BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms

BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms

Lab One

Microscopy

Ubiquity of Microorganisms

 

Microscope– coordinated system of lenses arranged to produce an enlarged (magnified), focusable image of a specimen.

  • The microscopes used in lab are expensive instruments and should be treated as such.  Please take care when transporting, using, and storing them! BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms

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Components of the compound microscope

  • You should review each of the components of the compound microscope and be familiar with where they are located and their function as well:
    • Arm
    • Base
    • Mechanical Stage
    • Light Source/Illuminator
      • Voltage control/regulator
    • Lens Systems
      • Ocular(s)
      • Diopter adjustment ring
      • Rotating head
      • Objective(s) (4x, 10x, 40x, 100x)
        • k.a – low/scanning, high-dry, and oil immersion objectives
      • Nosepiece
      • Condenser
        • Iris diaphragm
      • Mechanical stage control
    • Adjustment Knobs
      • Coarse adjustment
      • Fine adjustment

 

Resoultion

  • The resolution (resolving power) of a lens, or microscope, refers to its ability to distinguish between two separate points.  The limit of resolution for most light (compound) microscopes is 1000x.
  • Magnification increases resolution, ability to distinguish two points as separate points. The better the resolution, the sharper or crisper the image! BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
    • Resolving power of the human eye = 0.1mm (meaning our eyes can distinguish two points that are at least 0.1mm apart)
    • Resolving power of the light microscope = 0.2µm (anything closer than this to the objective would not be distinguishable)

Magnification

    1. The apparent increase in size of the specimen that occurs due to the microscope
    2. Determining total magnification: MagTot = MagObj X MagOcu
  1. Ocular magnification = 10X
  2. Objectives = 4X, 10X, 40X and 100X(oil immersion)
    1. Parfocal – image remains relatively focused when changing objectives
    2. Parcentered – the center of the image is still the center when changing objectives

Other terms related to microscopy to know

    1. Field of view – the circle of light you see when looking into the microscope
    2. Depth of field – the distance through which you can move the specimen and still have it remain in focus
    3. Working distance – the space between the objective lens and the surface of the slide
    4. Contrast – how well the details of the specimen stand out against the background of the image.  In many cases, staining improves contrast. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
    5. Qualitative observation – a subjective observation (in other words, one that is not defined by a numerical value).  For example “he is tall” or “the surface is hot”.
    6. Quantitative observation – observation that involves measurement or a numerical value.  For example, “he is 6 feet tall” or “the surface is 96 degrees”.

 

Instructions for Using the Light Microscope

  • Please see your lab manual for correct usage of the light microscope. You are responsible for understanding the correct manner in which the microscope should be used. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms

 

Slides available for viewing

 

Letter “e”

Silk Threads

Various slides of microbes for further practice using the microscope.

 

***Each student should view the letter ‘e’ and silk threads to get accustomed to the microscope.  The other prepared slides of the organism are for you to get acquainted with focusing on specific organisms.  You DO NOT need to view them all at this time.  Any of the slides will be sufficient to provide you with practice on focusing the microscope.*****

 

Steps for properly storing the microscopes.

  • Click the 4X objective into place.
  • Bring the stage to its lowest level.
  • Remove the slide and dispose of it appropriately.  Wipe prepared slides until ALL oil/dirt is removed (top and bottom).
  • Clean objective lenses with lens paper, and, if using oil, liquid lens cleaner. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
  • Clean eyepieces with dry lens paper.
  • Clean condenser lens with lens paper
  • If necessary, clean the stage with a damp lens paper.
  • Turn the light switch OFF.
  • Rotate the head so that the eyepieces are facing away from the stage (or toward the arm).
  • Replace the dust cover.
  • Return the microscope to the appropriate location in the cabinet

 

Other Types of Microscopes used in Microbiology (see lab manual)

 

