Aims not. Gram-negative bacteria are stain pink, due to

Aims of the

Perform a variety of biochemical tests used in

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Explain how these tests work and what you are

Identify your unknown organism with



Bacteria are resilient prokaryotic microorganism which can survive and
reproduce asexual in varying conditions. Bacteria can be beneficial and
harmless to humans, such as gut flora but several are pathogenic. Pathogenic
bacteria cause disease which can be fatal to humans. So, identifying the right micro-organisms
is important when it comes to treating the diseases. 2

It is important to classify and identify bacteria to distinguish one specie
from another, grouping similar organisms together for the ease of future
identification. Bacteria are classified by species. Within a species of
bacteria, there will be different strains and subgroups which vary from each
other by the disease they cause or difference in phenotypes.  DNA relatedness can be used to group strains
on their genetics. 2

Species can be identified by morphological traits and biochemical
tests. We will be using a variety of tests to differentiate and identify an
unknown bacterium.

Gram Stain is a method used to differentiate
gram-positive and gram-negative bacteria.  Gram-positive are stained violet as they retain
the crystal violet dye whereas gram-negative do not.  Gram-negative bacteria are stain pink, due to
the counterstain (safranin). Gram positive bacteria have a lard peptidoglycan layer
in their cell wall whereas gram-negative bacteria only contain a small layer
which dissolves in alcohol.

Catalase test is used to test if a bacterium
possesses catalase. Hydrogen peroxide added to bacterium, if bubbles are
produced then the organism is catalase-positive if no bubbles are produced then
the organism is catalase-negative.

Oxidase test is used to identify bacteria that
produce cytochrome c oxidase, as enzyme part of the electron transport chain.

Oxidase-positive bacteria contain cytochrome c oxidase, using aerobic
respiration to produce energy. Whereas oxidase-negative bacteria do not contain
the enzyme and uses anaerobic or another cytochrome to transfer electrons to
produce energy.

Coagulase test used to differentiate Staphylococcus aureus (positive) from Coagulase
Negative Staphylococcus. Coagulase is a bacterial enzyme that coagulates blood
or plasma, by converting fibrinogen to fibrin. Coagulase positive bacteria will
cause agglutination whereas with coagulase negative bacteria there will be no agglutination.

Motility Test is used to see if the bacterium can move by using their
flagella. Positive results indicate bacteria can propel themselves through
liquids or medians by their flagella.

Phenol Glucose Test, used to see if the micro-organism uses glucose as
its energy source. If glucose is used by the bacteria, acidic by-products will accumulate
giving a positive result, there will be a colour change in the medium from red
to yellow. Negative results, colour change from red to pink due to alkaline
by-products. Also, gasses produced by the bacteria will be trapped in the
durham tubes.

Urease test uses a urease agar, differential
medium, testing if bacteria produces urease (hydrolyses urea to ammonia and
carbon dioxide). Positive result will change the colour of the agar plate to
pink, with a negative result there will be no colour change.

Methyl red test used to identify bacteria
producing acids by fermentation of glucose. Positive result is indicated when
MR-VP broth turns red, negative result indicated when MR-VP broth stays yellow.

Apart from biochemical tests, bacteria can be differentiated and
identified in other ways. Pathogenic strains can be identified by the presence
of specific plasmid and their plasmid profile. Modern molecular biologically
techniques have arisen, which enable the identification of species and strains
by identifying specific gene or genetic sequences.

Differentiating and identifying bacteria is important. Bacteria
vary and are so different from one another. Some are pathogenic and may cause a
disease. Being able to differentiate and identify bacteria makes it easier to
treat such bacteria. They have different characteristics and the same course of
treatment for one bacteria may be redundant on another species.


Gram stain, spread a drop of water onto a microscope slide. Using a
sterile loop take a sample of bacteria from a plate and emulsify in the water.

Let it dry at room temperature. Heat-fix the bacteria by passing the glass
slide thrice over a bunsen flame. Then lay slide on a staining rack and pipette
crystal violet solution over the slide, leave for a minute. Wash with water and
drain the excess. Pipette iodine solution over the slide, leave for a minute
and wash smear with water. Drain excess water. Add acetone-alcohol to
decolourise, allow solvent to run over the smear until it flow colourlessly.

Wash with water and drain the excess. Add safranin to the glass slide to
counterstain, leave for a minute. Wash with water and drain the excess. Examine
the glass slide under oil-immersion.

Catalase Test, used to test for catalase. Using a sterile loop, take a
sample of a single colony and smear onto a microscope slide. Add a drop of
hydrogen peroxide, observe and record the result.

Oxidase Test, place an oxidase detection strip onto a colony. Leave
for five seconds and observe. Coagulase Test, with a sterile loop take a sample
of 1 to 3 colonies and place onto a circle on the test card. Then add a drop of
test reagent, mix the contents and look for agglutination.

Motility test, dip a culture needle into broth culture and stab to
about 75% of the agar. Carefully remove the needle. Phenol glucose test, dip a
sterile loop into the broth and dip into the phenol glucose test tube. Methyl
red test, dip a sterile loop into the broth and dip into the MR-VP broth.


Gram Stain





Phenol Glucose


Methyl red










AG- Acidic and gas produced.

From the biochemical tests, I can conclude that the organism we experimented
on was PROTEUS VULGARIS. The gram
stain result was negative, proteus
vulgaris belongs to a genus of gram-negative class.  Catalase test was positive, P.Vulgaris does contain catalase.

Coagulase test was negative as its located mainly in the gut. Motility test was
positive, so P.Vulgaris uses its
flagella to move. Phenol Glucose test was acidic and gases wore present, P.Vulgaris uses glucose as its primary
source of sugar. Ureases test was positive as agar turned pink, P.Vulgaris produces urease. Methyl red
test was positive, MR-VP broth turned red as P.Vulgaris uses glucose fermentation. The oxidase test was
positive, but P.Vulgaris is supposed
to give a negative result. Not sure what happened as it’s a pretty straight
forward procedure to conduct. The organism is a small rod shaped bacteria.