Biochemical reactions


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Dr.saumya singh
P.G.3rd yr

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Biochemical reactions

  1. 1. Dr. Saumya Singh
  2. 2. Order of Biochemical Tests  Test for enzymes  Test for metabolism of carbohydrates and related products  Test for specific break down products  Test to show ability to utilize a specific substance  Test for metabolism of protein and amino acids
  3. 3. Test for Enzymes  Catalase test  Oxidase test  Urease test  ONPG (ß- galactosidase)  Nitrate reduction
  4. 4. Catalase test Principle: Demonstrate the presence of an enzyme catalase. Catalase is an enzyme that decomposes hydrogen peroxide into water and oxygen. Chemically,it is a hemoprotein structurally similar to hemoglobin 2H2o2 catalase 2H2o + o2 (gas bubbles)
  5. 5. • Catalase is present in most cytochrome containing aerobic and facultative anaerobic bacteria (except streptococcus spp). Anaerobic bacteria eg. clostridium spp. possess the peroxidase enzyme in lieu of Catalase. • Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate metabolism. If this is allowed to accumulate in the bacterial cells it becomes lethal to the bacteria. • Catalase thus helps in converting H2o2 to H2o and o2 • Optimal PH for Catalase action is 7.
  6. 6. Reagents: 3% / 30%hydrogen peroxide stored in dark brown bottle under refrigeration 18 to 24 hrs culture of the organism to be tested Quality control: Positive control - Staphylococcus aureus Negative control – Streptococcus spp Methods: •Slide method •Tube method •Direct plate method
  7. 7. Slide Method With an inoculating wire transfer growth from the centre of the colony to the surface of the glass slide. Add 1 drop of 30% H2O2 and observed for bubble formation. Tube Method Direct Plate Method Small amount of culture to be tested is picked from nutrient agar with a clean sterile glass rod/capillary tube and inserted into H2O2 solution held in a small clean tube. 1 ml of 3% H2O2 is poured over a 24 hour nutrient agar culture of the test organism directly. Result: The rapid and sustained appearance of bubbles/effervascence indicates a positive test.A few tiny bubbles forming after 20 to 30 sec is not considered a positive test. Precaution: Avoid colonies from blood agar( contains catalase ) Platinum wire / Nichrome wire should be used with caution.
  8. 8. Positive: Negative: •Micrococcus •Saphylococcus •Bacillus •Listeria monocytogenes •Corynebacterium (except C.pyogenes & C.haemolyticum) •Moraxella spp (except Kingella kingae) •Enterobacteriacae •Gonococcus & Meningococcus •Vibrio cholerae •Pseudo/Aero/Plesiomonas •Streptococcus •Clostridium •Erysipleothrix
  9. 9. Oxidase Test Principle: Determines the presence of bacterial enzyme cytochrome oxidase. Cytochromes are iron containing hemoproteins and in aerobic respiration they transfer electrons(H) to oxygen to form water. The cytochrome oxidase test uses certain reagent dyes such as p- phenylenediamine dihydrochloride,that substitute for oxygen as artificial electron acceptors. In the reduced state the dye is colourless; however in the presence of cytochrome oxidase and atmospheric oxygen,p- phenylenediamine dihydrochloride is oxidized forming indophenol blue.
  10. 10. Oxidase reagent: • 1% Kovac’s reagent(tetra methyl -p-phenylenediamide dihydrochloride ) • 1% Gordons & Mcleods reagent(Dimethly - p- phenylenediamide dihydrochloride) Modified Oxidase Test: • 6%TMPD Reagent in Dimethly sulfoxide (DMSO) used for differentiating micrococci spp (positive in 30 secs) from staphylococci. • Ewing Johnson Modification ( 0.1gm α-naphthol in 10ml95% ethanol, 0.1gm p-aminodimethylaniline oxalate in 10ml d.w). Quality control: • Positive: P.aeruginosa Negative: E.coli
  11. 11. Direct plate method Wet filter paper method (KOVAC’S METHOD) Dry filter paper method 2 to 3 drops of reagent is added directly to colonies growing on medium. (1gm in100ml distilled water) A strip of Whatman’s No. 1 filter paper soaked in freshly prepared 1% Oxidase reagent is smeared with test bacterium. A strip of Whatman’s No. 1 filter paper is soaked in freshly prepared 1% Oxidase reagent,drained for 30 sec, freez dried and stored in dark bottle tightly. For use, strip is removed, laid in Petri dish , moistened with distilled water and smeared with test bacterium. Result: A positive reaction is indicated by a Deep blue colour appearing within 10 sec. Precautions: 1) Stainless steel or Nichrome inoculating loop and wires should not be used. 2) Tetra methyl derivative of pPD is recommended. 3) Do not perform the test from colonies growing in Macconkey medium.