  • The Darkfield Microscope
  1. Purpose of Darkfield Microscopy
    1. Used to view delicate, transparent, living organisms
    2. Principally used to view spirochetes from syphilitic lesions ( pallidum)
  2. Mechanism
    1. Dark-filed filter is placed within condenser
      1. Filter alters the wavelength of light passing through the condenser
      2. Light rays are taken out phase, so that the background will look completely dark/black BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
  • As rays pass through specimen, the rays are refracted and brought into phase so that the specimen appears brighter
  1. Causes background to appear dark, while specimen typically are white or may fluoresce due to staining

 

  • Phase-Contrast Microscope
  1. Purpose of phase-contrast microscopy
    1. To observe living cells without using staining techniques
    2. Vital to observing internal structures within microorganisms
    3. Allows cells to remain alive and achieves contrast of structures within the cell as well
  2. Mechanism
    1. Light waves passing through specimen are brought into phase with one another to produce a brighter image (coincidence)
    2. Light waves passing through specimen that are out of phase with one another lead to darker image (interference)

 

 

The Ubiquity of Microorganisms

Microorganisms are the most widely distributed life forms in the world.  They can be found in varying locations such as the digestive tract of human beings, hot springs, the air around us, rivers, deep within the earth, oceans, and soil.  Some are potentially pathogenic (disease causing) while other perform functions vital to the maintenance of our environment such as producing oxygen and eliminating carbon dioxide.  Although small in size, microorganisms are diverse in structure and in function.  In today’s exercises we will observe several microorganisms and you will see the diversity amongst sizes, shapes, functions, and characteristics.

 

Cells are divided into two major groupings based on their characteristics.  There are Prokaryotes and Eukaryotes respectively.  Prokaryotes are those cells that lack a membrane-delimited nucleus.  In contrast, Eukaryotes have a membrane-delimited nucleus.  Microorganisms (and all living organisms) are divided based upon these characteristics, in part, into the three domains of life.  These three domains are based upon the genetic similarity that exists between organisms.  The three domains are Bacteria, Archaea, and Eukarya.  Bacteria (Eubacteria) are those organisms that have a cell wall composed of peptidoglycan (a polysaccharide).  Many of these organisms are present in our everyday environment.  In contrast there are also Archaea (Archaeabacteria) that lack peptidoglycan in their cells walls.  They are usually found in more extreme environments such as thermal vents on the ocean floor, hot springs, and Antarctica.

 

We will look at the shape and morphology of a variety of eubacteria in this lab.

 

 

The Prokaryotes

  1. Cyanobacteria (“Blue-Green Algae”)
    1. Characteristics
  1. Do not contain an organized nucleus
  2. Do NOT contain chloroplasts
  • Not necessarily blue or green in color; others do exist
    1. Example:  Oscillatoria

 

Ubiquity of Bacteria

  1. Bacterial characteristics
    1. Bacteria usually contain a cell wall that separates them from their external environment
  1. Depending on the characteristics of the cell wall, bacteria can usually be separated into two groups based on their staining properties: Gram-negative vs. Gram-positive
    1. The cell wall typically contains peptidoglycan
    2. They are prokaryotic organisms
    3. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
  1. They lack a membrane bound nucleus and organelles
  2. Do contain ribosomes for protein synthesis, however, these ribosomes are different from eukaryotic ribosomes
    1. Contain 70S ribosomes vs. the 80S ribosomes of eukaryotic organisms
  1. Common Bacteria shapes
    1. Cocci – spherical
  1. Streptococci – chains, of spherical organisms
  2. Staphylococci – grape-like clusters
  • Diplococci – groups of two spherical organisms. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms
  1. Tetrads – groups of four organisms in square-like arrangement
  2. Sarcina – 8 cocci arranged into a cube
    1. Bacilli – rod-shaped
  1. Coccobacillus – short rods with curved ends resembling a cocci
  2. Palisade – bacilli that are “stacked” together
  • Streptobacilli – bacilli arranged in a chain-link manner
    1. Spirals
  1. Spirilla – Rigid helices
  2. Spirochetes – Flexible helices
    1. Vibrio
  1. Curved rods
    1. Pleomorphic
  1. Variable shape
  2. BIOL-2010-01 Lab One Microscopy Ubiquity of Microorganisms

 

 

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