Biochemical tests for identification of organism

Biochemical tests for identification of bacteria: 

1 Biochemical tests for identification of bacteria Dr. Prabesh Kumar Choudhary Resident, 2 nd Year Dept. Of Pathology Bir Hospital, NAMS

Preparation of inoculum and test medium: 

2 Preparation of inoculum and test medium Single, well separated colony from pure culture. Subculture into a tube of broth if large number of test media have to be inoculated. Incubate for 3-4 hours or overnight. Control for test Test for sterility - incubate one or two uninoculated tubes with the test medium. Test for performance of medium- Known positive and negative culture along with the test .

Bile solubility test: 

3 Bile solubility test Principle: The bile salt, Sod deoxycholate dissolves the S. pneumoniae in the emulsified saline preparation of the growth within 10-15mins.(Clearing of the turbidity) Usually done to differentiate S. pneumoniae from other alfa haemolytic streptococci (Viridan streptococci)

Method: 

4 Method Tube method is recommended over slide Emulsify several colonies of the test organism in a tube containing 2ml sterile saline (Turbid suspension) Divide the suspension in to two tubes Add 2 drops of Sod deoxycholate to one tube and mix Add 2 drops of D/W to the other tube Incubate the tubes at 35-37C for 10-15Mins Look for clearing of the turbidity in the tube containing Sod deoxycholate

Catalase test: 

5 Catalase test Principle: catalase producing bacteria will produce O2 bubbles when mixed with H2O2. Methods: 1. Slide method 2. Tube method

PowerPoint Presentation: 

6 1. Slide method: A drop of 3 percent hydrogen peroxide is put on a slide in a petri dish and the bacteria is emulsified in the drop. The dish is immediately covered to prevent contamination from active bubbling. 2. Tube method: Several colonies of the test organism is picked up with wooden stick or glass rod and immersed in to the tube containing 2-3ml of 3%H2O2 **Care must be taken when colonies are obtained from blood agar>> false positive reaction.**

Catalase test: 

7 Catalase test Catalase positive- Staphylococcus spp Catalase negative- Streptococcus spp.

Coagulase test: 

8 Coagulase test Principle: coagulase converts fibrinogen to fibrin & clot the plasma. Two types of coagulase: 1.Free coagulase: requires activation by coagulase reacting factor in the plasma. Detected by clotting in the test tube. 2.Bound coagulase/clumping factor : directly converts fibrinogen to fibrin. Can be detected in slide method also. **Tube test must be always performed when slide test is not clear or is –Ve** **Citrated plasma: citrate utilizing bacteria e.g. enterococci, pseudomonas, & serratia may cause clotting of the plasma**

Method: 

9 Method 1.Slide method: Place a drop of D/W on two ends of a slide Emulsify with the test colony and add a loopful of plasma to one of it; look for clumping within 10s 2.Tube method Take 3 tubes and label as “T”,“Pos” & “Neg”. Add 0.2 ml of plasma in to each. Add 0.8ml of test broth to “T”, 0.8ml of S. aureus culture to “Pos” & 0.8ml of sterile broth to “Neg”. Incubate trhe tubes at 35-37C and examine for clotting after 1 hr, if -ve, check after 3hrs; if -ve again check after overnight incubation at RT.

Oxidase Test: 

10 Oxidase Test This test is used to determine the presence of the cytochrome oxidase. Principle: Oxidase producing oraganisms will oxidize the reagent in the filter paper e.g. phenylenediamine and will be colorized to deep purple or blue. **Acidity inhibits oxidase enzyme activity, the test should not be performed on colonies that produce fermentation on carbohydrate containing media e.g. TCBS or MacConkey agar. Subinoculation in nutrient agar is recommended.**

Method: 

11 Method Place a filter paper in a clean petridish Add 2-3 drops of freshly prepared oxidase reagent . Remove a small portion of colony to be tested with a platinum wire or wooden stick or glass rod Smear it on the filter paper Observe for a color change to blue or purple within 10 seconds.

