IMVIC TEST.pptx

Dr.P.B.PRAVEENKUMAR
FIRST YEAR POST GRADUATE
DEPARTMENT OF MICROBIOLOGY
THANJAVUR MEDICAL COLLEGE
THANJAVUR

1. IMViC TEST Dr. P.B.PRAVEENKUMAR FIRST YEAR MICROBIOLOGY PG RESIDENT GOVERNMENT THANJAVUR MEDICAL COLLEGE

2. INDOLE TEST - PRINCIPLE To determine the ability of an organism by means of trytophanase enzyme to split indole from the tryptophan molecule .

3. LIST OF INDOLE +VE AND –VE ORGANISMS INDOLE POSITIVE Escherichia coli Proteus vulgaris K.oxytoca & Ornitholytica Vibrio cholerae Edwardsiella All Proteus species except Proteus mirabilis Bacillus alvei Haemophilus haemoglobinophilus and Haemophilus influenzae Pasteurella multocida and Pasteurella pneumotropica INDOLE NEGATIVE Klebsiella species except two Proteus mirabilis Pseudomonas Salmonella All bacillus species except Bacillus alvei Remaining Haemophilus species Pasturella hemolytica and Pasteurella ureae

4. MECHANISM OF INDOLE TEST

5. MEDIA USED FOR INDOLE TEST 1. Tryptophan (or) Peptone broth *Contains basal medium(Peptone water and Nacl ) *Tryptophan 1% Commercial products *BBL: Trypticase Peptone broth ( Tryptophanase broth) * Difco tryptone broth

6. METHOD OF PREPARATION IN PEPTONE BROTH Fill the test tube with 4 ml of peptone water Autoclave it at 121 degree celsius for 15 minutes. Light inoculum is added into tube from 18 to 24 hour pure culture / Kligler’s iron agar(KIA) Incubate at 35 degrees celsius for 24 to 48 hours. Add kovac’s or Ehrlich’s reagent.

7. MEDIA USED FOR INDOLE TEST (cont) 2. Casein medium *A pancreatic digest of casein solution may be prepared. * Protein casein contains 1.2 grams of tryptophan per 100 grams . *Dissolve by moderate heating and tube contains 4 ml *Autoclave it at 121 degree celsius for 15 minutes

8. INGREDIENTS OF CASEIN MEDIUM PANCREATIC DIGEST OF CASEIN 2 GRAMS SODIUM CHLORIDE 0.5 GRAMS DISTILLED WATER 100 ml

9. REAGENTS USED FOR INDOLE TEST 1. Ehrlich’s indole test p- DIMETHYL AMINO BENZALDEHYDE 2 GRAMS ABSOLUTE ETHYL ALCOHOL 190 ml CONCENTRATED HCL 40 ml

10. HOW TO UTILISE EHRLICH’S INDOLE REAGENT???? Add 1 ml of ether or xylene to a 24 hour or 48 hour incubated broth tube Shake well Wait for the ether to rise to the surface of the medium. Add 0.5 ml of Ehrlich’s reagent to the tube and tilt it.

11. REAGENTS FOR INDOLE TEST (cont) 2. Kovac’s indole test PURE AMYL / ISOAMYL / BUTYL ALCOHOL 150 ml p- Dimethyl amino benzaldehyde 10 grams CONCENTRATED HCL 50 ml

12. METHOD OF PREPARATION & UTILIZATION OF KOVAC’S REAGENT Dissolve the aldehyde in the alcohol Slowly add the acid in the aldehyde -alcohol mixture Add 5 drops of kovac’s reagent directly into 24 hour or 48 hour incubated tube Shake the tube gently.

