Starch agar differentiates organisms based on whether they can hydrolyze starch. Starch can be hydrolyzed by bacteria via excreting enzymes that break down starch, such as amylase. The prepared solution is light amber in color. Organisms are grown at the optimal temperature for the species. Then iodine is added to the solution. Iodine turns dark purple or blue in the presence of starch. A clearing around the bacterial colonies indicates starch degradation. If organisms can hydrolyze starch, the area around the colonies becomes colorless. A negative result has a blue or purple color formation with no clearing around the colonies.
In Oxidative Fermentation media, gram negative bacteria are identified and differentiated by using the type of fermentation utilized. Using carbohydrates, such as sucrose, lactose, or glucose, bacteria are categorized into oxidative fermentative, anaerobic fermentative, or no acid production using carbohydrates. If acid from the breakdown of carbohydrate is produced in the broth media, the broth will become yellow in color. If bacteria that cannot breakdown the carbohydrate are present, then the solution will remain green, or turn blue due to the utilization of protein in the solution, which increases the pH. To differentiate between oxidative fermentation and anaerobic fermentation, mineral oil is added onto the media. The mineral oil is used to prevent air from interacting with the media.
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To perform the test, two tubes of media are obtained. Using an inoculating needle, the target bacteria is stabbed through the media until the needle is 1/4th away from the bottom of the tube. After adding mineral oil to the top of one tube, the tubes are inoculated for 48 hours at 35 +/- 2Ëš C. Afterwards, the results can be seen in most bacteria. Some species of bacteria may require up to four days of incubation for accurate results.
The Citrate Simmons test is a differential test for gram negative bacteria. Bacteria that can utilize sodium citrate as the exclusive carbon and energy source, along with dihydrogen phosphate (NH4H2PO4) as the single nitrogen source grow in this media. When the two compounds are used, the bacteria produces an alkaline reaction and pH increase that changes the pH indicator, bromthymol blue, from dark or medium green to blue. This test is also useful for differentiating members of the genus enterobacter, especially when used as a part of a group of test, known as IMViC tests. IMViC is an abbreviation for indole, methyl red, Vogus-Proskauer, and citrate (little i is used for pronunciation purposes). A positive result is blue in color, and a negative result remains green.
To inoculate the medium, pure colony is lightly streaked across the slant at room temperature. A light amount of bacteria should be used. Extra cellular material could result in a false positive. The top of the test tube should be set to allow oxygen in, since an aerobic atmosphere is needed. The test tube is then incubated at 35 +/-2ËšC for 4 days. If the media turns blue, then the organism is able to use citrate as a carbon and energy ate as a carbon and energy source. Bacteria that cannot use citrate as a carbon and energy source will not increase the pH, and the media will remain green in color.
The Urea Hydrolysis, or Urease test differentiates organisms that can produce the enzyme Urease. This coenzyme hydrolyzes urea into ammonia and carbon dioxide. The ammonia causes the pH to increase. The broth media used contains pH buffers, urea, small amounts of nutrients, and the pH indicator phenol red. Phenol Red turns pink when the pH of the solution is greater than 7. The indicator can also turn yellow when the solution is acidic. Because the ammonium produced via urease breakdown of urea makes the phenol red turn pink, pink coloration of the solution indicates citrate positive results. Yellow color indicates citrate negative results.
Enteric bacteria, which belong in the family Enterobacteriaceae, are gram negative bacteria that are normally found in the intestines of animals. These bacteria have the ability to hydrolyze urea, some more rapidly than others. This feature can be used to differentiate between Enterbacteriaceae species. The components of the media include a high buffer concentration so that bacteria that rapidly utilize media are the best for use in the test. The high buffer also prevents delayed positives.
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For the experiment, a control should be used that has the same media without urea so that the results can be ruled out for false positives. Using at least two loops of bacteria, the broth is inoculated with the target bacteria by streaking the bacteria back and forth over the slant. The broth is then shaken to suspend bacteria and incubated at 35+/-2ËšC for 2, 4, 6, 18, 24, and 48 hours.
The Bile Esculin test is both a selective and differential test. It is selective because it allows only Enterococcia and Streptococcus bovis to grow, inhibiting the growth of all other gram positive organisms; it is differential because the reactions of esculetin and ferric citrate makes the media black, and is indicative of bacteria that can hydrolyze esculin. The media is made up of nutrients, esculin, oxgall, and ferric citrate. Oxgall is an inhibitor of gram positive bacteria other than Enterococci and S bovis. The Enterococci and S bovis that can hydrolyze glycoside to esculetin and dextrose produce a dark brown or black color when the esculetin reacts with ferric citrate.
To inoculate the media, two or the colonies are added to the media, or streaked onto a slant. For a slant, more than half of the media should be black for a positive test. Less than half blackening is considered negative. In other media, any blackening is considered positive.
Phenol red is a differential test that show if a particular bacteria is fermentative. It uses carbohydrates, peptone, phenol red as an indicator, and Durham tube to collects gas, if produced by fermentation. Typically, three test tubes are used, each containing a different carbohydrate: glucose, lactose, or sucrose. If the organisms, after being inoculated and incubated for 1824 hourse at 32 +/- 2ËšC, can ferment the sugars and produce acidic products, the color of the color of the media will turn yellow. If gas is produced, then an air bubble will form in the Durham tube. If the bacteria cannot utilize the carbohydrates, sometimes the bacteria can utilize the peptone in the media, which then become fuchsia colored.