Examples: Pseudomonas, Neisseria, Vibrios, Brucella, Pasteurella: 

12 Examples: P seudomonas, Neisseria, Vibrios, Brucella, Pasteurella Controls Positive : Pseudomonas aeruginosa Negative : Escherichia coli

Urease test: 

13 Urease test Principle: Urease splits urea into ammonia and carbon dioxide increasing the pH which will be detected by the change of color of an indicator to pink-red Method: The test organism is inoculated into 3ml urea broth that contains phenol red, a pH indicator, and has a pH of 6.8 . Incubation in water bath is done at 35-37c for 3-12hrs. At this pH phenol red is salmon color . However, when the pH rises above 8.1 phenol red turns pink

Proteus & Yersinia are +ve; Salmonella & Shigella are -Ve: 

14 Proteus & Yersinia are +ve; Salmonella & Shigella are -Ve

Indole test: 

15 Indole test Principle: Test organism producing tryptophanase cleaves trytophan in the media to produe indole which is detectedby Kovac’s (preferred) or Ehrlich’s reagent. *Kovac’s reagent: p-dimethylaminobenzaldehyde* **The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs**

PowerPoint Presentation: 

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Method: 

17 Method Inoculate the test organism in a bottle containing 3ml of tryptone water Incubate at 35-37C for 48hrs Add 0.5ml of Kovac’s reagent, shake gently and check for red color in the surface layer within 10mins .

E. coli, P. vulgaris, P. rettgeri and Providencia: 

18 E. coli, P. vulgaris, P. rettgeri and Providencia

Citrate utilization test: 

19 Citrate utilization test Principle: The test is based on the ability of an organism to use citrate as its only source of carbon. Simmon's media contains bromthymol blue , which is yellow at pH 6, green at pH 6.9, and gradually changes to blue at more alkaline pH's (around 7.6).

Method: 

20 Method Prepare slope of the medium Using sterile straight wire, first streak the slope with a saline suspension of the test organism and then stab the butt Incubate at 35C for 48hrs Look for bright blue color in the medium

Enterobacter and Klebsiella: 

21 Enterobacter and Klebsiella

Oxidative fermentation of glucose: 

22 Oxidative fermentation of glucose Principle: For differentiating Gram-negative bactereria. Bacteria that ferment oxidatively turn the medium yellow. Phenol red -pH indicator [Andraide’s indicator] -Neutral or basic = red color -Acidic = yellow Gas production - Usually CO2 Collects in Durham tube appears as a bubble Sugars are a carbon source and source of ATP It requires different enzymes to ferment each sugar. Pseudomonas aeruginosa : Yellow (+ve) Acinetobacter lwoffii : Remains green(-ve)

Sugar fermentation tests: 

23 Sugar fermentation tests

PowerPoint Presentation: 

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IMViC: 

25 IMViC IMViC>>indole , methyl red, Voges-Proskauer , and citrate . To obtain the results of these four tests, three test tubes are inoculated: 1. tryptone broth (indole test), 2. methyl red - Voges Proskauer broth (MR-VP broth), 3. citrate.

Indole test: 

26 Indole test

The Methyl Red and Voges-Proskauer Tests : 

27 The Methyl Red and Voges-Proskauer Tests The MR & VP tests are read from a single inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes. One tube is used for the MR test; the other is used for the VP test. MR-VP media contains glucose and peptone

The Methyl Red and Voges-Proskauer Tests : 

28 The Methyl Red and Voges-Proskauer Tests Enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. MR and VP tests determine what end products result when the test organism degrades glucose. E. coli produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test).

Methyl Red test; E. coli: 

29 Methyl Red test; E. coli

The Methyl Red Test: 

30 The Methyl Red Test Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. Methyl red indicator is a yellow color (-ve).

Voges-Proskauer Tests: 

31 Voges-Proskauer Tests Reagents for VP test 1. Barritt's A (alpha-napthol) and 2. Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (+ve VP test). This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.

VP test; Klebsiella & Enterobacter: 

32 VP test; Klebsiella & Enterobacter

Citrate utilisation test; Enterobacter & Klebsiella: 

33 Citrate utilisation test; Enterobacter & Klebsiella

IMViC Summary: 

34 IMViC Summary >E.coli gives ++-- results on the IMViC tests > Enterobacter and Klebsiella give the reverse: --++

Nitrate reduction test: 

35 Nitrate reduction test Principle: The identification of some bacteria is determined whether it can reduce nitrate (NO3) to nitrite (NO2) or another nitrogenous compound such as ammonia (NH3) or nitrogen gas (N2). NO3 ----> NO2 ----> NH3 or N2 Method: Test organism is inoculated into nitrate reduction broth , an undefined medium that contains large amounts of nitrate (KNO3). After incubation, alpha-napthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, (+ve nitrate reduction test).