  12. 12. Positive •Neisseria spp •Haemophillus sps •Pseudomonas spp(except P.maltophilia) •Aeromonas spp •Alcaligenes •Vibrio •Campylobacter •Plesiomonas shigelloides •Micrococcus spp •Moraxella Negative •Enterobacteriaceae •Acenitobacter spp •Brucella canis •Bordetella parapertusis •Franscisella tularensis •Gardenella vaginalis
  13. 13. Urease Test Principle : To determine the ability of the organism to split urea forming 2 molecules of ammonia by the action of the enzyme Urease with resulting alkalinity Urease NH2.CO.NH2 + H2O 2NH3 + CO2 phenolphthalein ( Colourless) ph < 8.1 Ammonia phenolphthalein ( pink red) ph > 8.1
  14. 14. Stuarts Urea broth (pH 6.8) • Monopotassium phosphate-9.1gm •Disodium phosphate-9.5gm •Urea-20gm •Phenol red indicator-0 .02gm •Yeast extract-0.1gm •Distilled water- 1l Christensens urea agar (pH 6.8) •Peptone-1gm •Glucose-1gm •Sodium chloride-5gm •Monopotassium phosphate-2gm •Urea-20gm •Phenol red indicator-0.012gm •Agar-15gm •Distilled water-1l Quality control • Positive – Proteus spp & Klebsiella spp(weak positive) • Negative – E.coli
  15. 15. Procedure: The surface of the agar slant is streaked with the test organism / the broth medium is inoculated with the loopful of pure culture of the test organism and incubated at 37 c for 18-24 hrs. Interpretation: Rapid hydrolysers gives +ve results in 1-2 hrs, less active sp. require 3 or more days. Stuarts broth – Positive red colour through out the broth. Christensens agar–Rapid urea splitters red through out the medium, Slow urea splitters red colour initially in slant only & then gradually entire tube. No urea hydrolysis-Negative yellow or straw colour
  16. 16. Precaution: Both the Urease test medium depend upon the demonstration of alkalinity i.e. not specific for Urease i.e. even protein hydrolysis may result in alkalinity hence false positive may be seen in Pseudomonas . To eliminate such false positivity control test using the same medium without urea should be tested.
  17. 17. Elek’s test: Reagent : • Urea-4gm • KH2PO4-50ML • NaOH-35ML • Distilled water -115ml • Nessler’s reagent(50gm KI in 50ml of H2O,add Hgcl2 sol.until a perm.precipitate appears.add 200ml NaOH and make upto 1lt with water). Method : Emulsify the organism in 0.5ml of substrate tube,place the tube in water bath at 37 c for 3hr.Remove the tube and add 0.1ml Nessler’s reagent and read after 3mins. Positive-yellow to dark brown precipitate. Negative –control and negative tubes are colourless.
  18. 18. Alternate tests: Reagent impregnated urease strip test (2hrs) Key urease tablets test Urea discs Ewings Urea R broth (pH 6.9) Positive Proteus Klebsiella pneumoniae Actinobacillus sp. Bacillus lentus Enterobacter Cryptococcus Helicobacter pylori Bordetella bronchoseptica Negative Escherichia Providencia Aeromonas Edwarseilla S.typhi
  19. 19. 0-Nitrophenyl-ß-D-Galactopyranoside (ONPG) Test Principle: To demonstrate the presence or absence of the enzyme ß Galactosidase Biochemistry: O – Nitrophenyl – ß – D – galactopyranoside is structurally similar to lactose except that orthonitrophenyl has been substituted for glucose H2o ONPG (colorless) Galactose+ Orthonitrophenol(yellow) galactosidase H20 Lactose Galactose + Glucose galactosidase It differentiate late lactose fermenting organism and is of particular use in identification of enterobacteria
  20. 20. • Disaccharide compound composed of glucose and galactose connected through an oxygen linkage known as galactoside bond. • Lactose fermentation depends upon the 2 enzymes ß-galactoside permease and ß-galactosidase. • ß-galactoside permease permits the transport of ß-galactoside such as lactose into the bacterial cell wall. • ß-galactosidase cleave the ß-galactoside bond after it enters into the bacterial cell liberating glucose and galactose. •Non lactose fermenting bacteria are devoid of both the enzymes. •Late lactose fermenters lack the enzyme permease but have ß- galactosidase activity and so the permeases activity occurs sluggishly and lactose fermentation is delayed by 2-8 days. In these instances a +ve ONPG test may provide a rapid identification of delayed lactose fermentors .
