OXIDASE, CATALASE, COAGULASE TESTS.pptx

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

1. BIOCHEMICAL TESTS ( Oxidase test, C atalase test, Coagulase test) Dr. P.B. PRAVEENKUMAR FIRST YEAR MICROBIOLOGY PG RESIDENT GOVERNMENT THANJAVUR MEDICAL COLLEGE

2. OXIDASE TEST PRINCIPLE : To determine the presence of Cytochrome oxidase enzyme which catalyses the oxidation of reduced cytochrome by molecular oxygen.

3. HISTORY Originally developed to identify all neisseria species but later used to separate the P seudomonadaceae from oxidase (-) of Enterobacteriaceae . Mostly G(+) are oxidase (-) Mostly G(-) will give variable reactions except ENTEROBACTERIACEAE.

4. IDENTIFICATION PURPOSE Oxidase (+) Moraxella Neisseria and Vibrio Plesiomonas shigelloides ( Aeromonas ) All brucella species(Except two) All pseudomonas species (Except one) Alcaligenes Branhamella (N. Catarrhalis ) Oxidase (-) Acinetobacter Enterobacteriaceae Brucella neotomae Brucella ovis Pseudomonas maltophilia Yersinia

5. BACTERIA RESPIRATION All aerobic bacteria obtain their energy by respiratory chain( Electron transport chain ) which is responsible for production of various substrates. O 2 is the final hydrogen acceptor from either it’s water or hydrogen peroxide depending on bacterial species.

6. ELECTRON TRANSPORT CHAIN

7. OXIDASE REMOVES HYDROGEN USE ONLY OXYGEN! All oxidase (+) organisms are either aerobic or facultative except Vibrio fetus (Campylobacter pylori) which requires microaerophilic conditions.

8. ACCORDING TO GORDON AND MCLEOD.... Positive oxidase reactions are only limited to those organisms capable of growing in presence of oxygen and ability to produce catalase . 2H 2 O 2 2H 2 O + O 2

9. OBLIGATE ANAEROBIC ORGANISMS Only survive without oxygen So lack cytochrome oxidase activity DEIBEL AND EVANS showed this in Lactobacillus. NO OXYGEN SURVIVING...NO OXIDASE ACTIVITY

10. STILL WE ARE BELIEVING OXIDASE ENZYME IS TWO SEPARATE ENTITY???? Previously we will consider that Neisseria species were thought to produce indophenol oxidase ....... Pseudomonas species were thought to produce cytochrome oxidase ....... After spectrophotometric studies both oxidases are same.............

11. OTHER NAMES OF CYTOCHROME OXIDASE Atmungsferment Cytochrome C oxidase Cytochrome Alpha 3 oxidase Cytochrome Alpha oxidase GABY AND HADLEY

12. FOUR BACTERIAL PIGMENTS ACT AS TERMINAL OXIDASE Cytochrome oxidase alpha 1 Cytochrome oxidase alpha 2 Cytochrome oxidase alpha 3 Cytochrome O (CO binding pigment) Derived by Castor and Chance by means of photochemical methods. A mixture of cytochrome alpha and alpha 3 having same protein.. So complex formed ( Cytochrome c oxidase / Cytochrome alphaalpha 3 )

13. CYTOCHROME C OXIDASE Having two molecules of heme A Each heme A having one Fe atom which go and come back between Ferric and Ferrous ions Cytochrome-Fe3+ + e- Cytochrome-Fe2+ ( Oxidised removal ( Reduced gain of of electrons ) electrons )

14. RESPIRATORY ENZYME (Important bichemical reaction) Two reduced cytochrome c + 2H+ + 1/2H 2 O Cytochrome C Oxidase Two oxidized cytochrome c + H 2 O

15. REAGENTS 1. Kovac’s reagent 1% solution of tetramethyl -p- phenylene - diamine (Less toxic but more expensive comparing with dimethyl ) imparts oxidase (+) lavender colour initially purplish black gradually. Solution is colourless.

