BIOCHEMICAL TESTING

Distinguishing Enterobacteriaceae Large family of bacteria Gram negative rods Capable of fermenting various sugars Many found in the intestines of human or other mammals Varrying pathogenicity: Commensals, opportunists or pathogens Some found in the environment Example species: Escherichia coli Klebsiella pneumoniae Citrobacter freundii Enterobacter aerogenes Proteus mirabilis Salmonella typhi Shigella dysenteriae Yersinia enterocolitica

Previous Experience with Enterobacteriaceae: EMB Media Large amounts of acid from lactose fermentation cause the dyes to precipitate on the colony surface, producing a black center or a “green metallic sheen” (E. coli) Smaller amounts of acid production result in pink coloration of the growth (E. aerogenes) Nonfermenting enterics do not produce acid so their colonies remain colorless or take on the color of the media (P. vulgaris)

Biochemical Testing Triple Sugar Iron (TSI): fermentation of sugars, sulfur reduction IMViC: Indole: Break down the amino acid Tryptophan Methyl Red: Glucose oxidation Voges-Proskauer: Production of neutral end products Citrate: Citrate fermentation Urease: Hydrolyzation of Urea Phenylalanine Deaminase: converts the amino acid phenylalanine to phenylpyruvic acid Nitrate Reductase: Reduction of nitrate (NO3) to nitrite (NO2)

TRIPLE SUGAR IRON TEST (TSI) Used to differentiate among the different groups of Enterobacteriaceae based on their ability to ferment glucose, lactose and/or sucrose Also differentiates between groups capable of reducing sulfur to hydrogen sulfide (Sodium Thiosulfate -> Hydrogen sulfide)

TSI Results: Red slant/Red butt = no fermentation Red slant/Yellow butt = only glucose fermentation Yellow slant/yellow butt = lactose and/or sucrose fermentation Dark color: Hydrogen Sulfide produced Sodium thiosulfate reduced P 190

IMViC TESTS A series of four tests consisting of: Used to differentiate the Enterobacteriaceae We will look at each test individually Indole: Break down the amino acid Tryptophan Methyl Red: Glucose oxidation Voges-Proskauer: Production of neutral end products Citrate: Citrate fermentation

Indole Test (SIM: Sulfide,Indole,Motility) Identifies bacteria capable of producing indole Some bacteria are capable of converting tryptophan (an amino acid) to indole and pyruvic acid by using the enzyme tryptophanase Pyruvic acid can be converted to energy or used to synthesize other compounds required by the cell Tryptophan Indole Ring Pyruvic Acid Ammonia

Procedure: Obtain 4 SIM Deep tubes Inoculate by the stab method with the following organisms: E.coli, P.vulgaris, E. aerogenes K. pneumoniae

Indole Test Results: If indole is produced, upon addition of Kovac’s Reagent (10 drops), a “cherry-red” band forms on the surface of the media Motility (if present) can be seen as growth of the bacteria away from the stab line Sulfur in the media may be reduced to hydrogen sulfide (H2S); this appears as a “blackening” within the media

Methyl Red Test Used to determine the ability of a bacteria to oxidize glucose and produce stable acid end products Methyl red is a pH indicator (red at pH less than 4.4 and yellow at a pH greater than 6) The combination medium used for this test is the MR-VP (methyl red/Voges-Proskauer) broth Acid production: positive methyl red End products of neutral pH : positive Voges-Proskauer

Procedure: Obtain 3 MR-VP broth tubes Inoculate (using a loop) with the following organisms: E.coli K.pneumoniae E.aerogenes

Results: From the 3 MR-VP broths that you inoculated, transfer 2 mLs from each and place into 3 separate clean tubes (set aside these aliquots for the VP test) To the remaining, original tubes that you inoculated add 5 drops of methyl red indicator A red color indicates that glucose has been oxidized

Methyl Red Test Results: A red color indicates that glucose has been oxidized. Methyl red positive tube on the right Methyl red negative tube on the left

Voges-Proskauer Test Used to determine the ability of microbes to produce nonacidic or neutral end products Remember that the MR-VP broth is a combined medium used for two tests— Methyl Red and Voges-Proskauer You have already inoculated the 3 MR-VP broth tubes from the previous procedure (Methyl Red Test) with E.coli K.pneumoniae E.aerogenes

Voges-Proskauer Procedure: To the aliquots of each broth culture separated during the methyl red test, add: 10 drops of Barritt’s Reagent A; shake 10 drops of Barritt’s Reagent B; shake Reshake the culture every 3 to 4 min. It can take as long as 15 min. for a color change to occur

