Unknowns Tests Micro

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CLO852
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211975
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Unknowns Tests Micro
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2013-04-07 15:10:53
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Unknowns Microbiology
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Micro
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  1. Motility
    • A. Motility Medium with TTC
    • The medium contains 0.4% agar and 2, 3, 5 – Triphenyl-Tetrazolium Chloride (TTC). 
    • Motile bacteria will be able to “swim” through the medium and will reduce TTC to form a red precipitate called formazan

    • B. Procedure:
    • (1) Use a needle to inoculate by making a single stab about two thirds down and then pulling out the needle along the same path.
    • (2) Incubate at the optimum temperature for 24 - 48 hours. 

    • C. Interpretation:
    • Motile -the tube will appear red and cloudy and usually the organism will spread over the top of the medium.
    • Non-Motile -the organism will grow along the streak line only; the medium will NOT be cloudy.
  2. OXYGEN REQUIREMENTS
    • A. Fluid Thioglycollate Medium
    • The medium contains glucose, cystine, and sodium thioglycollate to lower the oxidation-reduction potential.
    • The oxygen tension is high at the surface of the medium (allowing aerobes to grow) and decreases toward the bottom of the medium (for anaerobic growth).
    • Resazurin (a dye) causes the medium to turn pink in the presence of oxygen. 
    • B. Procedure:
    • (1) Boil medium for 5 minutes with screw cap loose.
    • (2) Tighten cap and allow medium to cool to body temperature or below.
    • (3) Inoculate medium with the organism using a wire loop. Do not shake the medium.
    • (4) Incubate at optimum temperature for 24 hrs. 
    • C. Interpretation:
    • Aerobe - growth at the top of the medium
    • Facultative - growth throughout the medium
    • Anaerobe - growth at the bottom of the medium
  3. GELATINLIQUEFACTION TEST
    • A. Nutrient Gelatin
    • Used to determine the ability of an organism to produce the enzyme gelatinase, which liquefies gelatin. 
    • Gelatinase hydrolyses (breaks down) gelatin, a large protein, into smaller components, which can then enter the organism and be metabolized.                                           Gelatinase Gelatin + H2O                                                 polypeptides                                                       Gelatinase             Polypeptides + H2O                                        amino acids 
    • C.        Procedure:(1)        Stab gelatin with organism using a straight wire.(2)        Incubate at optimum temperature for 24 – 48 hours.(3)               Place tubes in refrigerator for at least 30 minutes. 
    • D.        Interpretation:Positive -  gelatin is liquefied Negative - gelatin is solid(Note: continue incubation of negative tubes for another 4 or 5 days to see if gelatinase is produced slowly.)
  4. CARBOHYDRATE FERMENTATION
    • (Glucose, Lactose, Mannitol,Maltose, and Sucrose Broths)
    • A.          Used to determine the ability of an organism to ferment a specific carbohydrate with or    without the production of gas.
    • B.           Acid ProductionPhenol red is used as an indicator in each medium. At a neutral pH, the medium is red; at a pH of less than 7 (acid), the medium is yellow.  Fermentation of the carbohydrate produces acid, causing the medium to change from red to yellow. 
    • C.           Gas ProductionThe inverted tube in the broth, called a Durham Tube, captures some of the gas the organism produces, allowing gas production to be seen. If a bubble forms within the Durham Tube the organism is positive for gas production. 
    • D.        Procedure:(1)        Inoculate each medium with the organism using a wire loop.(2)               Incubate at the optimum temperature for 24-48 hours.      
    • E.     Interpretation:Acid ProductionPositive - medium turns yellow Negative - medium remains red (notes:  Continue incubation of negative tubes for up to 2 weeks to detect "slow" fermenters. To avoid alkaline reversion, do not continue incubation of positive tubes.)
