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Enterobacteriaceae Skills in Bacteriology

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Enterobacteriaceae Skills in Bacteriology

Enterobacteriaceae Skills in Bacteriology

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  • 1. Dr.T.V.Rao MD Dr.T.V.Rao MD 4/13/2012
  • 2. Dr.T.V.Rao MD 4/13/2012
  • 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 be pathogenic. as in UTI after catheterization. Dr.T.V.Rao MD 4/13/2012
  • 4. • Are facultative anaerobes• If motile, motility is by peritrichous flagella• Many are normal inhabitants of the intestinal 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 media including 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 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) Dr.T.V.Rao MD 4/13/2012
  • 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) Dr.T.V.Rao MD 4/13/2012
  • 8. Gram negative 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 pathogens • Salmonella spp. • Shigella spp. • Yersinia spp. • Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC) Most members 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 MD 4/13/2012
  • 10. Enterobacteriaceae Lactose fermenters Non-lactose fermenter E. coli, Citrobacter, Salmonell, Shigella Klebsiella, Enterobacter Proteus, YersiniaThere 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 In addition to Triple Sugar 4/13/2012 Dr.T.V.Rao MD Iron (TSI) agar
  • 11. Oxidase Test Negative Positive Enterobacteriaceae Pseudomonas MacConkey’s agar & TSI  O/F test: O+/F-  Nitrate test: +ve furtherPink colonies on MacConkey colorless colonies on MacConkey reduction to N2& acidic butt and slant on TSI & acidic butt alkaline slant onTSI  Growth on cetrimide agar:Lactose fermenter Lactose non-fermenter Pale colonies with green pigmentation IMViC test No H2S production H2S production & EMB (no blacking in TSI) (blacking in TSI)IMViC IMViC Urease production++ - - - - ++ Shigella& black colonies +ve -vewith metalic Motilityshines on EMB SS agar Not motile colorless colonies with black centers E.coli Motile Proteus Dr.T.V.Rao MD 4/13/2012 Salmonella
  • 12.  MacConkey agar is selective & differential medium for Enterobacteriaceae MacConkey Agar ContainsBile salts Crystal violet Lactose Neutral red pH indicatorInhibit growth of G+ve bacteria Cause of differential Acidic: PinkCause of selectivity Lactose feremnters Lactose non feremnters Pink coloniesDr.T.V.Rao MD colorless colonies 4/13/2012
  • 13. Enterobacteriaceae Lactose Fermenters Lactose Non-Fermenters No acid AcidNeutral red Colorless colonies Pink colonies Escherichia coli Salmonella spp Klebsiella spp Schigella spp Enterobacter spp Proteus spp Citrobacter spp Dr.T.V.Rao MD Yersinina spp 4/13/2012
  • 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 1 2 3 4 Flame & Cool 5 Dr.T.V.Rao MD 4/13/2012 Flame & Cool
  • 15. Colorless colonies Pink coloniesUninoculated plate Lactose non feremters Lactose feremters Salmonella, Shigella, E. coli, Citrobacter Proteus Klebsiella, Enterobacter Dr.T.V.Rao MD 4/13/2012
  • 16. Growth of Enterobacteriaceae on SS agar Both 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. Principle GlucoseAcidic pathway Or Neutral pathway Acety methyl carbinol (ACETOIN) Mixed acids pH less than 4.4 Barrit’s A Methyl Red Barrit;s B indicator MR positive VP positive Klebsiella Pink color Red color E. coli Dr.T.V.Rao MD 4/13/2012
  • 19. Indole, Methyl Red, Voges- Prosakaur, Citrate (IMViC) Tests: • The following four tests comprise a series 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 MD 4/13/2012
  • 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 Kovacs reagent. TryptophanaseTryptophane Indole + Pyurvic acid + NH3amino acids Kovac’s Reagent Red color Dr.T.V.Rao MD organic layer` in upper 4/13/2012
  • 21.  Method: Inoculate tryptone water with the tested microorganism Incubate at 37 C for 24 hours After incubation interval, add 1 ml Kovacs reagent, shake the tube gently and read immediately Dr.T.V.Rao MD 4/13/2012
