Citrate Utilization

2. Citrate Utilization • Tests for the ability of bacteria to convert citrate (an intermediate of the Krebs cycle) into oxaloacetate(another intermediate of the Krebs cycle). • This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon. • Some intestinal bacteria are (+)citrate as Klebsiella,Citrobacter, Enterbacteraerogenes ;while others is(-) citrate as E. coli .

3. Principle • Organisms that metabolize citrate utilize the ammonium salts • releasing ammonia and increasing the pH of the medium . • Bromthymolblue is present in the medium as the indicator • dye, while turns from green at neutral pH (6.9) to blue at pH • higher than 7.6 (basic or alkaline). • Simmon's citrate agar: sodium citrate as sole carbon source, • ammonium ion as the sole nitrogen source and Bromthymol • blue.

4. Procedures • Using a sterile inoculating needle, streak one organism over the surface of the agar slant, then stab the butt. Repeat with the second organism. • Incubate the tubes at 37oC for 48 hours. • Examine for growth. Is there a change in the indicator dye in the agar?

5. Examine for growth (+) . Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue. Result

6. Oxidase Test • The Oxidase Test is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase. • Cytochrome c oxidase is the terminal enzyme in the electron transfer chain , the enzyme which reduces molecular oxygen to water to compete the last link in chain of aerobic respiration .

7. Oxidase producing bacteria • Pseudomonas • Neisseria • Vibrio • Aeromonas • Pseudomonas aeruginosa is a gram-negative, aerobic rod having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor and thus is oxidase positive. • Enterobacteriaceae is Oxidase negative bacteria .

8. Reagent • oxidase test reagent or test strip (tetramethyl-p-phenylenediaminedihydrochloride or an Oxidase Disk, p aminodimethylaniline)

9. Procedures • Pick a colony for testing using a sterilewooden toothpick. • Transfer the colony to the surface of one of the four grid areas on the Oxidase Test slide. • The test slide surface is impregnated with the oxidase reagent,This reagent causes a dark purple color to appear in the presence ofcytochromeoxidase. • Observe the color change. The reaction color will change from pink to maroon to dark purple. • Read the test results within 20 seconds. • An alternate procedure is performed by placing some oxidase reagent directly on the colony on the agar.

10. Notes • Pick your inoculum, not with a metal loop (reagent may react with the metal :this will cause false positives), but with a wooden stick. • We keep the Oxidase reagent either frozen or unopened intubes until needed. If old reagent is sitting out on the bench and is Purple, ask for a new tube from the instructor • Use a young culture, preferably less than 24 hrs old. • Use fresh reagent, less than a couple of hours old (it is taken out of the freezer). • Read the reaction within 20 seconds (NOT after), usually it will change in less than 15 seconds. The oxygen will change the reagent color as time passes, so it must be read quickly.