13. INDOLE TEST HYPOTHESIS ACCORDING TO GADEBUSH & GABRIEL Addition of Isoamyl alcohol makes Kovac’s reagent more stable. With ether reagent in Ehrlich’s method, dissolving aldehyde in solvent require gentle heating. ACCORDING TO EWING AND DAVIS Use Kovac’s for Indole and Ehrlich’s for nonfermenters and anaerobes

14. Why we want to separate 2ml from peptone broth before we adding reagent after 24 hours?? Remove 2ml aseptically from 4ml containing peptone broth to check with reagent. Positive means ok.........Finish the test with interpretation. If no change while mixing with reagent means, then incubate that 2ml broth for further 24 hours and retested .

15. QUALITY CONTROLS

16. STORAGE OF INDOLE REAGENT Store it in refrigerator when not in use Each week quality control check should be done and then discarded. Stored in brown bottles with glass toppers.

17. CHEMISTRY OF REAGENT Due to pyrrole structure in indole Quinoidal red violet compound Due to tryptophan splitting of acid If concentrated Hcl is used no heating is required

18. INTERPRETATION INDOLE TEST +VE INDOLE TEST -VE

19. INTERPRETATION IN DETAIL POSITIVE TEST RED RING AT THE SURFACE OF THE MEDIUM NEGATIVE TEST TAKES THE COLOUR OF KOVAC’S REAGENT ORANGE COLOUR DUE TO FORMATION OF SKATOLE WHICH IS A PRECURSOR FOR INDOLE

20. RAPID INDOLE TESTS Indole spot test Indole spot test for anaerobic bacteria Indole microtechnique Reagent impregnated indole test strip

21. INDOLE SPOT TEST ( Method of Vracko and Sherris ) KOVAC’S REAGENT DMABA DMACA 3ml OF AMYL ALCOHOL IN 1ml OF CONCENTRATED Hcl 1% SOLUTION IN 10% v/v CONCENTRATED Hcl 1% SOLUTION IN 1% v/v CONCENTRATED Hcl PREPARE FRESH EVERY FOUR MONTHS; DETECTS 6-12 MICROGRAM OF INDOLE PER ml PREPARE FRESH EVERY TWO MONTHS; DETECTS 3 MICROGRAMS OF INDOLE PER ml MOST STABLE AND MOST ECONOMICAL REAGENT MOST SENSITIVE AGENT

22. PREPARING INOCULUM AND PROCEDURE Take a 18 to 24 hour pure culture from sheep blood agar Preferably Trypticase soy blood agar(5% SBA) Take a whatmann no.1 filter paper and put it in petri dish Moisten the filter paper with 1 to 1.5 ml of reagent. Smear inoculum onto moistened filter paper.

23. INDOLE SPOT TEST INTERPRETATION INTERPRETATION KOVAC’S REAGENT DMABA DMACA INDOLE +VE BROWN REDDISH-PURPLE RED COLOUR PINK TO RED COLOUR BLUE OR GREEN COLOUR INDOLE -VE WHITE TO YELLOW COLOUR WHITE TO YELLOW COLOUR WHITE TO YELLOW COLOUR

24. INDOLE SPOT TEST FOR ANAEROBIC BACTERIA Same procedure as previously mentioned Exception is that 1% DMACA in 10% Concentrated Hcl . Positive : Immediate formation of blue colour around growth. Negative : No colour change or pinkish colour.

25. INDOLE MICRO TECHNIQUE (Method of Arnold and Weaver) BROTH 1 BROTH 2 Tryptone 1% Tryptophane 0.03% Beef extract 0.3% Peptone 0.1% Dipotasium phosphate 0.5%

26. INDOLE MICRO TECHNIQUE (cont) Preheated broth tubes are used. Adding heavy inoculum from tryptone agar. Using kovac’s reagent Waterbath at 37 degree celsius for 6 minutes to 2 hours. Same interpretation as previously mentioned slide............

27. REAGENT IMPREGNATED INDOLE TEST STRIP Results within 4 hours incubation Many false negatives especially when the inoculum is taken from TSI broth. Occasional false positives

28. Why glucose containing broth not suitable for indole test????? Because glucose inhibits indole production . Formation of indole will occur with organisms which is fermenting carbohydrates but not utilising carbohydrates .

