Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Lab-11
Bacterial Identification
Biochemical Tests: Part-I
First Semester 2020-2021

Instructors:
Dr. Rasha Khayyat, Dr. Alaeddin Abu-zant, Dr. Motasem Almasri, Dr. Walid Basha

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Introduction:
Many bacterial species may share some structural and nonstructural properties (such as Gram-
reaction, cell morphology and arrangement, motility…... )). Accordingly, it is not possible to
distinguish one bacterial species from another based on these common properties.

Indeed, identification of a particular bacterial species is based on identifying several properties that
will characterize and distinguish this bacterial species from other ones.
The properties that must be examine during the identification process of a particular bacterial species
may include:

A-Growth characteristic: such as colony characteristics, pigment production, culture odor, .. etc.

B-Structural properties such as:

1- Cell morphology and arrangements
2- Gram-reaction
3- Acid-fast reaction (if needed)
4- Presence or absence of certain structural component such as: capsule, endospore and flagella.

C- Physiological properties: motile versus non-motile.

D-Physical properties: growth in preferences of O2, pH, temperature or salt concentration.

E- Biochemical and enzymatic properties

During the previous labs, you learned the followings:
Types of bacterial culture media and how to prepare bacterial culture media.

How to culture a clinical or an environmental sample using the four quadrant streaking method to
obtain separate colonies and to subculture these colonies to obtain pure cultures.

How to indentify colony morphology and initial growth characteristics (some of the initial growth
characteristics you learned reflect some of the biochemical properties of the grown bacterium, which
can be concluded based on the used culture media such as fermentation of some sugars included in the
used culture media such as Lactose, Sucrose and Manitol and hemolysis of Red blood cells in blood
agar)
How to conduct simple staining and to identify bacterial cell morphology and arrangements.

How to conduct and identify the Gram-reaction and Acid-fast reaction.

How to identify the presence or absence of certain bacterial structural components such as the
capsule and the endospore.

During this lab and the next lab, you will be learning additional methods to identify more and
more properties (biochemical, enzymatic, physical) of some bacterial species and how to use the
properties in the identification process (diagnosis process) by either using bacterial identification
flow-charts (diagnostic charts) or bacterial identification (diagnostic) tables.
Biochemical Tests Used in Bacterial Identification

These tests are based on:

1- The detection of a particular enzyme. Such as Catalase, Coagulase and Oxidase.

2- The detection of complete metabolic pathway(s), which involves the utilization of one or more
enzyme(s). Examples:

A- Carbohydrate oxidation and/or fermentation

B- Amino acid degradation. Such as:

Tryptophan deaminase
 Decarboxylases (such as Lysine, Ornithine and Arginine decarboxylases)
Phenylalanine deaminase

C- Single substrate utilizations. Such as Urease

Catalase Test:

This test is used to identify whether the examined bacterium produces the enzyme catalase or not.

Catalase is an enzyme that detoxifies hydrogen peroxide by breaking it down into water and oxygen gas
(bubbles):

H2O2 + catalase  H2O + O2

Although many types of bacteria produce this enzyme, Catalase test is commonly used as a a key test in
indentifying Gram-positive cocci. While all Staphylococcal species are Catalase positive (produce this
enzyme), all Streptococcal species are Catalase negative (do not produce this enzyme).

To conduct this test, a fresh colony of the bacterial

species being examined is mixed with a drop of 3%
H2O2 on a glass slide. Formation of bubbles (which
indicates the release of O2 gas) indicates that the
bacterium being examined produce this enzyme.

Examine: Staphylococcal species ( such

Staphylococcus aureus) and a Streptococcal species
Coagulase Test:

Coagulase is an enzyme that converts fibrinogen to fibrin to result in clotting of plasma.

Coagulase Test is utilized to identify certain bacterial species that produce this enzyme
and it is considered a key test to differentiate between Staphylococcal species.

