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Carbohydrate Fermentation
The identification of some bacteria is aided by determining what nutrients the
bacteria can utilize and what end products will be produced in the process.
These characteristics are controlled by the enzymes which the bacteria
produce. Because the type of enzyme(s) bacteria produce is genetically
controlled, the pattern of sugars fermented may be unique to a particular
species or strain. Fermentation products are usually acid (lactic acid, acetic
acid etc.), neutral (ethyl alcohol etc.), or gases (carbon dioxide, hyrogen,
etc.).
To determine the products of sugar fermentation, a carbohydrate
fermentation broth is prepared at pH 7.4. This broth contains 3 essential
ingredients: 0.5%-1.0% of the carbohyrate to be tested (e.g. lactose or
glucose), nutrient broth, and the pH indicator phenol red. The nutrient broth,
which is a light red color, supports the growth of most organisms whether
they are able to ferment the sugar or not.
The test organism is inoculated into a broth containing the test sugar and
incubated. A bright yellow color indicates the production of enough acid
products from fermentation of the sugar to drop the pH to 6.9 or less.
Production of gas is determined with a Durham tube
, a small inverted vial filled with the carbohydrate fermentation broth. If gas
is produced during fermentation of the sugar, it is trapped at the top of the
Durham tube and appears as a bubble. Slow fermenters may take a week or
more to cause color changes detectable by the human eye. Positive (yellow
color or yellow color with gas bubble) and negative results (red color, no gas
bubble)
Catalase Test
Catalase is an enzyme that converts hydrogen peroxide into water and
oxygen. The presence of catalase can be easily detected by the slide method.
A drop of 3 percent hydrogen peroxide is put on a slide and the bacteria is
emulsified in the drop. The presence of bubbles is evidence of the production
of oxygen. Alternatively, bacteria may be tested by dropping hydrogen
peroxide directly onto colonies growing on a plate. However, colonies
growing on blood agar should not be tested in this way because blood cells
contain catalase and would cause a false positive reaction. Staphylococcus,
Micrococcus and most aerobic organisms produce catalase, while
Streptococcus and most anaerobic bacteria are unable to produce it.
Oxidase
The identification of some bacteria is aided by detecting their ability to
produce the enzyme cytochrome c oxidase (more commonly referred to
simply as "oxidase"). Pseudomonas aeruginosa, Neisseria gonorrhoeae and
Campylobacter jejuni are oxidase-positive pathogens frequently encountered.
During cellular respiration, electrons are transferred through a series of
oxidation-reduction reactions to a terminal electron acceptor such as oxygen.
The terminal link in the electron transport chain is cytochrome oxidase, an
enzyme that mediates the transfer of electrons from the reduced cytochrome
c to molecular oxygen
Oxidase positive organisms are detected by the use of oxidase reagent
(N,N,N',N'-tetramethyl-p-phenylenediamine
dihydrochloride).
Oxidase
reagent is colorless in its reduced state and dark purple in its oxidized state.
Applying oxidase reagent directly to an oxidase-positive colony will cause it
to change color. Commercial products such as swabs or plastic slides are also
available that contain oxidase reagent and will turn purple when a heavy
inoculum of an oxidase-positive organism adheres to the product. The
images below show a negative (no color change) and positive (dark purple)
oxidase reaction using Difco's DrySlide oxidase test. The color change occurs
within 20 seconds if the test bacteria is oxidase positive.
The oxidase test determines whether a microbe can oxidize certain aromatic
amines, for example, p -aminodimethylaniline, to form colored end products.
This oxidation correlates with the cytochrome oxidase activity of some
bacteria, including the genera Pseudomonas and Neisseria. While a positive
oxidase test is important in the identification of these genera, the test is also
useful in characterizing the enteric bacteria ( Enterobacteriaceae), which are
oxidase-negative.
IMViC
Enterobacteriaeae (enterics) are Gram-negative bacteria that grow in the
intestinal tract of humans and other animals. The IMViC tests are frequently
employed for identification of this group of microbes which includes such
organisms as Klebsiella, Enterobacter, and Escherichia coli. The presence of
E. coli is used by public health officials as an indicator of fecal contamination
of food and water supplies. While Enterobacter and Klebsiella resemble E.coli
in being lactose fermenters, their presence does not necessarily indicate fecal
contamination because they are widespread in soil and grass. The IMViC
tests can be used to differentiate these three organisms.