    • Gas ProductionPositive – a bubble forms with the Durham TubeNegative – no bubble forms
  5. CITRATE UTILIZATION
    • (Simmons Citrate Agar)
    • A.    Used to determine if an organism is capable of using citrate as the sole source of carbon with production of enzyme citratase.                         Citratase Citrate                                                             Oxaloacetate and Acetate  Oxaloacetate                                                   Pyruvate + CO2  Pyruvate                                                          Acetate + Formate B.        The medium contains sodium citrate as the carbon source, ammonium salts as the nitrogen source, and brom thymol blue as the pH indicator. An organism that uses citrate breaks down the ammonium salts to ammonia, which creates an alkaline pH. C.        Procedure:(1)        Stab/streak Simmons Citrate Agar slant with straight wire inoculum of             organism.(2)        Incubate at optimum temperature for 24-48 hours.  D.        Interpretation: Positive - alkaline pH causes medium to change from green to Prussian blue Negative - no color change
  6. NITRATE REDUCTASE TEST
    • (Nitrate Broth)
    • A.        Used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2) by production of the enzyme nitratase. 
    • B.        The reduction of nitrate to nitrite or nitrogen gas takes place under anaerobic conditions in which an organism derives its oxygen from nitrate.                                                     Nitratase                              (further reduction)                         Nitrate (NO3)                          Nitrite (NO2)                                       N2  + NH3            
    • C.        Procedure:            (1)        Boil medium for 5 minutes with screw cap loose.(2)        Tighten cap and allow medium to cool to body temperature or below.(3)        Inoculate nitrate broth with the organism using a wire loop.(4)        Incubate at the optimum temperature for 24-48 hours.(5)        Add 5 drops of Nitrate Reagent A (dropper bottle) and 5 drops of Nitrate             Reagent B (ampule) to the tube.
    • D.        Interpretation: Positive - red color; nitrate reduced to nitrite; test is complete Negative - no color change; do Confirmation Test by adding a small pinch of zinc powder Interpretation of Confirmation Test: Positive -no color change; organism reduced nitrate completely to ammonia and nitrogen gas. Negative - red color; nitrate reduced by zinc, not the organism (confirms first negative).
  7. CATALASE TEST
    • A.                Hydrogen peroxide (H2O2) is formed as an end product of the aerobic breakdown of sugars. When H2O2 accumulates, it becomes toxic to the organism. Catalase decomposes H2O2 and enables the organism to survive. Only obligate anaerobes and aerotolerant bacteria such as Streptococci lack this enzyme.        Catalase                         2 H2O2                                                 2H2O + O2     
    •  C.        Procedure:(1)        Transfer a loopful of the organism from the working slant onto a clean slide.(2)        Add 2 – 3 drops of 3% H2O2 to the cells and mix with an inoculating loop. 
    • D.        Interpretation:Positive - bubbling (O2 gas is liberated from the H2O2)Negative - no bubbling
  8. METHYL RED TEST
    • (MR-VP Broth)
    • A.                Used to determine the ability of an organism to produce mixed acid end products from glucose fermentation. 
    • B.                 Some organisms produce large amounts of various acids (lactic, acetic, succinic, formic) plus H2 and CO2. The large amounts of acids lower the pH to less than 5.0.   
    • C.                 These organisms also produce great amounts of gas due to the presence of the enzyme formic hydrogen lyase.                                                                                                                   Formic Hydrogen Lyase                                     Formic Acid                                                                CO2  +  H2 
    • D.                Procedure:(1)               Label a tube of MR-VP broth with “MR Test”. (2)               Inoculate MR-VP broth with the organism using a wire loop.(3)        Incubate at optimum temperature for 3 – 5 days.(4)        Add 3 - 4 drops Methyl Red Reagent to the tube. E.                 InterpretationPositive - red color developsNegative - yellow color develops
  9. VOGES-PROSKAUER TEST
    • (MR-VP Broth)
    • A.                Used to determine the ability of an organism to produce acetoin (acetylmethyl carbinol), 2,3 butanediol, and ethanol which cause less lowering of the pH than methyl red positive organisms. 
