BCH1200 Discovery in Biology

Practical 1
Microbiology Lab: Identification of Bacteria

I. Bacterial features and identification

Morphological features of bacteria

Bacteria are prokaryotic cells. They are very small organisms (ca. 10-6 m in length) that cannot
be seen or identified using our naked eyes. Using a light microscope with a magnification of
×1000, you can see some characteristics of bacteria; they come in a wide variety of shapes and
sizes. The most common shapes are rod-like or spherical forms, but they also form spirals, ovals
and branched structures.

All bacterial cells have cell walls. This cell wall structure falls into two general types, which is
used as the major criterion for distinguishing different groups of bacteria. By staining the
bacteria with “Gram stain”, bacteria with a thick wall layer and do not have an outer membrane
can stain purplish blue and are called “Gram positive” (see Fig 1b). Bacteria with a thin cell
wall layer and an outer membrane do not retain the Gram stain after rinsing with alcohol but can
take up a red stain subsequently are called “Gram negative” (see Fig 1a).

Figure 1a. Gram stain of Gram-positive Figure 1b. Gram stain of Gram-negative
bacteria bacteria

(http://filebox.vt.edu/users/chagedor/biol_4684/Methods/cellwalls.html )

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Biochemical features of bacteria
Besides the cell wall structure, biochemical features of bacteria are usually used for
identification purpose. One of the examples is the presence or absence of oxidase, an enzyme
used to catalyze oxygen (O2) to water (H2O) or hydrogen peroxide (H2O2). This feature can be
identified using the “oxidase test”. Bacteria with the presence of oxidase can turn the oxidase
test reagent into dark purple are referred to as “oxidase positive” while the bacteria without
oxidase fail to turn the oxidase test reagent into purple (remain colorless) are referred to as
“oxidase negative”.

Different functional groups of bacteria have different nutritional requirements. They grow best
when specific types and optimal amount of sugars (e.g., glucose, adonitol, lactose, arabinose,
sorbitol, dulcitol), amino acids (e.g., lysine, ornithine, phenylalanine, tryptophan), peptides and
vitamins are provided. All these properties can be used for their identification based on their
fermentation (use) of these sugars and amino acids. These reactions will be illustrated in detail in
this practical.

II. Assembly of an identification kit: use of a fermentative-based commercial kit

for bacterial identification
In this practical, an exercise on bacterial identification using a commercial kit will be carried out.
Four unknown bacterial species belonging to one family, Enterobacteriaceae, isolated from
toilets or food samples will be examined and identified using the EnteroPluri-Test system. Before
this particular EnteroPluri-Test system is used, it is necessary to confirm that the test bacteria is
either oxidase positive or negative, as different diagnostic tubes are made for the identification of
different groups.

Distribution of Enterobacteriaceae and their importance to man

Enterobacteriaceae is a family of rod-shaped, Gram-negative and oxidase-negative bacteria.
They are widely distributed in nature. They are part of the intestinal flora (i.e., commonly found
in human intestines), and some are important human pathogens. The most common of these are
Escherichia, Enterobacter, Klebsiella, Proteus, Providentia, and Salmonella spp. Less
pathogenic Enterobacteriaceae include Citrobacter, Edwardsiella, Erwinia, Hafnia, Serratia,
Shigella, and Yersinia spp. To safeguard public health and food safety, the ability to accurately
identify and detect these bacterial species are of paramount importance in public health and
infection management.

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Principle of EnteroPluri-Test system
EnteroPluri-Test system (see Fig. 2) is a self-contained, compartmented plastic tube containing
12 different media that allows for the determination of 15 biochemical reactions.

Figure 2. EnteroPluri-Test system

Biochemical reactions include:

 Glucose - Glucose fermentation
 Gas - Gas production from glucose
fermentation
 Lysine - Lysine decarboxylase
 Ornithine - Ornithine decarboxylase
 H2S - Hydrogen sulfide (H2S) production
 Indole - Indole formation
 Adonitol - Adonitol fermentation
 Lactose - Lactose fermentation
 Arabinose - Arabinose fermentation
 Sorbitol - Sorbitol fermentation
 Voges-Proskauer (VP) test
 Dulcitol - Dulcitol fermentation
 Phenylalanine - Phenylalanine deaminase
 Harnstoff Urea / Uree - Urea hydrolysis
 Citrate - Citrate utilization

