Download Identification of GNB – IMViC Tests

Biochemical Activities of
Bacteria
Bacteria accomplish their various biochemical activities
(growth and multiplication) using raw materials (nutrients)
obtained from the environment. The biochemical
transformations that occur both inside and outside of bacteria
are governed by biological catalysts called enzymes.
Fermentations
are
energy-producing
biochemical
reactions in which organic molecules serve both as electron
acceptors and donors. The ability of microorganisms to
ferment carbohydrates and the types of products formed are
very useful in identification.

For example, if fermenting bacteria are grown in a liquid
culture medium containing the carbohydrate glucose, they
may produce organic acids as by-products of the
fermentation. These acids are released into the medium and
lower its pH. If a pH indicator such as phenol red or
bromcresol purple is included in the medium, the acid
production will change the medium from its original color
to yellow
Gram Negative Bacilli-Enterobacteriacae
Oxidase test :
Used to distinguish fermenters (Oxidase negative) from non-fermenters(Oxidase
positive).
Testing for the presence of the enzyme indophenol oxidase.
The reagent (Tetramethyl-para-phenylenediamine) will be oxidized in the
presence of oxygen by the enzyme indophenol oxidase producing a dark-purple
product called indophenol.
Production of a dark purple product (within 10 – 30 seconds) is a positive ( + )
test for oxidase.
Urease test :
Testing for the presence of the enzyme
urease.
This enzyme will break down urea to
ammonia and carbon dioxide.
CO(NH2)2

NH3
+ CO2
The ammonia produced increases the pH of
the media (becomes more alkaline) and
causes the indicator present in the media
(Phenol red) to change from a yellow-orange
color to a hot pink color.
A hot-pink color is an indication of a
positive test for Urease.
Triple Sugar Iron (TSI) Test
Test conditions:
 Provides information about sugar fermentation and the possible by-products produced by
the organisms tested.
 Three sugars are detect, glucose, lactose, and sucrose are present in the media at different
concentrations (Glucose at 0.1%, Lactose at 1.0%, and Sucrose at 1.0%).
 Fermentation is an anaerobic process.
 When sugar is fermented there may be an acid end-product produced.
 Phenol red is again used in the media as a pH indicator and will change from red to
yellow under acidic conditions.
 Some organisms also may produce hydrogen gas (H2) as a by-product of fermentation.
 Also present in the media is ferrous sulfate, which will react with any hydrogen sulfide
(H2S) produced as a another possible by-product to form a black precipitate (ferrous
sulfide). H2S production is an anaerobic process so the black precipitate produced will
only appear in the butt of the tube.
Procedure:
Work in pairs
You will be testing three organisms:
1.Pseudomonas aeruginosa
2.Escherichia coli
3.Proteus vulgaris
You will need one TSI agar slant for each organism/group (3 tubes).
The tests require anaerobic conditions, therefore the organisms must be
stabbed into the agar media. For this reason you will use sterile inoculating
needles (not loops) for inoculating the tubes.
The best method is to obtain one of the organisms, preferably on a nutrient
agar plate, and roll the tip of a sterile inoculating needle in the organism.
Next stab the agar slant nearly to the bottom of the agar tube and when
removing the inoculating needle, lightly streak the surface of the slant.
Follow the same procedure for each organism tested.
The tubes are placed in the incubator for 18 - 24 hours at 37 C.
Interpretation of Results:
 Many of the enteric organisms will ferment glucose with the production of acids which will change
the color of the medium in the butt and along the slant from red to yellow because of a reduction in
the pH (within the first few hours).
 However, since the glucose is present in small amounts (0.1%), the supply is soon exhausted and the
organisms growing on the surface of the slant in the presence of oxygen are forced to catabolize
peptones and amino acids in the media for their energy supply. Alkaline end-products ( NH4OH) are
produced from these substances which revert the pH of the slant to an alkaline pH and thus change
the color of the agar slant back to red (after 18-24 hours).
 Organisms such as Salmonella spp. or Shigella spp. and other organisms which attack glucose but
do not ferment lactose or sucrose will produce an alkaline slant and acid butt in TSI slants in 18 to
24 hours. Since metabolism is progressing at a slower rate in the butt, this reversion does not usually
take place in the butt until 48 hours or longer.
 If the glucose is metabolized to CO2, the gas will be seen as bubbles or cracks in the agar butt. If
hydrogen sulfide is formed during growth, a gray or black streak of iron sulfide is seen originating
where the inoculating needle entered and throughout the agar butt.
 Organisms which attack lactose and/or sucrose, such as Escherichia, will produce acid slants and
acid butts usually with the formation of gas. In these cases, the acid slants do not revert to an
alkaline status because lactose (1%) and sucrose (1%) are being fermented and are present in
concentrations ten times that of glucose.
 Some organisms (e.g., Pseudomonas, Acinetobacter) fail to ferment even glucose, and because they
are strictly aerobic, they fail to grow in the butt of the tube. In these cases, the butt will be
unchanged in color, and the slant either alkaline or unchanged.
TSI Test Results
Control
Pseudomonas aeruginosa
Shigella
Salmonella enteritidis
Eschericihia
Proteus vulgaris
Klebsiella
Enterobacter
Recording the Results of the TSI Tests
K = alkaline = Red; A = acid = Yellow; NC = No change; G = gas produced;
H2S = hydrogen sulfide produced
Acid or alkaline results in the slant are reported first, followed by the butt results (e.g., K/A would be read as "K over
A" or "alkaline over acid" and refers to an alkaline slant and acid butt).
Slant result / Butt result
K/A
A/A
K/K
K/NC
NC/NC
A/AG
A/A + H2S
A/AG + H2S
K/AG
K/A + H2S
K/AG + H2S
Only Glucose fermented; peptone utilized