IMViC is an acronym that stands for indole, methyl red, Voges-Proskauer,
and citrate. To obtain the results of these four tests, three test tubes are
inoculated: tryptone broth (indole test), methyl red - Voges Proskauer broth
(MR-VP broth), and citrate.
Indole Test
The test organism is inoculated into tryptone broth, a rich source of the
amino acid tryptophan. Indole positive bacteria such as Escherichia coli
produce tryptophanase, an enzyme that cleaves tryptophan, producing indole
and other products. When Kovac's reagent (p-dimethylaminobenzaldehyde)
is added to a broth with indole in it, a dark pink color develops. The indole
test must be read by 48 hours of incubation because the indole can be
further degraded if prolonged incubation occurs. The acidic pH produced by
Escherichia coli limits its growth.
The Methyl Red and Voges-Proskauer Tests
The methyl red (MR) and Voges-Proskauer (VP) tests are read from a single
inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP
broth is split into two tubes. One tube is used for the MR test; the other is
used for the VP test.
MR-VP media contains glucose and peptone. All enterics oxidize glucose for
energy; however the end products vary depending on bacterial enzymes.
Both the MR and VP tests are used to determine what end products result
when the test organism degrades glucose. E. coli is one of the bacteria that
produces acids, causing the pH to drop below 4.4. When the pH indicator
methyl red is added to this acidic broth it will be cherry red (a positive MR
test).
Klebsiella and Enterobacter produce more neutral products from glucose (e.g.
ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the
bacteria is not inhibited. The bacteria thus begin to attack the peptone in the
broth, causing the pH to rise above 6.2. At this pH, methyl red indicator is a
yellow color (a negative MR test).
The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's
B (potassium hydroxide). When these reagents are added to a broth in which
acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive
VP test). This color may take 20 to 30 minutes to develop. E. coli does not
produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.
The Citrate Test
The citrate test utilizes Simmon's citrate media to determine if a bacterium
can grow utilizing citrate as its sole carbon and energy source. Simmon's
media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6.
Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes
to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate
agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria
in the media leads to development of a Prussian blue color (positive citrate).
Enterobacter, Klebsiella, Salmonella, Citrobacter and Providencia are citrate
positive while Shigella and Morganella is negative.
Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and
Klebsiella give the reverse: --++
Urea
Some bacteria produce urease, an enzyme that hydrolyzes urea, a common
metabolic waste product of vertebrates that contains nitrogen and is excreted
in the urine. Urease splits urea into ammonia and carbon dioxide, making the
two products available for bacterial use. The test for urease production relies
on the fact that the ammonia produced upon hydroysis is alkaline.
The test organism is inoculated into a urea broth that contains phenol red, a
pH indicator, and has a pH of 6.8. At this pH phenol red is salmon color.
However, when the pH rises above 8.1 phenol red turns a cerise (hot pink)
color. Organisms that produce urease will turn cerise due to the ammonia
produced upon hydrolysis of urea (a positive result). Organisms that are
unable to synthesize urease will not produce ammonia and thus will not
experience the subsequent rise in pH. Thus a negative test is indicated by the
continuance of a salmon color in the urea broth. The urease test is useful for
differentiating Salmonella, which is urea negative, from Proteus, which is
urea positive.
Hydrogen sulfide production
Many proteins are rich in sulfur-containing amino acids such as cysteine.
When these proteins are hydrolyzed by some bacteria, the amino acids are
released and taken up as nutrients. Cysteine, in the presence of the enzyme
cysteine desulfurase, loses its sulfur atom through the addition of
hydrogen from water to form hydrogen sulfide gas
Gaseous hydrogen sulfide may also be produced by the reduction of inorganic
sulfur-containing compounds such as thiosulfate (S O
sulfite (SO
3
2-
2
3
2-
), sulfate (SO
2-
4
) or
). For example, when certain bacteria take up sodium
thiosulfate, they can reduce it to sulfite using the enzyme thiosulfate
reductase with the release of hydrogen sulfide gas Such a reduction occurs
during anaerobic respiration in which respiring cells use something other
than oxygen (such as thiosulfate) as the final electron acceptor in the
respiratory electron transport chain.
