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

DISCUSSION

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.

 

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.

1. diplococcus: a pair of cocci
- Photomicrograph of a diplococcus
- Scanning electron micrograph of a Streptococcus pneumoniae, a diplococcus
- Scanning electron micrograph of a Neisseria, a diplococcus; courtesy of Dennis Kunkel's Microscopy

2. streptococcus: a chain of cocci
- Photomicrograph of a streptococcus
- Scanning electron micrograph of Streptococcus pyogenes; courtesy of Dennis Kunkel's Microscopy
- Transmission electron micrograph of Streptococcus

-Scanning Electron Micrograph of Enterococcus

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

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).

staphylococcus: cocci in irregular, often grape-like clusters
- Photomicrograph of a staphylococcus, negative image
-
Scanning electron micrograph of Staphylococcus aureus; courtesy of Dennis Kunkel's Microscopy
- Scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA); courtesy of CDC

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. bacillus: a single bacillus (see Fig 2A and Fig 2B)
- Photomicrograph of a bacillus
- Scanning electron micrograph of a bacillus

- Scanning electron micrograph of Escherichia coli O157H7, a bacillus; courtesy of CDC

b. streptobacillus: bacilli in chains (see Fig 2A and Fig 2C)
- Photomicrograph of a streptobacillus


c. coccobacillus: oval and similar to a coccus (see Fig 2A, 2D, and 2E)

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):

a. vibrio: an incomplete spiral or comma-shaped (see Fig. 3A and Fig. 3B)
- Photomicrograph of a vibrio
-
Scanning electron micrograph of Vibrio cholerae; courtesy of Dennis Kunkel's Microscopy

b. spirillum: a thick, rigid spiral (see Fig. 3A and Fig. 3C)
- Photomicrograph of a spirillum

c. spirochete: a thin, flexible spiral (see Fig. 3A and Fig. 3D)
-
Photomicrograph of a spirochete
- Scanning electron micrograph of the spirochete Leptospira
; courtesy of CDC
- scanning electron micrograph of the spirochete Treponema pallidum; courtesy of CDC

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.

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

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. 6). 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.

 

  • Focusing With The 10X Objective - Olympus CX31 Microscope (see Fig. 7)

1. Rotate the yellow-striped 10X objective until it locks into place. This will give a total magnification of 100X.

2. Using the iris diaphragm lever under the stage (see Fig. 6A), reduce the light by sliding the lever most of the way to the right.

3. Turn the coarse focus control (larger knob; see Fig. 7) all the way away from you until it stops.

4. Look through the eyepieces and turn the coarse focus control (larger knob) towards you slowly until the specimen comes into focus.

5. Get the specimen into sharp focus using the fine focus control (smaller knob; see Fig.7) and adjust the light for optimum contrast using the iris diaphragm lever (see Fig. 6A) and/or the light intensity control (see Fig. 6).

 

SPECIMENS

Prepared slides of the following bacteria:

  • Staphylococcus aureus
  • Escherichia coli
  • Treponema pallidum
  • Spirillum species

On-line demonstration slides of the following bacteria:

  • Micrococcus luteus
  • Neisseria gonorrhoeae
  • Streptococcus pyogenes
  • Bacillus megaterium

Broth culture of Saccharomyces cerevisiae

Human hair

 

PROCEDURE

 

TIPS FOR MICROSCOPIC OBSERVATIONS

Move the slide around until you see an area representing the true arrangement of each organism. Also remember:

  • In the process of making the slide, some of the coccal arrangements will clump together and others will break apart. Look carefully to determine the true arrangement.
  • Small bacilli (such as Escherichia coli) that are dividing or have just divided by binary fission will look similar to cocci. Look carefully for bacilli that are not dividing and are definitely rod-shaped as well as bacilli in the process of dividing to confirm the true shape.
  • Bacilli do not divide so as to form clusters. Any such clusters you see are artifacts from preparing the slide.
  • Look carefully to see the spirochetes as they are the thinnest of the bacteria. When seen microscopically, spirochetes resemble extremely thin, wavy pencil lines.