16. REAGENTS (cont.) 2. Gordon and Mcleod’s reagent 1 to 1.5% dimethyl -p- phenylenediamine hydrochloride/N,N- dimethyl -p- phenylene diamine monohydrochloride (HCL)/p- aminodimethylaniline monohydrochloride ) Solution is light purple .

17. Why p- aminodimethylaniline oxalate reagent having advantage over others??? Extremely stable both in powder or solution forms ( Atleast 6 months!!!! ) No black precipitate while comparing with Hcl combination of Gordon’s reagent.

18. REAGENTS (Cont.) 3. Gaby and Hadley reagent modified by Ewing and Johnson/ Indophenol oxidase reagent Original Nadi reaction from na phthol and di amine Reagent A 1% naphthol in 95% Ethyl alcohol Reagent B 1% p- aminodimethylaniline Hcl / dimethyl -p- phenylenediamine oxalate

19. Why Gaby and Hadley reagent A alone is an exception?????? While preparing other reagents we will take 1 gram of reagent in 100 ml of distilled water. But for this reagent A of Gaby and Hadley we will take 1 gram of naphthol in 100 ml solution of 95% ethyl alcohol........... Warm gently and store it in darker place for 15 minutes before usage.........

20. REAGENTS(cont.) 4. Carpenter, Suhrland and Morrison reagent contains 1% p- aminodimethylaniline oxalate. 5. Commercial reagent: Cepti seal Oxidase reagent 6. Oxidase impregnated disc (p- aminodimethylaniline reagent) : (6a) Difco : Bacto -differentiation discs (6b) BBL: Taxo N discs

21. METHODS OF UTILIZATION OF REAGENT SOLUTIONS (A) Direct slant procedure ( Indophenol reagent) *Using nutrient agar slant and incubate it for 18 to 24 hours. *Put positive control as Aeromonas species. *Add 2 to 3 drops of both reagents A and B and tilt it. *Observe for change in colour to blue.

22. REAGENTS SOLUTIONS (cont.) (B) Direct plate procedure *Add 2 to 3 drops of reagent directly to few suspected colonies alone without flooding the entire plate . *For Kovacs reagent, colour reaction within 10 to 15 seconds. *For Gordon and Mcleod’s reagent, colour reaction within 10 to 30 minutes.

23. REAGENTS SOLUTIONS (cont.) (C) Kovac’s indirect filter paper method *Place a 6 square centimetres of Whatmann No.1 filter paper in a petri dish. *Add 2 to 3 drops of Kovac’s reagent to the centre of the paper. *With the usage of platinum wire inoculating needle smear a loopful of the suspected colony in paper 3 to 6 cm long. *Positive reaction within 5 to 10 seconds.

24. How is it looks like???????

25. OXIDASE DISCS (A) Direct plate procedure *Moisten the impregnated disc with sterile distilled water. *Place disc on few suspected colonies in plate medium. * Incubation at 35 degrees for 20 to 30 minutes. *Initially rose to purple but after 30 minutes red to black .

26. OXIDASE DISCS (B) Indirect procedure *Moisten disc with sterile distilled water. *Place it in a petri dish. *Add a loopful of colony growing on a solid medium. *Positive reaction occurs within seconds. * Pink to black.

27. QUALITY CONTROLS Escherchia coli (ATCC 25922) – Good control for negatives Neisseria , Pseudomonas(ATCC 27853) and Aeromonas – Good control for positives Easily available and no problems when we maintained as stock cultures.

28. STORAGE AND REPLACEMENT Store it in refrigerator and warm it before use. According to Barry and Bernshon reagents should be freezed at -20 degree . We have to thaw the reagent before 3 to 4 hrs of usage . Once thawed we may use that reagent till one day. ONLY REFRIGERATED REAGENTS we can keep it till maximum 14 days. Regular quality control checks should be done and discard it if comes as negative.

