Faculty of Medicine and Health Sciences

General Microbiology Lab (7105404)

Lab 6-1
Bacterial Identification
Biochemical Tests: Part-I

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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 identify 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
Plasma clotting is examined at several time points
clumping.
(( 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

-ve

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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
Faculty of Medicine and Health Sciences
General Microbiology Lab (7105404)

Lab 6-2
Bacterial Identification
Biochemical Tests: Part-II
First Semester 2023-2024

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Triple Sugar Iron Agar (TSI):

This test is used to differentiate between certain bacterial species on the basis of differences in their:
1- Sugar fermentation
2- Gas Production and
3- H2S production
TSI agar is orange-red in color, which reflects the color of the included
pH indicator at neutral pH.

Note: after being prepared, TSI is poured into glass tubes. After
sterilization of the tubes by autoclaving, the tubes are allowed to cool
and solidify at a slanted angle. So, the tubes of the prepared TSI agar
appear as shown in image.
TSI agar contains:

 Lactose, Sucrose and Glucose sugars with 1% concentrations for both Lactose and Sucrose
and 0.1% concentration for Glucose.

Phenol Red: it is a pH indicator that serves to detect fermentation of one or more of the sugars
included in the TSI agar. If the bacterium being tested ferments one or more of these sugars, the
color of this pH indicator turns from Red to Yellow (so does the color of the TSI agar) due to the
acid generated by the fermentation process.
 Sodium Thiosulfate: If the bacterium being tested reduces this substance, H 2S is generated.

Ferrous sulfate: It is used for the detection of H2S by the bacterium being tested. The generated
H2S reacts with Ferrous Sulfate to produce a black precipitate (Ferrous Sulfide).

Peptone: it serves as a nutrient to support the growth of the bacterium being tested. Its aerobic
utilization by the tested bacterium produces basic (have alkaline pH) byproducts.
TSI test is mainly used to differentiate between
bacterial species that belong to family
Enterobacteriaceae , however it can be used for other
bacterial species that belong to other bacterial families.

Inoculation of TSI agar:

TSI is inoculated with the bacterium being tested

using a sterile needle so that, the needle is initially
stabbed straight down into the butt of the agar medium,
and then pulled straight up.
After that, the bacterium is streaked on the surface of
the agar slant.
 The inoculated tubes are then incubated at 37 ºC for
18-24h.
Results: Reading and interpretation:
1- Red Slant (K) and Yellow Butt (A) ((K/A)): this indicates that the tested
bacterium ferments Glucose only. Because of the low concentration of Glucose
in TSI agar (0.1%), fermentation of this sugar generates a small amount of
acid.
The acidic pH changes the color of the pH indicator (Phenol Red) into yellow,
thus the color of the TSI agar turns from Red to yellow as well.
K/A
However, under aerobic conditions (at the Slant part of the TSI agar),
peptone metabolism generates alkaline products, which in turns neutralize the
acid produced due to Glucose fermentation. Accordingly, this will prevent
changing the color of the pH indicator at Slant part of the test medium, which
remains Red.

2- Yellow Slant (A) and Yellow Butt (A) ((A/A)): this indicates that the
tested bacterium ferments Glucose, Sucrose or/and Lactose.

This results in the production of a large amount of acid which will change the
color of the pH indicator (Phenol Red) into yellow and so that generated A/A
alkaline products due to peptone metabolism (at the Slant part of the
medium), will not be able to neutralize the large amount of acid generated by
the fermentation process, so that the entire test medium (Slant and Butt),
becomes yellow.
 K/K
3- Red Slant (K) and Red Butt (K) ((K/K)): this indicates that the
tested bacterium does not ferment any the three sugars found in the
medium.

Gas production: in addition to the production of acid due to sugar

fermentation, the fermentation process MAY generates a gas too. The
production of a gas in the TSI agar can be detected by noticing a A/A
cracking in TSI gar or the TSI agar is pushed up. Gas +ve
H2S production: in case that the tested bacterium generates H 2S
due to reduction of thiosulfate included in the TSI agar, the generated
H2S is detected by the formation of a black precipitate, (Ferrous
Sulfide) that results due to a reaction between H 2S and the Ferrous
Sulfate.

((Examples of H2S producing -bacteria: Salmonella, Proteus,

Citrobacter and Edwardsiella species))
K/A
H2S +ve
A/A
H2S +ve
+
Citrate Utilization Test (Simmon’s Citrate Test)

Citrate agar contains:

Sodium Citrate ((as the sole (the only)source of carbon))

Ammonium dihydrogen phosphate
A pH indicator (Bromthymol blue).

This test is used to examine the ability of the tested bacterium to utilize Citrate as a sole source of
carbon.

 Any bacterial species that is able to use Citrate as a sole source of Carbon has a transporter
(known as citrate permease) that internalizes Citrate from the surrounding environment into its
cytoplasm, where it can be utilized as a nutrient enabling the bacterium to replicate and grow.

