Triple Sugar Iron (TSI) Agar. Purpose: Determine fermentation of glucose and lactose or sucrose and H 2 S production.

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1 Triple Sugar Iron (TSI) Agar Determine fermentation of glucose and lactose or sucrose and H 2 S production. TSI contains three sugars glucose, sucrose, and lactose. Phenol Red is the indicator that turns yellow at an acid ph resulting from fermentation. The glucose concentration is 0.1% and the lactose and sucrose concentrations are 1%. If an organism ferments glucose only, the reaction will be K/A, or alkaline slant over acid butt. The reason for this reaction is that fermentation takes place in the butt, which results in enough acid to lower the ph here and turn the butt yellow. However, in the slant, aerobic oxidation or respiration takes place and not enough acid is produced to lower the ph, so it remains red. Also, alkaline amines from protein degradation neutralize the small amount of acid formed in the slant resulting from respiration. It is important to keep the caps loose on TSI medium to allow this differential in ph to be visualized. If sucrose or lactose is fermented, the reaction will be A/A (acid slant over acid butt). Because of the high concentration of these sugars, enough acid is produced by fermentation in the butt to lower the ph of both the butt and the slant, turning both yellow. Gas production, sometimes resulting from fermentation, can also be detected as bubbles trapped in the agar or actually splitting or pushing the agar upwards. H 2 S production is detected by incorporating ferrous ammonium sulfate into the medium. It reacts with the H 2 S gas, forming ferrous sulfide (FeS), an insoluble black precipitate. Sodium thiosulfate is the source of sulfar incorporated in the TSI slant. For the formation of FeS to occur, the medium must be acidified, se we can assume that H 2 S production occurs in and makes the acid butt. Interpretation: 1. A/A: ferments glucose and either sucrose, lactose, or both. 2. K/A: dose not ferment lactose or sucrose; dose ferment glucose. 3. K/K: a non-fermenter. 4. Black precipitate in stab: produces H 2 S (and ferments glucose).

2 Urease Detects whether an organism possesses the enzyme urease, which hydrolyzes urea. Urea agar contains phenol red indicator, which turns red at an alkaline ph. When urea is hydrolyzed it releases ammonia, which causes an alkaline reaction, and a vivid pink color develops. 1. Streak the slant portion of a urea agar slant (pale yellow). 2. Cap loosely. 3. Incubate at 35C. 4. A positive reaction shows the development of a pink color often starting at the slant. 5. Rapid urea splitters such as Proteus produce a positive reaction in 1-2 hours. Slow splitters take hours.

3 Methyl Red Voges-Proskauer (MR-VP) MR Determines if glucose is fermented by the mixed acid pathway. If glucose is fermented via the mixed acid pathway, strong acids are produced and a low ph of 4.0 is maintained for at least 48 hours. This low ph is detected by adding MR, which turns yellow to red at ph 4.4. Incubate organism hours in MR-VP broth. Remove 1 ml of broth for VP determination. To remainder of the tube add two drops of MR indicator. Development of a cherry red color after MR is added indicates a positive reaction. Determines if glucose is fermented by the butylenes glycol pathway by the detection of acetylmethylcarbinol (one of the products). VP If glucose is fermented by the butylenes glycol pathway, the main product is acetylmethylcarbinol with small amounts of mixed acids. The resulting ph of the medium is not as acidic as in the mixed acid pathway, and the MR test is negative. The principle of the VP test is to detect the presence of acetylmethylcarbinol. After adding alpha-naphthol and 40% KOH, the carbinol is converted to diacetyl, which is a red complex. 1. Incubate MR-VP broth for 48 hours. 2. Pipette 1 ml of broth to separate tube. 3. Add six drops of alpha-naphthol (reagent A) and then 40% KOH (reagent B). 4. Let stand for minutes. 5. A red color at the top is positive.

