BIO 320

INTRODUCTION TO BIOLOGICAL
DIVERSITY

Title of Practical: Eubacteria

Group: A4AS1204_03
Group Members:
Name Matric No Result Score
Judith Somiyar Raymond 2017641874
Muhammad Hazwan 2017681342
Hamim bin Shahfar Amil
Muhammad Danial bin 2017854412
Zulkepli

Date of experiment: 18th March 2019

Lecturer’s Name:
1.0 Title of Experiment:
Eubacteria

2.0 Objectives
1. To define coccus, bacillus, spirillum, Gram stain.
2. To describe and explain characteristics of eubacteria.
3. To identify and classify the organisms studied in this exercise.
4. To distinguish Gram-positive and Gram-negative bacteria, indicating their
susceptibility to certain antibiotics.

3.0 Introduction

Eubacteria, or “true” bacteria, are single-celled prokaryotic microorganisms that

have a range of characteristics and are found in various conditions throughout all parts of
the world. Since eubacteria is so common, it comprises one of the three main domains of
life, along with the Archaea and the Eukarya.

Eubacteria are prokaryotes, meaning their cells do not have nuclei in which
their DNA is stored. Eubacteria are enclosed by a cell wall. The wall is made of cross-
linked chains of peptidoglycan which is a polymer that combines both amino acid and
sugar chains. This gives the wall of the bacteria the strength needed to maintain its shape
and size during changing environments. Unlike the eukaryotes, bacteria have cholesterol
present in the membrane to enhance permeability of the membrane and increase stiffness.

Eubacteria are typically classified into five different phyla: Chlamydia,

Cyanobacteria (Blue-green algae), Gram-positive bacteria, Proteobacteria, and
Spirochetes. Other than that, bacteria commonly take on one of three shapes: bacillus,
coccus and spirillum. Bacillus have a rod shape, coccus have a spherical shape, and
spirillum have a spiral or wave shape. Their shape was often taken or used as a
classification system until recently.
4.0 Methodology

4.1 Experiment 1: Bacteria (Heterotrophic Eubacteria): Are bacteria Present in

the Lab?

1. Four petri dishes containing sterile nutrient agar has been labelled as “Dish 1:
Control”, “Dish 2: Dry Swab”, “Dish 3: Treatment A,” and “Dish 4: Treatment
B”.
2. The surface of the dish can in the lab was swabbed using a sterile cotton swab.
3. The lid of Dish 2 were lifted slowly as little as possible to run the swab over
the surface of the agar without ruining it.
4. The lid was securely taped to half of bottom of the dish.
5. Two different tissue paper were soaked with Liquid A (tap water) and Liquid B
(70% ethyl alcohol) respectively.
6. Tissue paper soaked with liquid A was used to wipe the surface of the sterile
nutrient agar in Dish 3 while the other tissue paper soaked with Liquid B was
used to wipe the agar surface of Dish 4.
7. Step 2 until step 4 was repeated with different surface areas such as the surface
under table for Dish 3 and the surface of the sink in the lab for Dish 4.
8. The cultures were placed in an incubator oven for 2 days.
9. After 2 days, the culture was scraped by using needle in preparing a wet mount
slide to observe the shape of the bacteria under the compound microscope by
starting with the medium-power objective up to the highest magnification
(1000x).
8. The morphology of the bacterial colonies was observed under dissecting
microscope and light microscope. Observations were recorded.

4.2 Experiment 2: Bacteria (Heterotrophic Eubacteria): Bacteria Shape and

Sensitivity to Antibiotics

1. Samples of Gram-stained bacteria was observed under light microscope

starting from the medium-power magnification up till the highest
magnification (1000x) using oil immersion.
 2. The species that was observed were in Table 3.2 below according to their
staining characteristics.

Bacterial Species Gram Reactions (+ or -)

Mixed Coccus Gram Stained +

Gram Negative Spirillum -

Gram Negative Bacillus -

Table 4.2: Gram Stain Reaction of Various Bacteria

3. Shapes of the Gram-stained bacteria was drawn.

4.3 Experiment 3: Cyanobacteria (Blue-Green Algae)

4.3.1 Oscillatoria
1. The demonstration slides were examined using light microscope
starting with the medium-power objective up till the highest
magnification. The individual cell was observed in presence of the
filament.
2. Portion of the filament was drawn and labelled.

