(6-8+2H→O2+H2O2) (enzyme: Superoxide dismutase)

The assignment content discusses the importance of oxidase test in identifying bacteria with cytochrome C oxidase activity and understanding oxidation and fermentation reactions. It highlights the use of substrate H2O2 or oxygen emitted as bubbles to detect catalase production and activity. The text also explains the principle of the oxidation-fermentation (OF) test, which differentiates between oxidative and fermentative bacteria. Furthermore, it describes the process of fermentation in food processing industries, involving microorganisms like yeast or bacteria that employ anaerobic respiration.

Running head: IDENTIFICATION OF AN UNKNOWN SAMLE OF BACTERIA
IDENTIFICATION OF AN UNKNOWN SAMPLE OF BACTERIA
Name of the Student
Name of the University
Author Note

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Title: Identification of an unknown sample of bacteria.
Abstract
The experiment aims to determine the morphology and the metabolic profile an unknown
sample of bacteria. A sample bacterium had been taken along with the known sample of
Bifidobacterium, Pseudomonas fluorescens and Escherichia coli. A differential plating and
staining had been done in order to distinguish between gram negative and gram positive
bacteria. Further confirmatory tests like using of selective media such as Eosin methylene
blue agar media and mannitol agar media had been used to determine the bacteria based on
their nutrient utilization. The unknown sample showed the growth with a green metallic
sheen in the eosin methylene blue agar that indicates that the bacteria are Escherichia coli.
The catalase oxidation and the oxidase confirmatory test also gave positive result, which
indicates the presence of the facultative anaerobe in the sample. The oxidation fermentation
result gave a yellow color, which indicated the presence of a glucose oxidizing, and glucose
fermenting bacteria, where oxidation and fermentation had taken place at the top and no
oxidation or fermentation has been on the bottom. The gram staining of the unknown bacteria
helped us to determine whether the type of the bacteria is Gram positive and gram negative,
depending upon the composition of the peptidoglycan.
INTRODUCTION
Background and Significance of the Study
One of the important aspects of the microbiology is identification of the unknown
bacterial sample. One of the basic and the sole pillar of the bacterial identification, which
have been used in microbiology since time immemorial is Gram’s staining. It classifies
bacteria into two groups, gram positive and gram negative based on their cell wall

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composition. However, with the advancement of technology and research, several different
methods and procedures have been elucidated into order to classify the bacteria on the basis
of their shape, presence of flagella, endospore, fermenting capability and also on the basis of
oxygen requirement or respiration (aerobic and aneorobic bacteria).
Aim of the Study
The aim of the study is to elucidate identify unknown bacterial sample based on the
application of the several microbiogical identification techniques.
Objectives
The aim objective of the study is to identify the gram positive/negative strain of the bacteria,
Identify the external outline of the bacteria, Identify the lactose fermenting capacity of the
bacteria, Identify the peroxidase activity of the bacteria, Identify whether the bacteria is
facultative anaerobe, anaerobe, obligate anaerobe or aerobic bacteria. Differential staining
and the use of differential media help to identify specific group or bacteria amidst different
strains of bacteria. A bacterium can react differently with nutrient s provided in the
differential media and thus helps in the identification. Differential techniques to identify
bacteria had been used, like Eosin methylene blue plates, Mannitol agar tests, catalase
oxidation and the oxidase tests, depending upon the ability of the unknown bacteria to
perform the catalase activity. The experiment further focuses on the oxidative fermentation
of the facultative anaerobe that helps to identify the bacteria. The fermentative property of the
bacteria has a wide range of application in the fermentative industry. They have a wide range
of application in the food, beverage, and bakery and breweries industry. The process of
fermentation is used in the food processing industry for converting carbohydrates into
alcohol. Microorganisms such as yeast or bacteria that employ anaerobic respiration are used
in this process. Widely consumed fermented foods that are processed by bacteria are vinegar,

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cheese, yoghurt. Escherichia coli are used for the production of ethanol, lactate, succinate and
acetate.
METHODS
In this method the bacteria has been subjected to differential staining. Differential staining is
normally done to identify a specific strain of bacteria in comparison to the other bacteria.
Different types of bacteria can have different types of reactions with the particular type of
stains used. Few techniques that have been used in the identification of the unknown bacteria
have been provided.
Confirmatory tests
Eosin methylene blue plates- Eosin Methylene blue is used to determine the gram negative
enteric rod shaped bacteria hence gram positive and gram negative bacteria can be
distinguished from the following differential staining. Eosin and methylene blue helps in
inhibiting the growth of the gram positive bacteria and helps to identify the gram negatives
(Wills et al.2014). The two dyes present in this stain helps to indicate the lactose fermentors
and the non lactose fermenters. The lactose fermentors produce colonies with clear borders
and dark centers. The non lactose fermentors form colorless colonies.
Mannitol agar- The mannitol agar can be used to identify the staphylococci group of
bacteria. Staphyloccoccus bacteria cannot use mannitol as their nuytrient and hence produce
colorless colonies (Hemraj et al.2013). The group of bacteria that can utilize mannitol can
turn the mannitol agar plate yellow and hence can be identified.
Catalase oxidation- Some bacteria produces flavoprotiens that can bring about reduction of
oxygen with the production of hydrogen peroxide and superoxide. These free radicals are
harmful and they can destroy the bacterial cells (Hemraj et al.2013). There are certain