  21. 21. Media and reagents: pH 7.0-7.5 Sodium phosphate buffer-(0.01mol/lt,ph7.5) 100ml O-Nitrophenyl- ß-D-galactopyranoside(ONPG)-0.6gm Toluene Physiologic saline 0.85% Quality control: Positive – E.coli Negative – Proteus spp
  22. 22. TUBE METHOD A loopful bacterial growth (KIA,TSI agar) is emulsified in 0.5 ml of physiologic saline. 1 drop of toluene is added and vigorously mix. An equal quantity of buffer ONPG solution is added and mixture is placed at 37 waterbath. DISC METHOD A loopful of bacterial suspension is added to the ONPG substrate ( 1 ml of distilled water to a tablet in test tube , placed in 37 waterbath. Interpretation: Yellow color within 5-10 mins - Positive Colorless after 24 hrs - Negative Precaution: •Glucose containing medium should not be used. •Do not use if medium yellow. •Store away from direct sunlight, ONPG is light sensitive
  23. 23. Nitrate reduction Principle: To determine the ability of the organism to reduce nitrate to nitrites or free nitrogen gas in the presence of enzyme nitrate reductase. Purpose: Aid in the identification of Enterobacteriaceae(+) except certain biotypes of Pantoa agglomerans and certain species of Serratia and Yersinia(-). Also aids in identifying members of Haemophilus,Neisseria,Moraxella species.
  24. 24. Media and Reagent: A. Nitrate Broth or Nitrate agar (Slant) Beef extract-3gm Peptone-5gm Potassium nitrate-1gm Agar (nitrite-free)-12gm Distilled water -1lt B. Reagent A α – Naphthylamine -5gm Acetic acid (5N), 30%-1lt C. Reagent B Sulfanilic acid-8gm Acetic acid (5N) , 30%-1lt Quality Control: Positive control - E.coli Negative control - Acinetobacter baumannii
  25. 25. Procedure: Inoculate the medium and incubate at 37 C for 18 to 24 hours.At the end of incubation add 1 ml of each of reagent A and B to the test medium. Result: Positive – Development of red colour within 30 sec. Negative-No colour change * Negative reaction(True)- Nitrates not reduced * Negative reaction(False)- Reduction of products other than nitrites ( test reaction detects only nitrites). Development of red colour after adding of zinc dust indicates the presence of residual nitrates and confirms true negative reactions. Note- α Naphthylamine is potentially carcinogenic.
  26. 26. Order of Biochemical Tests  Test for enzymes  Test for specific break down products  Test to show ability to utilize a specific substance  Test for metabolism of protein and amino acids  Test for metabolism of carbohydrates and related products
  27. 27. Test for Break down products  Methyl Red Test  Voges-Proskauer (Acetoin Production) Test
  28. 28. Methyl Red Test Principle: To test the ability of the organism to produce and maintain stable acid end products (lactic,acetic,formic) from glucose fermentation and to overcome the buffering capacity of the system. This is a qualitative test for acid production. Biochemistry:  Methyl red is a pH indicator with a range between 6(Y) and 4.4(R).  The pH at which the MR detects acid is considerably lower than the pH of other indicators.  Thus to produce a colour change the test organism must produce a large quantity of acid from the substrate being used.
  29. 29. Media & Reagents :  MR/VP Broth (glucose phosphate peptone water): Peptone-5 gm K2HPO4-4 gm Water-1 L Glucose 10%-50 ml  MR pH indicator : MR 0.1 g in 300ml of 95% Ethanol & Distilled water 200ml. Quality Control: Positive : E.coli Negative : E. aerogenes
  30. 30. Procedure: Inoculate the MR/VP broth with a pure culture of the test organism and incubate at 37 for 48 to 72 hrs Add 5 drops of MR reagent to the broth. Interpretation: Positive : red color (ph 4.4 or less) at the surface of the medium. Negative : Yellow color (pH 6.0) at the surface of the medium.
  31. 31. Positive Negative: E.coli Klebseilla Yersinia spp Enterobacter aerogenes Enterobacter cloacae Listeria monocytogenes Gram negative non enteric bacilli Precaution: orange colour reaction is considered negative.
  32. 32. Voges Proskauer Test Principle : To determine the ability of the organisms to produce neutral end product acetyl methyl carbinol (acetoin) from glucose fermentation. In presence of atmospheric oxygen and KOH, acetoin is converted to Diacetyl and αnaphthol (serves as a catalyst) along with arginine to produce red complex. Quality control : Positive : Enterobacter aerogenes Negative : E.coli
  33. 33. Media & Reagents: MR/VP Broth : pH 6.9 Peptone – 1.5 g Glucose 10% solution – 50 ml K2HPO4-5 g water-1 L 0’Meara reagent:(40g KOH & 0.3g creatine in 100ml distilled water)/ VP (A) : α naphthol 5% (color intensifier) ( α- naphthol 5g in 100ml Absolute ethyl alcohol) VP (B) : 40% potassium hydroxide (oxidising agent) ( KOH 40g in 100ml Distilled water)
  34. 34. Procedure: Inoculate pure culture of the test organism into MR/VP broth and incubate for 24 hrs at 37 c Aliquot 1 ml of the broth to a sterile test tube and add 0.6ml of VP(A) followed by 0.2ml of VP(B)/O’Meara rg(0.5ml) Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 mins. Intrepretation: Positive : Pinkish red colour at the surface of the medium. Negative :Yellow colour at the surface of the medium.