29. REAGENT ACTION Two molecules of oxidized cytochrome c + Reagent Coloured compound

30. REAGENT ACTION.........

31. INTERPRETATION FOR OXIDASE(+) Pink...............to...........Maroon.............to................. Finally black colour...... Pink colour - Viable, able to subculture Black colonies – Non viable

32. INTERPRETATION FOR OXIDASE(-) No colour change in colonies Only background will be black.

33. DELAYED OXIDASE TEST MEANS.......... Within 10 seconds colour formation – O(+) Within 10 to 60 seconds colour formation – Delayed oxidase test After 60 seconds colour formation – O(-)

34. REAGENTS IMPREGNATED OXIDASE TEST STRIPS Cytochrome oxidase test strip Barry and Bernsohn method ( Whatmann No.1 filter paper cut down into strips and dry rapidly) We can test many number of colonies simultaneously...... Positive test – Red colour

35. Why kovac’s reagent more sensitive than Gordon and Mcleod ?????? Ellingsworth first found that Haemophilus influenzae is negative with Gordon but positive with Kovac’s .......... Reason – Tetramethyl -p- phenylenediamine is more sensitive than Dimethyl -p- phenylenediamine .......

36. Don’t do oxidase test in glucose containing medium.....Ok....But why?? Because it’s fermentation will inhibit oxidase enzyme activity and results in false negatives . OTHER FALSE NEGATIVES : In mixed culture of Pseudomonas and Neisseria , inhibitory substance produced by pseudomonas interferes with oxidase production by Neisseria

37. FALSE POSITIVES...... Bordetella pertussis may show false(+) when cultivated in high conc. of blood . Platinum loop needle may contain trace amount of iron can catalyse oxidase reagent.....leads to false positive

38. Why can’t we call kovac’s reagent as simply kovac’s reagent???? Because there is also a reagent called kovac’s indole reagent. So just to avoid the confusions.....

39. ROLE OF VITAMIN C AND SUNLIGHT IN KOVAC’S REAGENT To avoid auto-oxidation in air , we will add ascorbic acid in kovac’s reagent Colour of the reagent turns into deep blue if it exposed to sunlight. So keep it in dark place......

40. MODIFIED OXIDASE METHOD Reagent in Dimethyl sulfoxide (DMSO) 6% solution used for identification of micrococci species (Positive reaction within 30 seconds)

41. OXIDASE TEST IN SINGLE LINE Oxidase test is an oxidising reaction and oxygen must reach colonies; after flooding growth or a portion of growth, tilt slants or plates to permit the reagent to run, exposing colonies to air..........

42. CATALASE TEST PRINCIPLE : To detect the presence of enzyme catalase in the bacteria which acts on Hydrogen peroxide to release oxygen ........

43. PURPOSE AND DIFFERENTIATION CATALASE(+) Staphylococcus Micrococcus Bacillus Listeria monocytogenes Corynebacterium (except two) All moraxella species (except two) CATALASE(-) Streptococcus Clostridium Erysipelothrix Corynebacterium pyogenes Corynebacterium haemolyticum Moraxella bovis Kingella kingae ( Moraxella kingii )

44. HISTORY AND MECHANISM The enzyme catalase is present in most cytochrome containing aerobic and facultative anaerobic organisms. Organisms lacking cytochrome system also lack the catalase ..... According to Doelle , catalase may interfere with peroxidase enzymes.....

45. CATALASES AND PEROXIDASES Peroxidases are plant enzymes but also found in milk and leucocytes.... Catalases are present in animals and plants. Iron contained catalases which retain its oxidized state during enzymatic activity. According to Burrows and Moulder non iron containing catalases are also there..... Dacre and Sharpe – Strong and Weak catalases .

46. Whittenbury Observation Two types of catalases : Classical Catalase and Pseudocatalase Classical Catalase : Which decomposes Hydrogen peroxide and nonsensitive to acid conditions. Pseudocatalase : Which lacks heme prosthetic group and sensitive to acid pH. According to Johnston and Delwiche both types are present in lactic acid organisms.

