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Biochemical Media Casitone Broth-White Cap with Black Circle. Loop Transfer Biochemical Media Casitone Broth-white cap with black circle. Loop transfer. (Use care tops may be very loose) (Do not put caps on the wrong tubes during the inoculation procedures). Can the organism split tryptophan into indole, pyruvic acid and ammonia? Test for the presence of indole (unique to this process) by adding 5 drops of Kovacs Test Reagent. If indole is present, a ruby red color will form at the top of the test tube. The media name for casitone comes from the word casein. Casein is a milk protein rich in the amino acid tryptophan. Result sheet recorded with a “pos” or “neg” Phenol Red Carbohydrate Fermentation Broths (Phenol Reds) Red capped broth with durham tube-Phenol Red with Lactose Clear capped broth with durham tube-Phenol Red with Glucose Yellow capped broth with durham tube-Phenol Red with Sucrose Blue capped broth with durham tube-Phenol Red with Maltose Green capped broth with durham tube-Phenol Red with Mannitol All tubes are inoculated with loop transfers. Can the organism ferment the carbohydrates listed above? Each comes with a separate sugar, a durham tube for gas collection and the acid-base indicator called phenolphthalein. Fermentation will produce acidity and sometimes gas. Gas alone is not produced by the fermentation process. Phenolphthalein will turn yellow in the presence of acid, red in an alkaline environment. Gas production can be observed by the presence of air bubbles captured in the durham tube. For each result both an acid and gas response should be indicated. For a large amount of acid (yellow) use “A”. For a small amount of acid (yellow/orange) use “a”. For no acid production at all use the symbol “Ø”. For a large amount of gas (durham tube rises to the top) use “G”. For a small amount of gas (bubble in durham tube) use “g”. For no gas production at all use ”Ø”. Sample responses include; AG, Ag, AØ, ag, ØØ, aØ. Methyl Red-Voges Proskauer Broth (MR-VP) -broth with red cap. Loop transfer. 2 separate test results from this one broth. Voges-Proskauer (VP)-can the organism produce acetylmethylcarbinol (acetoin)? Note, run VP test first. To check for this metabolic product, use a pipet and transfer 1.0 ml. (20 drops) of MR- VP broth to a sterile test tube. Add 15 drops of VP A (5% alphanaphthol in absolute ethanol) followed by 10 drops of VP B (40% KOH). A positive test is the development of a cherry red color in 15 to 30 minutes. On the result sheet use a “pos” for a positive result and a “neg” for a negative. Methyl Red (MR)-can the organism produce acid from the glucose in the media? With the remainder of the MR-VP broth (original tube), add 5 drops of methyl red test indicator. This is an acid-base indicator. Watch the drops as they land on the top of the broth. If the drops stay red, the appropriate acid environment from glucose metabolism exists and this is considered a positive test. If the drops turn yellow or orange this is considered a negative test. Use “pos” or “neg” on the result sheet. Tryptic Nitrate Broth-broth with white cap and black dot. Loop transfer. Can the organism reduce nitrate to nitrite? Check for the presence of nitrite in the grown broth. Add 5 drops of Nitrate A (sulfanilic acid and acetic acid) and 5 drops of Nitrate B (dimethyl –β-naphthylamine and acetic acid). If nitrite is present a red color will appear in the media after approximately 5 minutes. This is a positive test and a “pos” can go on the result sheet. If a red color does not appear there are two possible conclusions. First, the organism did not reduce nitrate to nitrite and we would have a negative test. Second, the organism did indeed reduce nitrate to nitrite and the nitrite left the test tube in the form of a gaseous byproduct (such as N2, or NH3 or NO). This would be a positive test for nitrate reduction. To determine which of these two possibilities occurred add a pinch of powdered zinc to the broth. Zinc will actually catalyze the conversion of nitrate to nitrite. If the organism never in the first place reduced nitrate to nitrite, nitrate should still be in the broth. Addition of zinc will produce nitrite from the unused nitrate and the media (reagents A and B already added) will turn red after approximately 5 minutes. If this occurs enter a “neg” on the result sheet, indicating that the organism does not have the ability to reduce nitrate to nitrite. If on the other hand a red color does not develop after approximately 5 minutes after having added the zinc, this indicates that