Procedure: the bacterial species being examined is mixed with citrated-plasma (Rabbit plasma) and then the
plasma is examined for clump (clot) formation. There are two methods for detection of coagulase enzyme:

1- The slide method (which detects the surface-bound coagulase (known as the clumping factor)

2-The tube method ( which detects free coagulase).

Initially, carryout the slide method. If negative, confirm by conducting the tube method.
The Slide test: The Tube test:

A colony of the bacterial species being About 0.5 ml of citrated-plasma is mixed with a
examined is mixed with a drop of citrated- loopful of bacterial species being examined.
plasma on a glass- slide.
Then the mixture is incubated at 35 ◦C.
Then, the mixture is examined for
clumping. Plasma clotting is examined at several time points
(( 0.5, 1, 2, 4 h and over night)).
 Bacterial Identification Flowchart

-Ve Gram Stain +ve

Morphology Morphology

Cocci Bacilli Branching Cocci Bacilli

filaments
Oxidase (Cytochrome Oxidase) Test:

This test is used to identify bacterial species that produce Cytochrome-C Oxidase, which is one of the
enzymes of the electron transport chain of certain bacterial species.
Procedure:

This enzyme is detect by placing a loopful of the

bacterial species being examined on a strip or a filter-
paper impregnated (or moistened) with as substrate
that functions as a redox indicator for Cytochrome-C
Oxidase.
The presence of this enzyme in the bacterial species
being tested results in the development of a dark-
violet/blue color (positive-result) due to the reduction
of the redox substrate in the filter paper.

If the dark-violet/blue color does not develop, this

means that the tested bacterium does not produce this
enzyme (negative-result).

Examine Pseudomonas aeruginosa and E. coli

Note: the used redox indicator is tetramethyl-p-
phenylenediamine or dimethyl-p-phenylenediamine).
Phenol Red Broth Test
This test is used to detect fermentation of a particulate CHO (sugar) as well as the production of
gas by the tested bacterium.

The test tube contains:

Phenol Red Broth with a particular single sugar
A pH indicator (Phenol Red), and
A small inverted tube (known as Durham tube used for the detection of
gas that may be generated during the fermentation process).
Procedure:

Several Phenol Red Broth tubes are prepared, each of which contains a single sugar
Each of these tubes is inoculated aseptically by the bacterium intended to be tested
Then the inoculated tubes are incubated for 24 hours at 37 ◦C.

Upon the fermentation of any of the sugars by the tested bacterium, the generated acid (due to
the fermentation process) changes the color of the pH indicator from red to yellow without or
with gas production (in case of gas production, the produced gas is detected by the Durham
tube).

Examine: E. coli, P. aeruginosa

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MRVP (Methyl Red-Vogues Proskauer Test:

Certain types of bacteria are able to convert the acid produced during the fermentation into
a neutral compound (such as acetylmethylcarbinol). In such cases, the usage of the pH
indicator will not be useful.

So, the main purpose of MRVP is to detect whether a bacterium has a fermentation
pathway that generates an acid, or a fermentation pathway that generates a neutral
compound, or non.

Methyl Red Vogues Proskauer test consists of two main parts:

1- The Methyl Red (MR) part: is used to determine if the tested bacterium ferments
glucose and produces acidic compounds. If not, the second part must be conducted.

2- The Vogues Proskauer (VP) part: it is used to determine whether tested bacterium
ferments glucose to generate neutral compounds (acetylmethylcarbinol).

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Procedure:

The MRVP tests are performed by inoculating 2 tubes of MRVP broth with the bacterium to be
tested. Then, the inoculated tubes are incubated for 3-5 days at 37 ºC.

To one tube, few drops of Methyl Red pH indicator are added:
 Positive result: Red (indicating that the pH is below 6)
 Negative result: Yellow (indicating no acid production)

To the other tube, few drops of the VP reagent are added:
Positive result: Red (indicating the presence of acetylmethylcarbinol)
Negative result: No color change

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Identification Chart for Enterobacteriaceae