29. Which organism will break down indole rapidly as soon as it is formed????? CLOSTRIDIUM SPECIES

30. Then what’s the optimal pH for indole production???? SLIGHT ALKALINE (7.4 TO 7.8)

31. Indole producing organisms interferes with antimicrobial activity than non- indole producing organisms??? YES ( ACCORDING TO MARTIN AND KLEIN )

32. METHYL RED TEST - PRINCIPLE To test the ability of an organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system.

33. LIST OF METHYL RED +VE AND –VE ORGANISMS METHYL RED +VE Escherichia coli Yersinia species Listeria monocytogenes METHYL RED -VE Enterobacter aerogenes Enterobacter cloacae Klebsiella Other gram negative Non enteric bacilli

34. MECHANISM OF METHYL RED TEST Based on the use of pH indicator , methyl red to determine the hydrogen ion concentration (pH) present when an organism ferments glucose. HYDROGEN ION CONCENTRATION IS DEPENDS ON GAS RATIO 1:1  MR +VE 2:1MR -VE

35. Why we have to incubate the broth atleast for 48 hours???? If we test it before 48 hours by adding methyl red reagent, false positives will occur...... Since we have to check the capability of bacteria to retain it’s acid producing capability even in the presence of phosphate buffer Just wait for 48 hours and then check with reagent

36. CHARACTERISTICS OF MR +VE AND MR –VE ORGANISMS METHYL RED +VE FERMENTED PRODUCTS PRODUCES ACIDIC METABOLITES AFTER TWO TO FIVE DAYS MORE ACIDS LOW TERMINAL pH MAINTAINING ACIDITY IN PHOSPHATE BUFFER METHYL RED -VE FERMENTED PRODUCTS DECARBOXYLATION NEUTRAL ACETYL METHYL CARBINOL (ACETOIN) HIGH TERMINAL pH NEUTRAL pH

37. METHYL RED +VE 1:1 GAS RATIO 2 Glucose + H2O 2 Lactic acid + Acetic acid + Ethanol + 2Co2 + 2 H2

38. METHYL RED –VE 2:1 GAS RATIO Glucose + ½ H2O 2,3 butanediol + H2O + 2CO2 Acetoin 2,3 butanediol

39. CLARK AND LUBS MEDIUM (MR/VP BROTH) POLYPEPTONE OR BUFFERED PEPTONE 7.0 GRAMS DEXTROSE (GLUCOSE) 5.0 GRAMS DIPOTTASIUM PHOSPHATE BUFFER 5.0 GRAMS DISTILLED WATER 1000 ml

40. INOCULUM AND INCUBATION Light inoculum from 18 to 24 hours pure culture of kligler’s iron agar in 5 ml broth (MR and VP) Incubated at 35 degree celsius for 48 hours (or) Incubated at 30 degree celsius for 3 to 5 days

41. REAGENT PREPARATION AND USAGE Dissolve 0.1 gram of methyl red in 500 ml of 95% ethyl alcohol . Add 200 ml of distilled water to the alcohol indicator mixture Add 5 drops of methyl red indicator into aliquot.

42. QUALITY CONTROL FOR METHYL RED TEST POSITIVE CONTROL Escherichia coli (ATCC 25922) NEGATIVE CONTROL Klebsiella (ATCC 13883)

43. INTERPRETATION OF MR TEST COLOUR pH INTERPRETATION RED LESS THAN OR EQUAL TO 4.4 METHYL RED POSITIVE ORANGE (Delayed reaction) 5 TO 5.8 FI (FURTHER INCUBATION) FOR 4 DAYS AND REPEAT THE TEST YELLOW AT A pH of 6.0 METHYL RED NEGATIVE

44. RAPID TEST (Method of Barry, Bernsohn , Adams and Thrupp ) 0.5 ml aliquot into small test tubes Pick a centre of colony from either EMB/MAC/BAP Incubate it for 18 hours After incubation add one drop of MR reagent in each tube setup Interpretation is same as previously mentioned.