  35. 35. Alternate Tests:  Gas liquid chromatography measure of diacetyl.  Electron capture gas liquid chromatography.  Gas chromatography – chemical ionization mass spectrography.  Calorimetric method of measurement of diacetyl. Rapid test:  Reagent impregnated VP strip. Precautions:  Organism like Hafnia alvei & Proteus mirabilis may give both MR & VP positive results although VP reaction is delayed.  Excess KOH may mask a weak VP positive reactions.  The test should not be read after 1hr.
  36. 36. Positive: Negative:  Klebseilla pneumoniae  E.coli  Enterobacter  Micrococcus  Staphylococcus  K.ozanae  Hafinia alvei(25 +ve)  K.rhinoscleromatis  Hafinia alvei(37 variable)  Y.enterocolitica 37  Yersinia enterocolitica 25  Listeria monocytogenes
  37. 37. Test to show ability to utilize a specific substrate.  Citrate utilization Test  Malonate utilization Test
  38. 38. Citrate utilization test  Principle: To determine the ability of an organism to utilize citrate as sole carbon and energy source for growth and an ammonium salt as the sole source of nitrogen.  Citrate is a salt of citric acid, one of the metabolites in the Krebs cycle.  Bacteria that can use citrate ,can also extract nitrogen from the ammonium salt with the production of ammonia, leading to alkalinization of the medium, from conversion of the NH3²⁺ to NH4OH. Sodium citrate Bromothymol blue (green) ph 6.9 citritase Alk.metabolic products- Bromothymol blue (blue) ph7.6 ph
  39. 39. Koser’s medium (Ph 6.9) Simmon’s citrate medium (ph 6.9) Sodium chloride - 5 g Magnesium sulphate - 0.2 g Ammonium dihydrogen ph. – 1 g Potassium dihydrogen ph. – 1 g Sodium citrate – 5 g Distilled water – 1 L Koser’s medium – 1 L Agar - 20 g Bromothymol blue 0.2 % - 40 ml Quality control: Positive: Enterobacter aerogenes Negative: E.coli
  40. 40. Procedure: Inoculate pure culture of the test organism into Koser’s medium / streak on citrate agar tube , incubate at 37 C for 24 - 48 hrs. Interpretation :  Koser’s medium Positive: Turbidity Negative: No Turbidity  Simmon’s citrate medium Positive: Blue colour and streak of growth. Negative: Original green colour and no colour. A positive test may also be read without a blue colour if there is visible colony growth along the inoculation streak line & it can be confirmed by inoculating tube for additional 24 hrs.
  41. 41. Precaution:  For koser’s medium a positive test should be subcultured into a second tube to eliminate false positive due to an excessive initial inoculum.  Avoid using a large inoculum to streak the slant; an inoculum that is too heavy may result in a false-positive test. Positive Klebsiella Salmonella typhimurium Citrobacter Proteus Enterobacter Pseudomonas Providencia Serratia Morganella Negative E.coli Salmonella typhi Edwardsiella Hafnia
  42. 42. Malonate utilization test Principle: Determines the ability of an organism to utilize sodium malonate as its sole source of carbon. If an organism is capable of utilizing sodium malonate as its sole carbon source, and the same time utilizes ammonium sulphate as its nitrogen source, an alkaline reaction is produced by the formation of NaOH , resulting in a colour change from green to blue in medium. It is widely used for the differentiation of Enterobacter and Escherichia coli based on the use of malonate. Quality control: Positive : Enterobacter aerogenes Negative: Yersinia ruckeri
  43. 43. Malonate agar: (ph7.4) Yeast extract-1g Ammonium sulphate-2g Dipotassium dihydrogen sulphate-0.6g Potassium dihydrogen phasphate-0.4g Sodium chloride-2g Sodium malonate-3g Glucose-0.25g Bromothymol blue-0.025g Distilled water-1L Agar-25g Procedure: Inoculate pure culture of the test organism into medium , incubate at 37 C for 48 hrs.
  44. 44. Interpretation: Positive: Deep blue Negative: Green Positive Negative Enterobacter K.pneumoniae Arizona C.koseri Escherichia K.ozaenae Serratia C.amalonaticus Salmonella P.vulgaris Precaution:   Reaction should be read after 48 hrs. Some bacteria produces slight alkalinity,it is useful to compare the test to an uninoculated tube.
  45. 45. Test for metabolism of protein and amino acids  Indole test  Gelatin liquefaction  Phenylalanine deaminase test  Amino acid decarboxylase and arginine dihydrolase tests
  46. 46. Indole test Principle: To determine the ability of the organism to split Indole from the tryptophan molecule. Biochemistry: Indole is one of the metabolic degradation product of the amino acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of Indole, Pyruvic acid and ammonia. Indole is then tested for by a colorimetric reaction with p-dimethylaminobenzaldehyde (active chemical in Kovac / Ehrlich reagent) resulting in formation of red complex.
