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Carbs
Carbs
All bacteria must utilize the energy sources in their environment in order to produce
ATP. ATP is required for all of the biosynthetic processes that bacteria use for their
maintenance and reproduction. Bacteria produce enzymes that allow them to oxidize
environmental energy sources; however, the energy sources that different bacteria use
depends on the specific enzymes that each bacteria produce.
Heterotrophic bacteria often use carbohydrates as energy sources. Many bacteria
use glucose, a monosaccharide or simple sugar, because many bacteria possess the enzymes
required for the degradation and oxidation of this sugar. Fewer bacteria are able to use
complex carbohydrates like disaccharides (lactose or sucrose) or polysaccharides (starch).
Disaccharides and polysaccharides are simple sugars that are linked by glycosidic bonds;
bacteria must produce enzymes to cleave these bonds such that the simple sugars that result
can be transported into the cell. If the bacteria cannot produce these enzymes then the
complex carbohydrate is not used. For example, lactose is a disaccharide consisting of
monomeric glucose and monomeric galactose linked by a glycosidic bond. Bacteria that
use lactose must first produce the enzyme lactase (beta-galactosidase) to break the
glycosidic bond between these monomers. Starch is a large polysaccharide consisting of
long chains of monomeric glucose linked by glycosidic bonds. Bacteria that use starch
produce an exoenzyme, alpha amylase, that break these bonds such that free monomeric
glucose is produced.
Each bacterium has its own collection of enzymes that enable it to use diverse
carbohydrates; this is often exploited in the identification of bacterial species. One can
determine if a given bacterial species can utilize a given carbohydrate by checking for the
presence of byproducts that are produced by the oxidation of these carbohydrates. To this
end, pH indicators may be added to the media to detect metabolic acids that have been
produced by bacteria after the oxidation and fermentation of sugars. (Phenol Red with
Durham tubes, Citrate agar slants) Alternatively, reagents may be added to media after the
bacteria have grown, these reagents react with specific byproducts or intermediates in a
metabolic pathway. (Methyl Red/Vogues-Proskauer test)
In this exercise you will inoculate glucose or lactose broth containing Durham
tubes, Methyl Red-Voges Proskauer media and Simmons citrate agar with selected
bacteria and observe these media for the evidence of carbohydrate utilization by these
bacteria.
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that the tubes are closed at the top and open at the bottom. When gas is produced in these
media a small bubble representing trapped gas can be found in the tube.
There are four possible reactions in glucose or lactose broths with Durham tubes:
2. No reaction (-) with growth: the broth remains red in color but the
medium becomes turbid or cloudy—the organism can grow in the media but is
not using the carbohydrates; these organisms are using the supplied peptone as
the energy source.
3. Acid production (A): the broth turns yellow in color--the organism is able
to use the carbohydrate and produces organic acids that lower the pH of the
medium.
4. Acid and Gas production (A/G): the broth turns yellow in color and a
small bubble is present in the inverted Durham tube—the organism is able to
use the carbohydrate and produced organic acids and gas in the process.
There are four possible reactions one can observe with the MR/VP tests
+/- -/+ +/+ -/-
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SIMMONS CITRATE AGAR: Simmons citrate agar is used to determine if bacteria are
able to utilize citrate as a sole carbon source. Citrate (citric acid) is an intermediate of the
Kreb’s cycle (citric acid cycle) used in respiratory metabolism. A pH indicator, brom
thymol blue is incorporated into the medium. Brom thymol blue is green at pH 7 and turns
blue at a pH above 7.6. Therefore, if the citrate (citric acid) has been used as a sole carbon
source, then the pH of the medium will increase imparting a blue color to the pH indicator.
There are two possible reactions that can be observed with the Simmons Citrate Agar
1. Citrate (+) –the medium turns blue—the bacteria have used citric acid as a
sole carbon source, thus raising the pH of the medium.
2. Citrate (-)—the medium remains green—the bacteria are not able to use
citric acid as a sole carbon source; the pH of the medium remains the same
Laboratory Period 1:
Glucose or Lactose broths with Durham tubes: Glucose or lactose utilization will be
tested on the following bacteria: Staphylococcus aureus, Bacillus subtilis, Enterobacter
aerogenes, Serratia marcescens, Shigella flexneri, Pseudomonas aeruginosa, Klebsiella
pneumoniae and Citrobacter freundii. Each student will be assigned ONE of these bacteria.
The student should aceptically transfer bacteria from an overnight culture into 1. the glucose
broth with a Durham tube and 2. the lactose broth with a Durham tube. These cultures will
be incubated for 24-48 hours at 37 degrees Celsius then held at 4oC and saved for analysis at
the next laboratory session.
Simmons citrate agar slants: Each student will be supplied with one slant containing
Simmons citrate agar and ONE of the following bacteria: Staphylococcus aureus, Bacillus
subtilis, Enterobacter aerogenes, Serratia marcescens, Shigella flexneri, Pseudomonas
aeruginosa, Klebsiella pneumoniae and Citrobacter freundii. Each student should streak the
bacteria onto the surface of the slant with a loop as shown by the instructor. These tubes will
also be incubated at 37 degrees Celsius for 24-48 hours; the tubes will be analyzed at the next
lab period.
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Laboratory Period 2: Analysis of results for carbohydrate utilization by bacteria.
Glucose or Lactose broths with Durham tubes: Use a + or – in the table below to indicate
whether the bacteria utilize glucose and/or lactose to produce acid and/or gas.
How do you know which organisms produced acid? Gas? How do these results compare to
a control tube—an un-inoculated tube?
Simmons Citrate Agar: Use + or – in the table below to indicate whether the bacteria utilize
citrate. How do you know which organism utilized the citrate? Why?
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MR test: Add 3 drops of methyl red to the appropriately labeled test tube and mix. A
color change should occur almost immediately.
VP test: Add 12 drops of alpha napthol and 6 drops of 40% KOH (potassium
hydroxide) to the appropriate test tube. Vortex the tubes and set aside. Vortex the
tubes every 3 minutes for up to 20 minutes. Use a + or – sign to indicate how the
organisms reacted with the reagents in each test.
Additional notes.