MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #1: CARE AND USE OF THE MICROSCOPE

NAME, ID #:_______________________________________________

NAME of TA:________________________________________

REVISED: 08/24/99

Glossary of Microscope Terminology

INTRODUCTION TO EXERCISE:

Figure 1. Relative Size of Microbes. E.M. refers to the Electron Microscope.

A general definition of MICROBES includes all those living organisms that can not be viewed (seen) in any detail by the human eye. Alternatively, a MICROBE is any living creature that must be examined with a magnifying lens in order to see its unique physical characteristics (size, shape, motility, color). Magnifying systems come in two general forms; SIMPLE magnifying glasses or lenses that consist of a single lens of polished, rounded glass or plastic and COMPOUND MICROSCOPES that use two lens, an OBJECTIVE LENS and an EYEPIECE LENS to magnify the sample (Fig. 1). The best single lenses are limited to magnifications of 500 Xs or less and most only magnify 10 to 25 Xs. The better compound microscopes are capable of magnifications of up to approximately 1,200 Xs, but the average compound microscope only magnifies 1,000 Xs. As you've learned in lecture the first man credited with seeing microbial life was #Anton van Leeuwenhoek. Leeuwenhoek performed this amazing feat with simple lenses and a keen eye.

In this exercise you will learn the parts of the compound microscopes and their purpose. You will learn to care for and to use the compound microscope. Following a lecture and demonstration by the TA on the care and use of the microscope, you will practice with it by viewing live microbes. You will observe the relative size and shape of the microbes in the various microbe-containing samples provided. These samples will contain bacteria, algae, fungi and protozoa. You will draw at least five different microbes, including one of each group. For lots of information on various microscopes view this site.

View the following URLs to get an idea how microbes appear under the microscope:

View the "Rumen Ciliate" (fairly large file) & consider why these bacteria would be growing on the surface of the ciliate.

• You should see paramecia in your pond or aquarium sample. The TA may have some yeast stained with Congo Red to feed your pet paramecia.

To get an idea of what sort of magnifications you are working with go to the following URL, scroll to the "Stage Micrometer" and view the 100X, 400X and 1000X in order. You may also want to view some diatoms. This site allows you to compare the magnification of an item. Choose the "Onion Root Mitosis" and look at the 25, 100 & 1,000 magnifications.

This site shows pictures mostly of Cyanobacteria.

For a discussion of microscopes, magnification and microscopic techniques, including photography, view these sites.

PURPOSE OF LABORATORY:

1. To learn how to use and care for a microscope.
2. To view and study the morphology, motility and other distinguishing characteristics of bacteria, algae, fungi/yeast and protozoa.
3. To gain an understanding of the size of various microbes and their numbers in the world around you.
4. To learn how to use the 100X and 400X objective lens.
5. To learn how to use the 1000X oil-immersion lens required to view prokaryotes.

RELATIONSHIP TO LECTURE MATERIAL

• LECTURE: Prokaryotes vs. Eukaryotes; Bacterial Architecture.
• NetText101/102: CHAP. I , II & III.

GENERAL INSTRUCTIONS:

1. Draw what you see in the circles below and have the TA verify that you are truly seeing microbes.

Figure 2. Parts of the typical microscope.

PROCEDURE:

FIRST LAB:

1. Record the purpose or function of each of the parts of the microscope as described by your TA.
2. Record the steps required to clean up the microscope prior to putting it away.
3. Practice viewing the mark of a Marking Pen, cross-hairs slides or news paper print on a slide at 10X, and 40X before looking at the living samples.
   • Always view your sample with the 10X objective first, then the 40X and finally with the oil-immersion 100X objective (we won't use the oil objective this time).
4. Prepare a microscope slide as demonstrated by your instructor and illustrated in the figures below; clean the slide if necessary.
5. Obtain your samples from the material provided at the front/side of the room or at your desks. Prepare wet mounts as described below. Draw four different microbes, including one eukaryotic algae, one protozoa and one yeast and a mold. Include the MAGNIFICATION that each was viewed at and indicate WHICH SAMPLE the organism was in. In one of the drawings indicate the relative size of bacteria to any larger cells present in the sample.

