Enterobacteriaceae Identification

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Enterobacteriaceae Identification

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Enterobacteriaceae Identification

  1. 1. Dr.T.V.Rao MD06/14/13Dr.T.V.Rao MD 1
  2. 2. BacteriaGram positive Gram negativeCocci Bacilli Cocci Rods06/14/13Dr.T.V.Rao MD2
  3. 3.  Commonly present 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 bepathogenic. as in UTI aftercatheterization. 06/14/13Dr.T.V.Rao MD3
  4. 4. • Are facultative anaerobes• If motile, motility is by peritrichousflagella• Many are normal inhabitants of theintestinal tract of man and other animals• Some are enteric pathogens and othersare urinary or respiratory tract pathogens• Differentiation is based on biochemicalreactions and differences in antigenicstructure
  5. 5. • Most grow well on a variety of labmedia including a lot of selective anddifferential media originally developedfor the selective isolation of entericpathogens. Most of this media is selective byincorporation of dyes and bile salts thatinhibit G+ organisms and may suppress thegrowth of nonpathogenic species ofEnterobacteriaceae. Many are differential on the basis ofwhether or not the organisms ferment
  6. 6.  All Enterobacteriaceae• Gram-negative rods• Ferment glucose with acid production• Reduce nitrates into nitrites• Oxidase negative Facultative anaerobic Motile except Shigella and Klebsiella Non-capsulated except Klebsiella Non-fastidious Grow on bile containing media(MacConkey agar) 06/14/13Dr.T.V.Rao MD6
  7. 7.  All Enterobacteriaceae• Gram-negative rods• Ferment glucose with acid production• Reduce nitrates into nitrites• Oxidase negative Facultative anaerobic Motile except Shigella and Klebsiella Non-capsulated except Klebsiella Non-fastidious Grow on bile containing media(MacConkey agar) 06/14/13Dr.T.V.Rao MD7
  8. 8.  Gram negative bacilli or coccobacilli Non-spore forming Colony morphology on BAP or CA of littlevalue, as they look the same, except forKlebsiella Selective and differential media areused for initial colony evaluation (ex.MacConkey, HE, XLD agars)
  9. 9.  Some Enterobacteriaceae are true pathogens• Salmonella spp.• Shigella spp.• Yersinia spp.• Certain strains of E.coli (ETEC, EPEC, EIEC, EHEC) Most members of the Enterobacteriaceae areopportunistic or cause secondary infections ofwounds, the urinary and respiratory tracts, andthe circulatory system e.g. E.coli. Enterobacteriaceae divided into TWO maingroups according to action on LACTOSE• Lactose Fermenters (LF) E.coli,Citrobacter, Klebsiella,Enterobacter• Lactose Non-Fermenters (LNF) Salmonella,Shigella, Proteus,Yersinia06/14/13Dr.T.V.Rao MD9
  10. 10. EnterobacteriaceaeLactose fermentersE. coli, Citrobacter,Klebsiella, EnterobacterNon-lactose fermenterSalmonell, ShigellaProteus, YersiniaThere are several selective and differential media used toisolate distinguishes between LF & LNFThe most important media are:MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn addition to Triple Sugar Iron (TSI) agar06/14/13Dr.T.V.Rao MD10
  11. 11. Oxidase TestOxidase TestPositiveNegativePseudomonasEnterobacteriaceae Nitrate test: +ve furtherreduction to N2 Growth on cetrimide agar:Pale colonies with greenpigmentationMacConkey’s agar& TSILactose fermenterPink colonies on MacConkey& acidic butt and slant on TSIcolorless colonies on MacConkey& acidic butt alkaline slant onTSILactose non-fermenterIMViC test& EMBIMViC++ - -& black colonieswith metalicshines on EMBE.coliIMViC- - ++No H2S production(no blacking in TSI)H2S production(blacking in TSI)ShigellaUrease production+veProteus-veSS agarcolorless colonies with black centersSalmonella O/F test: O+/F-MotilityNot motile Motile06/14/13Dr.T.V.Rao MD11
  12. 12. MacConkey AgarContainsBile salts Crystal violet Lactose Neutral red MacConkey agar is selective & differential medium forMacConkey agar is selective & differential medium for EnterobacteriaceaeEnterobacteriaceaeInhibit growth of G+ve bacteriaCause of selectivityCause of differentialpH indicatorAcidic: PinkLactose feremntersPink coloniesLactose non feremnterscolorless colonies06/14/13Dr.T.V.Rao MD12