Glucose and Lactose/Sucrose fermented

No sugars fermented, only peptone utilized

No sugars fermented, Peptone used aerobically only

No growth, neither sugars or peptone used

Glucose and Lactose/Sucrose fermented, gas produced

Glucose and Lactose/Sucrose fermented, H2S produced

All sugars fermented and both H2S and gas produced

Only Glucose fermented; peptone utilized, gas produced

Only Glucose fermented; peptone utilized, H2S produced

Only glucose fermented; Peptone utilized, both gas and H2S
produced
Motility Test


To test for bacterial motility a diluted agar
is used. Normally the agar concentration is
1.5 g of agar per mL of nutrient media
broth. Motility agar is usually 0.7 g agar
per mL of nutrient broth.
When the lower concentration agar
solidifies it is less gel-like and more fluid
allowing bacteria with flagella to move
into the surrounding media. Additionally,
a special reagent, Triphenyltetrazonium
chloride, or TTC, is used to make the
motility more visual in the tube. TTC is
colorless and soluble in it’s oxidized form,
but becomes insoluble and dark red in
color when in it’s reduced state. Any
metabolic process, such as the expenditure
of energy for flagellar movement, will
reduce the TTC and cause the appearance
of the red, reduced TTC in areas where
motile bacteria are present.
Identification of GNB – IMViC Tests
Mnemonic for the four Biochemical tests performed on the Gram Negative Bacilli
being studied
1.
2.
3.
4.
Indole test
Methyl Red test
Voges-Proskauer test
Citrate utilization test
These tests divide the Enterobacteriaceae into two major groups:
1. Escherichia coli group
2. Enterobacter-Klebsiella group
Indole test:
Tests for the production and secretion of the enzyme tryptophanase.
If the organism can produce tryptophanase and break down tryptophan to Indole the
Indole will react with Kovac’s reagent (p-dimethylaminobenzaldehyde) forming a pinkcolored chemical complex.
We use commercially prepared slides impregnated with Kovac’s reagent
Methyl Red Test
Some organisms produce acid from the metabolism of glucose (fermentation) in a
sufficient quantity to alter the pH of the media to about 4.4. These are stable acids
and are not further metabolized.
Methyl red indicator is used to detect the presence of these acids in the MRVP
broth medium. Methyl red indicator at this pH (~ 4.4) changes to a bright cherry
red color.
Voges-Proskauer Test

Some microorganisms produce organic acids from glucose metabolism, but
further metabolize the acid produced to various neutral end products, like
acetoin, and 2,3-butanediol.