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obligate and facultative anaerobes that contain the enzyme superoxide dismutase that helps in
the destruction of the super oxide and they can also produce catalase or peroxidase that
neutralizes the hydrogen peroxide (Borisov et al.2013)
This catalase activity can be detected by the addition of the hydrogen peroxide a substrate to
tryptic soy agar slant. A bacteria producing catalase will be able to produce oxygen case
which can be determined by the bubbles released. This is recognized as a useful method in
detecting the catalytic activity of bacteria.
2O2- + 2H+ → O2 + H2O2 (Action of Superoxide dismutase)
2H2O2 → 2H2O + O2 (Action of Peroxidase)
Oxidase- The oxidase cytochrome test involves addition of an oxygen test strip containing
tetramethyl-p-phenylenediaminedihydrocholoride or an Oxidase Disk, p-amino-
dimethylaniline. The oxidase test reagent is light pink in color and acts as the substrate and
donates electrons to the cytochrome oxidase produced by the bacteria, which can turn the
strip to dark purple due to the presence of free oxygen. A dark purple coloration is the
indication of positive result.
Oxidation fermentation Test- This test was done to differentiate between the gram positive
and the gram-negative bacteria on the basis glucose fermentation (Hemraj et al. 2013).
Further concentration of the bacteria has been measure by diluting them and measuring the
optical density at 660 nm.

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Results-
General Observation
The nutrient agar plate over which the unknown bacterial sample was grown overnight at 37
degree centigrade in the incubator showed that the bacteria colonies are circular in shape with
a convex elevation and a smooth outer margin.
Gram staining observation of the unknown sample
Pink coloured colonies had been found which determines gram-negative bacteria.
Metabolic profiling: In the Eosin methylene blue agar, the unknown sample showed
growth with a greenish metallic sheen, which indicates Escherichia coli bacteria, which has
been able to ferment lactose.
In the Mannitol agar plate, the unknown inoculums showed no change in color.
Catalase activity: Bubbles were formed on the plates as soon as it is reacted with the
hydrogen peroxide indicating catalase activity of the bacteria.
Oxidase activity:
The oxidase test reagent showed a dark purple color which indicated a positive test for the
bacteria having the oxidase enzyme.
Oxidation/Fermentation test:
Table 1
If the open assay produces yellow coloration then it gives indication that the organism has
oxidized as well as fermented. A green coloration signifies no reaction with the glucose. For
closed assay, yellow coloration indicates fermentation and a green colour signifies that the

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bacteria has not reacted with the glucose and has not oxidized. Yellow result is obtained in
the unknown sample both in the open and in the closed assay which is same as that of
Escherichia coli which indicates that the bacteria is Escherichia coli.
No Oil (open assay) Oil (closed assay) Result
yellow green Oxidation of glucose
yellow yellow Fermentation of glucose
green green no action on glucose
Table 2
It can be seen that the colonies obtained from the unknown sample is same as that of the
Escherichia coli. The orange colored colonies are due to the production of gluconic acid. The
orange colour at the top indicates that the fermentation has occurred at the top due to
oxidation and no glucose utilization had taken place at the bottom.
Name of the bacteria With paraffin Without Paraffin
Bifidobacterium Orange colour, colonies
visible
All green colored colonies
Pseudomonas fluorescens Green colonies, no colour
visible
Orange top , green bottom
Escherichia coli Green on bottom, orange on
top colonies
Orange on top, green at the
bottom,

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Unknown bacteria Green on bottom, orange on
top, Escherichia coli colonies
visible
Orange on top and green at
the bottom , colonies visible
Table 3
The spectrometric results obtained are as follows-
It can be seen as the dilution increases the absorbance at 600 nm decreases as the
concentration of bacteria decreases.
Dilution Absorbance(600 nm)
½ 0.224
¼ 0.141
1/8 0.100
1/16 0.075
DISCUSSION
Results
The unknown bacterial sample showed round colonies with convex elevation and a
continuous margin. This signifies that the margin of the unknown bacterial sample is
smooth and continuous (Harrigan, Wilkie and Margaret 2014). The gram staining of the
isolated single concave bacterial colony provided pink color, indicating gram-negative