  47. 47. Media & Reagents: Tryptophan 1% Peptone – 2g Sodium chloride – 0.5 g Distilled water – 100 ml Ehrlich’s(other GNB) p - dimethyl aminobenzaldehyde - 2g Abs. ethyl alcohol – 190 ml Conc. HCL – 40 ml Kovac’s(Enterobacteriaceae) p - dimethyl aminobenzaldehyde – 2g Pure amyl or Iso amyl alcohol – 150 ml Conc.HCL – 50 ml Quality control: Positive – Elizabethkingia meningoseptica Negative – CDC group EO-2 Positive – E.coli Negative – K.pneumoniae
  48. 48. Procedure: Inoculate tryptophan broth with the test organism and incubate for 18 to 24 hrs at 37 c. Add 15 drops of Indole reagent down the inner wall of the tube. If Ehrlich reagent is used it should be preceded by addition of 1 ml of xylene, this is not necessary for Kovac reagent. Intrepretation: Positive:Development of bright fuchsia red color at the interface of the interface of the reagent and the broth within seconds after adding the reagent . Negative : No colour change after the addition of the reagent
  49. 49. Positive Negative  E.coli  Salmonella  P.vulgaris  K.pneumoniae  P.penneri  Enterobacter  K.oxytoca  P.mirablis  K.ornitholytica  Pseudomonas  Citrobacter diversus  Hafnia  Citrobacter amalonaticus  Serratia  E.tarda  Providencia  Morganella morgagni  Morgenella  S.dysenteriae sero type 2  S.sonnei  S.dysenteriae sero type 1
  50. 50. Other media which can be used for indole production Sulphide indole motility agar Motility indole ornithine agar Indole rapid tests Indole spot test(filter paper) Indole spot test for anaerobic bacteria Indole microtechnique(indole test strip) Precautions Medium containing glucose should not be used
  51. 51. Gelatin liquefaction / Hydrolysis Principle:  Determines the ability of the organism to produce the proteolytic enzyme (gelatinases) that liquefy gelatin.  Gelatinases hydrolyses gelatin into polypeptides and individual amino acids.  It destroy the structure of gelatin(incorporated into the media ,is free of preservatives and heavy metals) and converts semisolid agar into a liquid. Quality control : Positve : P.vulgaris Negative: Enterobacter aerogenes
  52. 52. Nutrient gelatin (ph-6.8) Pancreatic digest of gelatin – 5 g Beef extract – 3 g Gelatin – 120 g Distilled water – 1 L Gelatin,0.4 %(ph-7.0) Gelatin – 40 g Distilled water – 1 L Procedure :    Inoculate a tube of solidified (refrigerated) nutrient gelatin by stab technique. Incubate the inoculated tube and a control gelatin tube at 37º C at which temperature ,the gelatin will liquefy. At the end of each 24-hour incubation period, place the inoculated tube and the control tube of liquefied gelatin in a refrigerator, for a sufficient length of time (2 week) to determine whether digestion of gelatin has occurred.
  53. 53. Interpretation: Positive test : If a portion of gelatin liquefies. Negative test : If the gelatin remains solid. Positive Negative Bacillus spp. Staphylococcus P.vulgaris P.mirabilis Enterobacter cloacae Clostridium perfringens Pseudomonas Serratia Salmonella B.fastidiosus Morganella Providentia spp. E.coli Klebsiella Precaution: Tube should not be disturbed. A gelatin conc. of 12-15 % may inhibit growth of some microorganisms .
  54. 54. Phenylalanine deaminase test Principle: Determines the ability of the organism to deaminate phenylalanine with the production of phenyl pyruvic acid , which will react with ferric salt to give green colour. Ferric chloride acts a chelating agent and combines with the phenyl pyruvic acid ( end product of deamination ). PPA is a keto acid and forms a green colour in the presence of ferric chloride in the tube. Quality control : Positive: P.vulgaris Negative : E.coli
  55. 55. Phenylalanine agar medium (ph-7.4) 10%ferric chloride,acidified Yeast extract – 3 g DL -phenylalanine – 2g Disodium hydrogen phosphate – 1 g Sodium chloride – 5 g Agar – 12 g Distilled water – 1 L D.w -97.5ml Conc.HCL-2.5ml Ferric chloride-12g Procedure : Inoculate medium with the test organism (BHI broth) and incubate for 1824hrs at 37 c. Add 4-5 drops of aqueous ferric chloride to the slant.
  56. 56. Interpretation: Positive: Green colour develops on slant. Negative: Slant remains original colour. Positive Negative Proteus Morganella Providencia E.Cloacae Serratia Hafnia E.coli Klebsiella Salmonella Shigella Precaution: Test should be interpreted within 5 min.
