.Tryptophane amino acidsTryptophanaseIndole +
Pyurvic acid + NH3 Kovacs ReagentRed color Dr.T.V.Rao MD organic
layer` in upper 2/20/2014 21. Method: Inoculate tryptone water with
thetested microorganism Incubate at 37 C for 24 hours After
incubation interval, add 1 mlKovacs reagent, shake the tubegently
and read immediately Dr.T.V.Rao MD2/20/2014 22. Negative test e.g.
KlebsiellaResult:A bright pink color in the top layer indicates the
presence of indoleThe absence of color means that indole was not
produced i.e. indole is negativeSpecial Features:Used in the
differentiation of genera and species. e.g. E. coli (+) from
Klebsiella (-).Dr.T.V.Rao MD2/20/2014Positive test e.g. E. coli 23.
Inoculate the tested organism into One tube of MRVP brothIncubate
the tubes at 37 C for 24 hoursAFTER INCUBATION: Pour 1/3 of the
suspension into a clean nonsterile tube:Run the MR test in the tube
with 2/3, and the VP test in the open tube with1/3. For methyl red:
Add 6-8 drops of methyl red reagent.For Voges-Proskauer: Add 12
drops of Barritt's A (-naphthol), mix, 4 drops of Barritt's B (40%
KOH), mixLet sit, undisturbed, for at least 1hour Dr.T.V.Rao
MD2/20/2014 24. ResultsVoges-Proskauer testMethyl Red testPink:
Positive VP (Klebsiella)Red: Positive MR (E. coli) Yellow or
orange: Negative MR (Klebsiella)No pink: Negative VP (E.
coli)Dr.T.V.Rao MD2/20/2014 25. Principle: CitrateCitrate
Utilization TestPyruvateNa2CO3CO2 + Na + H2OAlkaline,pHSimmones
Citrate media Contains Citrate as a sole of C sourceBromothymol
blue Positive testBlue colourPositive test: Klebsiella,
Enterobacter, Citrobacter Negative test: E. coliDr.T.V.Rao
MD2/20/2014 26. Method Streak a Simmon's Citrate agar slant withthe
organism Incubate at 37 C for 24 hours.Dr.T.V.Rao MD2/20/2014 27.
Result Examinefor growth (+) Growth on the medium is accompanied by
a rise in pH to change the medium from its initial green color to
deep blue Positive Klebsiella, Enterobacter 2/20/2014 Dr.T.V.Rao
MDNegative E. coli 28. Principle UreaUrea agar contains urea and
phenol red Urease is an enzyme that catalyzes the conversion of
urea to CO2 and NH3 Ammonia combines with water to produce ammonium
hydroxide, a strong base which pH of the medium. in the pH causes
phenol red r to turn a deep pink. This is indicative of a positive
reaction for urease UreaseH2OCO2 + NH3NH4 OH in pH Phenol RedMethod
Streak a urea agar tube with the organism incubate at 37 C for 24 h
Dr.T.V.Rao MD2/20/2014Pink Positive test 29. Result If color of
medium turns from yellow to pink indicates positive test. Proteus
give positive reaction after 4 h while Kelebsiella and Enterobacter
gave positive results after 24 h Positive test Dr.T.V.Rao
MD2/20/2014Negative test 30. TSI containsThree different types of
sugars Lactose (10 part) Glucose (1 part) Sucrose (10 part)Phenol
red (acidic: Yellow)TSI dispensed in tubes with equal butt &
slant Principle To determine the ability of an organism to attack a
specific carbohydrate incorporated into a basal growth medium, with
or without the production of gas, along with the determination of
possible hydrogen sulphide production. Dr.T.V.Rao MD2/20/2014 31.
Method:Inoculate TSI medium with an organism by inoculating needle
by stabbing the butt and streaking the slant Incubate at 37 C for
24 hoursDr.T.V.Rao MD2/20/2014 32. Reaction on TSI Butt colorSlant
colorYellowRedYellowRedYellowYellowRedRedH2 S Negative Positive
black in buttNegativeNegativeResult s ResultExampleA/Alk/(Glucose
fermented)LNF e.g. ShigellaA/Alk/+ (Glucose fermented with H2S)LNF
e.g. Salmonella & ProteusA/A/(three sugars are fermented)LF
e.g. E. coli, Klebsiella, EnterobacterAlk/Alk/(No action on
sugars)Non fermenter e.g. Pseudomonas 33. Results ExampleReaction
on TSI Butt color RedSlant color RedH2S NegativeNegative
YellowYellowYellowRedRedYellowPositive black in
buttNegativeResultAlk/Alk/(No action on sugars) A/Alk/(Glucose
fermented without H2S) A/Alk/+ (Glucose fermented with H2S)Non
fermenter e.g. PseudomonasLNF e.g. Shigella LNF e.g. Salmonella
& ProteusLF e.g. E. coli, Klebsiella, Dr.T.V.Rao MDEnterobacter
2/20/2014A/A/(three sugars are fermented) 34. Summary of
morphology, cultural characteristics, and biochemical reactions of
Enterobacteriaceae Gram stainOxidaseNitrate reductaseO/FMacCon
keySSEMBE. coli-ve rod-ve+veO+/F+LFLFMetallic sheenCitrobacter-ve
rods-ve+veO+/F+LFLFDarkKlebsiella-ve
rods-ve+veO+/F+LFLFDarkEnterobacter-ve
rods-ve+veO+/F+LFLFDarkSalmonella-ve rods-ve+veO+/F+NLFNLF/
H2SColorlessShigella-ve rods-ve+veO+/F+NLFNLFColorlessProteus-ve
rods-ve+veO+/F+NLFNLF/ H2SColorlessDr.T.V.Rao MD2/20/2014 35.
Summary of morphology, cultural characteristics, and biochemical
reactions of Enterobacteriaceae
TSIIndoleMRVPCitrateUreaseMotilityE.
coliA/A/-+ve+ve-ve-ve-veMotileCitrobacter
freundiiA/A/-+ve+ve-ve+ve-veMotileKlebsiella
pneumoniaeA/A/--ve-ve+ve+ve+veNon motileEnterobacter
cloacaeA/A/--ve-ve+ve+ve+veMotileSalmonella typhiA/Alk/+
-ve+ve-ve+ve-veMotileShigella boydiiA/Alk/--ve+ve-ve-ve-veNon
motileProteus mirabilisA/Alk/+ -ve+ve-ve+ve+veMotile
SwarwingDr.T.V.Rao MD2/20/2014 36. ProgrammeCreated from Web
Sources for skills in Identification ofEnterobacteriaceae email
[email protected] Dr.T.V.Rao MD2/20/2014