In this exercise, the SIM medium (named after J.S. Simmons in 1926)
contains peptones and sodium thiosulfate as substrates, and ferrous
ammonium sulfate, Fe(NH )SO , as the H S indicator. Cysteine is a
4
4
2
component of the peptones used in SIM medium. Sufficient agar (about
0.4% instead of the usual 1.5%) is present to make the medium semisolid.
Once H S is produced, it combines with the ferrous ammonium sulfate
2
(ferrous sulfate will also work), forming an insoluble, black ferrous sulfide
precipitate that can be seen along the line of the stab inoculation
Lysine decarboxylase
The decarboxylase tests are used primarily to aid in the identifi cation
of organisms within the family Enterobacteriaceae, but may be used to
differentiate other gram-negative bacilli as well.
The decarboxylase test detects the enzymatic ability of an organism to
decarboxylate an amino acid to form an amine, which results in alkaline byproducts in the media and a pH change.7 Bacteria that possess specifi c
decarboxylase enzymes are capable of attacking amino acids, yielding an
amine, or diamine, and carbon dioxide.
Decarboxylases are induced enzymes and are formed only in an acid
environment and in the presence of a specifi c substrate containing the
amino acid. In the test, the organism is grown in a media containing a specifi
c amino acid and dextrose, and is overlaid with mineral oil to create an
anaerobic environment. All unbound oxygen is utilized by the organism in the
growth phase, and an acid environment is created due to the fermentation of
dextrose. The reduced pH induces the decarboxylase enzyme if present in
the organism. Decarboxylation takes place, and the pH shifts to the alkaline
range as amines and NH3 are produced. This causes the pH indicator to
change to a purple or gray-purple color. Organisms, which do not possess
the enzyme, will utilize the dextrose, dropping the pH to acid, changing the
indicator to yellow.
The breakdown of arginine is a two-step process involving two enzyme
systems. Arginine is fi rst broken down by a dihydrolase enzyme. An NH3
group is removed from arginine to form citrulline. Citrulline is broken down
further to ornithine, which is then decarboxylated by the other enzyme
system to the fi nal end products that result in an alkaline pH.
Positive and negative reactions in the lysine decarboxylation test. This test
involves two tubes. One containing lysine (Lysine Decarboxylase Broth - LDB)
and the other (Decarboxylase Control Broth - DCB), a control, having the
identical medium, but no lysine. DCB and LDB are both rich media
supplemented with glucose. Enterics will ferment the glucose causing an
acidic reaction. Enterics that can decarboxylate lysine will cause a net
alkaline reaction, and turn the medium purple. 1 - a non-enteric, note the
inability to ferment glucose in DCB. 2 - a positive reaction. DCB turns acidic
due to the fermentation of glucose, while LDB turns purple due to
carboxylation of lysine. 3 - a negative reaction. Both broths are yellow due to
fermentation of glucose, but lysine is not decarboxylated.
Phenylalanine Deamination
The enzyme phenylalanine deaminase catalyzes the removal of the amino
group (–NH ) from the amino acid phenylalanine
2
The resulting products include the organic acid phenylpyruvate, water and
ammonia. Certain enteric bacteria (e.g. Proteus, Morganella and Providencia)
can use the phenylpyruvate in biosynthesis reactions. In addition the
deamination detoxifies inhibitory amines.
The phenylalanine deaminase test can be used to differentiate among
enteric bacteria such as Escherichia coli and Proteus vulgaris. Proteus
vulgaris produces phenylalanine deaminase. When ferric chloride is added to
the medium it reacts with the phenylpyruvic acid reaction product, forming a
green compound. Since E. coli does not produce the enzyme, it cannot
deaminate phenylalanine. Therefore when ferric chloride is added to an E.
coli culture there is no color change.
Positive and negative in phenylalanine agar. Ec- E. coli showing a negative
reaction Mn- Morganella morgarii showing positive result
Phenylalanine Agar. Add about one-half dropperful of the FeCl3 solution. If
deamination of phenylalanine has taken place, the reagent will react with the
phenylpyruvic acid formed, and a dark green color will appear in the slant
region.