4 Decarboxylation of Amino Acids Differentiate organisms based on their ability or inability to decarboxylate (or dihydrolate) the amino acids lysine, arginine, and ornithine. If an organism posses a specific decarboxylase enzyme, it is capable of decarboxylating (removing the carboxyl portion) a specific amino acid, which results in the formation of alkaline amines and CO2. Lysine, arginine, and ornithine are the amino acids routinely used for identification of Enterbacteriaceae. Utilization of arginine involves an initial dihydrolase enzyme that removes NH2. It is then converted to ornithine and decarboxylated. Decarboxylase enzymes are only activated at an acid ph and under anaerobic conditions. Moeller broth is used; it contains the specific amino acid, glucose and bromocresol purple, which is purple at an alkaline ph and yellow at acid ph. An organism is capable to decarboxylate the amino acid only after fermenting glucose, creating an acid ph, after which the decarboxylase enzymes are activated and decarboxylation takes place. The tube is purple before it is inoculated, then turns yellow after glucose is fermented, and then finally turns black to purple when decarboxylation takes place. A control tube with Moeller broth devoid of amino acid should also be run to ensure the initial fermentation reaction takes place. 1. Inoculate Moeller base containing specified amino acid. 2. Overlay with mineral oil to create an anaerobic conditions. 3. Incubate for 24 hours. 4. A positive reaction is purple, indicating decarboxylation. 5. A negative reaction is yellow, indicating only fermentation of glucose. 6. A control tube of decarboxylase base Moeller (DBM) with no amino acid added should always be run to ensure that initial fermentation of glucose has occurred. The control tube should also be overlaid with oil and the reaction should always be yellow.

5 Deoxyribonuclease The purpose of the deoxyribonuclease (DNase) test is to determine if an organism produces the enzyme DNase, which can degrade extracellular DNA. This test is useful in differentiating Serratia, which is DNase-positive, from many other members of the Enterobacteriaceae that are DNase-negative. Principle and DNase agar contains DNA in complex with a green dye that makes the plate a light green color. The organism is inoculated on the plate as a single streak and incubated for 24 hours at room temperature. (DNase enzymes are more active at this temperature.) If the organism produces the enzyme DNase, the agar around the streak will appear clear because the DNA complex has been degraded and the dye released. A negative reaction would show no clearing around the streak. Positive and negative controls should be run on the same plate to make interpretation easier. Motility Determines if an organism is motile. By inoculating motility medium that contains a low concentration of agar, motility can be observed as diffuse growth spreading out from the line of inoculation. Some organisms produce flagella better at lower temperatures. Therefore, motility may be better observed at 25C rather than 37C. Citrate Determines if an organism is capable of utilizing sodium citrate as a sole source of carbon. If sodium citrate is utilized, alkaline products including NaOH are produced. This is indicated by bromothymol blue, which is a blue color at alkaline ph. 1. Inoculate the surface of Simmons citrate slant in a single streak. 2. Cap loosely and incubate for 24 hours. 3. A positive test shows development of deep blue color or visible growth along with the streak. It some cases, re-incubation is necessary for the development of a blue color.

6 Orthonitrophenyl-β-D-Galactopyranoside (ONPG) Test (β-galactosidase Test) Detection of the presence of the enzyme β-galactosidase, which hydrolyzes lactose into glucose and galactose. This test is used to differentiate slow lactose fermenters and non-lactose fermenters from lactose fermenters. ONPG is an analog of lactose that can be hydrolyzed by the enzyme β-galactosidase, as lactose is, but the resulting product is yellow and visually detectable. ONPG is not dependent on the enzyme permease for entry into the cell, so it detects only β-galactosidase activity independent of permease activity. Lactose fermentation is a two-step procedure for most bacteria that requires two enzymes: 1. Permease - transports lactose into the cell. 2. β-galactosidase - hydrolyzes lactose to glucose and galactose. Non-lactose fermenters are devoid of one or both enzymes. Slow lactose fermenters may appear as non-lactose fermenters initially because they are deficient in permease, but since they possess β-galactosidase they can utilize lactose once it is inside the cell. Demonstration of β-galactosidase in these organisms permits differentiation from nonlactose fermenters that are β-galactosidase-negative. 1. Place an ONPG-impregnated filter paper disk in 0.5 ml sterile saline. 2. Inoculate heavily with bacterial isolate. 3. Incubate at 35C for l-6 hours. 4. A positive test turns pale yellow, which indicates production of orthonitrophenyl from ONPG by β-galactosidase. 5. A negative test is indicated by no color change. Nitrate Reduction Test Nitrate Reduction test is used to determine the ability of an organism to reduce nitrate. The test is useful for the identification of Enterobacteriaceae. The reduction of nitrate to nitrite is determined by adding sulfanilic acid and alpha-naphthylamine. The sulfanilic acid and nitrite react to form a diazonium salt. The diazonium salt then couples with the alpha-naphthylamine to produce a red, water-soluble azo dye. If there is no color change, zinc dust is added. Zinc reduces the remaining nitrate to nitrite to form a red color. If nitrate was reduced all the way to nitrogen gas, however, no color change occurs.