2018

GEBZE TECHNICAL
UNIVERSITY
MICROBIOLOGY LABORATORY
TURKEY

EBRU AKHARMAN
142204026
18.05.2018
 BIOCHEMICAL ACTIVITIES OF BACTERIAS
AIM
Microorganisms must be separated and identified for a wide variety of reasons, such as:
1. Determination of pathogens responsible for infectious diseases.
2. Selection and isolation of stains of fermentative microorganisms necessary for the industrial production of
alcohols, solvents, vitamins, organic acids, antibiotics, and industrial enzymes.
3. Isolation and development of suitable microbial stains necessary for the manufacture and enhancement of
quality and flavor in certain food materials such as yogurt, cheeses, and other milk products.
4. Comparison of biochemical activities for taxonomic purposes.
To accomplish these tasks, the microbiologist is assisted by the fact that, just as human beings possess a
characteristic and specific set of fingerprints, microorganisms all have their own identifying biochemical
characteristics. These so-called biochemical fingerprints are the properties controlled by the cells’ enzymatic
activity, and they are responsible for bioenergetics, biosynthesis, and biodegradation. The sum of all these
chemical reactions is defined as cellular metabolism, and the biochemical transformations that occur both
outside and inside the cell are governed by biological catalysts called enzymes. The aim of this experiment is
control of catalase activity, control of oxidase activity and thioglycollate test.

Image 1: Catalase Test Image 2: Oxidase Test

Image 3: Thioglycollate Test

 RESULTS
FOR CATALASE TEST:

Image 4: The Catalase Test for S. epidermidis, E. coli, E. feacalis, X bacteria

This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from
hydrogen peroxide (𝐻2 𝑂2 ). It is used to differentiate those bacteria that produces an enzyme catalase, such as
staphylococci, from non-catalase producing bacteria such as streptococci. Normally 3% 𝐻2 𝑂2 is used for the
routine culture while 15% 𝐻2 𝑂2 is used for detection of catalase in anaerobes.

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of
the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and
the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble
production. The culture should not be more than 24 hours old.

Bacteria thereby protect themselves from the lethal effect of hydrogen peroxide which is accumulated as an
end product of aerobic carbohydrate metabolism. In this experiment, Staphylococcus epidermidis uses
catalase enzyme strongly (+++++), E. coli and X bacteria use catalase enzyme middle level (+++) but
Enterococcus feacalis does not use catalase enzyme(-). The reason of this situation will explain in discussion
part.
FOR OXIDASE TEST:

Image 5: The Oxidase Test for E. coli, X bacteria and P. aeruginosa

The oxidase test detects the presence of a cytochrome oxidase system that will catalyse the transport of
electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. The
dye is reduced to deep purple color. This test is used to assist in the identification of Pseudomonas, Neisseria,
Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of which produce the enzyme
cytochrome oxidase. Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase
enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as part of their
respiratory chain are oxidase-positive and turn the reagent blue/purple. Organisms lacking cytochrome c as
part of their respiratory chain do not oxidize the reagent, leaving it colorless within the limits of the test, and
are oxidase-negative.

Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron containing
haemoprotein). Both of these catalyse the transport of electrons from donor compounds (NADH) to electron
acceptors (usually oxygen). The test reagent, N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride
acts as an artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the coloured
compound indophenol blue. The cytochrome system is usually only present in aerobic organisms which are
capable of utilising oxygen as the final hydrogen receptor. The end product of this metabolism is either water
or hydrogen peroxide. Only P. aeruginosa can convert color from white to purple.
FOR THIOGLYCOLLATE TEST:

Image 6: Thioglycollate Test for P. aeruginosa, E. coli and X bacteria

Thioglycollate broth is the enrichment broth most frequently used in diagnostic bacteriology. This broth
supports growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms. It contains many
nutrient factors, including casein, yeast, and beef extracts, and vitamins to enhance the growth of most
medically important bacteria.

The addition of a small amount of agar in Thioglycollate Medium aids in the initiation and growth of small
inocula and anaerobes by impeding the diffusion of oxygen into the medium. It also retards the dispersion of
CO 2 and the reducing substance from the microenvironment surrounding the inoculum. Sodium
Thioglycollate is a reducing agent which maintains a low oxygen tension by removing molecular oxygen from
the environment. Peroxides, which may be lethal to many anaerobic organisms, are not formed under this
condition. Cystine and casein supply carbon and nitrogenous compounds, dextrose is added as another energy
source, and sodium chloride maintains osmotic equilibrium.

Certain additives are incorporated into the Thioglycollate Medium as desired. Resazurin is an oxidation-
reduction indicator that turns pink when increased oxidation has occurred. Yeast extract or papaic digest of
soybean meal are added as growth enhancers. In this experiment, P. aeruginosa grows on top of broth
medium. The E. coli and X bacteria grow under surface of broth medium. The reason of this situation will
explain in discussion part.
 Drawings of Results

Image 4: The Catalase Test for S. epidermidis, E. coli, E. feacalis, X Image 5: The Oxidase Test for E. coli, X bacteria and P. aeruginosa
bacteria

Image 6: Thioglycollate Test for P. aeruginosa, E. coli and X bacteria

 DISCUSSION
The identification of higher plants and animals involves the observations of the structural differences, both
internal and external, which exist among them. Most of these structural differences are visible to naked eye.
Even the identification of microscopic plants and animals involves the observations of their structural
differences under a microscope.

Such identification based on structural differences is not possible in case of bacteria, because structural
differences, which may differentiate one species of bacteria from the other, are not discernible even under a
microscope. The structural differences with respect to shape, size and arrangement of bacteria only help in
the process of identification, because there are many species of bacteria having similar shape, size and
arrangement. Therefore, ultimately, the identification of bacteria is mostly based on the differences in their
biochemical activities.

In catalase test, the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen
peroxide (𝐻2 𝑂2 ). It is used to differentiate those bacteria that produces an enzyme catalase, such as
staphylococci, from non-catalase producing bacteria such as streptococci. In this catalase test, Staphylococcus
epidermidis occurs very much bubbles. Thus, we say that, Staphylococcus epidermidis can use catalase
enzyme strongly. E. coli and X bacteria occur some bubbles. We say that, E. coli and X bacteria can use
catalase enzyme in middle level but Enterococcus feacaalis does not occur bubbles so it can not use catalase
enzyme.

The oxidase test detects the presence of a cytochrome oxidase system that will catalyse the transport of
electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. The
dye is reduced to deep purple color. E. coli and X bacteria can not convert color from white to purple but P.
aeruginosa can convert color from white to purple. Thus, we say that, E. coli and X bacteria have not to
cytochrome oxidase system but P. aeruginosa has cytochrome oxidase system.

In thioglycollate test, Sodium Thioglycollate is a reducing agent which maintains a low oxygen tension by
removing molecular oxygen from the environment. Peroxides, which may be lethal to many anaerobic
organisms, are not formed under this condition. The P. aeruginosa grows on the surface of broth medium, E.
coli and X bacteria grow under the surface of broth medium. Thus, we say that, P. aeruginosa is aerobic but E.
coli and X bacteria are anaerobic.

RESOURCES
https://microbiologyinfo.com/catalase-test-principle-uses-procedure-result-interpretation-with-
precautions/

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/FluidThioglycollateEnriched.html

http://microbiologyrocks.blogspot.com.tr/2010/03/thioglycollate-broth-is-really-cool.html

http://www.mikrobiyoloji.org/TR/Genel/BelgeGoster.aspx?F6E10F8892433CFFAAF6AA849816B2EF33440F
A0A3040406