4.3.2 Anabaena
1.The demonstration slides has been examined using the compound
microscope in starting with the medium-power objective and finally
with the highest magnification. The individual cell has been
observed in presence of the filament.
2. The observation was drawn and labelled.
3. Heterocyst was identified within the filament and labelled in the
drawing.
5.0 Results

5.1 Experiment 1
Characteristics of each of the colony that exist first in four of the petri dishes.

Characteristics Petri Dishes Containing Sterile Nutrient Agar

A: Control B: Dry swab C: Treatment D: Treatment
A B
Shape No Filamentous Punctiform Circular
Margin No Filamentous Undulate Entire
Elevation No Umbonate Raised Convex
Size No 30 mm 1-2 mm 5-6 mm
Texture No Friable Friable Butyrous
Optical Property No Translucent Opaque Opaque
Colour No White White Yellow
Shape of bacteria No Bacillus Bacillus Coccus
under light
microscope
(Total
magnification:
1000x)

Table 5.1: Bacterial Colony Morphology

5.2 Experiment 2
The Gram-stained bacteria were drawn together with its reaction towards the
gram stain and the total magnification used under light microscope.

5.3 Experiment 3
Shape of Oscillatoria and Anabaena were drawn and labelled together with the
total magnification used under light microscope.
6.0 Discussion

6.1 Experiment 1

Petri Dish A was labelled as a control variable because the sterile nutrient
agar in Dish A was not swabbed or treated. The lid of Dish A was not lifted too to
avoid any bacterial organisms effecting the agar. Dish A was used as a baseline to
allow comparison between other dishes that was swabbed or treated.

In Petri Dish B, the dry sterile cotton swab that was swabbed over the
surface of dish can in the laboratory produced a filamentous bacterial colony with
a filamentous edge. The elevation of the colony was umbonate which made the
colony elevate unevenly. The colony took almost one fourth of the agar surface,
approximately 30 mm. The white translucent colony have a friable texture which
is dry and powdery. When observed under light microscope with total
magnification of 1000x, the shapes of the bacteria were rod-shaped.

Petri Dish C was treated with tap water, the sample was taken from under
the table. The colony obtained from the sample was in punctiform with an
undulate edge. The elevation of the bacterial colony was raised with
approximately between 1.0 mm to 2.0 mm diameters. The texture of the colony
was dry and powdery with an opaque optical property. The white colony was rod-
shaped in shape when observed under light microscope with total magnification
of 1000x.

In Petri Dish D, which was treated with 70% ethyl alcohol, yellow circular
bacterial colonies were obtained. The sample was taken from the laboratory sink.
The colonies were approximately 5.0 mm to 6.0 mm in size with an entire edge.
The butyrous (buttery texture) opaque colonies were elevated convexly. A shape
of the bacterial colonies was spherical in shape when observed under light
microscope with total magnification of 1000x.

In this experiment, the non-pigmented (white) colonies were mainly

bacillus colonies while the pigmented (yellow) colonies were coccus colonies.
6.2 Experiment 2

In this experiment, bacteria were identified through their shapes under light
microscope and how they react to gram stain. After staining, the purple bacteria
were classified as gram-positive while pink bacteria were gram-negative.

The Mixed Coccus Gram Stained was classified as gram-positive because it

retains the purple colour of the gram stain. Under total magnification of 1000x of
light microscope, the stained bacteria were spherical in shape in forms of either
pairs or clusters. Both Gram-negative Spirillum and Gram-negative Bacillus
changes the colour of the purple stain into pink, thus both were gram-negative
bacteria. Under light microscope, gram-negative Spirillum were spiral in shape
while the Gram-negative Bacillus were rod-shaped.

The gram-positive bacteria were able to retain the purple colour of the gram
stain because gram-positive bacteria have very thick cell walls consisting of
several layers of peptidoglycans held by amino acids. Alcohol which involves in
gram staining process acts as a decolourizer. Alcohol dehydrates gram-positive
bacteria thus causes the pores of the cell walls to shrink, tightens and trap the
crystal violet dye.

