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PURPOSE OF THE TEST: to differentiate bacteria based on their ability to oxidize or ferment specific sugars
RESULTS:
Fermentative results: Positive result: -production of acid from both tubes sealed and unsealed with water agar
Oxidative results -Organisms that are able to oxidize only will turn the unsealed medium yellow(or partially yellow) and leave the sealed medium green or blue.
Genera of bacteria (e.g. Escherichia, Salmonella, Proteus, and Aeromonas ferment glucose to promote large amount of acids (e.g. lactic, acetic, succinic, and formic acids) plus CO2, H2, and ethanol.
Accumulation of these acids lowers the pH of the medium
They produce the enzyme formic hydrogenase, which splits formic acid into equal parts of CO2 and H2
Citrate is generated by many bacteria. Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO2.
Under alkaline conditions, pyruvate is metabolized to acetate and formate.
RESULTS:
D. MALONATE UTILIZATION
used to determine whether an organism can use malonate as its sole source of carbon, with resulting alkalinity
Malonate (malonic acid), which can be added to growth media, is similar enough to succinate to replace it as the substrate in the reaction. This competitive inhibition of succinate dehydrogenase, in combination with the subsequent build up of succinate in the cell, shuts down the Krebs cycle and will kill the organism unless it can ferment or utilize malonate as its sole remaining carbon source.
D. MALONATE UTILIZATION
D. MALONATE UTILIZATION
RESULTS: If the organism utilizes malonate, it will alkalinize the medium and change the indicator from green to deep blue. Deep blue is positive. If an organism cannot utilize malonate but manages to ferment a small amount of glucose, it may turn the medium slightly yellow or produce no color change at all. These are negative results.
F. 2-KETOGLUCONATE PRODUCTION
used for testing the ability of an organism to oxidize gluconate to 2-ketogluconate The basis of the test is the change from gluconate (a nonreducing compound) to 2-ketogluconate (a reducing compound) when tested with a suitable reagent
F. 2-KETOGLUCONATE PRODUCTION
RESULTS:
Organisms capable of oxidative metabolism use potassium gluconate as their sole carbon source, leading to the accumulation of 2-ketogluconate in the medium. 2-ketogluconate reduces copper sulphate, when heated, to an insoluble cuprous oxide, which is precipitated out as yellow to orangeto-orange red precipitate. The colour produced depends on the amount of 2- ketogluconate accumulated, the greater the amount, the more orange-to-orange red the colour becomes.
ANSWERS TO QUESTIONS:
1. An enzyme capable of utilizing a certain carbohydrate should be possessed by a microorganism for it to have a positive result. Some organisms which appear negative on certain tests on utilization of carbohydrates simply means they do not possess the enzyme needed for the certain carbohydrate to be utilized.
ANSWERS TO QUESTIONS:
2. The importance of vaspar or water agar to one of the inoculated tubes of Hugh and Leifson medium is to promote anaerobic growth and fermentation.
ANSWERS TO QUESTIONS:
3. The MR test is designed to detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH.
The Voges-Proskauer test was designed for organism that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol.
Reference:
Leboffe,M.J. and Pierce, B.E. 2011. A Photographic Atlas for the Microbiology Laboratory. 4th ed. USA: Morton Publishing Company.