There is an initial pH drop in the MRVP broth, but the neutral end product
raise the pH so that the methyl red test will be negative.
The presence of acetoin, and 2,3-butanediol is tested for using a-naphthol
which reacts with these two neutral products to produce a mahogany red color.


Citrate Test
Citrate may be used as a carbon source by some microorganisms.

The Citrate test uses an agar medium with citrate and the pH indicator
Bromothymol blue present. At the pH of the un-inoculated medium the color is
Blue-green.

If the organism can utilize Citrate as a carbon source the breakdown of citrate
releases bicarbonate ions (HCO3-) into the medium. The bicarbonate ions raise
the pH of the medium above 7.4 pH. This causes the Bromothymol blue
indicator to turn dark blue in color.
Catalase test
Principles
 Some bacteria contain flavoproteins that reduce O2, resulting in the production of
hydrogen peroxide (H2O2) or superoxide (O2 –). These are extremely toxic
because they are powerful oxidizing agents and destroy cellular constituents very
rapidly. A bacterium must be able to protect itself against such O2 products or it will
be killed. Many bacteria possess enzymes that afford protection against toxic O2
products. Obligate aerobes and facultative anaerobes usually contain the enzymes
superoxide dismutase, which catalyzes the destruction of superoxide, and either
catalase or peroxidase, which catalyze the destruction of hydrogen peroxide as
follows:


Most strict anaerobes lack both enzymes and therefore cannot
tolerate O2.
Catalase production and activity can be detected by adding the
substrate H2O2 to an appropriately incubated (18- to 24-hour)
tryptic soy agar slant culture. If catalase was produced by the
bacteria, the above chemical reaction will liberate free O2 gas.
Bubbles of O2 represent a positive catalase test; the absence of
bubble formation is a negative catalase test. Catalase activity is
very useful in differentiating between groups of bacteria. For
example, the morphologically similar Enterococcus (catalase
negative) and Staphylococcus (catalase positive) can be
differentiated using the catalase test
Coagulase test


Principles
Coagulases are enzymes that clot blood plasma by a mechanism
that is similar to normal clotting. Although coagulase activity is not
required for pathogenicity, this enzyme is a good indicator of the
pathogenic potential of S. aureus. Coagulase-producing staphylococci
(termed coagulase positive) form a fibrin clot around themselves
and avoid attack by the host’s defenses. In the coagulase test,
coagulase positive staphylococci will cause the plasma to clot by
using coagulase to initiate the clotting cascade. Citrate and EDTA are
usually added to act as anticoagulants and prevent false-positive
results. Cultures should be considered coagulase negative if they are
unclotted after 4 hours.
Oxidase test

Principles

Oxidase enzymes play an important role in the operation of the electron
transport system during aerobic respiration. Cytochrome oxidase (aa3 type)
uses O2 as an electron acceptor during the oxidation of reduced cytochrome
c to form water and oxidized cytochrome c. The ability of bacteria to
produce cytochrome oxidase can be determined by the addition of the
oxidase test reagent or test strip (an Oxidase Disk) to colonies that have
grown on a plate medium. Or, using a wooden applicator stick, a bacterial
sample can either be rubbed on a Dry Slide Oxidase reaction area, on a KEY
test strip, or filter paper moistened with the oxidase reagent. The light pink
oxidase test reagent (Disk, strip, or Slide) serves as an artificial substrate,
donating electrons to cytochrome oxidase and in the process becoming
oxidized to a purple and then dark purple compound in the presence of free
O2 and the oxidase. The presence of this dark purple coloration represents a
positive test. No color change or a light pink coloration on the colonies
indicates the absence of oxidase and is a negative test.