LABORATORY #2: DOMAIN BACTERIA
Required materials:
1. Light Microscope & lens paper
2. Microscope slides & coverslips
3. Prepared slides
a. Neisseria gonorrhea – cocci
b. Treponema pallidum & Spirillum voluntans - spiral
c. Mycobacterium tuberculosis – bacillus
d. Nostoc – filamentous/colonial cyanobacteria
e. Anabaena – filamentous/colonial cyanobacteria
4. Wet mount supplies and wet mount samples of cyanobacteria
Prokaryotes are a large and varied group of single-celled organisms that are commonly known as
bacteria. However, today’s biological classification schemes have taken “true bacteria” and placed
them into a large clade called Domain Bacteria. This classification scheme has also identified
single-cell organisms that share many characteristics with bacteria but are distinct enough to
warrant placing into another domain. These relatives to bacteria are now placed into the Domain
Archaea. Together the Bacteria and Archaea can be grouped together into a “superdomain”
called the Prokaryotes.
Prokaryotic cells do not possess a well-defined nucleus. Instead, the genetic material is found in
the nucleoid region of the cell’s cytoplasm. The DNA is organized and some texts refer to it as a
chromosome, but technically it is called a genophore. Only eukaryotic cells have DNA organized
by histones into the form known as a chromosome.
In addition to being surrounded by a plasma membrane, prokaryotic cells are also surrounded by
a cell wall. This cell wall is not like the cell wall of a plant or a fungus. It is a unique structure
comprised of two sugars (NAG and NAM) cross-linked to one another by peptide chains for
strength. Without the cell wall, bacteria die due to a rapid influx of water from their surrounding
environment. Many antibiotics work by preventing the production of a cell wall when bacteria
divide. The cell wall of bacteria can be thin and relatively simple in construction (gram-positive
bacteria) or can be thicker because of the lipopolysaccharide layer found outside of the cell wall.
These bacteria are called gram-negative bacteria because they resist staining using a technique
called…… Gram staining!
Gram staining is only one way biologists can classify bacteria. Another well-used mechanism is
to classify them by cell shape. This scheme gives three shape, round or coccus; rod-shaped or
bacillus or spiral. Frequently, microbiologists classify bacteria using both cell shape and gramstaining.
Figure 2.1: Bacteria shapes
Laboratory Exercise #2.1: Kingdom Eubacteria
In this exercise, you will examine some representative members of Domain Bacteria, the largest
of the three domains on planet Earth. This domain is made up of one kingdom – Kingdom
Eubacteria which means “true bacteria”. While the taxonomic group known as a “Kingdom” is
falling out of favor, with many biologists suggesting that Domains are divided up into Phyla, we
will keep this classification group for now.
Kingdom Eubacteria contains most of the unicellular prokaryotes that were originally in
Kingdom Monera. Those prokaryotes that are not considered eubacteria are now grouped into
Domain Archaea. You will not be observing these in class.
The Eubacteria are the most primitive and smallest forms of cellular life, measuring only 1 – 10
um in diameter. Many bacteria will be barely visible with the compound light microscopes you
use in lab. These bacteria are likely the descendants of the first cells that evolved on our planet
billions of years ago!
The taxonomic classification of Eubacteria currently identifies 29 distinct phyla. Since there is
still some argument about this, the figure below shows some of the accepted phyla that have
been created based on the DNA sequences found in bacterial ribosomes.
Some well-known bacterial phyla are represented in the figure below, including the grampositive bacteria and several gram-negative bacteria such as the cyanobacteria, the purple-sulfur
bacteria (i.e. proteobacteria) and the chlamydiae.
In this lab exercise, you will observe slides of some well-known gram-positive and gram-negative
bacteria that are cocci, bacilli or spiral in shape. You will have to use the oil immersion lens in
order to see these bacteria. Refer to lab #1 to remember the oil immersion protocol.
Figure 2.2: The phylogenetic tree of life. Some of the accepted phyla in Domain Bacteria are
shown, including the gram positive bacteria, the purple-sulfur bacteria (now known as
proteobacteria) and the chlamydiae.
A. Observe the prepared slides of Neisseria gonorrhea, Treponema pallidum &
Mycobacterium tuberculosis using the 40X dry and the 100X oil immersion lenses. Draw
what you see in your field of view in the spaces below with respect to cell shape and size.
