Bio206 Microbiology

Lab 2: Microscopic Observation of Microorganisms

Updated 10 January 05

I. Cleaning microscope slides:

You can save time and make better observations if you prepare very clean slides for both wet mounts and smear preparations.

1. Wet your first two fingers and apply cleanser to them.
2. Clean the slide on both sides by rubbing.
3. Rinse the cleanser off and inspect the slide.
4. Repeat these steps if the slide is not clean.
5. Wipe clean slides thoroughly with paper towels.
6. Handle the clean slide only by the edges.

Now you can test the cleanliness of the slide. Place a small drop of tap water on the slide. Spread the drop out to the size of a quarter with an inoculating loop. If the water does not coalesce and remains spread out, you have a clean slide.

Prepare enough clean slides at one time to use for the several activities scheduled for today.

II. Observing living microorganisms:

To observe living microorganisms they must be kept in a liquid environment. Both wet mounts and hanging drop preparations can be used to observe living microbes. See pages 56, 57, & 64 in your text. *Also go to the BioLab Mac and see Phase Contrast and Motility in "Identibacter Interactus". You will use wet mount preparations to see the following:

1. The size and shape of individual organisms.
2. The characteristic arrangement or groupings of cells.
3. Whether the organism is motile or nonmotile.

The stained smear preparation you will be using most often later can distort the size and arrangement of cells and make observation of motility impossible.

Wet mount preparation.

1. Using a pipette or a loop, place a small drop of culture fluid at the center of the slide.
2. Carefully place a clean coverglass over the drop.
3. Place your wet mount preparation on your microscope stage, and reduce the amount of light by closing the iris diaphram (These adjustments do not apply on a phase contrast microscope).
4. Use the low-power objective to locate the microbes.
5. Switch to the high-power objective, focus with the fine adjustment knob, and adjust the light.
6. Make drawings and descriptions in your notebook
7. Discard the wet mount into a disinfectant solution.

III. Observing stained microorganisms

Many microorganisms are so small, transparent and sometimes motile that they are difficult to see in wet mounts. Especially for bacteria, we devise ways to still their motion and treat them so they become more visible. Bacterial suspensions are smeared on clean slides, heat fixed and stained with dye. See pages 57 - 61 & 91. Also see Gram Stain in Identibacter Interactus.

Smear preparation

1. Obtain a clean slide and transfer drops of cell suspension from a broth culture to the middle of the slide.
2. Using the loop spread the drops out to the size of a dime.
3. Allow the smear to air dry. After drying you should be able to see a thin white film. If not, add more drops of cell suspension and spread as before until a dry white film is obtained.
4. Heat-fix the cells to the slide by passing it through the Bunsen flame three times or until the slide feels hot to the back of your hand.

Gram stain: Please review the background information related to Gram stain in your text. Use the following procedure to observe the samples available.

1. Prepare fixed smears of several different specimens on one slide. Always include Gram + and Gram - controls.
2. Stain the smears as follows:

   a. Flood the smears with crystal violet for about 1 min.

   b. Drain the dye into the sink and gently rinse off with tap water.

   c. Flood smears with iodine solution for 1 min.

   d. Rinse thoroughly in tap water.

   e. Decolorize with drops of acetone-alcohol until no more color washes off. Be careful not to over decolorize.

   f. Rinse briefly with tap water.

   g. Apply safranin for 30-60 sec.

   h. Rinse off with tap water.

   i. Drain slide and gently BLOT dry.

3. Label the dry slide with your name, date, organisms and stain.
4. Examine prep under oil with 100 X lens.
5. Record observations as sketches and notes in your lab book.

Expected results and conclusions: Indicate what results you expect and then comment on how the actual results compare. Include sketches of what you see on the slides at 1000 X. How do you account for any differences?

"Pet" Culture: Please review the Streak Plate Method in your text or other source.

1. Retreive the tube culture you started last week or use another source of your assigned pet microbe.
2. Obtain a petri dish of nutrient medium.
3. Label appropriately (on the bottom of the dish).
4. Make a streak plate of your organism.
5. Place in the incubator for at least 24 hr.
6. Examine for growth and remove after you can observe significant growth.
7. Look for isolated colonies on the dish.
8. In your lab book sketch and label what you obeserve.
9. Show the instructor your isolated colonies.
10. Prepare a Gram stain of a single isolated colony to verify purity.
11. If no isolated colonies are found, repeat the technique until successful.
12. Transfer cells from the "pure" colony to a slant for storage after incubation.

A group database containing characteristics of our microbial cultures will be essential for the unknowns project. Obervations on your pet microbes need to be collected and made available for everyone.

Questions

1. How can you tell if a slide is clean enough to make a smear?
2. What are the advantages and disadvantages of staining a sample?
3. How does true motility differ from brownian movement?
4. Why are microorganisms hard to see in wet preparations?
5. List at least three morphological types of bacteria whose names reflect their shapes and arrangements, and state the meaning of each name. Cite your source of this information.
6. Suppose you are viewing a Gram stained field of red rods and purple cocci through the microscope/ what do you conclude?
7. On the basis of Gram reaction, can you distinguish between Staphylococcus and Streptococcus? Escherichia and Klebisella? Escherichia and Bacillus? Explain.
8. For which pair of organisms in #5 would a capsule stain be useful? How do you know? Cite the source of your information. Help may be found in Chapter 11 of your text, Bergey's Manual, or Identibacter Interactus software.
9. What is the purpose of a streak plate?