47. H 2 O 2 IS TOXIC TO BATERIA......?? Yes of course.....Hydrogen peroxide accumulation is very much toxic to bacteria. It is an Oxidative end product of aerobic breakdown of sugars. Since it’s toxic, bacteria uses it’s catalase enzyme to breakdown H 2 O 2 .

48. CATALASES VS PEROXIDASES At present, it is believed that peroxidases more predominates than catalases in elimination of Hydrogen peroxide. Optimal pH for catalase activity is 7.0

49. REAGENTS 1. Hydrogen peroxide 30% *Store in a dark bottle *Keep refrigerated at all times when not in use.

50. Reagents (cont.) 2. Phosphate buffer – pH 7.0 INGREDIENTS QUANTITY Potassium dihydrogen phosphate 1.361grams Anhydrous Disodium hydrogen phosphate 1.420 grams Distilled water 1000 ml of distilled water

51. Reagents (cont.) 3. Tween 80,10% *Add 1 ml of concentrated tween 80 to a small volume of distilled water in a 10 ml volumetric flask * Polyoxyethylene derivative of sorbitan mono- oleate ; a surface active compound

52. QUALITY CONTROLS POSITIVE CONTROLS NEGATIVE CONTROLS Staphylococcus (ATCC 25923) Streptococcus Blood agar plate (d/t catalase in RBCs) Chocolate agar d/t no RBCs Note : Hydrogen peroxide should undergo quality control check daily or immediately prior to it’s use............

53. ROUTINE CATALASE TESTS At room temperature 25 degree celsius . Slide method (Routinely used) Tube method

54. SLIDE CATALASE TEST With an inoculating needle, pick the centre of an 18 to 24 hour pure colony on glass slide. Exception is colony from blood agar. Add a drop of 30% Hydrogen peroxide over organism on slide. Observe for immediate bubbling (Gas liberation) and Discard the slide. These are DO’S IN SLIDE CATALASE TEST.

55. DONT’S IN SLIDE CATALASE TEST Do not reverse the order of procedure Do not mix the culture and Hydrogen peroxide ( Just add a drop of H2O2 on colony and then just observe!!!! )

56. How it looks like???? Here we go!!!!

57. TUBE CATALASE TEST Directly add 1 ml of 3% H 2 O 2 to an 18 to 24 hour heavily inoculated pure agar slant culture. Observe for immediate bubbling(Gas liberation) and observe.

58. Tube catalase test

59. Catalase test for differentiation of Mycobacterium species (Under controlled pH and temperature) Perform procedure under bacteriological hood ( Strict aseptic conditions ) Add 0.5 ml of phosphate buffer into sterile screwcap tube. Add several loops of Mycobacterium growth from slant culture 3 to 4 weeks old Screw cap down loosely. Incubate for 20 to 30 minutes/In 68 degrees celsius Water bath.

60. Catalase test for Mycobacterium species(Cont.) Cool at room temperature Prepare a 1:1 mixture of 10% Tween 80 and 30% H 2 O 2 Add this mixture in cooled suspension Observe for bubble formation. Note : ALL MYCOBACTERIUM SPECIES ARE CATALASE POSITIVE for Routine catalase test except Isoniazid Resistant strains .

61. But what’s the interpretation when we will do under controlled pH and temperature?? Positive Mycobacterium avium Negative Some J subgroups of III nonphotochromogens Mycobacterium kanasasii Mycobacterium tuberculosis Mycobacterium bovis

62. ALTERNATE TESTS Capillary tube technique (Method of Fung and Petrishko ) Commercial capillary tubes 10 ml of 3% Hydrogen peroxide in 50 ml beaker Touching centre of colony No bubbles + Few bubbles ++ Many bubbles +++ Gas forcing H2O2 to tip of tube

63. Disadvantages of capillary tube method Vigorous catalase activity (+++) – Serratia , Proteus, Providencia Moderate catalase activity(++) – Salmonella Weak catalase activity/Negative catalase activity - Enterobacteriaceae

64. Alternate test (cont.) Cover slip technique (Method of Taylor and Achanzar ) Primarily used for identification of Enterobacteriaceae .