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strain of bacteria (Carter et al 2012). The stained slide when viewed under the light
microscope, it showed a tubular structure and no motility was detected. The gram-negative
rod was then subjected to metabolic profiling in order to detect the lactose fermentation.
When plated over Eosin Methylene Blue (EMB) Agar, the gram-negative bacterial sample
provided metallic green to blue colour colonies (positive result). The blue color signified
that the gram-negative bacterial sample is successful in fermenting lactose. This lactose
fermenting gram negative rod was then plated over the mannitol agar and no colour change
was observed. This test acts as a second confirmatory test, proving that is a gram negative rod
and not a gram negative Staphylococcus coccus (Bautista-Trujillo et al. 2013). The lactose
fermenting gram negative rod is then checked for the catalaze activity and it showed positive
results with the emission of bubbles, indicating the bacterial sample has active catalaze or
peroxidase enzyme enzyme and hence do not fall into the category of the strict anerobes
(Iwase et al. 2013). The oxidation fermentation test proved that the bacterium is facultative
anaerobe. The unknown bacterial sample reacted in the same way as that of the Escherichia
coli in the both the open (no paraffin oil attached at the top) and at the closed (paraffin oil
taached) systems. This indicates that the unknown bacterial sample could be Escherichia coli.
Important Findings
The importance of the findings showed that the bacteria is gram negative in nature
and that means it has thin layer of peptidoglycan with an outer layer of thick Lipo-
polysaccharide membrane (LPS). In EMB agar, the Eosin Y and methylene blue are pH
indicator it distinguish between lactose fermenting and nonlactose fermenting bacteria. The
lactose fermenters produce dark colonies with clear borders while the non-lactose fermenters
produce colourless colonies.

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The importance of Mannitol salt agar test lies in the fact that it hsowed that the sample
is not Staphylococci. Mannitol-using bacteria produce yellow colour and the bacteria which
do not use Mannitol shows no change in the colony (De Visscher et al. 2014).
The importance of catalse test is, the bacteria, which contains flavoproteins reduce
oxygen to produce hydrogen peroxide (H2O2) or superoxide (O2-). H2O2 is an extremely
toxic oxidising agent that destroys the cellular constituents. Obligate aerobes/facultative
anaerobes contains enzymes, superoxide dismutase that catalyses H2O2 into harmless water
and oxygen.
2O2- + 2H+ → O2 + H2O2 (enzyme: Superoxide dismutase)
2H2O2 → 2H2O + O2 (enzyme: Catalase or peroxidise)
Such catalase production and activity can be detected by using substrate for H2O2 or the
oxygen thus produced emits bubbles giving the positivity of the test (Hemraj et al. 2012).
The importance of the oxidase test helps in the identification of the bacteria that has
cytochrome C oxidase activity and finally the importance of the oxidation and fermentation
reaction. The oxidative-fermentative (OF) test was first developed by the famous
microbiologist Hugh and Leifson in the year of 1953. They developed OF media in order to
differentiate between the oxidative bacteria (that is capable of producing acid from
carbohydrates (glucose to pyruvic acid) under strict aerobic condition or supply of oxygen
only) and fermentative bacteria (that is capable of producing acid both under aerobic and
anaerobic conditions). The principle of PF test is the gram-negative rods metabolize glucose
into pyruvate via fermentation or by aerobic respiration (oxidatively). During the anaerobic
respiration, pyruvate is converted into weak acids (pyruvic acids) depending on the type of
fermentation. This high concentration of acid produced as a result of the fermentation, turns

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the bromthymol blue indicator in the OF media from green to yellow. The non-fermenting
gram-negative bacteria metabolize glucose via aerobic respiration and thus produce weak
acids during glycolysis and hence produce difference in colour. They also fail to survive in
the absence of oxygen and fail to undergo fermentation (Bhuyar et al. 2012).
The process of fermentation is used in the food processing industry for converting
carbohydrates into alcohol. Microorganisms such as yeast or bacteria that employ anaerobic
respiration are used in this process. Widely consumed fermented foods that are processed by
bacteria are vinegar, cheese, yoghurt. Escherichia coli are used for the production of ethanol,
lactate, succinate and acetate (Förster, Andreas and Johannes et al. 2014).
Conclusion
Thus from the above study, it can be concluded that the unknown bacterial sample is
Escherichia coli. The bacteria is gram negative and features rod shape. The bacterium is also
show to give positive result in the oxidation/fermentation reaction.
Gaps in the research
There are several gaps in the identification study. In the future research procedure,
there must be provision or steps to determine the external configuration of the bacteria with
the help of the background staining or negative staining (Murray et al. 2015), determination
of the flagella (live staining) and endospore (with the help of the endospore staining) (Mahon
et al. 2014).

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