    • B.        VP test detects the presence of acetoin, which is a precursor to 2,3 butanediol 
    • C.        Procedure:(1)               Label a tube of MR-VP broth with “VP Test”.  (2)        Inoculate MR-VP broth with the organism using a wire loop.(3)        Incubate at optimum temperature for 3 – 5 days.(4)        Pipet 1 ml (20 drops) of culture into a clean test tube. (5)        To the extracted culture, add 18 drops Barritt's Solution A and 18 drops Barritt's Solution B.(6)        Agitate vigorously for 1-2 minutes. Let stand for 30 minutes. 
    • D.          Interpretation: Positive - wine red (burgundy) color develops Negative - brown color develops
  10. STARCH HYDROLYSIS
    • (Starch Agar Plate)
    • A.                Used to determine the ability of an organism to hydrolyze (break down) starch. 
    • B.                 The enzyme amylase breaks starch down into components more easily metabolized by the organism.                                                                         Amylase                         Starch + H2O                                                              Maltose + Glucose + Dextrins 
    • C.                 Procedure:(1)        Use a wire loop to make a single streak of the organism on a starch agar             plate.(2)               Incubate at the optimum temperature for 24 – 48 hours.(3)               Drop a small amount of IKI (Gram’s Iodine) onto the plate and rotate the plate gently.  (Iodine is an indicator of starch; in the presence of starch the iodine will turn blue/black.) 
    • D.                Interpretation:            Positive – a zone of clearing appears adjacent to the streak line.            Negative  - no clearing; only a blue/black area surrounding the streak line.
  11. CASEIN HYDROLYSIS
    • (Skim Milk Agar)
    • A.               Used to determine the ability of an organism to produce the enzyme caseinase which hydrolyzes (breaks down) casein (a white protein in milk) to more soluble products.                                                                                  Caseinase                         Casein (Milk Protein) + H2O                                                  Polypeptides                                                                                  Caseinase                         Polypeptides + H2O                                                                Amino Acids 
    • B.        Procedure:(1)               Use a wire loop to make a single streak of the organism on a skim milk             agar plate.(2)               Incubate at the optimum temperature for 24-48 hours. 
    • C.        Interpretation: Positive -a zone of clearing occurs along the streak line Negative -no zone of clearing
  12. TRYPTOPHAN HYDROLYSIS/ “INDOLE TEST”
    • (Tryptone Broth)
    • A.                Used to determine the ability of an organism to split indole from the amino acid tryptophan using the enzyme tryptophanase.                   Tryptophanase Tryptophan + H2O                                                      Indole + Pyruvic Acid                         
    • B.     Procedure:(1)               Inoculate broth with organism using a wire loop.      (2)        Incubate at optimum temperature for 24-48 hours.      (3)        Add 10-12 drops of Kovacs Reagent. 
    • C.        Interpretation:Positive - pink layer forms on surface of the mediumNegative - yellow layer forms on surface of the medium
  13. UREA HYDROLYSIS
    • (Urea Broth)
    • A.        Used to determine the ability of an organism to split urea to form ammonia (an alkaline end product) by the action of the enzyme, urease. The medium also contains the pH indicator phenol red, which turns an intense pink at alkaline pH.          Urease Urea + H2O                                                                 2 ammonia  +  CO2      
    • B.                Procedure:       (1)        Inoculate urea broth with the organism using a wire loop.(2)         Incubate at optimum temperature for 24-48 hours. 
    • C.        Interpretation:Positive - intense pink color                          Negative - no color change(Note:  continue incubation of negative tubes for a total of 7 days to check for slow urease producers.)
