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  Section: Biotechnology Methods » Microbiology
 
 
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Introduction to Microscope & Comparison of Size & Shape of Microorganisms

 
     
 
Content
Microbiology
  Introduction
  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Bacterial Shapes and Arrangements
Bacteria are unicellular prokaryotic microorganisms. There are 3 common shapes of bacteria: the coccus, bacillus, and spiral. Bacteria divide by binary fission, a process by which 1 bacterium splits into 2.


Coccus
A coccus-shaped bacterium is usually spherical, although some appear oval, elongated, or flattened on one side. Cocci are approximately 0.5 micrometers (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:
  1. Division in one plane produces either a diplococcus or streptococcus arrangement.


    Figure 2 Arrangements of cocci.

    (i) Diplococcus: pair of cocci

    Figure 1

    (ii) Streptococcus: chain of cocci




    Figure 1

  2. Division in 2 planes produces a tetrad arrangement.
    A tetrad: square of 4 cocci
  3. Figure 5 Tetrad arrangement (appears as a square of
    4 cocci shown by arrows).

  4. Division in 3 planes produces a sarcina arrangement. Sarcina: cube of 8 cocci.
  5. Division in random planes produces a staphylococcus arrangement. Staphylococcus: cocci in irregular, often grape-like clusters.
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. The correct form, however, should predominate. Also remember that each coccus in an arrangement represents a complete, single, one-celled organism.


Bacillus (rod)
A bacillus or rod is a hotdog-shaped bacterium having one of the following
arrangements:
  1. bacillus: a single bacillus.
  2. streptobacillus: bacilli in chains-Streptobacillus arrangement.

    Figure 6 Bacilli in chains.

  3. Coccobacillus: oval and similar to a coccus. A single bacillus is typically 0.5–1.0 mm wide and 1–4 mm long. Small bacilli or bacilli that have just divided by binary fission may at first glance be confused for cocci, so they must be observed carefully. You will, owever, be able to see bacilli that have not divided and are definitely rod-shaped, as well as bacilli in the process of dividing.

    Figure 7 Arrangements of bacilli.
Spiral
Spiral-shaped bacteria occur in one of 3 forms.

Spiral forms.
Figure 8 Spiral forms.

  1. vibrio: an incomplete spiral, or comma-shaped.
  2. spirillum: a thick, rigid spiral.
  3. spirochete: a thin, flexible spiral.
    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.
Yeasts
Yeasts, such as the common baker’s yeast Saccharomyces cerevisiae, are unicellular fungi. They usually appear spherical and have a diameter of 3–5 µm. Yeasts spirochete commonly reproduce asexually by a process called budding. Unlike bacteria, which are prokaryotic, yeast are eukaryotic.

Saccharomyces cerevisiae (Budding
Figure 9 Saccharomyces cerevisiae (Budding
yeast shown by arrows).

Measurement of Microorganisms
The ocular micrometers provided are calibrated so that when using 1000X oil immersion microscopy, the distance between any 2 lines on the scale represents a length of approximately 1 micrometer. Remember, this does not hold true when using other magnifications.

Ocular micrometer.
Figure 10 Ocular micrometer.

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

Focusing with Oil Immersion
  1. Before you plug in the microscope, turn the voltage control dial on the righthand side of the base of the microscope to 1. Now plug in the microscope and turn it on.
  2. Place the slide in the slide holder, center the slide using the 2 mechanical stage control knobs under the stage on the righthand side of the microscope, and place a rounded drop of immersion oil on the area to be observed.
  3. Rotate the white-striped 100X oil immersion objective until it is locked into place. This will produce a total magnification of 1000X.
  4. Turn the voltage control dial on the righthand side of the base of the microscope to 9 or 10. Make sure the iris diaphragm lever in front under the stage is almost wide open (toward the left side of the stage), and the knob under the stage on the lefthand side of the stage controlling the height of the condenser is 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 lens just touches the slide. Do this by turning the coarse
    focus (larger knob) away from you until the spring-loaded objective lens just begins to spring upward.
  6. While looking through the eyepieces, turn the fine focus (smaller knob) toward 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 and the lens is starting to come out of the oil, you missed the specimen when it went through focus. Simply reverse direction and start turning the fine focus away from you.)
  7. Using the iris diaphragm lever, adjust the light to obtain optimum contrast.
  8. When finished, wipe the oil off the oil immersion objective with lens paper, turn the voltage control dial back to 1, turn off the microscope, unplug the power cord, and wrap the cord around the base 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 light as needed. This procedure is discussed in the introduction to the lab manual.