• Place a cover slip on each of the samples and view them first with the 10X lens and then with the 40X lens:
  • The paramecium culture: It contains a large paramecia and a smaller paramecia and other protozoa and bacteria.
  • The aquarium algae: scrape chunks of green material off the rocks with the edge of a slide. Pick up a chunk of algae you can see and place it on the slide: look for a variety of protozoa of all sizes; eukaryotic algae; different type of diatoms, Cyanobacteria & bacteria.
  • The straw/grass culture: Many bacteria of different shapes; most highly motile. If you can pick the slime from the bottom of the container you may find ameba (FIRST ONE IN EACH LAB TO FIND AN AMEBA GETS 2 EXTRA POINTS).
  • Pond water: A complete microbial world of bacteria, green algae, cyanobacteria, protozoa and fungi. You may see something no one else has ever seen.

SECOND LAB:

USE OF THE OIL IMMERSION LENS

1. Continue examining samples taken from the sources provided. Be sure and describe to your instructor what you are viewing and ask him/her to also view the material.
2. Continue viewing the various samples, especially the bacteria from the grass/hay infusion and from the Winogradsky column. The Instructor will help you collect samples from the latter source.
3. View the bacteria first through the 40X lens and then through the oil immersion lens. To use the oil immersion lens proceed as follows:

• Prepare a wet mount as before.
• Focus the 40X lens on the sample and examine it. The hay infusion sample should consist mostly of bacteria with a few protozoa darting back 'n forth. Generally the bacteria will be highly motile and will appear as faint tiny spots moving about rapidly.
  • Decrease the amount of light if you are having difficulty seeing something.
• Turn the barrel of the microscope so that the 40X and 100X lens is 1/2 way between and directly over the coverslip.
• Place a drop of oil on the middle of the coverslip.: Fig. 3 A.
• Carefully turn the objective lens until the 100X lens contacts the oil and snaps into position: Fig. 3 B.
• Examine the lens; it should almost touch the coverslip and the oil should fill the space between the coverslip and the lens: Fig. 3 C.
• If it touches the coverslip carefully back the lens off until it is about 1 mm above the coverslip.
• While looking through the eyepiece use the FINE ADJUSTMENT knob to move the lens up and down in tiny increments; this action should bring the organisms into focus.
  • If you can't see anything initially try varying your lighting.
  • If you are still having trouble seeing something try to center a piece of debris directly under the lens by eye and then focus on it. Once that is done carefully move away and examine other areas of the slide.

Figure 4. Setting up oil immersion lens to view a slide.

Figure 5. Ameba engulfing food. Taken by Mr. Steve Durr.

Figure 6. Large Spirochete. These bacteria are often found in ponds containing a lot of organic pollution. Image taken using a phase contrast X40 objective by Mr. Steve Durr.

The above are pictures of some microbes you may see in the various samples.

RELATED URLs

Index page on microscopes and microscopy.

History of the microscope; view some of the early scopes and read the chapter on the 17th century microscopes and be able to answer the following questions:

1. Who was Robert Hooke and what were his contributions?
2. How did Leeuwenhoek's scopes work?
3. How did Leeuwenhoek improve the lens so as to see more?

Compare what you've seen in the aquarium water with that from pond water. The first one to see an AMEBA in each lab gets two extra points. See how many green algae (hint: they are usually relatively large and green) you can identify in your samples.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• Distinguish between the OBJECTIVE and EYEPIECE lens of the microscope and explain what each one does.
• What are the MAGNIFICATIONS of the various eyepiece and objective lenses?
• Define COARSE and FINE adjustment.
• What is the SIZE DIFFERENCE between a eukaryotic algae and a prokaryotic algae?
• What does the IRIS DIAPHRAGM do?
• Why do you hold the microscope at the bottom when carrying it around?
• Why do you let in more light when you go from a low magnification objective to a higher magnification objective?

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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