Voges-Proskauer Results: The presence of a deep rose color after 15 minutes is indicative of non-acidic / neutral metabolic end products and a positive VP test result. Voges-Proskauer positive on the right negative on the left

Citrate Utilization Test Used to determine if an organism is capable of fermenting citrate and using that citrate as its sole carbon source The ability of an organism to utilize citrate occurs via the enzyme citrase

Procedure: Obtain 3 Simmons Citrate agar slants Inoculate these slants using the stab and streak method (the same way you inoculated the TSI media using a needle) with the following organisms: E.coli K.pneumoniae E.aerogenes

Citrate Test Results: Simmon’s Citrate agar utilizes sodium citrate as its sole carbon source Bromthymol blue is included as a pH indicator; the medium initially is green Organisms capable of using citrate as a carbon source turn the media “Prussian blue”. - + - +

Summary of IMViC Reactions Page 199

Urease Test Used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease The pH indicator, phenol red, is used to detect the breakdown of urea and the production of ammonia which is used by bacteria to produce amino acids and nucleotides

Procedure: Obtain 2 urea broth tubes Inoculate with the following organisms: E.coli P.vulgaris

Urease Test Results: Urinary tract pathogens from the genus Proteus may be distinguished from other enterics urease Urea + H2O CO2 + H20 + NH3 As the alkaline end products build, phenol red turns from yellowish gold to pink—a positive result

Urease Test Results As the alkaline end products build, phenol red turns from yellowish gold to pink—a positive result Urease positive organism on the right Urease negative organism on the left

Phenylalanine Deaminase Test Used to identify bacteria possessing the enzyme phenylalanine deaminase Phenylalanine deaminase converts the amino acid phenylalanine to phenylpyruvic acid + NH3

Procedure: Obtain 2 phenylalanine agar slants Inoculate (with a loop on the surface) with the following organisms: E.coli P.vulgaris

Results Phenylpyruvic acid produced by some organisms is colorless After inoculation and incubation, 10% ferric chloride, an oxidizing agent, is added to the surface of the slants Ferric chloride (FeCl3) reacts with the phenylpyruvic acid (if present) and changes color from yellow to green—a positive result

Phenylalanine Deaminase Results: Positive Negative Ferric chloride (FeCl3) reacts with the phenylpyruvic acid (if present) and changes color from yellow to green — a positive result Positive Negative

Nitrate Reductase Test Used to detect the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or some other nitrogenous compound, such as molecular nitrogen (N3) using the enzyme nitrate reductase NO3 NO2 Nitrate Reductase - Forms red color once solutions A and B are added.

Procedure: Obtain 3 Nitrate broth tubes Inoculate (with a loop) those tubes with the following organisms: E.coli A.faecalis P.aeruginosa

Results: (Pay close attention to this test; its one of the hardest test to read) After inoculation and incubation, the ability of an organism to reduce nitrate to nitrite (or molecular nitrogen) is detected by adding two reagents: Solution A (sulfanilic acid) Solution B (α-naphthylamine) If a red color appears after addition of solution A and B, this is considered a positive result NO3 NO2 Nitrate Reductase - Forms red color once solutions A and B are added.

Results continued: If there is no color change occurs after additions of solutions A & B, two possibilities must be considered: nitrates were not reduced by the organism the organism possessed such potent nitrate reductase enzymes that nitrates were reduced beyond nitrites to ammonia or even molecular nitrogen NO3 NO3 - NO3 NO2 Nitrate Reductase - NH3+ (Ammonia) N2 (Nitrogen Gas) P 219

Results Continued: Next, solution C (zinc) is added to the tubes that showed no color change after addition of Nitrate A & B Zinc, a reducing agent, is capable of converting nitrate to nitrite; the development of a red color after addition of Nitrate C means that nitrates were present and reduced by the zinc not the organism; this is a negative result If the addition of zinc does not produce a color change, the nitrates were reduced beyond nitrites to ammonia or nitrogen gas; this is a positive reaction NO3 NO3 - NO3 NO2 Nitrate Reductase - NH3+ (Ammonia) N2 (Nitrogen Gas)

Solutions A and B have been added to these tubes Solution C has been added to these tubes C. xerosis - Reductase Negative NO3 NO3 - Unreactive tubes P. aeruginosa Reductase Positive E. coli - Reductase Positive NO3 NO2 Nitrate Reductase - NH3+ (Ammonia) N2 (Nitrogen Gas) NO3 NO2 Nitrate Reductase -