the organism did indeed reduce nitrate to nitrite and the nitrite left the tube in the form of a gas. On the result sheet write “pos “(zinc added). Simmons Citrate Agar Slant- slant with green cap. To inoculate, streak the slant with the loop. Can the organism use citrate when it is provided in the form of its sodium salt, as the sole source of carbon? Organisms capable of utilizing the citrate produce alkaline metabolites which raises the pH of the media. Bromothymol blue is an acid-base indicator in the media. When acid the bromothymol blue is green to yellow (lower pH) in color. When alkaline, a deep blue color develops (Prussian blue). The appearance of any Prussian blue color in the media after growth should be interpreted as a positive test (“pos”). Lack of this color is “neg”. Urea Agar Slant-slant with yellow cap. To inoculate, streak the slant with the loop. Can the organism produce the enzyme urease and hydrolyze urea producing alkaline ammonia. The media is made with urea and the acid-base indicator phenolphthalein. If the test is positive alkaline ammonia will be produced and cause the phenolphthalein to become a pink/red color. Place a “pos” on the result sheet. Lack of a pink/red color should be interpreted as a “neg”. Triple Sugar Iron Agar Slant (TSI) - slant with red cap. To inoculate, stab the butt region with the loop. Continue the inoculation by now streaking the slant region with the loop. There are 3 boxes on the result sheet for TSI. TSI-sl, TSI-butt and TSI-H2S. In determining the results we want to know if the organism can ferment any or all of the three sugars that have been placed in the media. The 3 sugars are; glucose (dextrose), sucrose and lactose. TSI has 1/10 the amount of glucose as lactose and sucrose. The acid-base indicator phenolphthalein has been placed in the media. As mentioned above, when acid it is yellow and when alkaline it is red. Fermentation of the sugars produces acid and sometimes gas. Never gas alone. A yellow color is observed to indicate the acid from fermentation. When gas is also produced from the fermentation reaction, this can be noted by the appearance of bubbles within the media, or splitting of the media or the raising of the media. On the result sheet both an acid and a gas result should be placed in both the TSI slant and the TSI butt boxes. (The butt region is the portion of the media below the slant). For a large amount of acid (yellow) use “A”. For a small amount of acid (yellow/orange) use “a”. For no acid production at all use the symbol “Ø”. For a large amount of gas (the media splits or rises) use “G”. For a small amount of gas (a few bubbles in the media) use “g”. For no gas production at all use ”Ø”. Sample responses include; AG, Ag, AØ, ag, ØØ, aØ. Yellowing and perhaps gas production as well in the butt region alone, indicates the fermentation of glucose only. Fermentation is anaerobic and starts in the butt region. Acid metabolites will diffuse from the butt region into the slant. Since there is only 1/10 the amount of glucose as the other two sugars, those organisms that ferment glucose only will produce 1/10 the amount of acid. There will not be enough acid produced to spread upwards to the slant region leaving the butt with the yellow color and perhaps signs of gas production as well. Members of the genus Salmonella and the genus Shigella are important pathogens which do not ferment sucrose or lactose but do ferment glucose. TSI is also used to determine if an organism can produce H2S (hydrogen sulfide) from sulfur that is split off of the sulfur containing amino acids. The sulfur containing amino acids include cysteine and methionine. Iron sulfate is placed in the media. Any H2S that is produced will react with the iron sulfate and produce iron sulfides. Iron sulfides are noticeable as black precipitates. The appearance of a black precipitate should be interpreted as a positive result for H2S production. Place a “pos” in the TSI H2S box. Lack of any black precipitate should be interpreted as a “neg” for H2S production. Sometimes there is an excessive amount of black precipitate in the media. This may interfere with the reading of the fermentation results in the TSI media. If this happens and the fermentation results are hidden and obscured by the black precipitate, place the word “black” in the appropriate TSI slant or butt boxes. IMViC Tests This is a series of tests that are primarily used to distinguish between the coliform bacteria. The letters stand for Indole, Methyl red, Voges-Proskauer and Citrate reactions.
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