Be sure to note your magnification. Classify these bacteria by shape and by gram-staining.
Laboratory Exercise #2.2: Cyanobacteria
Most eubacteria are heterotrophs (“other feeders”). They acquire organic food from outside
sources by internalizing it. This is in contrast to organisms that produce their own food from
sunlight. These organisms are called autotrophs. You are very familiar with autotrophs. You
know them as plants. However, there are autotrophic bacteria in Kingdom Eubacteria. They
used to be called blue-green algae but are now known as the Cyanobacteria.
There are more than 1,500 species of cyanobacteria found in both marine and fresh-water
environments. Most of the disagreeable tastes and odors of drinking water are caused by these
types of bacteria. Some cyanobacteria produce hepatotoxins or neurotoxins. There is a link
suggesting exposure to the neurotoxins of some cyanobacteria can result in the development of
ALS. However, most cyanobacteria are not harmful to human health.
Cyanobacteria are able to undergo photosynthesis because they possess light absorbing pigments
Figure 2.3: Photosynthesis in plants and
cyanobacteria
that are used in photosynthesis. These pigments are found in a light-harvesting complex (a
phycobilisome) that is very similar to the photosystem II complex found in photosynthetic plants
and algae. In fact, the chloroplasts of algae and plants are thought to be have descended from
cyanobacteria engulfed by these cells – a process called endosymbiosis. Cyanobacteria can
undergo photosynthesis because they possess chlorophyll a (although some will possess
chlorophyll b). Chlorophylls are green-colored pigments (“chloro” means green) that use the
energy in sunlight to power the conversion of atmospheric CO2 into sugar through a process
known as carbon fixation. In plants, this sugar becomes part of the plant’s biomass, available for
use in a wide variety of metabolic processes. In cyanobacteria, it is also stored – mainly as a
source of cellular energy (i.e. ATP) for their metabolic reactions. Cyanobacteria possess
numerous types of pigments in addition to chlorophylls. Therefore, they can appear yellow,
orange, red or purple, depending on these other possess.
Cyanobacteria are unicellular or colonial. In colonial cyanobacteria, as the cells divide, an outer
slime layer causes the individual cells to stick together forming a colony. Different species of
cyanobacteria will exhibit different colonial forms, such as filamentous or spherical or mat-like.
The cyanobacteria will be observing, Nostoc and Anabaena form long filamentous colonial forms.
In this exercise, you will observe the prepared slides of the cyanobacteria, Nostoc and Anabaena.
1. Nostoc: Under high power (400X), you may notice that a small fraction of the cells in the
Nostoc colony are different from the majority you see in the colony. These larger, rounder
cells are called heterocysts. These cells remove nitrogen (N2) from the environment (i.e.
the surrounding water) and convert it into nitrogen-containing compounds through a
process called nitrogenfixation. These compounds are
used by the plant to grow. In
addition to heterocysts, you
will see that the majority of the
colony is made of round cells.
These cells are called
vegetative cells and are the
ones that undergo
photosynthesis and divide to
increase the size of the colony.
Draw the Nostoc colony in the
space given to you and label
the different types of cells.
2. Anabaena: Observe the colonial cyanobacteria Anabaena under high power. Like Nostoc,
this cyanobacteria has round, dividing vegetative cells and heterocysts. But they also have
another type of cell called an akinete. The akinete is a dormant cell. When a colony breaks
apart and a new colony starts to grow it is the akinete that creates the new colony by
undergoing very rapid division. Under the microscope, the akinete is larger than the
heterocyst and its inside has a granular appearance.
Draw the Anabaena colony in the space below and label the different types of cells in your
drawing and the one given to you.
Figure 2.4: Anabaena
Review Questions
1.
How do the cyanobacteria differ from bacteria?
2.
Write the overall equation for photosynthesis performed by cyanobacteria
3.
Make a drawing of a bacillus bacteria
4.
Make a drawing of a coccus bacteria
5.
What does the heterocyst of a cyanobacteria do?
6.
What are the three Domains of life?
7.
To what domain does an animal belong?
NOTES: Use this space to make any notes regarding this laboratory