Exercise 13

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


SUGARS USED IN THE EXERCISE:Glucose, sucrose, mannitol,

lactoseMedium used:

HUGH AND LEIFSON MEDIUM

Bromthymol blue- pH indicator


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.


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


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


RESULTS:Red color is the only true

indication of a positive result. Yellow is negative


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


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.


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.


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.


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.

Exercise 13

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