It is specifically designed for the identification of Enterobacteriaceae based on their

characteristic biochemical reactions during growth.
 Enterobacteriaceae ferment a variety of sugars (including glucose, adonitol, lactose,
arabinose, sorbitol, and dulcitol). Fermentation of sugars results in the production of acidic
end products, the presence of which can be detected by the pH indicator in the medium
resulting in a color change.
 Some Enterobacteriaceae produce gas during glucose fermentation – either CO2 alone or a
mixture of H2 and CO2. H2 is insoluble and can be detected by bubble formation inside the
medium chamber.
 Enterobacteriaceae also metabolize a variety of amino acids (including lysine, ornithine,
phenylalanine, and tryptophan). They can be further distinguished by their capability to

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 produce H2S (hydrogen sulfide), hydrolyze urea (produce ammonia) and/or to utilize citrate
as a carbon source.

The resulting combination of the 15 biochemical reactions together with the Interpretation Guide
(codebook), allow identification of clinically significant Enterobacteriaceae.

Intended Learning Outcomes

Upon completion of this exercise, students should be able to:

1. Identify some common features in bacteria.
2. Use a commercial test kit to identify bacteria.
3. Differentiate the different principles of selected biochemical tests used for bacteria
identification.

Materials Provided:

 Four unknown bacteria, isolated from toilet or food samples, labeled A, B, C or D are
provided for identification.
 Filter papers (Whatman No.1 or equivalent)
 Sterilized toothpick
 Oxidase test Reagent Droppers
 Inoculated EnteroPluri-Test systems
 Indole test Reagent Droppers (modified Kovacs' reagent)
 Fine-point forceps
 Color chart
 Data record sheet
 Interpretation Guide (Codebook)

Procedure:

Before the experiment:

1. Students are divided into groups of 4. Each group is provided with some unknown bacterial
isolates and EnteroPluri-Test systems.

2. One day before this practical, each EnteroPluri-Test system has been pre-inoculated with one
unknown bacteria by a technical officer using an inoculating wire. This allows inoculation of
all compartments in one step from one or a few single colonies of an isolate (see video

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 demonstration). This pre-inoculation allows the bacteria to grow for one day and hence
sufficient time for the biochemical reactions to develop during their growth (Table 1).

During the experiment:

3. Oxidase test
Before checking the results of the EnteroPluri-Test system, make sure that your unknown
bacteria isolated is oxidase-negative (i.e., absence of the enzyme, oxidase) by following the
procedure described below:
a. Add a few drops of Oxidase test reagent to a strip of filter paper (Whatman No.1)
b. Streak a small amount of bacteria onto the reagent-saturated paper with a sterilized
toothpick.
c. Positive reaction – the bacteria turns dark purple within 30 seconds.
Negative reaction – the bacteria remains colorless or turns light pink/light purple after 30
seconds.

4. Indole test
You still need to perform the final biochemical test, Indole test in the inoculated EnteroPluri-
Test system. It is a presumptive test to check whether the bacteria can utilize tryptophan (an
amino acid) to produce Indole. The procedures are shown as below:
a. Break the thin Mylar plastic film that covers the flat surface of the H2S/indole
compartment of EnteroPluri-Test system with a syringe or a fine-point forceps.
b. Inject 1 or 2 drops of Indole test reagent onto the surface of the medium in the
H2S/indole compartment.
c. Positive reaction – the reagent added turns red within 10 seconds.
Negative reaction – the reagent added remains colorless.

5. Reading results
a. After the tests, check positive test results for each EnteroPluri-Test system according to
the Color Chart given in Fig 3.

Negative
test result

Positive
test result

Figure 3. Color differences between uninoculated (negative) and inoculated (positive) tests.

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 b. Record and circle the number below those tests of positive results on the Data Record
Sheet (see Fig. 4) provided.

Sample A

Circle the number

if the test has a
positive result.

Figure 4. Data record sheet

6. Tabulation and interpretation of results

a. After circling the numbers of the positive tests on the Date Record Sheet, add up the
numbers of each bracketed series to determine the 5-digit code number (= ID value).
b. Refer to the Interpretation Guide (Codebook) for identification of the unknown using this
5-digit code number.

Example:
Sample A

Add up the
numbers of each
bracketed series
3 4 3 6 3

The “ID Value” 34363 can be found by thumbing the pages of the Interpretation
Guide provided. The listing is as follows:

Conclusion: The unknown organism is identified as Klebsiella pneumoniae.

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Table 1. Biochemical reactions measured by the EnteroPluri-Test system

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Table 1 (continued).