Biology 213
Lab 1: Prokaryotic Diversity
Objectives:
o Describe prokaryotic cell shape and structure
o Learn to use the oil-immersion lens on a compound microscope
o Describe prokaryotic colony structure: morphology, shape, growth patterns
o Learn the process of gram-staining and how to identify bacterial specimens based on the results of
gram-staining
General Introduction:
Microorganisms are everywhere, in the air, soil, and water; on plants and rock surfaces, and even in hot
springs and ice. Millions of microorganisms are found living on and with animals, in places such as the skin, the
mouth, the nose and the intestines. In this lab, we will exam the diversity of the two prokaryotic kingdoms.
Using prepped slides, we will exam the variety of shapes and sizes of prokaryotic cells. We will also sample
material from our bodies and from our surroundings to determine what microorganisms are around us.
Prokaryotic organisms are typically unicellular organisms in one of two domains: Archaea and
Eubacteria. In this lab, we will exam bacteria from the latter domain. Prokaryotic cells are simpler than
eukaryotic cells in that they lack a nucleus, as well as other membrane-bound organelles. These cells typically
have a cell wall (often used to distinguish one cell from another. Often they live in colonies that may also have
distinguishing characteristics.
One word of caution: Some of the microorganisms that we will grow are pathogens (capable of
causing disease), so once you close the petri dish for the day, do not open it again.
A) Cell Morphology
Examination of individual prokaryotic cells reveals a diversity of size and shape. Most prokaryotic cells
can be described by their basic cellular shapes: bacillus (rod shaped), coccus (spherical), and spirillum (helical
or spiral). In some species, cells clump together to form aggregate colonies (each cell still maintains metabolic
and reproductive independence). These colonies can be described by how the cells are positioned next to
each other: chains of cells are described using the prefix “strepto-“, and clusters of cells are described using the
prefix “staphylo-“.
Procedures:
1. Using the “mixed” bacteria slides, focus in on the individual bacterial cells (using the oil immersion
lens). Note that using oil immersion requires both training and effective clean-up!
2. Find and draw representative bacteria for the shapes and colonies described above in your lab
notebook.
3. As always, you should label your drawings with the total magnification, shape, color, and colony
description. Make sure you identify critical structures in every sketch you make!
B) Colony Morphology (Culturing common prokaryotes)
A colony of bacteria grows from a single cell dividing through binary fission to become millions of cells.
Each colony has a characteristic size, shape, consistency, texture, and color. These characteristics can be
used as we classify bacterial species. Use Table 1 below to familiarize yourself with some of the terms used for
colony description.
To grow microorganisms, we must provide a proper medium for them to grow on. We will use a nutrient
agar that is a gelatinous (jelly-like) medium containing nutrients that the microorganisms use as food.
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Temperature is also important, and we will grow our microorganisms at the appropriate temperature (37 C for
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body microorganisms and 25 C for “environmental” microorganisms). The microorganisms will eat, grow, and
reproduce on the agar so that after a few days we will be able to see, without a microscope, colonies of
bacteria, mold and other microorganisms that we have "captured". There may be fungal colonies as well on
your agar plates. The fungal colonies appear fuzzy when compared to bacterial colonies. If you have fungal
colonies, Do Not Open your agar plates as the colony may release pathogenic spores into the air. We will use
these plates to study fungal colonies in a later lab exercise.
Lab 1: Prokaryotic Diversity
Table 1: Terminology used in describing bacterial colonies
Colony Characteristic:
Terms and Definitions:
Colony shapes
Punctiform (under 1 mm in diameter), Round, Filamentous, Irregular
Colony edges
Smooth, Curled, Wavy, Filamentous
Colony surfaces
Smooth, Concentric, Wrinkled, Contoured
Colony color
White, Cream, Yellow, Orange, Red (and others)
Materials: each individual (or pair) needs
2 agar plates, sterile swabs (as needed), 1 tube of sterile water, 1 marking pen or grease pencil
Procedures (Set-up):
1. Each student (or pair of students) will receive 2 petri dishes. (Notice that the lid of the dish fits loosely
over the top.)
2. Label the bottom of the plates with your name, class and the date.
3. Divide the plate into halves, by drawing a line down the center of the bottom of the plate. (There
will be an example in the front of the room or you can ask your instructor if you have questions.)
4. Once you get the plate labeled, you can inoculate the agar with microorganisms.
5. For the “environmental” plate, you can sample from anywhere in the room. One suggestion though
is not to sample from metallic surfaces, as not many microorganisms grow on these surfaces. To
sample an area, dip a swab in the sterile water (remember to replace the lid!), and then wipe the
swab on any surface you want. Lightly, touch the swab to one of the halves of the environmental
plate. Then label the plate according to the area you sampled. Then taking another swab, sample
another area. Some suggestions include: the bottom of your shoe, a corner of the room, your
desktop, your neighbor's desktop or wherever else you can think of (be creative).
