Observing Bacterial Specimens Using the Light Microscope

IB HL Biology 2 Objectives: Upon completion of this lab activity, you will be able to: Outline the diversity of Eubacteria, including shape and cell wall structure. Compare the structure of the cell walls of Gram-positive and Gram-negative Eubacteria. To understand an infectious disease, one has to study microbes intensely. This is essential to identifying which specific microbe causes a disease. Among the many microbes one might find living in a sick patient's body, only one is usually causing the disease, and the rest are usually harmless. Sometimes distinguishing one micro-organism from another requires the use of dyes or stains that highlight various structural features of the organism. Many stains colour some kinds of microbe, while leaving others alone. The Gram stain (actually a series of staining and washing techniques) is often used to help classify bacteria. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. The difference between gram-positive (blue) and gram-negative (pink) bacteria lies in the ability of the cell wall of the organism to retain a stain called crystal violet. When bacteria are heat-fixed and stained they tend to clump together. At high dry power individual cells often can't be detected because the space between cell walls is within the limits of resolution of the microscope due to diffraction. To properly view stained bacteria it is necessary to use oil immersion microscopy. Most bacterial species are rod-shaped or round (known as cocci), although some are curved, spiral-shaped, or irregularly shaped. Bacterial cells form clumps which have specific arrangements; paired, in chains, in clusters or simply random. This experiment was modified from the following source: http://www.chemheritage.org/educationalservices/pharm/antibiot/activity/stain.htm Aims 1. To observe bacterial cells from a live specimen under a light microscope. 2. To learn the gram stain technique for the identification of different bacterial species. 3. To complete accurate scientific drawings of bacterial cell arrangements. Materials and Equipment Light Microscope Microscope slides Lens Tissues Prepared slides of Bacteria: 3 Types Yogurt Culture: better if it is close to its use-by date Microcentrifuge tube Toothpicks

Method PART A: Observation of Bacterial Cells under oil-immersion. 1. Obtain the prepared slide labelled Bacteria: 3 Types. Mount the slide with smear up and focus at low power on the etching. Move the slide so that the lens is over the smear itself. Now move the objective away from the slide until the upper surface comes into focus.
Observing Bacteria Using a Light Microscope Adapted from http://www.accessnano.org/files/teaching-modules/health-medicine/nanogold/HM_NanoGold_Experiment_1_Guide.doc

3. A bacterial smear at low power looks like a patch of dirt. Focus on the mess, first at 40x then at 100x and finally 400x, all in bright field mode. Note that you can see some detail at 400x, but the shapes and colors of the bacteria are somewhat distorted. 4. Complete IB quality drawings of both specimens and describe the shape and arrangement of cells. PART B: Examination of bacteria from fresh yogurt cultures. 1. Clean slides and cover slips for dust and other particles. 2. Using a toothpick, place a very small portion of plain yogurt onto the slide. You will want to spread a very thin layer of yogurt on the slide. 3. Add one drop of water to the yogurt and place the cover slip on top. 4. Under low power, find a section where the yogurt is very thin; this is where you will find the bacteria. Sketch what you see in your field of view. 5. Switch to high power (400X for most microscopes) for a better view of the bacteria. Sketch what you see in your field of view. 6. Put a small amount of yogurt in a microcentrifuge tube and put it aside in a dark, relatively warm area, such as a classroom cabinet. Do not close the tube. Leave undisturbed for at least 24 hours. 7. After the time has passed, take a small sample with a toothpick and place on a slide. If the sample seems too thick, dilute with a drop of water. 8. Place a cover slip on top and observe the bacteria at low power (100X) to find a good place to start looking. Be sure to adjust the diaphragm so that a minimal amount of light enters your eyepiece, otherwise the bacteria will be very difficult to view. 9. Switch to the highest power to identify the bacteria according to arrangement. Identify any bacteria you might find. 10. Dispose of the microcentrifuge tube and slide in the appropriate manner according to your teacher.

LAB QUESTIONS
1. 2. 3. 4. 5. What types of bacteria were you able to observe on your prepared specimen slide? Describe the shapes and arrangements of the specimens you viewed. What visual differences do you observe between Gram-negative bacteria and Gram-positive bacteria? What can the differences between Gram-negative and Gram-positive bacteria be attributed to? Describe the composition of the cell wall in these bacteria that makes them stain different colors. Of what importance is it to know whether bacteria are Gram-negative or Gram-positive? What shape and arrangement do the yogurt bacteria you observed on Day 1 have? Does this differ from the shape and arrangements you observed on Day 2? If so, how?

Observing Bacteria Using a Light Microscope Adapted from http://www.accessnano.org/files/teaching-modules/health-medicine/nanogold/HM_NanoGold_Experiment_1_Guide.doc