  57. 57. Decarboxylase Test (Moeller’s method) Principle: Determines the enzymatic ability of an organism to decarboxylate or hydrolyze an amino acid to form an amine with the liberation of carbon dioxide. The production of these decarboxylases is induced by a low ph and as result of their reaction ph rises to neutrality or above . The lysine and ornithine reaction are truly decarboxylases test but the arginine reaction is more correctly recognized now as a dihydrolase test. Quality control: Positive Negative Lysine – Ornithine Arginine Base - E.cloacae K.pneumoniae K.pneumoniae K.pneumoniae K.pneumoniae E.cloacae E.cloacae
  58. 58. 1) Moeller Decarboxylase Broth (Control) (pH 5.2) / Agar (3 g) Peptic Digest of Animal Tissue - 5 g Beef Extract - 5 g Bromcresol Purple - 5 ml Glucose - 0.5 g Cresol Red – 2.5 ml Pyridoxal - 5 g Distilled water – 1 L (2) Moeller Arginine Dihydrolase Broth: Same as (1) with the addition of 10.0 g of L-Arginine HCl. (3) Moeller Lysine Decarboxylase Broth: Same as (1) with the addition of 10.0 g of L-Lysine HCl. (4) Moeller Ornithine Decarboxylase Broth: Same as (1) with the addition of 10.0 g of L-Ornithine HCl.
  59. 59. Procedure : Inoculate each of the three decarboxylase broths / agar medium with the test organism (BHI broth), add 4 mm layer of sterile mineral oil and incubate for 4 days (glucose fermenting organisms), 7 days (glucose non-fermenting organisms) at 37 c. Interpretation : Positive: Alkaline (Purple) colour change compared with the colour tube.(ph 6.8) Negative : No colour change or acid ( yellow) colour in test and control tube.(ph 5.2)
  60. 60. Positive Negative Lysine - E.coli , Klebsiella Hafnia , E.aerogenes Salmonella ,Serratia . Citrobacter E.cloacae, E.amnigenus Proteus, Morgenella, Providentia,Yersinia. Ornithine - E.coli , Hafnia Morgenella, P.mirabilis Citrobacter, Providentia P.vulgaris , P.penneri Arginine – E.cloacae , P.aeruginosa P.fluorescens , P.putida S.anginosus , S.sanguis Plesiomonas,E.faecalis Proteus, Providentia Morgenella , Serratia Hafnia , E.aerogenes E.avium Precaution: Mineral oil is applied to surface of each inoculated broth medium .
  61. 61. Test for metabolism of carbohydrates and related products  Oxidation and Fermentation test (Hugh & Leifson)  Fermentation test
  62. 62. Oxidation / fermentation Test (Hugh & Leifson) Principle : To determine the oxidative or fermentative metabolism of a carbohydrate or its non utilization Biochemistry:  This test employs a semi-solid medium in tubes containing the carbohydrate under test (usually glucose) and a pH indicator.If acid is produced only at the medium surface (aerobic), attack on the sugar is oxidative.  Oxidation is a aerobic process and bacterial oxidisers are usually strict aerobes.  If acid is found through out the tube including the lower layer (anaerobic) ,breakdown is fermentative.  Fermentation is a anaerobic process and bacterial fermenters of carbohydrates are usually facultative anaerobes.
  63. 63. Medium: (ph – 7.1) Peptone – 2g Sodium chloride – 5 g Di potassium hydrogen phosphate – 0.3 g Bromothymol blue – 3 ml Agar – 3g Water – 1 L The carbohydrate to be added is sterilized separately and added to give final conc. of 1 %. Quality control: Fermenter Oxidizer E.coli P.aeruginosa Non utilizer A.faecalis
  64. 64. Procedure: Duplicate tubes of medium are inoculated by stabbing, one tube is covered with layer of sterile melted petroleum jelly to a depth of 5 – 10 ml ; Second left uncovered and both are incubated for 7 days at 37 C. Results:  Oxidising organisms, eg Pseudomonas species, produce an acid reaction in the open tube only  Fermenting organisms, eg Enterobacteriaceae, produce an acid reaction throughout the medium in both tubes  Organisms that cannot break down the carbohydrate aerobically or anaerobically (Non utilizer), eg Alcaligenes faecalis, produce an alkaline reaction in the open tube and no change in the covered tube Hugh and Leifson’s medium can also be used for recording gas production and motility
  65. 65. Fermenters E.Coli Staphylococcus Stomatococcus Vibrio Aeromonas Oxidizers Pseudomonas Micrococcus
  66. 66. Fermentation test Principle: It determines the ability of an organism to ferment a specific carbohydrate that is incorporated in a basal medium , there by producing acid with or without visible gas. Quality control: A. Peptone medium with andrade’s indicator Dextrose Positive ,with gas Positive , No gas Negative E.coli S.flexneri P.aeruginosa B. Heart infusion broth with bromocresol purple indicator Sorbitol Positive Negative S.mutans S.mitis
  67. 67. Fermentation Broth: Trypticase: 1g Carbohydrate: 0.5 g Sodium Chloride: 0.5 g Phenol red : 0.0189 mg Procedure: Inoculate each labeled carbohydrate broth with bacterial culture.(keep uninoculated tubes as control tubes) ,Incubate for 24 hours at 37oC. Interpretation: Acid production: Yellow color(ph 6.8) Acid and Gas production: Yellow colour and gas. Gas production can be detected by the presence of small bubbles in the inverted Durham’s tubes. Absence of fermentation: The broth retains the red colour(ph8.4).