Nitrate reduction test: 

36 Nitrate reduction test Method;contd: If no color change at this step, nitrite is absent. If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result. To distinguish between these two reactions, zinc dust must be added.

Nitrate reduction test: 

37 Nitrate reduction test Method;contd: Zinc reduces nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic  acid are already present in the tube. Red color after the zinc is added indicates the zinc found the nitrate unchanged. The bacteria was unable to reduce nitrate. This is recorded as a negative nitrate reduction test.

Nitrate reduction test: 

38 Nitrate reduction test Method;contd: If the tube does not change color upon the addition of zinc, then the zinc did not find any nitrate in the tube. That means the test organism converted the nitrate to nitrite and then converted the nitrite to ammonia and/or nitrogen gas. Thus no color change upon the addition of zinc is recorded as a positive nitrate reduction test.

PowerPoint Presentation: 

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Triple Sugar Iron Agar (TSI) and Kligler's Iron Agar (KIA): 

40 Triple Sugar Iron Agar (TSI) and Kligler's Iron Agar (KIA) Used to determine whether bacteria can ferment glucose, lactose and/or sucrose and whether they produce H2S or other gases. These characteristics help distinguish various Enterobacteriacae TSI: glucose, lactose and sucrose. Lactose and sucrose occur in 10 times the concentration of glucose (1.0% versus 0.1%). KIA: lactose and glucose Ferrous sulfate, phenol red (a pH indicator, yellow below pH 6.8 and red above it ), and nutrient agar are also present.

PowerPoint Presentation: 

41 If an organism ferment lactose and/or sucrose, the butt and slant will turn yellow and remain yellow for at least 48 hours because of the high level of acid products produced from the abundant sugar(s). If the gas being produced is H2S, it reacts with FeSO4 and preciptates as a black precipitate (ferric sulfide) in the butt. Organisms producing large amounts of H2S (e.g. Salmonella and Proteus ) may produce so much black precipitate that it masks the yellow (acid) color of the butt.

PowerPoint Presentation: 

42 Method: The tube is inoculated by stabbing into the agar butt and then streaking the slant in a wavy pattern. Results are read after 18 to 24 hours of incubation. Reading the Results A yellow slant : the organism ferments sucrose and/or lactose. A yellow butt: the organism fermented glucose. Black preciptate in the butt indicates H2S production. Production of gases other than H2S is indicated either by cracks or bubbles in the media or the media being pushed away from the bottom of the tube.

PowerPoint Presentation: 

43 Reading the result; contd: 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.

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Interpretation of the reaction in TSI: 

47 Interpretation of the reaction in TSI TSI Slant Butt Gas H 2 S Inference Orange red Orange red - - Control Pink Yellow - Slight Glucose fermentation e.g. Salmonella typhi Yellow Yellow + - Glucose, Lactose Sucrose fermentaton e.g. Escherichia coli Pink Black + Abundant Glucose fermentation production e.g Proteus vulgaris Pink Pink - - Oxidative Carboxylation e.g. Pseudomonas aeruginosa

PowerPoint Presentation: 

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Aminoacid decarboxylation tests Lysine, ornithine and arginine dihydrolysis tests: 

49 Aminoacid decarboxylation tests Lysine, ornithine and arginine dihydrolysis tests Principle Decarboxylases cleave the carboxyl group from amino acids so that amino acids are converted into amines with liberation of CO 2 Increased pH of the medium is detected by color change . Requirement Moeller decarboxylase base- 4 tubes With Lysine, ornithine and arginine hydrochloride 1% Control

PowerPoint Presentation: 

50 Method Inoculate the test medium overlaid with paraffin layer. Incubate and read daily for four days. Result Purple color-Positive Decarboxylation Yellow colour-Negative No decarboxylation

Controls : 

51 Controls Positive Lysine: Klebsiella pneumoniae Ornithine: Enterobacter cloacae Arginine: Enterobacter cloacae Negative Lysine: Enterobacter cloacae Ornithine: Klebsiella pneumoniae Arginine: Klebsiella pneumoniae

Thank you !: 

52 Thank you !