45. VOGES PROSKAUER TEST - PRINCIPLE Depends on the production of acetyl methyl carbinol ( Acetoin ) from pyruvic acid from glucose fermentation. In the presence of alkali and atmospheric oxygen, acetoin is oxidised to diacetyl which reacts with alpha-naphthol to give red colour.

46. LIST OF VP +VE AND VP –VE ORGANISMS VOGES PROSKAUER +VE Klebsiella pneumoniae Enterobacter Staphylococcus Klebsiella oxytoca Hafnia alvei ( Enterobacter alvei at 25 degree celsius ) Listeria monocytogenes with coblentz reagent Yersinia enterocolitica at 25 degree celsius Serratia group VOGES PROSKAUER -VE Escherichia coli Micrococcus Klebsiella ozaenae Klebsiella rhinoscleromatis Yersinia enterocolitica at 37 degree Listeria monocytogenes with O’Meara reagent

47. MECHANISM INVOLVED Same mechanism as already explained in methyl red test in exact opposite way (Acetyl methyl carbinol from BUTYLENE GLYCOL PATHWAY )

48. MEDIUM AND INOCULUM Same MR/VP broth.... Light inoculum for all except for Coblentz method where heavy inoculum is used.

49. VP REAGENTS REAGENTS BARRIT’S VP REAGENT COBLENTZ VP REAGENT O’MEARA’S REAGENT (MODIFIED SINGLE VP REAGENT) REAGENT A 5% ALPHA NAPHTHOL IN ABSOLUTE ETHANOL 5% ALPHA NAPHTHOL IN 95% ETHANOL REAGENT B 40% KOH 0.3% CREATINE IN 40% KOH REAGENT 0.3% CREATINE IN 40% KOH

50. METHOD OF UTILISATION ( In MR/VP broth ) REAGENTS ALIQUOT DIVIDING LEVEL REAGENT ADDING PROCESS SHAKING THE TUBE WAITING TIME BARRIT’S VP REAGENT (0.6 ml and 0.2 ml) 2.5 ml REAGENT A f/b REAGENT B FOR 30 SECONDS TO ONE MINUTE ATLEAST 10 TO 15 MINUTES COBLENTZ REAGENT (0.6 ml and 0.2 ml) 2.0 ml REAGENT A f/b REAGENT B FOR 30 SECONDS TO ONE MINUTE ATLEAST 10 TO 20 MINUTES O’MEARA’S REAGENT 1.0 ml Add one ml of reagent FOR 30 SECONDS TO ONE MINUTE 35 degrees/ RT Final reading only after 4 hours, If equivocal Repeat at 25 degrees incubation

51. BIOCHEMICAL MECHANISM

52. COMPOUNDS CONTAINING GUANIDINE NUCLEUS Arginine (Here it is present) Agmatine Creatine Dicyanamide Guanidine acetic acid

53. ROLE OF REAGENT CONTENTS IN VP TEST DIACETYL COMPOUND COLOUR FORMING AGENT ALPHA NAPHTHOL COLOUR INTENSIFIER KOH (POTTASIUM HYDROXIDE) OXIDIZING AGENT TO OXIDIZE ACETOIN TO DIACETYL

54. QUALITY CONTROLS Positive control – Escherichia coli (ATCC 25922) Negative control – Enterobacter cloacae Enterobacter aerogenes

55. HOW LONG WE CAN WAIT FOR VP TEST INTERPRETATION???? Maximum one hour otherwise after one hour copper like colour due to the action of KOH on alpha naphthol ( FALSE POSITIVE )

56. INTERPRETATION

57. ALTERNATE TESTS Gas liquid chromatography ( Diacetyl measurement) Electron capture gas liquid chromatography (EC-GLC) Gas chromatography chemical ionization mass spectrometry Colorimetric method for measurement of diacetyl