  68. 68. Precautions: After inoculation into a particular sugar, sterilize the loop in order to avoid cross contamination of the tube with other sugars. Do not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in breaking down of the molecules and form compounds with a characteristic colour and flavour. The process is known as caramelization of sugar (the browning of sugar).
  69. 69. Peptone medium with andrade’s indicator (for enteric and coryneforms). Positive – Pink with or without gas formation in Durham tube.(ph5.5). Negative – Growth but no change in colour. Heart infusion broth with bromocresol purple indicator (streptococci and enterococci ). Positive – yellow(ph 5.2) Negative – Growth, but no change in colour (ph6.8)
  70. 70. Combined tests  Egg yolk agar; Test for lipase and lecithinase.  Triple sugar iron agar test for H2S production/Kligler iron agar test.
  71. 71. Egg yolk agar test Principle :  Lecithinase are enzyme released by bacteria that destroy animal tissues. Lecithin is a normal component of egg yolk in EYA . lecithinase  Lecithovitellin     phosphorylcholine + diglyceride Diglyceride forms a ppt in the medium, this ppt appears as a white opaque halo surrounding the colony that produce lecithinase. Bacterial lipase hydrolyze the breakdown of triglyceride into glycerol and free fatty acids . Fatty acids are insoluble and cause opacity in EYA , producing an iridescent sheen on the colonies and surface. Lipase is not diffusible and the reaction occurs on the surface of agar in the immediate vicinity of the colony.
  72. 72. Quality control: Positive – B.cereus ( lecithinase) , S.aureus (lipase) Negative – E.coli Egg yolk agar medium: Nutrient agar – 85 ml Egg yolk suspension – 15 ml Procedure: Streak an area of agar surface to obtain isolated colonies examine for halo and iridescence. Incubate for 24 to 48 hrs /72 hrs for anaerobes at 37 C.
  73. 73. Interpretation: Lecithin: use transmitted light to observe halo. Positive: development of a milky white opaque halo around colony. Negative: no halo in the medium or around the colony. Lipase: hold the plate on an angle with good lightening. Positive: development of an iridescent sheen on the surface of the colony and the surrounding. Negative: no change in the medium.
  74. 74. Lecithinase positive: Lipase positive: B.cereus C.perfringens A.haemolyticum P.fluorescens Burkhholderia C.sporogenes G.vaginalis Lecithinase negative : Lipase negative: C.difficile P.putida C.difficile C.perfringens Precaution: Incubation can be required upto 2 weeks.
  75. 75. Triple sugar iron agar test Principle:  Bacteria that ferment any of the three sugars in the medium will produce by products .These by-products are usually acids, which will change the colour of the red pH-sensitive dye (phenol red) to a yellow colour.  Triple Sugar Iron Agar (TSI) are used to determine if bacteria can ferment glucose,sucrose or lactose and if they can produce hydrogen sulfide or other gases.  TSI contains three sugars: glucose, lactose and sucrose. Lactose and sucrose occur in 10 times the concentration of glucose (1.0% versus 0.1%).  Some bacteria utilize thiosulphate anion as a terminal electron acceptor, reducing it to sulphide.
  76. 76.  If this occurs, the newly-formed hydrogen sulphide (H2S) reacts with ferrous sulphate in the medium to form ferrous sulphide, which is visible as a black precipitate.  Blackening of the butt due to H2S production may mask the acid reaction (yellow) in the butt. .  Production of gases other than hydrogen suphide is indicated either by cracks or bubbles in the media or the media being pushed away from the bottom of the tube Quality control: A/A : E.coli K / A H2S⁺ : S.typhi K / NC : P.aeruginosa
  77. 77. TSI slant: Beef extract – 3 g Peptone – 20 g Lactose – 10 g Ferric citrate – 0.3 g Sodium thiosulphate – 0.3 g Phenol red – 12 ml Yeast extract – 3g Glucose – 1 g Sucrose – 10 g Sodium chloride – 5 g Agar – 12 g Distilled water – 1 L Procedure: Steak a heavy inoculum over the surface of the slope and stab into the butt , incubate at 37 C for 4 hrs.
