Environmental Microbiology & ET 1303

Department of Environmental Technology

Group 02
Practical number 08
Biochemical test for bacterial identification

Index number: 2021T01058

Student name: Thalgahawatta S.P
Date: 23rd of June 2023
Instructor name: Miss Samadhi
Objectives:
The objective of this experiment was to perform biochemical tests to identify bacterial species
based on their ability to produce bubbles when hydrogen peroxide (H2O2) is added to the
bacterial colony.

Introduction:
Biochemical tests for bacterial identification are a set of laboratory techniques used to
determine the metabolic characteristics of bacteria, which can aid in their classification and
identification. These tests involve introducing specific substrates or chemicals to bacterial
cultures and observing the resulting reactions. By analyzing the presence or absence of certain
enzymes and metabolites, as well as the products produced, scientists can make inferences
about the identity and properties of the bacteria under study. These tests provide valuable
information for medical diagnosis, epidemiological studies, and research on bacterial
physiology and genetics.

Materials:
• 3% Hydrogen Peroxide (H2O2)
• Sterilized glass rod
• Test tube
• Bacterial colony incubated for 24 hours

Procedure:
1. Add 4 to 5 drops of 3% H2O2 to a test tube.

2. Using a sterilized glass rod, collect a small amount of bacterial colony from a colony
incubated for 24 hours and place it into the test tube.

3. Place the test tube against a dark background and observe for immediate bubble
formation. The formation of bubbles indicates the presence of catalase activity in the
bacterial species under investigation.

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Observations:
Positive Result - Immediate bubble formation occurred, indicating the presence of catalase
activity in the bacterial species. The production of bubbles suggests that the bacteria have the
ability to break down hydrogen peroxide into oxygen gas and water.

Discussion:
• The experiment aimed to determine whether a bacterial colony possesses the enzyme
catalase, which aids in the breakdown of hydrogen peroxide. This test is commonly
employed in bacterial identification since catalase activity can differ among various
bacterial species.

• Hydrogen peroxide is introduced into a test tube containing the bacterial colony. If the
bacteria possess catalase, the enzyme breaks down hydrogen peroxide into water and
oxygen gas. The released oxygen gas appears as bubbles, which become visible against
a dark background.

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 • The immediate formation of bubbles upon adding hydrogen peroxide indicates the
presence of catalase-positive bacteria. The production of bubbles confirms that the
bacteria can effectively break down hydrogen peroxide into water and oxygen gas.

• Catalase activity is an important characteristic used to differentiate between different

groups of bacteria. Certain bacteria, such as staphylococci and micrococci, produce
catalase and exhibit immediate bubble formation. On the other hand, some bacteria like
streptococci and enterococci either lack or have reduced catalase activity, resulting in
the absence or delayed formation of bubbles. During our practical also, we could not
observe catalase-negative bacteria.

• While the catalase test is a helpful tool for bacterial identification, it may not be
applicable to all bacterial species. Some bacteria might exhibit weak or delayed catalase
activity, leading to inconclusive results. Therefore, it is essential to use other
biochemical tests and techniques in conjunction with the catalase test to obtain a more
comprehensive identification of the bacteria.

• Using a sterilized glass rod is crucial during the transfer of the bacterial colony to
prevent contamination. Contamination from external sources may yield inaccurate
results and misidentification of the bacteria. By ensuring sterilization, we maintain the
integrity of the experiment and increase the reliability of the obtained data.

Conclusion:
Based on the observations, the bacterial species can be categorized into two groups: those with
catalase activity (positive result) and those without catalase activity (negative result). Catalase
activity is a common characteristic exhibited by many bacterial species, but its absence can
also provide valuable information for bacterial identification.
It is important to note that this test alone cannot determine the specific bacterial species.
Additional biochemical tests and procedures are required for more accurate identification.
Further testing and analysis can help narrow down the potential bacterial species based on their
unique biochemical properties.

References:
➢ “11 BACTERIAL IDENTIFICATION TESTS” [online] Available From:
https://nios.ac.in/media/documents/dmlt/Microbiology/Lesson-11.pdf (Accessed by:
2023/07/05)

➢ “Biochemical Tests for Microbial Identification” [online] Available From:

https://www.news-medical.net/life-sciences/Biochemical-Tests-for-Microbial-
Identification.aspx (Accessed by: 2023/07/05)