 

1. Using oil immersion microscopy (1000X), observe and measure the bacteria listed below.

a. Staphylococcus aureus: Staphylococcus species, as the genus name implies, are cocci possessing a staphylococcus arrangement (cocci in irregular, often grape-like clusters). Measure the diameter of a single coccus.

b. Escherichia coli: Escherichia coli is a small bacillus. Estimate the length and width of a typical rod.

c. Treponema pallidum: Treponema pallidum is a spirochete - a thin, flexible spiral. On this slide you are examining a direct stain ofTreponema pallidum, the bacterium that causes syphilis. Measure the length and width of a typical spirochete.

d. Spirillum: Spirillum species appear as thick, rigid spirals. Measure the length and width of a typical spirillum.

When finished, remove the oil from the prepared slides using either a paper towel or a Kim wipe and return them to their proper tray.

 

2. Observe the on-line demonstration slides of the following bacteria:

a. Micrococcus luteus: Micrococcus luteus can appear as tetrads or cubes of 8. This strain is a coccus usually exhibiting a tetrad or a sarcina arrangement. Note the shape and arrangement.

b. Neisseria gonorrhoeae: Neisseria species are cocci usually having a diplococcus arrangement. Note the shape and arrangement.

c. Streptococcus pyogenes: Streptococcus species, as the genus name implies, are cocci that usually possess a streptococcus arrangement (cocci in chains). Note the shape and arrangement.

d. Bacillus megaterium: Bacillus megaterium appears as large bacilli in chains (a streptobacillus). Note the shape and arrangement.

 

3. Prepare a wet mount of baker's yeast (Saccharomyces cerevisiae).

a. Using a pipette, put a small drop of the yeast culture on a microscope slide and place a cover slip over the drop.

b. Using your iris diaphragm lever, reduce the light for improved contrast by moving the lever almost all the way to the right.

c. Observe using oil immersion microscopy. Measure the diameter of a typical yeast cell.

d. When finished, wash the slide and use it again for step 4. Discard the coverslip in the biowaste disposal containesra at the front of the room and under the hood.

 

4. Prepare a wet mount of your hair.

a. Remove a small piece of a hair from your head and place it in a small drop of water on a slide.

b. Place a cover slip over the drop and observe using oil immersion microscopy.

c. Measure the diameter of your hair and compare this with the size of each of the bacteria and the yeast observed in steps 1-3.

d. Discard the slide and coverslip in the biowaste disposal containers at the front of the room and under the hood.

 

5. At the completion of the lab, remove the oil from the oil immersion objective using lens paper and put your microscope away.

RESULTS

1. Make drawings of several of the bacteria from each of the four prepared slides and indicate their approximate size in micrometers.

 

Diameter = _____ micrometers

Shape =

Arrangement =

 

Length = _____ micrometers

Width = _____ micrometers

Shape =

 

 

 

Length = _____ micrometers

Width = _____ micrometers

Shape =

Form =

 

Length = _____ micrometers

Width = _____ micrometers

Shape =

Form =

 

 

 


2. Make drawings of several of the bacteria from each of the four demonstration slides and indicate their approximate size in micrometers.

 

Shape =

Arrangement =

 

Shape =

Arrangement =

 

 

 

Shape =

 

Arrangement =

 

 

 

 

Shape =

Arrangement =

 

 

 


3. Make a drawing of several yeast cells and indicate their size in micrometers.

 

Diameter = _____ micrometers

 

 

 

4. Make a drawing indicating the size of the bacteria and yeast observed above relative to the diameter of your hair.

 


hair

 

Diameter = _____ micrometers

PERFORMANCE OBJECTIVES FOR LAB 1

After completing this lab, the student will be able to perform the following objectives:

 

DISCUSSION

1. State three basic shapes of bacteria.

2. State and describe five different arrangements of cocci.

3. State and describe three different arrangements of bacilli.

4. State and describe three different spiral forms.

5. Describe the appearance of a typical yeast.

RESULTS

 

1. When given an oil immersion microscope, a prepared slide of a microorganism, and an ocular micrometer, determine the size of that organism in micrometers.

2. Using a microscope, identify different bacterial shapes, arrangements, and forms.

3. Differentiate a yeast from a coccus-shaped bacterium by its size.

4. Compare the size of the microorganisms observed in lab with the diameter of a hair when using oil immersion microscopy.

SELF-QUIZ

Contributors

  • Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)