  • 22. Negative test Positive test e.g. Klebsiella e.g. E. coli Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Dr.T.V.Rao MD 4/13/2012
  • 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 clean nonsterile tube: Run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3.  For methyl red: Add 6-8 drops of methyl red reagent.  For Voges-Proskauer: Add 12 drops of Barritts A (-naphthol), mix, 4 drops of Barritts B (40% KOH), mix  Let sit, undisturbed, for at least 1hour Dr.T.V.Rao MD 4/13/2012
  • 24. Results Methyl Red test Voges-Proskauer testRed: Positive MR (E. coli) Pink: Positive VP (Klebsiella)Yellow or orange: Negative MR (Klebsiella) No pink: Negative VP (E. coli) Dr.T.V.Rao MD 4/13/2012
  • 25. Citrate Utilization TestPrinciple:Citrate Pyruvate CO2 + Na + H2O Na2CO3 Alkaline,↑pH Simmone’s Citrate media Contains Citrate as a sole of C source Bromothymol blue Positive test Blue colourPositive test: Klebsiella, Enterobacter, Citrobacter Dr.T.V.Rao MD 4/13/2012Negative test: E. coli
  • 26. Method Streak a Simmons Citrate agar slant withthe organism Incubate at 37 C for 24 hours. Dr.T.V.Rao MD 4/13/2012
  • 27. Result Examine for 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 Negative Klebsiella, Enterobacter 4/13/2012 Dr.T.V.Rao MD E. coli
  • 28. Principle  Urea 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 Urease H2OUrea CO2 + NH3 NH4 OH ↑ in pH Phenol RedMethod Pink Positive test  Streak a urea agar tube with the organism  incubate at 37 C for 24 h Dr.T.V.Rao MD 4/13/2012
  • 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 Negative test Dr.T.V.Rao MD 4/13/2012
  • 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 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 MD 4/13/2012
  • 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 hours Dr.T.V.Rao MD 4/13/2012
  • 32. Result Reaction on TSI s Result ExampleButt color Slant color H2 S Negative A/Alk/- LNF Yellow Red (Glucose fermented) e.g. Shigella Positive LNF A/Alk/+ Yellow Red black in butt e.g. Salmonella & (Glucose fermented with H2S) Proteus LF A/A/- Yellow Yellow Negative e.g. E. coli, Klebsiella, (three sugars are fermented) Enterobacter Alk/Alk/- Non fermenter e.g. Red Red Negative (No action on sugars) Pseudomonas
  • 33. Results Reaction on TSI ExampleButt Slant Result H2Scolor color Non fermenter Alk/Alk/- Red Red Negative e.g. (No action on sugars) Pseudomonas Negative A/Alk/- LNFYellow Red (Glucose fermented e.g. Shigella without H2S) Positive LNF black in A/Alk/+ e.g. Salmonella &Yellow Red butt (Glucose fermented Proteus with H2S) LF A/A/- Negative e.g. E. coli,Yellow Yellow (three sugars are Klebsiella, fermented) Dr.T.V.Rao MDEnterobacter 4/13/2012
  • 34. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae Gram Oxidase Nitrate O/F MacCon SS EMB stain reductase keyE. coli -ve rod -ve +ve O+/F+ LF LF Metallic sheenCitrobacter -ve rods -ve +ve O+/F+ LF LF DarkKlebsiella -ve rods -ve +ve O+/F+ LF LF DarkEnterobacter -ve rods -ve +ve O+/F+ LF LF DarkSalmonella -ve rods -ve +ve O+/F+ NLF NLF/ Colorless H2SShigella -ve rods -ve +ve O+/F+ NLF NLF ColorlessProteus -ve rods -ve +ve O+/F+ NLF NLF/ Colorless Dr.T.V.Rao MD 4/13/2012 H2S
  • 35. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae TSI Indole MR VP Citrate Urease MotilityE. coli A/A/- +ve +ve -ve -ve -ve MotileCitrobacter A/A/- +ve +ve -ve +ve -ve MotilefreundiiKlebsiella A/A/- -ve -ve +ve +ve +ve Nonpneumoniae motileEnterobacter A/A/- -ve -ve +ve +ve +ve MotilecloacaeSalmonella A/Alk/+ -ve +ve -ve +ve -ve MotiletyphiShigella A/Alk/- -ve +ve -ve -ve -ve Nonboydii motileProteus A/Alk/+ -ve +ve -ve +ve +ve Motilemirabilis Dr.T.V.Rao MD 4/13/2012 Swarwing
  • 36. Programme Created from Web Sources for skills in Identification of Enterobacteriaceae email  doctortvrao@gmail.com Dr.T.V.Rao MD 4/13/2012

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