Posted: August 28th, 2023

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BSC2011C: Principles of Biology II

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Module 2 Lab: Prokaryotes


Read the information in your textbook and in the document below, then complete all the activities in this worksheet (Activities A through D).


As a group, the prokaryotes possess numerous metabolic pathways that are not found in the eukaryotes. For example: many can ‘fix’ nitrogen (convert nitrogen gas into ammonia – a form plants can use to build proteins and other molecules); others can use light energy to drive their metabolism, but they don’t necessarily use chlorophyll a or release oxygen; and, some are actually poisoned by the presence of oxygen gas.

As our understanding of these organisms increased, it became obvious that they are not all closely related. Current classification systems place the prokaryotes in two domains – Archaea and Bacteria – and all the eukaryotes in another. Again, this underscores their extreme amounts of difference, both when compared to each other and when compared to the eukaryotes.

In this lab, we will briefly survey the structural variability and some of the metabolic diversity of these groups.

Activity A: Use your textbook to review the general structure of prokaryotic cells. Fill in the table below to contrast them to typical eukaryotic cells:

Present in all cells

Present only in (some or all) prokaryotes

Present only in (some or all) eukaryotes




Cell membrane

Flagella made of microtubules


Membranous organelles

Cell wall

Divide by mitosis

Divide by binary fission

Part I: Domain Bacteria

The majority of prokaryotes belong to domain Bacteria, including all those known to cause human diseases as well as many that are either beneficial or harmless. Members of domain Bacteria occupy diverse habitats, including on and in other organisms, fresh and salt water and soil. Many form mutually beneficial relationships with eukaryotes, including ourselves.

Typical photosynthetic bacteria – the cyanobacteria

These bacteria are the only prokaryotic group that does ‘typical’ photosynthesis, using chlorophyll
a and releasing oxygen from water. They are the closest relatives of the eukaryotic chloroplast. They also include species with some of the largest prokaryotic cells and some which show limited cooperation between cells and specialization of function.


Oscillatoria (Figure 1)
has the cells arranged in elongate filaments.


Anabaena (Figure 2)
is another filamentous genus, which will have occasional cells that are enlarged and have thickened end walls. These large cells are
heterocysts, which fix nitrogen.


Nostoc (Figure 3)
is similar to

, but differs strongly in the amount of
gelatinous matrix that it secretes. When growing on moist soil, it actually forms macroscopic spheres.

Figure 1

Figure 2

Figure 3

Purple Non-sulfur Bacteria

This is one of several groups of bacteria that use chlorophyll-like molecules to capture light energy, but photosynthesize without producing O2. Reminder: O2 is generated by photosynthesis only when chlorophyll
a uses water as a source of H+ and electrons. These bacteria don’t generate O2 because they use different pigments and molecules other than water as sources of electrons.

A: Bacterial Identification by Shape

One of the ways that prokaryotes are categorized is by their shape:

Coccus Bacillus Spirillum

Although some prokaryotes live independently as individual cells, others remain joined together rather than separating after cell division. They form colonies that appear to be strings or grape-like clusters.

Diplococcus Streptococcus Staphylococcus

Some rod-shaped (bacilli) bacteria contain spores, called

These are formed when the cells are faced with adverse conditions, such as high temperature. When environmental conditions are more favorable for survival, the spores germinate to form new cells.

B: Bacterial Identification by Gram Stain

Gram staining is a common technique used to differentiate bacteria based on their different cell wall constituents. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safranin.

The Gram staining process is as follows:

1. Cells are stained with crystal violet dye. Next, a Gram’s iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.

2. A decolorizer, such as ethyl alcohol or acetone, is added to the sample; this dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram-positive bacteria. Conversely, the outer membrane of Gram-negative bacteria is degraded and the thinner peptidoglycan layer of Gram-negative cells is unable to retain the crystal violet-iodine complex and the color is lost.

3. A counterstain, such as the weakly water-soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram-positive cells. However, the decolorized Gram-negative cells are stained red.

In summary, due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram-positive and Gram-negative bacteria, Gram-positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram-negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process.

Activity B: Gram Stain Activity

· Click the hyperlink to view the Gram stain animation:

· Click “Start” and allow animation to run.

· Click “View Slide under the Microscope”

· Click “Examine Samples”

· Complete the table below

Sample Name

Physical Description

(What does it look like?)

Example species

Gram-positive cocci in clusters

Gram-positive cocci in chains

Gram-positive diplococci

Gram-positive pleiomorphic* rod

Gram-positive rod

Gram-negative rod

Gram-negative diplococci

Gram-negative pleiomorphic* rod

*Pleiomorphic: Variable in size or shape of cells.

Activity C: Survey of Prokaryotes

For each of the prokaryote species listed in the table below, fill in the shape, whether the species is Gram-positive or Gram-negative, and what disease condition is associated with the species.

Prokaryote Species


Gram-positive or Gram-negative

Disease Condition(s)

Pseudomonas aeruginosa

Staphylococcus aureus

Cornybacterium diphtheriae

Bacillus anthracis

Haemophilus influenzae

Vibrio cholerae

Escherichia coli

Yersinia pestis

Clostridium perfringens

Heliobacter pylori

Part II: Domain Archaea

The best known archaeans have highly specialized habitats (including, but not limited to, extremes of temperature, pressure and pH) and thus can be quite difficult to culture. Among the easiest to culture are the halophiles (salt-lovers), which typically thrive in solutions of 10 – 40% salt. For comparison, seawater is typically around 3.5% salt.

Halobacterium salinarum
. These organisms are aerobic heterotrophs and can generate ATP from absorbed organic molecules. However, oxygen is often not readily available in their environment (oxygen’s solubility in water declines with increasing salinity), and they have an alternative method of ATP generation. They use a purple pigment,
bacteriorhodopsin, which absorbs light energy and generates ATP.

Note that this is
NOT photosynthesis, because it does not result in the production of carbohydrates from carbon dioxide.

Activity D: Research the Archaea further. Select either one group or a specific species within the Archaea and discuss their relevance to humans.





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