  13. 13. EnterobacteriaceaeLactose Fermenters Lactose Non-FermentersEscherichia coliKlebsiella sppEnterobacter sppCitrobacter sppSalmonella sppSchigella sppProteus sppYersinina sppPink coloniesColorless coloniesAcidNeutral redNo acid06/14/13Dr.T.V.Rao MD13
  14. 14.  Method:• MacConkey agar is inoculated with tested organismusing 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 & CoolFlame & CoolFlame & Cool1 234506/14/13Dr.T.V.Rao MD14
  15. 15. Uninoculated plate Lactose non feremtersSalmonella, Shigella,ProteusLactose feremtersE. coli, CitrobacterKlebsiella, EnterobacterColorless colonies Pink colonies06/14/13Dr.T.V.Rao MD15
  16. 16. BothBoth are lactose fermentersare lactose fermentersBothBoth Salmonella sp. & ProteusSalmonella sp. & Proteus product H2Sproduct H2SPseudomonasPseudomonas colonies are nearly colorlesscolonies are nearly colorlessGrowth of Enterobacteriaceae on SS agar
  17. 17. Coli-type colonies are very dark,almost black e.g. E. coli
  18. 18. GlucoseAcidic pathwayMixed acids pH less than4.4Methyl RedindicatorRed colorPrincipleMR positiveE. coliOr Neutral pathwayAcety methyl carbinol(ACETOIN)Barrit’s ABarrit;s BPink colorVP positiveKlebsiella06/14/13Dr.T.V.Rao MD18
  19. 19.  Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:• The following four tests comprise aseries of important determinations thatare collectively called the IMViC seriesof reactions• The IMViC series of reactions allowsfor the differentiation of the variousmembers of Enterobacteriaceae.06/14/13Dr.T.V.Rao MD19
  20. 20.  Principle Certain microorganisms can metabolizetryptophan by tryptophanase The enzymatic degradation leads to theformation of pyruvic acid, indole andammonia The presence of indole is detected byaddition of Kovacs reagent.Tryptophaneamino acidsTryptophanaseIndole + Pyurvic acid + NH3Kovac’s ReagentRed color in upper organic layer`06/14/13Dr.T.V.Rao MD20
  21. 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 immediately06/14/13Dr.T.V.Rao MD21
  22. 22.  Result: A bright pink color in the toplayer indicates the presenceof indole The absence of color meansthat indole was not producedi.e. indole is negative Special Features: Used in the differentiation ofgenera and species. e.g. E. coli(+) from Klebsiella (-).Positive teste.g. E. coliNegative teste.g. Klebsiella06/14/13Dr.T.V.Rao MD22
  23. 23.  Inoculate the tested organism into One tube of MRVP broth Incubate the tubes at 37°C for 24 hours AFTER INCUBATION:  Pour 1/3 of the suspension into a cleannonsterile tube:  Run the MR test in the tube with 2/3, and theVP test in the open tubewith 1/3. For methyl red:  Add 6-8 drops of methyl red reagent. For Voges-Proskauer:  Add 12 drops of Barritts A (α-naphthol),mix, 4 drops of Barritts B (40% KOH), mix Let sit, undisturbed, for at least 1hour06/14/13Dr.T.V.Rao MD23
  24. 24. ResultsMethyl Red test Voges-Proskauer testRed: Positive MR (E. coli)Yellow or orange: Negative MR (Klebsiella)Pink: Positive VP (Klebsiella)No pink: Negative VP (E. coli)06/14/13Dr.T.V.Rao MD24
  25. 25. Citrate Utilization TestCitrate Utilization TestPrinciple:Citrate Na2CO3Alkaline,↑pHBlue colourBromothymol blueSimmone’s Citrate mediaPositive test: Klebsiella, Enterobacter, CitrobacterCO2 + Na + H2OPyruvatePositive testNegative test: E. coliContains Citrate as a sole of C source06/14/13Dr.T.V.Rao MD25
  26. 26.  Incubate at 37°C for 24 hours.MethodMethod Streak aStreak a Simmons Citrate agarSimmons Citrate agar slant withslant withthe organismthe organism06/14/13Dr.T.V.Rao MD26
  27. 27.  Examine for growth(+) Growth on the mediumis accompanied by arise in pH to changethe medium from itsinitial green color todeep blueResultResultPositiveKlebsiella, EnterobacterNegativeE. coli06/14/13Dr.T.V.Rao MD27
