Citrate Utilization Islamic University_Gaza Medical Technology Department Citrate Utilization

Introduction Tests for the ability of bacteria to convert citrate (an intermediate of the Krebs cycle) into oxaloacetate (another intermediate of the Krebs cycle). In this media, citrate is the only carbon source available to the bacteria.

Principle Of Citrate Test To determine if an organism is capable of utilizing citrate as sole carbon source for metabolism with resulting alkalinity. If organisms utilize citrate and ammonium salts, the medium becomes more basic and the bromthymol blue changes from green to blue as a result of an increase in the pH of the media. If it can not use citrate then it will not grow.

Significance Of Citrate Test A differential cultural test to identify genera within the bacterial family Enterobacteriaceae that are able to utilize sodium citrate as a sole source of carbon.

Materials Simmons Citrate agar which contains : sodium citrate (carbon source) . ammonium salts (nitrogen source). Bromthymol blue indicator. Inoculating needle .

Method Streak one organism over the surface of the agar slant, then stab the butt. Incubate the tube at37°C for 48 hours .

Result 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 Some intestinal bacteria are (+)citrate as: Klebsiella . Citrobacter. Enterbacter aerogenes .  while others is(-) citrate as: E. coli

Summary Klebsiella Enterbacter aerogenes Citrobacter E. coli (+) (-)

Oxidase Production

Oxidase Test This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram-negative bacteria. It is a hallmark test for the Neiserria . It is also used to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other Enterobacteriaciae Cytochrome oxidase, part of an intracellular system that perform the final step in electron transport, reducing oxygen to water.

PROCEDURES Pick a colony for testing using a sterile wooden 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 reagent tetramethyl-p-phenylenediamine dihydrochloride. This reagent causes a dark purple color to appear in the presence of cytochrome oxidase. Observe the color change. The reaction color will change from pink to maroon to dark purple. Read the test results within 20 seconds. Some organisms may show slight positive reactions after this period and such results are NOT considered definitive.

Remark Don't use an Iron inoculating loop to transfer cells; this will cause false positives.

Result

Result

Significance The oxidase test is negative for all the Enterobacteriaceae. It is positive for the oxidative non-fermenters, Pseudomonas and Burkholderia, Neisseria, Hemophilus and Moraxella,Vibrio, Campylobacter, and Helicobacter are other oxidase-positive bacteria.

Significance