58. RAPID TESTS REAGENT IMPREGNATED VP STRIP TEST RAPID VP TEST OF BARRY AND FEENEY Results within 4 hours of incubation Two step procedure False positives with proteus and klebsiella species Growth from MAC , BA, TSI and KIA Add it in MR/VP broth 35 degree celsius waterbath for 4 hours Add creatine solution 0.3% in 2 drops Add Barrit’s reagents A and B: 2 to 3 drops each Positive – Cherry red colour within 15 minutes

59. Sometimes acetoin positive organisms fails to give positive VP test.......What will we do???? GENTLE HEATING OF CULTURE CONTAINING VP REAGENT

60. What will happen if overnight incubation done more than 3 days (OR) reverse the pouring of reagents A and B?? WEAKLY POSITIVE OR FALSE NEGATIVE

61. WHICH ORGANISM GIVES BOTH MR AND VP POSITIVE??? PROTEUS MIRABILIS HAFNIA ALVEI (BUT STILL VP DELAYED)

62. Shall we use routine clark and lubs medium even for bacillus and staphylococcus??? No......In presence of phosphate and sodium chloride, it may interfere with acetoin production..... So use plain glucose peptone broth without Nacl and phosphate (ACCORDING TO ABD-ED-MALEK AND GIBSON)

63. Meat infusion broth should not be used for VP test....Why???? Because of the presence of acetoin , diacetyl and related substances in muscle extract FALSE POSITIVE

64. CITRATE TEST - PRINCIPLE To determine if an organism is capable of utilising citrate as the sole source of carbon for metabolism with resulting alkalinity .

65. LIST OF CITRATE UTILISED AND CITRATE UTILISED NOT ORGANISMS CITRATE UTILISED Klebsiella species Salmonella species except S.typhi Citrobacter species Enterobacter species Serratia liquefaciens Aeromonas hydrophilia Bordetella species except B.pertussis Proteus/ Providencia rettegeri Pseudomonas cepacia CITRATE NOT UTILISED Escherichia coli Edwardsiella Salmonella typhi Yersinia Actinobacillus equui Aeromonas salmonicida Moraxella species ( M.urethralis – variable) Morganella morganii (Proteus morganii ) Pseudomonas maltophilia

66. MECHANISM IN CITRATE TEST Citrate metabolism occurs via Tricarboxylic acid (TCA Cycle)/Krebs cycle/Citric acid cycle in presence of Coenzyme A . But in bacteria NO COENZYME A. Instead bacteria can use Citritase /Citrate oxaloacetate lyase /Citrate demolase . Citrate breakdown will happen well in basic pH comparing with acidic pH.

67. MECHANISM (cont.) CITRATE Mn2+ (or) Mg2+ Co2 + Formic acid + Acetate

68. BASED ON pH CITRATE TEST LOOKS LIKE......

69. This is how citrate test works??? Citrate utilised Citrate not utilised

70. Is this citrate test using citrate alone as a sole source??? NO.............It’s using citrate as a sole source for carbon. It is using ammonium salts also as a sole source of nitrogen which is giving alkalinity .........( By breakdown of ammonium salts to ammonia )

71. MEDIA USED FOR CITRATE TEST Simmons citrate medium – pH 6.9 (Modification of Koser’s medium with 1.5% agar and pH indicator bromothymol blue) Christensen’s citrate sulfate medium – pH 6.7

72. SIMMONS CITRATE MEDIUM Magnesium sulphate 0.2 Grams Monoammonium phosphate / Ammonium dihydrogen phosphate 1.0 Grams Dipottasium phosphate 1.0 Grams Sodium citrate (2.77 grams of sodium citrate in 5 ½ molecules of water) 2.0 Grams Sodium chloride 5.0 grams Agar 15 to 20 Grams Bromothymol blue (1.5% alcoholic) 0.08 Grams Distilled water 1000 ml