65. TWO METHODS OF INTERPRETATION IN COVER SLIP METHOD Frosted appearance of bubbles under cover slip indicates speed of reaction Seeing presence or absence of trapped bubbles

66. ORGANISMS POSITIVE IN 0.5% COVER SLIP METHOD Pseudomonas aeruginosa Serratia Staphylococcus

67. ALTERNATE TESTS (cont.) Colour reaction streak test ( Method of Hanker and Rabin ) For primarily to differentiate staphylococci and streptococci

68. REAGENTS Stock solutions DOPAMINE 20 mg/ml in 0.2M Phosphate buffer P- phenylenediamine dihydrochloride 1 mg/ml in 0.2M phosphate buffer

69. REAGENTS (cont.) Working reagent DOPAMINE 1 ml P-PHENYLENEDIAMINE DIHYDROCHLORIDE 1 ml 3% H2O2 2 ml Dimethyl sulfoxide 1 ml

70. INTERPRETATION OF RESULTS After streaking pure bacterial colony on clean slide and add 1 drop of working reagent Formation of highly coloured purple colour and it may lasts for days so that results may be rechecked ( Advantage! )

71. CHEMISTRY OF REAGENT FOR BOTH CATALASES AND PEROXIDASES FOR PEROXIDASES

72. CRITERIA FOR TAKING CULTURE IN CATALASE TEST It should be a pure culture. It must be performed in heavy inoculum .

73. Why 70% ethyl alcohol is needed while handling Hydrogen peroxide?? Just to avoid exposure to skin as painful burns may occur.

74. Why gentle shaking needed for Hydrogen peroxide before usage?? Hydrogen peroxide decomposition increases as the temperature increases due to dissolved oxygen.....It may create false positive reactions.

75. COAGULASE TEST PRINCIPLE : To test the ability of an organism to clot plasma by the action of enzyme coagulase ( Staphylocoagulase )

76. USES To differentiate staphylococcus aureus from other staphylococcus species. Used as an indication for virulence or pathogenicity .

77. NORMAL MECHANISM

78. NAMED HYPOTHESIS RELATED TO COAGULASE TEST Oginsky and Umbreit Smith Burrows and Moulder Duthrie Dubos and Hirsch Soulier , Tager and Zajdel

79. OGINSKY AND UMBREIT HYPOTHESIS Coagulase enzyme may induce alternate pathway of clotting mechanism either as an enzyme or or an plasma activator to convert fibrinogen to fibrin.

80. SMITH HYPOTHESIS

81. BURROWS AND MOULDER HYPOTHESIS Procoagulase + Plasma activator Coagulase Reason for clotting of plasma

82. DUTHRIE HYPOTHESIS

83. DUBOS AND HIRSCH HYPOTHESIS Every step is same as previously mentioned mechanisms except one thing......... Here PLASMA/CRF doesn’t require calcium ions to form a clot .

84. SOULIER,TAGER AND ZAJDEL HYPOTHESIS

85. OVERVIEW OF COAGULASE INDUCED FIBRIN COATING AND ANTIMICROBIAL DRUGS Basically serum is having bactericidal action against staphylococcus.......... But when coagulase induced fibrin coated staphylococcus is present in serum means, antimicrobial drugs cannot penetrate that coating...... BUT THIS MECHANISM WE CANNOT SEEN IN CONS.