  28. 28.  Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversionof urea to CO2 and NH3 Ammonia combines with water to produceammonium 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 ureaseUreaUreaseCO2 + NH3H2ONH4 OH ↑ in pHPhenol RedPinkPositive test Streak a urea agar tube with the organism incubate at 37°C for 24 hMethodPrinciplePrinciple06/14/13Dr.T.V.Rao MD28
  29. 29.  If color of medium turnsfrom yellow to pinkindicates positive test. Proteus give positivereaction after 4 h whileKelebsiella andEnterobacter gavepositive results after 24hResultPositive test Negative test06/14/13Dr.T.V.Rao MD29
  30. 30.  TSI contains• Three different types of sugars Glucose (1 part) Lactose (10 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 specificcarbohydrate incorporated into a basal growth medium, with orwithout the production of gas, along with the determination ofpossible hydrogen sulphide production.06/14/13Dr.T.V.Rao MD30
  31. 31.  Method:• Inoculate TSI medium with anorganism by inoculating needle bystabbing the butt and streaking theslant• Incubate at 37°C for 24 hours06/14/13Dr.T.V.Rao MD31
  32. 32. Reaction on TSIResultExampleButt color Slant color H2SYellow RedNegative A/Alk/-(Glucose fermented)LNFe.g. ShigellaYellow RedPositiveblack in buttA/Alk/+(Glucose fermented with H2S)LNFe.g. Salmonella &ProteusYellow Yellow Negative A/A/-(three sugars are fermented)LFe.g. E. coli, Klebsiella,EnterobacterRed RedNegativeAlk/Alk/-(No action on sugars)Non fermenter e.g.PseudomonasResults
  33. 33. Reaction on TSIResultExampleButtcolorSlantcolorH2SRed Red Negative Alk/Alk/-(No action on sugars)Non fermentere.g.PseudomonasYellow RedNegative A/Alk/-(Glucose fermentedwithout H2S)LNFe.g. ShigellaYellow RedPositiveblack inbuttA/Alk/+(Glucose fermentedwith H2S)LNFe.g. Salmonella &ProteusYellow YellowNegative A/A/-(three sugars arefermented)LFe.g. E. coli,Klebsiella,EnterobacterResults06/14/13Dr.T.V.Rao MD33
  34. 34. GramGramstainstainOxidaseOxidase NitrateNitratereductasereductaseO/FO/F MacConMacConkeykeySSSS EMBEMBE. coliE. coli -ve rod-ve rod -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF MetallicMetallicsheensheenCitrobacterCitrobacter -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDarkKlebsiellaKlebsiella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDarkEnterobacterEnterobacter -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ LFLF LFLF DarkDarkSalmonellaSalmonella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/H2SH2SColorlessColorlessShigellaShigella -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLFNLF ColorlessColorlessProteusProteus -ve rods-ve rods -ve-ve +ve+ve O+/F+O+/F+ NLFNLF NLF/NLF/H2SH2SColorlessColorlessSummary of morphology, cultural characteristics,Summary of morphology, cultural characteristics,and biochemical reactions ofand biochemical reactions of EnterobacteriaceaeEnterobacteriaceae06/14/13Dr.T.V.Rao MD34
  35. 35. TSITSI IndoleIndole MRMR VPVP CitrateCitrate UreaseUrease MotilityMotilityE. coliE. coli A/A/-A/A/- +ve+ve +ve+ve -ve-ve -ve-ve -ve-ve MotileMotileCitrobacterCitrobacterfreundiifreundiiA/A/-A/A/- +ve+ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotileKlebsiellaKlebsiellapneumoniaepneumoniaeA/A/-A/A/- -ve-ve -ve-ve +ve+ve +ve+ve +ve+ve NonNonmotilemotileEnterobacterEnterobactercloacaecloacaeA/A/-A/A/- -ve-ve -ve-ve +ve+ve +ve+ve +ve+ve MotileMotileSalmonellaSalmonellatyphityphiA/Alk/+A/Alk/+ -ve-ve +ve+ve -ve-ve +ve+ve -ve-ve MotileMotileShigellaShigellaboydiiboydiiA/Alk/-A/Alk/- -ve-ve +ve+ve -ve-ve -ve-ve -ve-ve NonNonmotilemotileProteusProteusmirabilismirabilisA/Alk/+A/Alk/+ -ve-ve +ve+ve -ve-ve +ve+ve +ve+ve MotileMotileSwarwingSwarwingSummary of morphology, cultural characteristics,and biochemical reactions of Enterobacteriaceae06/14/13Dr.T.V.Rao MD35
  36. 36.  Programme Created fromWeb Sources for skills inIdentification ofEnterobacteriaceaeemail doctortvrao@gmail.comDr.T.V.Rao MD 06/14/1336

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