  78. 78. Interpretation:  If an organism ferments glucose only, the entire tube turns yellow due to the effect of the acid produced on phenol red. Because there is a minimal amount of glucose present in the tube, the organism quickly exhausts it and begins oxidizing amino acids for energy. Ammonia is thus produced and the pH rises.  Within 24 hours the phenol red indicator reverts to its original red color on the slant. Because TSI/KIA media is poured as a deep slant, the butt has limited oxygen and bacteria are unable to oxidize amino acids there. The butt thus remains yellow.  If an organism can ferment lactose and/or sucrose, the butt and slant will turn yellow (as they do from glucose fermentation).  However, they remain yellow for at least 48 hours because of the high level of acid products produced from the abundant sugar(s).
  79. 79. Reading the results on TSI enables to identify the several pathogens
  80. 80. Results (slant/butt) Symbol Interpretation Red/yellow K/A Glucose fermentation only; Peptone catabolized Yellow/yellow A/A Glucose and lactose and/or sucrose fermentation Red/red K/K No fermentation; Peptone catabolized Red/no color change K/NC No fermentation; Peptone used aerobically Yellow/yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation; Gas produced Red/yellow with bubbles K/A,G Glucose fermentation only; Gas produced Red/yellow with bubbles and black precipitate K/A,G, H2S Glucose fermentation only; Gas produced; H2S produced Red/yellow with black precipitate K/A, H2S Glucose fermentation only; H2S produced Yellow/yellow with black precipitate A/A, H2S Glucose and lactose and/or sucrose fermentation; H2S produced No change/no change NC/NC No fermentation A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction
  81. 81. Organisms ATCC™ Slant *E.coli *S.enterica *S.flexneri *P.aeruginosa 25922 14028 12022 27853 Acid Alkaline Alkaline Alkaline Butt Gas H2S Acid Acid Acid Alkaline + +/– – – – + – –
  82. 82. Miscellaneous test  Detection of motility – Semi-solid agar  Coagulase test
  83. 83. Motility testing Principle: Motility agar is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark. The results of motility agar are often difficult to interpret. Generally, if the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile). Quality control: Positive : E.coli Method:  Hanging drop  Semisolid agar deep Negative: K.pneumoniae
  84. 84. Procedure: Hanging drop Place 1 drop of broth culture in centre of cover slip. Place small drop of immersion oil on cover slip corners. Invert the cover slip over the concavity of depression slide. Examine with 40 X objective. Semi-solid agar deep Stab a culture growing on agar medium to a depth of only 1/3rd to ½ inch in the middle of the tube. incubate at 37 C for 7 days. Interpretation: Hanging drop: Positive: organism change position with the respect to each other. Negative : organism remain in the same relative position to other organism. Semi-solid agar deep: Positive: Motile organism will spread out into the medium from the site of inoculation. Negative: Non motile organism remain at the site of inoculation .
  85. 85. Motile Non-motile E.coli Pseudomonas Salmonella spp. Proteus Citrobacter Planococcus Rhodococcus Klebsiella Shigella Acinetobacter S.gallinarum-pullorum
  86. 86. Coagulase test Principle:  Coagulase is an enzyme that clots blood plasma by catalyzing the conversion of a soluble protein (fibrinogen) to an insoluble protein (fibrin), resulting in a visible clot.  Coagulase is present in two forms bound and free (different property)  Bound coagulase (CF) : Is attached to bacterial cell wall and not present in culture filtrate, fibrin strands are formed between the bacterial cells when suspended in plasma, causing them to clump to visible aggregates.  Free coagulase: Is a thrombin like substance present in culture filtrate. When a suspension of coagulase producer is prepared in plasma , a visible clot forms as the result of coagulase reacting with a serum substance (CRF) to form the complex that in turn reacts with fibrinogen to produce the fibrin clot. Quality control: Positive: S.aureus Negative: S.epidermidis
  87. 87. Method: Slide test Tube test •Place a drop of plasma (rabbit plasma with EDTA) on a glass slide. •Place a drop of distilled water / saline next to the plasma drop as a control. •Emulsify a colony in each drop, mix well. •Rock the slide for 5-10 sec. 1)Emulsify several colonies in 0.5 ml of rabbit plasma to give a milky suspension, incubate at 37 C for 4 hrs. 2) Prepare 1:6 dilution of the plasma in saline and place 1 ml diluted plasma in test tube , emulsify a colony, incubate at 37 C upto 4 hrs. Examine for clot formation. Interpretation: Slide test: Positive : Macroscopic clumping in 10 sec or less in plasma drop and no clumping in control drop. Negative: No clumping in either drop. Tube test: Positive: Clot of any degree. Negative: No clot.
  88. 88. Coagulase: Positive S.aureus S.intermedius Negative S.saprophyticus S.epidermidis S.hominis S.capitis Precaution:  Rabbit plasma is preferably used over human plasma.  Plasma should be sterilized.