However, in gram-negative bacteria, alcohol penetrates readily into the thin

lipid-rich walls and causes the crystal violet stain to be released from the cell
walls. Cell walls of gram-negative bacteria consist of two layers which were a
thin layer of peptidoglycan and layer of thick outer membranes rich in
lipopolysaccharide. Another process in gram staining which involves a
counterstain – safranin, a red water-soluble stain was absorbed by the cell walls
of the decolourized gram-negative bacteria making it red or pink when observed
under light microscope.

It if found that gram-negative bacteria such as pathogenic strains of

Escherichia coli have higher resistance to antibodies and antibiotics than gram-
positive bacteria because of their lipopolysaccharide outer membrane. This shows
 that gram-positive bacteria such as Methicillin-resistant Staphylococcus aureus
(MRSA) have higher sensitivity towards antibiotics compared to gram-negative
bacteria and were easier to be treated because antibiotics were able to inhibit the
growth of bacteria.

6.3 Experiment 3

6.3.1 Oscillatoria

Oscillatoria is a genus of a filamentous cyanobacteria

classified as a heterotrophic bacterium which can synthesize their
own food by obtaining energy from light (photoautotroph). Under
light microscope, Oscillatoria can be seen in blue colour. It is
observed that the cells are arranged in elongate filaments. Between
several short cells, lies a separation disk. Oscillatoria cells look alike
except the end cell which is convex in shape.

There was differentiation of cells within the filament because

there was a formation of separation disk. These separation disks are
mucilaginous, pad-like and are similar in shape with its neighbouring
cells. Filament breaks apart at separation disk to release hormogonia.
Hormogonia are short segments of trichome consisting of few cells.

6.3.2 Anabaena

Under light microscope with total magnification of 1000x, it

can be seen that Anabaena is a blue-green cyanobacteria which is
cylindrical or barrel shaped of a long chain and has a glassy structure.
Anabaena is a genus of filamentous cyanobacteria. In between the
cells of Anabaena lies heterocyst, specialized cells impermeable to
oxygen providing anaerobic environment necessary for the operation
of nitrogen-fixing enzymes.

No nucleus was present in Anabaena as well as membrane

bound organelles which by then classifies them into Domain
 Prokaryote. Domain is the highest taxon in taxonomy. Anabaena have
trichomes but they are lack of endospores and exospores.
Anabaena live as symbionts within organisms. The association
between Anabaena and water fern Azolla is mutualistic symbiosis.
Both organisms benefit from living together providing a safe
environment for the cyanobacteria in exchange for nitrogen.

7.0 Conclusion

In this experiment, the objective was to define coccus, bacillus, spirillum, Gram
stain, describe and explain characteristics of eubacteria, to identify and classify the
organisms studied in this exercise and to distinguish Gram-positive and Gram-negative
bacteria, indicating the susceptibility to certain antibiotics.

First of all, from this experiment, we can define coccus is a shape of bacteria
which were spherical in shape. For bacillus, the shape the bacteria are rod-shaped. Next,
spirillum is the shape of bacteria that are coil-shaped with curved forms. Gram stain is a
differential staining technique in which cells either pink (gram-negative) or purple
(gram-positive) depending upon the structural composition of their cell walls.

Eubacteria were diagnosed as unicellular but some may be found in colonies or

filaments with specialized cell. Based on the observation, bacteria have various shape
such as spherical, rod and spiral/helical. Bacteria has lack of nuclear membrane and
membrane-bounded organelles are absent. In this exercise, three samples were observed
under light microscope categorizing between Gram-negative and Gram-positive. For
Gram-negative bacteria which have thin cell wall consisting of two layers of
peptidoglycan and outer membrane, Gram-negative Spirillum and Gram-negative
Bacillus are in this category. Mixed coccus gram stained are categorized in Gram-
positive due to its thick cell wall compromising of peptidoglycan.

Lastly, difference between Gram-positive bacteria and Gram-negative bacteria

was that Gram-positive retained crystal violet dye when washed with absolute alcohol
but Gram-negative bacteria decolourized. Gram-negative bacteria were then stained once
more absorbing a counter stain of red or pink.
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