  14. HYDROGEN SULFIDE PRODUCTION
    • (Triple Sugar Iron Agar)
    • A.        Used to determine the ability of an organism to hydrolyze (break down) the sulfur containing amino acid cysteine to produce pyruvic acid, ammonia, and hydrogen sulfide.  The initial step is the removal of the sulfide from cysteine which is catalyzed by the enzyme cysteine desulfurase.                                                                       Cysteine desulfurase Cysteine + H2O                                                    Pyruvic Acid + H2S + NH3   
    • B.        The medium contains ferrous salts that react with H2S to produce an insoluble black             precipitate, ferrous sulfate.  H2S + ferrous salts                                               ferrous sulfide (black precipitate)                                      C.        Procedure:(1)        Stab/streak TSI with straight wire inoculum of organism.(2)        Incubate at optimum temperature for 24-48 hrs. 
    • D.        Interpretation:Positive - black precipitate along stab lineNegative - no precipitate
  15. PHENYLALANINE DEAMINATION
    • (Phenylalanine Agar)
    • A.        Used to determine the ability of an organism to deaminate the amino acid phenylalanine resulting in the production of phenylpyruvic acid and ammonia.  This reaction is catalyzed by the enzyme phenylalanase.                                                            Phenylalanase                         Phenylalanine                                                  Phenylpyruvic Acid + NH3      
    • B.  Procedure:(1)                                       Streak phenyalanine agar slant with the organism using a wire loop.(2)                                       Incubate at the optimum temperature for 24 – 48 hours.(3)                                       Place 5 drops of 10% Ferric Chloride on the slant culture.(4)                                       Wait 5 minutes.            
    • C.        Interpretation:                                    Positive – a green color develops.                                    Negative – a yellow color develops.
  16. LYSINE DECARBOXYLASE TEST
    (Lysine Decarboxylase Broth)A.        Used to determine the ability of an organism to decarboxylate the amino acid lysine, resulting in the production of the alkaline end-product cadaverine, by producing the enzyme lysine decarboxylase.     Lysine decarboxylase Lysine                                                                               Cadaverine + CO2 B.        The medium contains lysine, glucose (as a substrate for fermentation), and the pH indicator brom cresol purple (purple at alkaline pH; yellow at acid pH) C.        The enzyme requires an acid pH (below 5) for activation. IF the organism is capable of glucose fermentation (check carbohydrate broth), AND has the enzyme lysine decarboxylase, the following events occur:(1)        Microbe ferments glucose, producing a low pH; indicator turns yellow.(2)        Lysine decarboxylase is activated.(3)        Cadaverine is formed, pH rises and the medium returns to its original purple color. D.        Procedure:(1)        Inoculate broth with the organism using a wire loop(2)        Incubate at optimum temperature for 24-48 hrs.             E.         Interpretation:Positive - purple (Verify organism ferments glucose by checking the Carbohydrate Fermentation Test, if the organism does NOT ferment glucose then a purple color is negative.)Negative – yellow
  17. ORNITHINEDE CARBOXYLASE TEST
    • (Ornithine Decarboxylase Broth)
    • A.        Used to determine the ability of an organism to decarboxylate the amino acid ornithine, resulting in the production of the alkaline end-product putrescine, by producing the enzyme ornithine decarboxylase.    Ornithine decarboxylase Ornithine                                                                           Putrescine + CO2 
    • B.        The medium contains ornithine, glucose (as a substrate for fermentation) and the pH indicator brom cresol purple (purple at alkaline pH; yellow at acid pH) 
    • C.        The enzyme requires an acid pH (below 5) for activation. IF the organism is capable of glucose fermentation (check carbohydrate broth), AND has the enzyme ornithine decarboxylase, the following events occur:(1)        Microbe ferments glucose, producing a low pH; indicator turns yellow.(2)        Ornithine decarboxylase is activated.(3)        Putrescine is formed, pH rises and the medium returns to its original purple color. 
    • D.        Procedure:(1)        Inoculate broth with the organism using a wire loop(2)        Incubate at optimum temperature for 24-48 hrs.            
    • E.         Interpretation: Positive - purple (Verify organism ferments glucose by checking the Carbohydrate Fermentation Test, if the organism does NOT ferment glucose then a purple color is negative.)Negative – yellow

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