Specimens
  • Prepare slides of the following bacteria:
    • Staphylococcus aureus
    • Escherichia coli
    • Borrelia recurrentis or Borrelia burgdorferi
    • Spirillum species.
  • Demonstration slides of the following bacteria:
    • Micrococcus luteus
    • Neisseria gonorrhea
    • Streptococcus species
    • Bacillus megaterium
  • Broth culture of Saccharomyces cerevisiae
  • Human hair.
Procedure
  1. Using oil immersion microscopy (1000X), observe and measure the bacteria that follow:
    Tips for Microscopic Observations
    Remember that in the process of making the slide, some of the coccal arrangements will clump together and others will break apart. Move the slide around until you see an area representing the true arrangement of each organism. Also, remember that small bacilli (such as Escherichia coli) that 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. Also, bacilli do not divide to form clusters. Any such clusters you see are artifacts from preparing the slide. Finally, you will have to look carefully to see the spirochetes, since they are the thinnest of the bacteria. When seen microscopically, spirochetes resemble extremely thin, wavy pencil lines.
    1. Staphylococcus aureus: Staphylococcus species, as the genus name implies, have a staphylococcus arrangement (cocci in irregular, often grape-like clusters). Measure the diameter of a single coccus.
    2. Escherichia coli: Escherichia coli is a small bacillus. Estimate the length and width of a typical rod.
    3. Borrelia recurrentis: Borrelia species are spirochetes (thin, flexible spirals). You are examining blood infected with Borrelia recurrentis. Measure the length and width of a typical spirochete and the diameter of a red blood cell.
    4. 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 a paper towel and return them to their proper tray.
  2. Observe the demonstration slides of the following bacteria:
    1. Micrococcus luteus: Micrococcus luteus can appear as tetrads, cubes of 8, or in irregular clusters. This strain usually exhibits a tetrad or sarcina arrangement. Measure the diameter of a single coccus.
    2. Neisseria gonorrhea: Neisseria species usually have a diplococcus arrangement. Measure the diameter of a single coccus.
    3. Streptococcus pyogenes: Streptococcus species, as the genus name implies, usually have a streptococcus arrangement (cocci in chains). Measure the diameter of a single coccus.
    4. Bacillus megaterium: Bacillus megaterium appears as large bacilli in chains (a streptobacillus). Measure the length and width of a single bacillus.
  3. Prepare a wet mount of baker’s yeast (Saccharomyces cerevisiae) by putting a small drop of the yeast culture on a microscope slide and placing a cover slip over the drop. Using your iris diaphragm lever, reduce the light for improved contrast by moving the lever almost all the way to the right and observe using oil immersion microscopy. Measure the diameter of a typical yeast.
    When finished, wash the slide and use it again for step 4. Discard the coverslip in the biowaste disposal container at the front of the room and under the hood.
  4. Remove a small piece of a hair from your head and place it in a small drop of water on a slide. Place a cover slip over the drop and observe using oil immersion microscopy. 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. 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 4 prepared slides and indicate their approximate size in micrometers.


  2. Make drawings of several of the bacteria from each of the 4 demonstration slides and indicate their approximate size in micrometers.


  3. Make a drawing of several yeast cells and indicate their size in micrometers. Saccharomyces cerevisiae
    diameter = _____ micrometers.
  4. Make a drawing indicating the size of the bacteria and yeast observed
    above, relative to the diameter of your hair.
    diameter = _____ micrometers.
Performance Objectives
Discussion
  1. Name 3 basic shapes of bacteria.
  2. Name and describe 5 different arrangements of cocci.
  3. Name and describe 3 different arrangements of bacilli.
  4. Name and describe 3 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 and arrangements.
  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.

 
     
 
 
     




     
 
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