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Objectives:: Questions To Answer in Lab Notebook

This document provides instructions for a laboratory experiment on bacterial cell morphology, colony structure, and gram staining. Students will observe bacterial cells under a microscope and describe their shapes. They will also learn how to gram stain bacteria and identify them as gram positive or gram negative based on staining. Students will take samples from the environment and their own bodies to culture on agar plates and observe different colony types, structures, and growth patterns. The purpose is to describe prokaryotic cell structure and identify bacteria using microscopic techniques.

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119 views3 pages

Objectives:: Questions To Answer in Lab Notebook

This document provides instructions for a laboratory experiment on bacterial cell morphology, colony structure, and gram staining. Students will observe bacterial cells under a microscope and describe their shapes. They will also learn how to gram stain bacteria and identify them as gram positive or gram negative based on staining. Students will take samples from the environment and their own bodies to culture on agar plates and observe different colony types, structures, and growth patterns. The purpose is to describe prokaryotic cell structure and identify bacteria using microscopic techniques.

Uploaded by

tv8
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Objectives:

Describe prokaryotic cell shape and structure Learn to use the oil-immersion lens on a compound microscope Describe prokaryotic colony structure: morphology, shape, growth patterns Learn the process of gram-staining and how to identify bacterial specimens based on the results of gram-staining

Part A. Cell morphology


Questions to answer in Lab Notebook: 1. Are all the prokaryotes seen in this lab the same shape?

2.

Are all the prokaryotes seen in this lab single celled?

3.

What do the terms strepto- and staphylo- mean and are there any examples seen in this lab?

Procedures: 1. Using the mixed bacteria slides, focus in on the individual bacterial cells (using the oil immersion lens). 2. Find and draw ALL representative bacteria seen on the slide. 3. Label your drawings with the total magnification, shape, color, and colony description.

Part C: Gram staining


Materials: each pair needs Bacterial specimens Micro kit (containing loops, fire source, microscope slides, crystal violet, distilled 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.

Questions to answer: (in the Discussion section of your lab notebook) 1. What is the purpose of the grams iodine in the gram staining procedure? 2. What is the purpose of the alcohol in the gram staining procedure?

3. Why dont gram-negative bacteria stain purple?

Part D: Metabolism
Questions to answer in Lab Notebook: 1. Do all the prokaryotes seen in this lab have the same metabolism?

2.

Do all prokaryotes seen in this lab produce ATP and acquire a carbon source using the same metabolic means?

For each question, propose a hypothesis that can be tested to help answer it using the procedure below. Procedures: 1. Make a wet mount slide of live cyanobacterium and draw a representative sample. 2. Describe any observations that might relate to metabolic capabilities of the organism.

Part B. Colony morphology


Materials: 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. a. 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. Place the swabs in the red biohazard bucket 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. When drawing the colonies and plates, use the space provided to draw the full plate, and try to draw the colonies in correct proportions. 10. Then draw representatives of each specific colony on the agar halves. 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.

Questions to answer: (in the Discussion section of your lab notebook) 1. Do colonies of bacteria represent multicellular organisms? Why or why not? 2. In general, how is colony size affected by density of organisms in an area? (hint: compare your plates of different densities) 3. Did your controls on your plates remain clear? If not, can you explain why?

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