CATALASE TEST Some bacteria can reduce diatomic oxygen to hydrogen peroxide or superoxide. Both of these molecules are toxic. A defense mechanism which can minimize the harm done by the two compounds is the production of enzymes: SuperOxide Dismutase and Catalase. Both enzymes together will convert the Super Oxide back into diatomic oxygen and water. The catalase test involves adding hydrogen peroxide to a culture sample. If the bacteria in question produce catalase, they will convert the hydrogen peroxide into water and oxygen gas. The evolution of gas, causing bubbles, is indicative of a positive test. PROCEDURE “SLIDE TEST” FOR CATALASE ACTIVITY: Step 1. Place one drop of hydrogen peroxide on a clean glass slide. Step 2. Using a toothpick, pick an isolated colony from your assigned species and slowly immerse the cells into the drop of Hydrogen Peroxide. Step 3. A positive test is read directly from the slide. Look for Oxygen Gas bubbles. CITRATE Purpose The citrate test screens a bacterial isolate for the ability to utilize citrate as its carbon and energy source. A positive diagnostic test rests on the generation of alkaline by-products of citrate metabolism. The subsequent increase in the pH of the medium is demonstrated by the color change of a pH indicator. The citrate test is often part of a battery of tests used to identify gram-negative pathogens and environmental isolates. Theory The citrate test is commonly employed as part of a group of tests, the IMViC tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products. In the most common formulation, citrate is the sole source of carbon in the Simmons citrate medium while inorganic ammonium salt (NH4H2PO4) is the sole fixed nitrogen source. When an organic acid such as citrate is used as a carbon and energy source, alkaline carbonates and bicarbonates ultimately are produced. The visible presence of growth on the medium and the change in pH indicator color due to the increased pH are the signs that an organism can import citrate and use it as a sole carbon and energy source; such organisms are considered to be citrate positive. PROTOCOL A. Inoculation of medium Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single isolated colony and lightly streak the surface of the slant. A needle is the preferred sampling tool in order to limit the amount of cell material transferred to the agar slant. Avoid using liquid cultures as the inoculum source. Citrate utilization requires oxygen and thus screw caps, if used, should be placed loosely on the tube. B. Incubation conditions Incubate at 35oC (+/- 2oC) for 18 to 48 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium. C. Interpretation of results Citrate negative: trace or no growth will be visible. No color change will occur; the medium will remain the deep forest green color of the uninoculated agar. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant. Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue. OXIDASE TEST Cytochrome oxidase is an enzyme found in some bacteria that transfers electrons to oxygen, the final electron acceptor in some electron transport chains. Thus, the enzyme oxidizes reduced Cytochrome C to make this transfer of energy. Presence of cytochrome oxidase can be detected through the use of Oxidase test reagent (1% tetra methyl-para –phenylene diamine dihydrochloride). This reagent acts as an electron donor to cytochrome oxidase. If the bacteria oxidize (remove electrons) the Oxidase test reagent the reagent will turn purple, indicating a positive test. No color change in one minute indicates a negative test. PROCEDURE Step 1. Touch one colony with sterile swab. Step 2. Place a drop of oxidase reagent (1% tetra methyl-para-phenylene diamine dihydrochloride) on swab. Results. A positive test is indicated by the rapid appearance of a purple color at this site. Void any test after 1 minute. Phenol Red Carbohydrate Fermentation Broth: Purpose Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. Fermentation patterns can be used to differentiate among bacterial groups or species. For example, all members of the Enterobacteriaceae family are classified as glucose fermenters because they can metabolize glucose. Theory During the fermentation process, an organic substrate serves as the final electron acceptor. The end-product of carbohydrate fermentation is an acid or acid with gas production. Various end-products of carbohydrate fermentation can be produced. The end-product depends on the organisms involved in the fermentation reaction, the substrate being fermented, the enzymes involved, and environmental factors such as pH and temperature. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen. Fermentation reactions are detected by the color change of a pH indicator when acid products are formed. This is accomplished by adding a single carbohydrate to a basal medium containing a pH indicator. Because bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH changes only when excess acid is produced as a result of carbohydrate fermentation. Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests because most of the end-products of carbohydrate utilization are organic acids. However, other pH indicators such as bromocresol/bromcresol purple, bromothymol/bromthymol blue, and Andrade’s can be used. PROTOCOL A. Inoculation of media Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. Make sure to avoid the Durham tube, if present. Swirl the tube gently to mix contents. Avoid contact of liquid with tube cap. B. Incubation Incubate tubes at 35 to 37°C for 18 to 24 hours. C. Interpretation of results 1. Fermentation results When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to reincubate the tube. 2. Gas production results Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production. No bubbles within the Durham tube indicate a non-gas-producing. 3. Negative results A reddish or pink color indicates a negative reaction. In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative. SULFATE INDOLE MOTILITY Indole is a by-product of the metabolic breakdown of the amino acid tryptophan used by some microorganisms. The presence of indole in a culture grown in a medium containing tryptophan can be readily demonstrated by adding Kovac’s reagent to the culture. If indole is present, it combines with the reagent to produce a brilliant red color. If it is not present, there will be no color except that of the reagent itself. This test is of great value in the battery used to identify enteric bacteria. Hydrogen sulfide is produced when amino acids containing sulfur are metabolized by microorganisms. If the medium contains metallic ions, such as lead, bismuth, or iron (in addition to an appropriate amino acid), the hydrogen sulfide formed during growth combines with the metallic ions to form metal sulfide that blackens the medium. The most convenient medium for testing indole and/or hydrogen sulfide production is SIM medium (SIM is an acronym for sulfide, indole, and motility). This is a tubed, semisolid agar that can also be used to demonstrate motility. It is inoculated by stabbing the needle straight down the middle of the agar to about one-fourth the depth of the medium and withdrawing the wire along the same path. For Motility, you are to examine the tubes in the line of inoculation. A motile species of bacteria will grow away from the stab line with general turbidity extending throughout the tube. A nonmotile species will only grow in the line of inoculation. PROCEDURE Step 1. “STAB” inoculate growth from your culture into one SIM’s medium. Step 2. Incubate tubes at 37C for 24 hours. Results. Record your results using the following interpretation. Hydrogen Sulfide. Examine the tube for evidence of hydrogen sulfide production (browning or blackening of the medium). Motility. Examine the stab line of inoculation for evidence of motility. Indole. Perform the Kovac’s test for indole as follows: 1. Using a reagent dropper, add 6 or 8 drops of Kovac’s Reagent to the SIM’s medium. 2. Allow tube to sit of about 1 minute. 3. Observe the reagent for a “Cherry Red” color. Red is positive for Indole production. Phenol Red Carbohydrate Fermentation Broth: Purpose Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. Fermentation patterns can be used to differentiate among bacterial groups or species. For example, all members of the Enterobacteriaceae family are classified as glucose fermenters because they can metabolize glucose. Theory During the fermentation process, an organic substrate serves as the final electron acceptor. The end-product of carbohydrate fermentation is an acid or acid with gas production. Various end-products of carbohydrate fermentation can be produced. The end-product depends on the organisms involved in the fermentation reaction, the substrate being fermented, the enzymes involved, and environmental factors such as pH and temperature. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen. Fermentation reactions are detected by the color change of a pH indicator when acid products are formed. This is accomplished by adding a single carbohydrate to a basal medium containing a pH indicator. Because bacteria can also utilize peptones in the medium resulting in alkaline by-products, the pH changes only when excess acid is produced as a result of carbohydrate fermentation. Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests because most of the end-products of carbohydrate utilization are organic acids. However, other pH indicators such as bromocresol/bromcresol purple, bromothymol/bromthymol blue, and Andrade’s can be used. PROTOCOL A. Inoculation of media Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. Make sure to avoid the Durham tube, if present. Swirl the tube gently to mix contents. Avoid contact of liquid with tube cap. B. Incubation Incubate tubes at 35 to 37°C for 18 to 24 hours. C. Interpretation of results 1. Fermentation results When using phenol red as the pH indicator, a yellow color indicates that enough acid products have been produced by fermentation of the sugar to lower the pH to 6.8 or less. A delayed fermentation reaction may produce an orange color. In such cases, it is best to reincubate the tube. 2. Gas production results Bubbles trapped within the Durham tube indicate the production of gas. Even a single bubble is significant and denotes evidence of gas production. No bubbles within the Durham tube indicate a non-gas-producing. 3. Negative results A reddish or pink color indicates a negative reaction. In negative tubes, the presence of turbidity serves as control for growth. A reddish or pink color in a clear tube could indicate a false negative. SULFATE INDOLE MOTILITY Indole is a by-product of the metabolic breakdown of the amino acid tryptophan used by some microorganisms. The presence of indole in a culture grown in a medium containing tryptophan can be readily demonstrated by adding Kovac’s reagent to the culture. If indole is present, it combines with the reagent to produce a brilliant red color. If it is not present, there will be no color except that of the reagent itself. This test is of great value in the battery used to identify enteric bacteria. Hydrogen sulfide is produced when amino acids containing sulfur are metabolized by microorganisms. If the medium contains metallic ions, such as lead, bismuth, or iron (in addition to an appropriate amino acid), the hydrogen sulfide formed during growth combines with the metallic ions to form metal sulfide that blackens the medium. The most convenient medium for testing indole and/or hydrogen sulfide production is SIM medium (SIM is an acronym for sulfide, indole, and motility). This is a tubed, semisolid agar that can also be used to demonstrate motility. It is inoculated by stabbing the needle straight down the middle of the agar to about one-fourth the depth of the medium and withdrawing the wire along the same path. For Motility, you are to examine the tubes in the line of inoculation. A motile species of bacteria will grow away from the stab line with general turbidity extending throughout the tube. A nonmotile species will only grow in the line of inoculation. PROCEDURE Step 1. “STAB” inoculate growth from your culture into one SIM’s medium. Step 2. Incubate tubes at 37C for 24 hours. Results. Record your results using the following interpretation. Hydrogen Sulfide. Examine the tube for evidence of hydrogen sulfide production (browning or blackening of the medium). Motility. Examine the stab line of inoculation for evidence of motility. Indole. Perform the Kovac’s test for indole as follows: 1. Using a reagent dropper, add 6 or 8 drops of Kovac’s Reagent to the SIM’s medium. 2. Allow tube to sit of about 1 minute. 3. Observe the reagent for a “Cherry Red” color. Red is positive for Indole production. BIO175- Virtual Labs ‘Unknown Organism ’Due 11/10/21 Here is a link that will be helpful. https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#catalase Carbohydrate (Sugar) Fermentation 1. 2. 3. 4. What is the purpose of the test? What is the pH indicator? What color is a negative and positive results? Give one example of an organism that would be positive and one that would be negative. Catalase 1. What is the substrate in the reaction? 2. What is the product we are looking for in a positive reaction? 3. Give one example of an organism that would be positive and one that would be negative. Oxidase 1. 2. 3. 4. What is the enzyme we are testing for? How long is the results valid? What color is a negative and positive results? Give one example of an organism that would be positive and one that would be negative. Sulfate Indole Motility (SIM) 1. What three results can be observed with this test? 2. Describe the positive result for each test. 3. List one organism that is positive for each result. Citrate (Simmon’s Citrate) 1. 2. 3. 4. What is the purpose of the test? What is the pH indicator? What color is a negative and positive results? Give one example of an organism that would be positive and one that would be negative. 
Purchase answer to see full attachment