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Lab 1: Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms - Biology

Lab 1: Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms - Biology


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In this lab, you will become familiar with the use of the microscope (particularly oil immersion microscopy) and will compare the relative size and shape of various microorganisms.

A. BACTERIAL SHAPES, ARRANGEMENTS, AND FORMS

Bacteria are unicellular prokaryotic microorganisms that divide by binary fission, a process by which one bacterium splits into two.

  • Scanning electron micrograph of Salmonella typhimurium undergoing binary fission.

There are three common shapes of bacteria:

  • coccus
  • bacillus
  • spiral.

The cocci come in 5 different arrangements; the bacilli in 3 different arrangements; and the spirals in 3 different forms.

1. Coccus

A coccus-shaped bacterium is usually spherical, although some appear oval, elongated, or flattened on one side. Most cocci are approximately 0.5 - 1.0 micrometer (µm) in diameter and may be seen, based on their planes of division and tendency to remain attached after replication, in one of the following arrangements (see Fig. 1A):

a. Division in one plane produces either a diplococcus (see Fig. 1A and Fig. 1B) or streptococcus (see Fig. 1A and Fig. 1C) arrangement.

b. Division in two planes produces a tetrad arrangement (see Fig. 1A and Fig. 1D).

  • tetrad: a square of 4 cocci
    - Photomicrograph of a tetrad
    - Scanning electron micrograph of Micrococcus luteus

c. Division in three planes produces a sarcina arrangement (see Fig. 1A).

  • sarcina: a cube of 8 cocci
    - Photomicrograph of a sarcina

It is difficult with a conventional light microscope to tell a tetrad arrangement (square of four cocci) from a sarcina arrangement (cube of eight) so in our lab, anytime you see ba square of four cocci, say it is either a tetrad or a sarcina arrangement.

d. Division in random planes produces a staphylococcus arrangement (see Fig. 1A and Fig. 1E).

As you observe these different cocci, keep in mind that the procedures used in slide preparation may cause some arrangements to break apart or clump together (see Figs. 1D and 1E). The correct form, however, should predominate. Also remember that each coccus in an arrangement represents a complete, individual, one-celled organism.

2. Bacillus (rod)

A bacillus or rod is a hotdog-shaped bacterium having one of the following arrangements (see Fig 2A):

A single bacillus is typically 0.5-1.0 µm wide and from 1- 4 µm long. Small bacilli or bacilli that are dividing or have just divided by binary fission may at first glance be confused for diplococci or cocci (see Fig. 2A) so they must be observed carefully. You will, however, be able to see bacilli that have not divided and are definitely rod-shaped as well as bacilli in the process of dividing.

3. Spiral

Spiral-shaped bacteria occur in one of three forms (see Fig. 3A):

The spirals you will observe range from 5-40 µm long but some are over 100 µm in length. The spirochetes are the thinnest of the bacteria, often having a width of only 0.25-0.5 µm.

To view a nice interactive illustration comparing size of cells and microbes, see the Cell Size and Scale Resource at the University of Utah.

B. YEASTS

Yeasts, such as the common baker's yeast Saccharomyces cerevisiae (see Fig. 4), are unicellular fungi. They usually appear spherical and have a diameter of 3 - 5 µm. Yeasts commonly reproduce asexually by a process called budding. Unlike bacteria, which are prokaryotic, yeasts are eukaryotic.

C. MEASUREMENT OF MICROORGANISMS

The approximate size of a microorganism can be determined using an ocular micrometer (see Fig. 5) , an eyepiece that contains a scale that will appear superimposed upon the focused specimen.

To view a nice interactive illustration comparing size of cells and microbes, see the Cell Size and Scale Resource at the University of Utah.

D. FOCUSING

  • Focusing With The 1000X Oil Immersion Objective - Olympus CX31 Microscope (see Fig. 7)

1. Before you plug in the microscope, turn the light intensity control dial on the right-hand side of the microscope to 1 (see Fig. 6). Now plug in the microscope and turn it on (see Fig. 6).

2. Place a rounded drop of immersion oil on the area of the slide that is to be observed under the microscope, typically an area that shows some visible stain. Place the slide in the slide holder (see Fig. 8) and center the slide using the two mechanical stage control knobs under the right-hand side of the stage (see Fig. 6).

3. Rotate the white-striped 100X oil immersion objective until it is locked into place. This will give a total magnification of 1000X.

4. Turn the light intensity control dial on the right-hand side of the microscope to 6 (see Fig. Make sure the iris diaphragm lever in front under the stage is set approximately at 0.9, (toward the left side of the stage; see Fig. 6A). Do not close the field iris diaphragm ring on the light source; that should remain fully open. The knob under the stage on the left-hand side of the stage controlling the height of the condenser should be turned so the condenser is all the way up.

5. Watching the slide and objective lens carefully from the front of the microscope, lower the oil immersion objective into the oil by raising the stage until the white-striped, 100X, oil immersion lens just touches the immersion oil on the slide (see Fig. 9). Do this by turning the coarse focus(larger knob; see Fig. 7) away from you until the 100X oil immersion objective lens just touches the oil. When the lens enters the immersion oil you will see the light scatter away from the lens. Do not push the spring-loaded portion of the lens up into the barrel of the oil immersion lens.

6. While looking through the eyepieces, turn the fine focus (smaller knob; see Fig. 7) away from you at a slow steady speed until the specimen comes into focus. (If the specimen does not come into focus within a few complete turns of the fine focus control, reverse direction and start turning the fine focus toward you - or go back and repeat step 5.

7. Using the iris diaphragm lever, adjust the light to obtain optimum contrast (see Fig. 6A).

8. When finished, wipe the oil off of the oil immersion objective with lens paper, turn the light intensity control dial back to 1, turn off the microscope, unplug the power cord, and wrap the cord around the cord holder on the back of the microscope.

An alternate focusing technique is to first focus on the slide with the yellow-striped 10X objective by using only the coarse focus control and then without moving the stage, add immersion oil, rotate the white-striped 100X oil immersion objective into place, and adjust the fine focus and the light as needed. This procedure is discussed in the Introduction to the lab manual.