O Describe Prokaryotic Cell Shape and Structure

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. Temperature is also important, and we will grow our microorganisms at the appropriate temperature (37oC for body microorganisms and 25oC 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.

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 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.