86. HEMOLYSIN PRODUCTION COWAN All C(+) strains produce either alpha or beta hemolysin or both BURROWS AND MOULDER Either alpha or gamma hemolysin associated with coagulase activity YOUNG AND LEITNER No correlation at all WECKMAN AND CATLIN Production of deoxyribonucleases along with coagulase

87. ANTIGENIC SUBTYPES RAMMELKAMP, DUTHRIE AND ZEN-YOJI Seven antigenic subtypes MIALE TYPES A,B,C,D SMITH TYPES I AND II

88. REAGENTS

89. METHOD OF PREPARATION OF REAGENT Commercial plasma Fresh plasma from whole blood ( Centrifuge Take supernatant Store it in sterile container) Fresh fibrinogen (Centrifuge  Take the precipitate  Make up(Dilution) to 5 times of sterile distilled water)

90. Why are we adding heparin or EDTA with plasma in special??? Simply, reason is that citrate utilising organisms creates delayed coagulase reaction which leads to false positive coagulase test(Clot formation). So by means of adding 5 ml of heparin  inhibit coagulation by citrate utilisation  Test is more specific for staphylococcus.

91. QUALITY CONTROL Positive quality control – Staphylococcus aureus (ATCC 25923) Negative quality control – Staphylococcus epidermidis

92. PLASMA COAGULABILITY CHECK Add one drop of 5% calcium chloride solution to 0.5ml aliquot of plasma (1:2 dilution) and a clot should form.

93. STORAGE Dehydrated plasma vials  Refrigerator (4 degree celsius ) Reconstituted plasma vials  Freezer (-20 degree celsius )

94. PLASMA QUALITY CHECK CRITERIA CRF/Plasma concentration is highest in human plasma followed by Pig>Rabbit>Horse> Bovine>Chicken>Sheep. MUST CONTAINS SUFFICIENT AMOUNT OF CRF CONCENTRATION AND FIBRINOGEN FAIRLY FREE OF FIBRINOLYTIC ACTIVITY ( BY MEANS OF STAPHYLOKINASE ENZYME AND MULLER FACTOR PLASMIN ) REASONABLY FREE OF INHIBITORS (PROTEASES)

95. WHY RABBIT PLASMA IS PREFERRED OVER HUMAN PLASMA?? Clot is firm and it’s rate of coagulation is faster (Rabbit plasma) Antibodies in human plasma will inhibit coagulase production by staphylococcus.

96. Why dilution of human plasma preferred?? Because bacteriostatic action against staphylococcus is eliminated by dilution of human plasma......

97. Why diabetics more prone to infections caused by staphylococcus??? Yearsly and Carter proved that a slight increase in coagulase activity in diabetic plasma over human plasma... So we should control diabetics as much as possible with the help of insulin/OHA.

98. SLIDE COAGULASE TEST Place a drop of distilled water in glass slide. Emulsify it using heavy suspension of 18 to 24 hour culture of staphylococcus Gently mix with small loop of fresh human plasma and mixture should be homogenous. Set up positive and negative controls in same slide Observe for clump formation

99. INTERPRETATION

100. TUBE COAGULASE TEST

101. INTERPRETATION (A) POSITIVE TEST : Clot or distinct fibrin threads formed. Complete clot: Clot throughout tube. Partial clot: Clot doesn’t extend throughout fluid column. (B) NEGATIVE TEST : No clot formation, suspension remains homogenous.

102. RAPID TEST: Staph strip Strips impregnated with lyophilized rabbit plasma with EDTA Results of both screening and tube tests with as single inoculum

103. ALTERNATE TESTS 1. Pour plate coagulase test Method of Parisi , Baldwin and Sottile * Detects coagulase production * BHI or YETS agar ( Trypticase soy broth) and 0.3% yeast extract * Plasma preferably swine pig plasma ; Alternatively rabbit. Correlation with standard test tube(Excellent) * No plasma – Parisi recommended not to use plasma

104. ALTERNATE TESTS (cont.) 2. Tube coagulase thermonuclease test Method of Barry, Lachica and Atchison * Detects free coagulase and heat stable nuclease * BHI broth, EDTA plasma and DNA agar TDA technique ( Toluidine Blue Deoxyribonucleic acid ) * Correlation with standard tube test.

105. ALTERNATE TESTS (cont.) 3. Coagulase-mannitol agar plate test * Detects free and bound coagulase * Mannitol fermentation * Contents : Human blood plasma and Mannitol * Disadvantage : Too many false positives

106. THANK YOU