73. METHOD OF PREPARATION OF MEDIA Weigh it properly Rehydrate it with distilled water Heat gently Place approximately 4 to 5 ml in each tube ( Long slant, short butt ) Autoclave it for 10-15 minutes Allow medium to solidify in a slanted position Light inoculum is used

74. CHRISTENSEN’S CITRATE SULFIDE MEDIUM Sodium citrate 3.0 Grams Glucose 0.2 Grams Yeast extract 0.5 Grams Cysteine monohydrochloride 0.1 Grams Ferric ammonium citrate (Will be eliminated if H2S didn’t formed) 0.4 Grams Monopotassium phosphate 1.0 Grams Sodium chloride 5.0 Grams Sodium thiosulfate (Will be eliminated if H2S didn’t formed) 0.08 Grams Phenol red 0.012 Grams Agar 15.0 Grams Distilled water 1000 ml

75. QUALITY CONTROL CHECKS FOR CITRATE TEST POSITIVE CONTROL – Klebsiella aerogenes (ATCC 13048) NEGATIVE CONTROL – Escherichia coli (ATCC 25922)

76. INTERPRETATION OF CITRATE TEST INTERPRETATION SIMMONS CITRATE MEDIUM CHRISTENSEN’S CITRATE SULFIDE MEDIUM CITRATE UTILISED INTENSE BLUE COLOUR ON THE SLANT PINK RED COLOUR ON THE SLANT CITRATE NOT UTILISED NO COLOUR CHANGE ( GREEN COLOUR ) NO COLOUR CHANGE

77. SIMMONS CITRATE TEST - INTERPRETATION

78. RAPID CITRATE BLOOD TEST ( Method of Cordaro and Sellers ) To test the ability of an organism to utilise the citrate present in citrated blood to form a clot .

79. CORDARO AND SELLER’S CITRATE BLOOD MEDIA INOCULUM DILUENT TEST MEDIUM DEHYDRATED BHI BROTH (12.5 GRAMS/LITER) FOR ONE BOTTLE OF BHI BROTH WE SHOULD ADD 15 ml OF OUTDATED CITRATED BLOOD BANK BLOOD CALCIUM CHLORIDE (CaCl2 100 mg) MIX ASEPTICALLY AND PIPETTE IT 1 ml AMOUNTS INTO SMALL STERILE TEST TUBES BASED ON OUR REQUIREMENT SODIUM CHLORIDE (2.5 g) REMAINING OUTDATED BLOOD WE CAN KEEP IT TILL 2 MONTHS IF IT’S REFRIGERATED

80. RAPID CITRATE TEST – INOCULUM AND AUTOCLAVING Growth from an 18 hour pure culture from TSI media ( Heavy inoculum ) Incubate it at 35 degrees for 1 to 6.5 hours ( Hourly monitoring is must )

81. INTERPRETATION OF RAPID CITRATE TEST Citrate utilised – Presence of firm clot Citrate not utilised – No clot formation

82. What will happen if large inoculum is used in citrate slant test??? (LIGHT YELLOW TO TAN COLOUR ON SLANT) FALSE POSITIVE

83. Why it is advisable to diluting the inoculum with normal saline prior to inoculating citrate??? TO PREVENT CARRY OVER OF OTHER CARBON SOURCES LIKE GLUCOSE, PEPTONE AND ACETATE FALSE POSITIVE

84. Sometimes organisms will grow on citrate slant but not producing colour change...... Intrepretation ???? CITRATE IS UTILISED ( POSITIVE CITRATE TEST ) ORGANISM ENTERED LOG PHASE SO ONLY GROWTH IS VISIBLE IF CARBON AND NITROGEN IS ASSIMILATED.......

85. Till how long we can wait to interpret citrate test??? TILL 7 DAYS ( For pH to change )

86. REFERENCES Bailey scott diagnostic microbiology Macfaddin biochemical reactions Mackie and Mcartney diagnostic microbiology

87. THANK YOU