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!

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

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

Field of view – the circle of light you see when looking into the microscope
Depth of field – the distance through which you can move the specimen and still have it remain in focus
Working distance – the space between the objective lens and the surface of the slide
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
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”.
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
Purpose of Darkfield Microscopy
Used to view delicate, transparent, living organisms
Principally used to view spirochetes from syphilitic lesions ( pallidum)
Mechanism
Dark-filed filter is placed within condenser
Filter alters the wavelength of light passing through the condenser
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
Causes background to appear dark, while specimen typically are white or may fluoresce due to staining

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

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

Ubiquity of Bacteria

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

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