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Metabolism of Carbohydrates and Organic Acids
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EXERCISE 13METABOLISM OF CARBOHYDRATES AND ORGANIC ACIDS
A. OXIDATION AND/OR FERMENTATION OF SUGARS
PURPOSE OF THE TEST:to differentiate bacteria based on
their ability to oxidize or ferment specific sugars
A. OXIDATION AND/OR FERMENTATION OF SUGARS
SUGARS USED IN THE EXERCISE:Glucose, sucrose, mannitol,
lactoseMedium used:
HUGH AND LEIFSON MEDIUM
Bromthymol blue- pH indicator
A. OXIDATION AND/OR FERMENTATION OF SUGARS
RESULTS:
A. OXIDATION AND/OR FERMENTATION OF SUGARS
Fermentative results:
Positive result:
-production of acid from
both tubes sealed and
unsealed with water agar
A. OXIDATION AND/OR FERMENTATION OF SUGARS
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.
A. OXIDATION AND/OR FERMENTATION OF SUGARS
Negative results:
-Organisms that are not able to
metabolize the sugar will either
produce no color change or turn
the medium blue because of
alkaline products from amino acid
degradation.
B. METHYL RED AND VOGES-PROSKAUER TEST
METHYL RED TEST Identifies bacterial ability to produce stable
acid end products by means of a mixed-acid fermentation of glucose
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
B. METHYL RED AND VOGES-PROSKAUER TEST
Positive result:
In addition of methyl red, the medium turns red which indicates that the organism is a mixed acid fermenter.
They produce the enzyme formic hydrogenase, which splits formic acid into equal parts of CO2 and H2
B. METHYL RED AND VOGES-PROSKAUER TEST
RESULTS:Red color is the only true
indication of a positive result. Yellow is negative
B. METHYL RED AND VOGES-PROSKAUER TEST
VOGES-PROSKAUER TEST
identifies organisms able to produce acetoin from the degradation of glucose during a 2,3-butanediol fermentation
Adding VP reagents to the medium after incubation oxidizes the acetoin (if present) to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color
B. METHYL RED AND VOGES-PROSKAUER TEST
B. METHYL RED AND VOGES-PROSKAUER TEST
RESULTS:
A positive VP result, therefore,
is red.
No color change (or development
of copper color) after the addition of
reagents is negative. The copper
color is a result of interactions
between the reagents and should
not be confused with the true
red color of a positive result.
C. CITRIC ACID UTILIZATION
used to determine the ability of an organism to use citrate as its sole source of carbon
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.
At pH 7.0 and below, lactate and acetoin are also produced.
C. CITRIC ACID UTILIZATION
C. CITRIC ACID UTILIZATION
RESULTS:
conversion of the medium
to blue is a positive citrate test result.
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.
E. STARCH HYDROLYSIS TEST
Starch is a polysaccharide made up of α-D-glucose subunits.
Organisms that produce and secrete the extracellular enzymes α-amylase and oligo-1,6-glucosidase are able to hydrolyze starch by breaking the glycosidic linkages between the sugar subunits.
Reagent iodine is used to detect the presence or absence of starch.
E. STARCH HYDROLYSIS TEST
RESULTS:
Iodine reacts with starch and produces a blue or dark brown color; therefore, any microbial starch hydrolysis will be revealed as a clear zone surrounding the growth.
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 orange-to-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.