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Carbohydrate Fermentation The identification of some bacteria is aided by determining what nutrients the bacteria can utilize and

what end products will be produced in the process. These characteristics are controlled by the enzymes which the bacteria produce. Because the type of enzyme(s) bacteria produce is genetically controlled, the pattern of sugars fermented may be unique to a particular species or strain. Fermentation products are usually acid (lactic acid, acetic acid etc.), neutral (ethyl alcohol etc.), or gases (carbon dioxide, hyrogen, etc.). To determine the products of sugar fermentation, a carbohydrate fermentation broth is prepared at pH 7.4. This broth contains 3 essential ingredients: 0.5%1.0% of the carbohyrate to be tested (e.g. lactose or glucose), nutrient broth, and the pH indicator phenol red. The nutrient broth, which is a light red color, supports the growth of most organisms whether they are able to ferment the sugar or not. The test organism is inoculated into a broth containing the test sugar and incubated. A bright yellow color indicates the production of enough acid products from fermentation of the sugar to drop the pH to 6.9 or less. Production of gas is determined with a Durham tube , a small inverted vial filled with the carbohydrate fermentation broth. If gas is produced during fermentation of the sugar, it is trapped at the top of the Durham tube and appears as a bubble. Slow fermenters may take a week or more to cause color changes detectable by the human eye. Positive (yellow color or yellow color with gas bubble) and negative results (red color, no gas bubble) are shown in this image.

Carbohydrate fermentation

Nitrate Reduction
The identification of some bacteria is aided by determining if the organism can reduce nitrate (NO3) to nitrite (NO2)or another nitrogenous compound such as ammonia (NH3) or nitrogen gas (N2). This reaction is expressed as: NO3 ----> NO2 ----> NH3 or N2 In order to determine if a bacteria can reduce nitrate, the test organism is inoculated into nitrate reduction broth, an undefined medium that contains large amounts of nitrate (KNO3). After incubation, alpha-napthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test. (Tube 2 in image below.) If there is no color change at this step, nitrite is absent. (Tube 1 below.) If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result. To distinguish between these two reactions, zinc dust must be added. Zinc reduces nitrate to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic acid are already present in the tube. (Tube 4 below.) Thus a red color after the zinc is added indicates the zinc found the nitrate unchanged. The bacteria was unable to reduce nitrate. This is recorded as a negative nitrate reduction test. If however, the tube does not change color upon the addition of zinc, then the zinc did not find any nitrate in the tube. (Tube 3 below.) That means the test organism converted the nitrate to nitrite and then converted the nitrite to ammonia and/or nitrogen gas. Thus no color change upon the addition of zinc is recorded as a positive nitrate reduction test. In the image below, alpha-napthylamine and sulfanilic acid were added to all four tubes. Subsequently, zinc dust was added to the third and fourth tubes. Click on the image to see an enlarged version.

nitrate reduction test

Gelatin causes liquids to solidify at temperatures below 28 degrees Celsius. At temperatures above 28 degrees C. gelatin is a liquid. Some bacteria produce gelatinase, a proteolytic enzyme that hydrolyzes gelatin. The hydrolyzed gelatin no longer has the ability to gel and thus remains a liquid, even if placed at temperatures below 28 degrees. The presence or absence of gelatinase can be used to aid in identification of certain bacteria. Two methods used to determine gelatinase production are the gelatin stab method and the gelatin strip method. The Gelatin Stab Method The gelatin stab method employs nutrient gelatin deep tubes that contain 12% gelatin. A heavy inoculum from a pure culture of the test organism is stabbed into the media. The gelatin media is incubated for at least 48 hours, and then placed into the refrigerator for approximately 30 minutes. If the gelatin is still intact (the bacteria did not produce gelatinase), the media will solidify in the refrigerator and a negative test result is recorded. If the organism has produced sufficient gelatinase, the tube will remain liquid (at least partially) and not solidify in the refrigerator. A positive test result is recorded. Some organisms may produce gelatinase in rather small quantities. Thus, a tube with a negative gelatinase result should be reincubated for up to two weeks. Whenever desired, the tube may be refrigerated and results observed. If the tube is still negative after two weeks of incubation (completely solidifies when refrigerated), it can be reasonably concluded that this organism does not produce gelatinase. The Gelatin Strip Method The gelatin strip method employs strips of clear blue plastic covered with a gray-green coating of gelatin (available from Key See Products, 2124 Sawtelle Blvd., Los Angeles, CA 90025). Upon incubation with a gelatinase producing bacteria, the gelatin coating is slowly hydrolyzed and the blue plastic strip becomes visible (a positive result). There will not be any blue plastic visible on the gelatin strip in an organism that is unable to produce gelatinase (a negative result). Some organisms may produce gelatinase in rather small quantities. Thus, a negative gelatin strip tube should be reincubated for up to two weeks. Positive and negative gelatinase results are shown below. Tubes in image 1 have been inoculated, incubated, and refrigerated for 30 minutes. Click on each image to see an enlarged version.

1. Gelatin Stab Method

2. Gelatin Strip Method

Starch Hydrolysis
Starch hydrolysis is a test used to detect the enzyme Amylase, which breaks down starch. After incubation, the plate is treated with Grams iodine. If the starch has been hydrolysed, then a reddish colour of clear zone will be formed around the bacterial growth. However, if the starch has not been hydrolysed, a blue or black area will be formed indicating the presence of starch. This test can be easily conducted by using an inoculating loop to spread bacteria on the plate surface. After it is incubated at an appropriate temperature, iodine will be added to the surface. Then, the colour change should be noted to know the results.

A. Absence of starch hydrolysis is indicated by the blue colour that completely surrounds the colony.

B. Starch hydrolysis is indicated by the reddish colour of clear

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