In addition, Citrate agar contains inorganic ammonium salts (ammonium dihydrogen phosphate),
which can be utilized as a sole source of Nitrogen. When ammonium salts are used by tested
bacterium ,the ammonium salts breakdown into NH 3, which increases the pH of medium.
Alteration of the pH of the agar medium into an alkaline pH, converts the color of the pH indicator
( Bromthymol blue) into a blue color

Note: At a neutral pH, Bromthymol blue is green. At pH 7.5 or above, Bromthymol blue turns blue

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Procedure:
The test bacterium is inoculated onto the slant of the agar test tube.
The inoculated tube in then incubated for 24h
After 24 h of incubation, if the inoculated medium turns blue, the bacterium is citrate positive. If
there is no color change, the color of the medium remains green, the bacterium is said to be citrate
negative

Note: the tested bacterium must be inoculated only onto the slant or the surface of the citrate agar,
because citrate utilization occurs only under aerobic conditions.

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Urease Test:

This test is used to examine the production of urease enzyme by the tested bacterium. The production
of this enzyme can be utilized as one of the diagnostic features in the process of bacterial
identification (diagnosis).

The test medium (Urea Agar), contains urea and a pH indicator. Degradation of urea by the urease
enzyme generates ammonium, which has an alkaline pH .

This implies that, if the tested bacterium produces the enzyme urease, by the end of the incubation
period, the generated ammonium converts the pH of the medium into Alkaline, which in turns changes
the color of the pH indicator from yellow to fuchsia (pink).
Procedure:
The test bacterium is inoculated into Urea Agar and then incubated for about 24h at 37 ºC.
The change in the color of the medium into fuchsia (pink), implies that the tested bacterium
produces urease enzyme.

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SIM Test:
SIM medium is used to examine three features of the tested- bacteria, which can be used in the
identification process (diagnosis).

1. H2S production (S)

2. The production of Tryptophanase enzyme, which degrades tryptophan to generate inodole (I)
3. Motility (M)

SIM medium contains:

 A bout half of the agar is used to a semisolid medium.
Ferrous Sulfate and Sodium thiosulfate
A large amount of the amino acid, Tryptophan

Procedure:

An SIM tube is inoculated with the bacterium

being tested using a sterile needle. The medium is
stabbed straight down an then pulled straight up.

The inoculated-tubes are then incubated at for 24 h

at 37 ºC.

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By the end of the incubation period:

1- Motility (M):

In case that the tested bacterium is non-motile, only a line of growth is seen along the stabbing line (as
seen in B of the image given below).

In case that he tested bacterium is motile, there will be a line of growth a long to the stabbing line
surrounded by a turbid or a hazy region, indicating that the tested bacterium is spreading through the
medium by virtue of its motility (as seen in C of the image given below).

2- H2S production:

In case that the bacterium being tested generates H 2S

(due to reduction of thiosulfate included in the medium),
the generated H2S reacts with the Ferric Sulfate
((included in the medium)) to generate a black
precipitate (Ferric Sulfide). The tested bacterium in this
case is said to be H2S positive (as seen in D of the image
given below).

If not, the tested bacterium is said to be H2S negative

(as seen in B and C of the image given below).

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3- Detection of Indole:
Some bacterial species produce the enzyme Tryptophanase, which hydrolyzes the amino acid
Tryptophan into Indole, Pyruvic acid, and Ammonia.

It is possible to know that the bacterium being tested has this enzyme by detecting the presence of
Indole in the medium (recall that SIM medium contains a large amount of Tryptophan)..

Indole can be detected by the addition of about 0.5 ml of a colorless solution known as Kovac’s
Reagent. This reagent contains a substance that reacts with Indole to generate a product that is
Red/Fuchsia in color.

In case that the tested bacterium generates Indole (Indole positive/has Tryptophanase), the added
Kovac’s Reagent becomes Red/Fuchsia in color within few minutes (as seen in C of the image given
in the next slide).

In case that the tested bacterium does not generate Indole (Indole negative/does not have
Tryptophanase), the added Kovac’s Reagent remains colorless (as seen in B and D of the image
given in the next slide)
The presence or absence of this enzyme is one of the features that can be used in bacterial
identification.
Note: Kovac’s reagent contains
Hydrochloric acid, p-
dimethylaminobenzaldehyde (DMABA),
and n-Amyl alcohol. DMABA reacts with
Indole to generate a Red to Fuchsia-
colored Quinoidal compound.

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A Follow Chart Based on Culture Characteristics & Biochemical testing
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Find the 7 digital Code for this results
 Chromogenic Technology

This technology is based on soluble colourless molecules (called chromogens), composed of a

substrate (targeting a specific enzymatic activity) and a chromophore.
When the target organism’s enzyme cleaves the colourless chromogenic conjugate, the chromophore
is released. In its unconjugated form, the chromophore exhibits its distinctive colour and, due to
reduced solubility, forms a precipitate.

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