Methyl Red-Voges Proskauer medium, a differential test, utilizes two different types of reagents to test for the type of fermentation a bacterial species uses. The methyl red pH indicator is used to test for bacteria that undergo the mixed acid fermentation pathway. This pathway creates end products that are both acidic (such as lactic or acetic acid) and neutral (such as ethanol), hence the mixed acid name. The majority of product is acidic, which reduces the pH in the media and allows the methyl red indicator to change the media to red. The Vogues-Proskauer test is used to test for bacteria that use the butylene glycol, or butanediol fermentation pathway. The end products of the butanediol pathway are acetoin (3- hydroxybutanone) as an intermediate (acetoin can be reduced to butanediol). The VP test has hydrogen peroxide in it, which oxidizes the acetoin to diacetyl, using creatine as a catalyst. The diacetyl produces a red color, which is indicative of a positive VP test.
The Methyl Red and VP reagents are used simultaneously because organisms cannot be positive for both. However, organisms can be negative for both. A double negative indicates that the organisms cannot ferment the sugar within the media. To perform the test, a light amount of bacteria are inoculated within a tube containing MRVP broth at 35 +/-2ËšC for a minimum of 48 hours. Then divide the media into two sterile test tubes. To one test tube, and the VP reagent, and to the other, add the methyl red reagent. For methyl red, a positive test is red in color; negative tests are yellow in color. For the VP test, a red color within five minutes of adding the reagent is a positive result.
The decarboxylase test uses differential media to test for the production of the enzyme decarboxylase. The broth contains the carbohydrate dextrose, pH indicators Bromcresol purple and cresol red, peptones and beef or yeast extract for nutrient supply, the decarboxylase cofactor Pyridoxal, and the amino acids lysine, ornithine, or arginine. It is used for members of the group Enterbacteriaceae. The test process can be broken down into two steps: whether the bacteria can ferment dextrose, and whether it can produce the specific decarboxylase enzyme for the specific amino acid.
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The media will turn yellow first because of the byproducts of dextrose fermentation. The byproducts lower the pH of the media, and the Bromcresol indicator will turn yellow as a result. Because of the acidity of the byproducts and presence of the specific amino acid, bacteria, if able, can remove the carboxyl group from the amino acid, thus raising the pH and turning the media purple. Because specific decarboxylase enzymes are made for specific amino acids, media are made explicitly for each amino acid. Negative tests are yellow in color or have no color change. Even though an organism can ferment dextrose, results are still considered negative if the decarboxylase is not made, which is indicated by purple colored media. Positive tests are purple in color.
To perform the test, broth media is inoculated with on-two colonies and mixed throughout the media. A thin layer of mineral oil (1 ml) is applied to encourage bacteria to ferment by creating an anaerobic environment, thus preventing false positives at the surface. Tubes are incubated with tightly capped caps at 35 +/-2ËšC and examined at 18-24, 48, 72, and 96 hours. Gray color maybe the reduction of the indicator, and more indicator can be added for interpretation.
The Nitrate (NO3) test is a differential test that shows if an organism can reduce nitrate to nitrite using the enzyme nitrate reductase. The reduction of nitrate is usually an anaerobic pathway, and this test is usually used on gram negative aerobic or facultative anaerobic bacteria. First, several colonies of bacteria are inoculated in the media, then incubated at 35-37ËšC. If the organism is able to reduce nitrate, which is in the form of potassium nitrate in the media, to nitrite, the nitrite will react to the sulfanilic acid (reagent A). The product of the reaction will then react to N, N-dimethyl-alpha-naphthylamine (reagent B) and produce a red color, which is considered a positive result.
Zinc dust, or reagent C, is used as a final step if the solution does not turn red. Zinc reduces any nitrate that was not reduced by the enzyme nitrate reductase. The nitrite produced by the zinc reaction then reacts to the reagent A and B that was previously added, forming a red coloring. If the solution does not turn red, then the nitrate was reduced completely into molecular nitrogen and ammonia. This type of reaction is called a complete positive.The Durham glass can collect nitrogen gas, which is a typical end product of the dentrification pathway.
After incubation, if gas is observed, then nitrate was reduced to nitrate gas, and the test is positive. Ten drops of both reagent A and B are added, and a red color formation within two minutes is a positive result. If the color does not change, then 20mg of reagent C is added. A red result is a negative result, while no color change within 5-10 minutes is a positive result.
The Fluid Thioglycollate test is used to differentiate bacteria based on their utilization of oxygen. The broth has the pH indicator methylene blue, the oxidation-reduction indicator resazurin, which turns pink when oxidized and colorless when reduced, nutrients for bacterial growth and replication, the reducing agent L-cystine, and sodium thioglycollate, which is a reducing agent as well. The sodium thioglycollate reacts with oxygen in the media, keeping oxygen levels low so that oxygen levels decrease going the down the tube. The bottom of the tube is completely absent of oxygen. Therefore, the tube is made into an environment where bacterial can only grow in, and are localized into areas where they can survive.
Obligate anaerobes fail to grow in the presence of oxygen, and therefore grow throughout the tube, except on the top layer. Obligate aerobes only grow where oxygen is present, and will therefore grow on the top layer of the tube. Aerotolerant organisms have uniform growth throughout the tube, while facultative organisms have uneven growth throughout the tube. Finally, microaerophiles grow throughout the upper and middle layer of the tube, since they require little oxygen.