6. For the “body” plate, you can use a swab dipped in sterile water to sample from parts of your body.
(One suggestion is not to sample from your mouth as the microorganisms in your mouth do not grow
very well on the plates.) You can sample from under your nails, your nose, your eyelids, your skin, or
wherever else you think of. Again label the halves of your "body" petri dish based on the area you
sampled.
One interesting experiment you can attempt for the body plate is:
Lightly touch your fingers to one half of the petri dish and label that half of the
"dirty" (or "prewash" if you prefer). Then wash your hands, and touch the other
half of the agar with your fingers, and label that half of the petri dish "clean" (or
"postwash" if you prefer). You can then see how well you wash your hands!
7. When you have finished sampling, return the labeled dish to the instructor for incubation. Be sure to
record your notes on your collection in your lab notebook! In the next session, we will examine what
we have grown!
Procedures (Observations): Again, I caution everyone to not open your plates as there may be pathogenic
organisms in the agar. We will see many colonies on the agar plates, but they can be separated into two types
of organisms: bacterial and fungal. The bacterial colonies will appear soft and glistening and often white,
cream-colored, yellow, or orange. The mold (fungal) colonies will appear large and fluffy (very scientific, I
know). As you find different colonies, share them (visually) with other students, and also with the instructor.
8. Observe your plates as well as the plates of those students around you. Try to get an idea of the
diversity of bacteria in our environment and the microhabitats where they are found.
9. Draw the bacterial and fungal colonies on each of your 4 agar plate “halves” in your lab notebook.
When drawing the colonies and plates, try to draw the colonies in correct proportions.
10. Then draw representatives of each specific colony on the agar “halves” in your notebook, taking
notes on the color and other details your sketch might not capture.
11. Label each colony as either a bacterial or fungal colony, and use the terms in Table 1 to describe
each colony.
12. Try to name each colony more specifically using the information you receive from your instructor.
C) Gram Staining
We will Gram stain several species of bacteria (for example: Escherichia coli, Staphylococcus
epidermidis, Bacillus cereus). It is very important that you follow the steps described in this lab as this is a very
precise procedure. Your lab instructor will also review the proper procedure specific for our equipment and
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Lab 1: Prokaryotic Diversity
materials. Most important is the order in which you use the stains: Crystal violet, water, Iodine, water, Alcohol,
water, Safranin. Use the bacterial cultures independently or as mixes based on the request of your instructor.
(Note: see Figure 27.3 for the “maps” of the gram-positive and gram-negative bacterial cell walls.)
Materials: each pair needs
Bacterial specimens
Micro “kit” (containing loops, fire source, microscope slides, crystal violet, water, iodine, decolorizer, safranin)
Procedures:
1. Heat-fix a smear of a bacterium as follows:
a) Using the dropper bottle of distilled water found in your staining rack, place 1/2 a drop of water on
a clean slide by touching the dropper to the slide.
b) Aseptically remove a small amount of the bacterium from the agar surface and mix it with the
water. Flame the loop and let it cool.
c) Using the loop, spread the mixture over the slide to form a thin film.
d) Allow this thin suspension to completely air dry.
e) Pass the slide (film-side up) through the flame 3 or 4 times to heat-fix.
2. Stain with crystal violet for one minute. Gently wash with water. Shake off the excess water but do not blot
dry between steps.
3. Stain with gram's iodine solution for one minute and gently wash with water.
4. Decolorize by adding gram's decolorizer drop by drop until the purple stops flowing and wash immediately
with water.
5. Stain with safranin for one minute and wash with water.
6. Blot dry and observe using oil immersion microscopy.
7. Record your results, observations, and conclusions in your lab notebook.
Repeat the above procedures with the second bacterial sample.
Be sure to record your observations in your notebook as you work!
Prelab: Answer the following questions on a separate piece of paper. These pre-labs are due at the start of
your lab period. As these questions are designed to prepare you for lab, no late prelabs can be accepted.
1. In your own words, describe the difference between the outer surface of a gram positive and gram
negative bacteria.
2. Describe the function of each of the following solutions in the gram-stain process: Crystal violet,
Iodine, Alcohol, Safranin.
Post-Lab Instructions: In our imaginary scenario, the cultures we worked with today were critical lab strains of
two different bacteria, Escherichia coli, and Staphylococcus epidermidis. Your biotech company needs to
continue their work on these two strains in order to keep the board of directors happy. Unfortunately, one of
the technicians in the lab forgot to label the two cultures, so now no one knows which is which, and all work on
the project has come to a sudden halt! The project manager has called on you to distinguish the two. For your
post-lab, write her a memo justifying your identification of these two strains, including as much data, and as
many observations as possible to create a compelling argument. Note that the project manager’s training is in
business. Thus you will need to explain the scientific basis of your work (including the gram staining process) to
her in your memo. Like all our post-labs, this typed assignment is due one week after you complete the lab.
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