Regents Prep: Living Environment: Laboratory:
Instrumentation

Stereo Microscopy

Stereo Microscope

The image at the left is that of a dissecting or stereo microscope.  Notice that it has only two sets of lenses, including the eyepiece.   The specimen to be observed is an opaque object (light does not pass through it).   The observer sees the surface of the dissection specimen or other specimen being studied.   This specimen is placed in a container on the stage of the microscope.

Light Microscopy 
Biologists use the light microscope to observe microscopic specimens  This microscope is also called the compound microscope is given its name because it has more than two sets of lenses. It has an eyepiece lens  (or ocular) and two or more sets of objective lenses (this microscope has three lenses) on a nosepiece that usually revolves.    This kind of microscope is also called a light microscope as it requires a source of light to pass through the specimen.  The specimen observed with this kind of microscope is usually microscopic and has to be translucent (allows light to pass through it).  The specimen to be observed is placed on the stage of this microscope.

Parts of the Light Microscope

  1. eyepiece or ocular
  2. body tube
  3. fine adjustment knob
  4. nosepiece
  5. high power objective
  6.  low power objective
  7. diaphragm
  8. mirror (many   microscopes have a light instead)
  9. base
  10. coarse adjustment 
  11. arm
  12. stage clip 
  13. inclination joint


Functions of the Light Microscope Parts

  1. eyepiece (ocular) - where you look through to see the image of your specimen. 

  2. body tube-the long tube that holds the eyepiece and connects it to the objectives (not labeled)

  3. fine adjustment knob-small, round knob on the side of the microscope used to fine tune the focus of your specimen after using the coarse adjustment knob

  4. nosepiece-the rotating part of the microscope at the bottom of the body tube; it holds the objectives

  5. high power objective -- used for high power magnification of the specimen (the longer objective lens)

  6. low power objective -- used for low power magnification of the specimen

  7. diaphragm-controls the amount of light going through to the specimen  

  8. light or mirror-source of light usually found near the base of the microscope; makes the specimen easier to see

  9. base-supports the microscope

  10. coarse adjustment knob -- used for focusing on low power 

  11. arm-part of the microscope that is grasped when one carries the microscope

  12. stage clips-shiny, clips on top of the stage which hold the slide in place
    (The specimen is placed on the stage for viewing.)

  13. inclination joint -is used to tilt the microscope

Some Microscope Usage Rules

  1. Always carry the microscope with two hands - one on the arm and one underneath the base of the microscope. Hold it up so that it does not hit other objects.
     
  2. Do not touch the lenses. If they are dirty, ask the teacher for special lens paper or ask your teacher to clean the lenses for you. 
     
  3. If using a microscope with a mirror, do not use direct sunlight as the light source. Blindness can result. If using a microscope with a light, turn off light when not in use.
     
  4. Notify teacher if a slide or cover slip breaks. Students should not handle broken glass.
     
  5. Always clean slides and microscope when finished. Store microscope set on the lowest power objective with the nosepiece turned down to its lowest position (using the coarse adjustment knob).  Cover microscope with dust cover and return it to storage as directed by your teacher.

Other Points About the Compound Microscope

1.   Always begin focusing on the lowest possible power.   Remember to center the specimen you
      are observing in the field of view before switching to a higher power.   Make certain that you
      move the objectives away from the specimen when focusing so their is no collision between
      the objective being used and the slide/cover slip which may damage the objective lens.

2.   As you switch from low to high power, the field of view becomes darker.   To deal with this
      the diaphragm needs to be opened to allow in more light.   (Frequently on low power the
      diaphragm needs to be partially closed as it is too bright.)

3.   As you switch from low to high power the field of view becomes smaller.
 

Images viewed under the light microscope are reversed (backward) and inverted (upside down).   This is a compound light microscope view of the letter F placed on a slide in its normal position.

Chromatography
Paper chromatography is a procedure used to separate substances in a mixture. In the Living Environment/Biology lab, this mixture is usually a solution of liquid plant pigments containing different kinds of chlorophylls and other colored photosynthetic pigments.  

A small concentrated sample of a mixture is placed on the chromatography paper above the line of a solvent mixture.   The paper is contact with a solvent solution at its bottom. This solvent moves through the paper due to capillary action and dissolves the mixture spot.  Some parts of the solvent mixture to be separated have a greater attraction for  the chromatography paper, so they move a lesser distance, while other parts of the solvent mixture have a lesser attraction, so they move a greater distance up the paper.

Paper Chromatography Apparatus

 

Completed Paper Chromatography of a Plant Pigment

The specific mixture placed on chromatography paper will separate into consistent patterns as long as the same solvent, paper, and amount of time allowed for the separation are not changed.. Different solvents will change the separation pattern of the mixture.  Mixtures that are colored can be separated into component colors by paper chromatography.

The Rf value of a pigment is a statistic often computed from a chromatography separation.   Each component of a solution.   Each pigment in the solution will have a specific Rf for the same solvent when the chromatography occurs for a specific length of time.

Calculation of Rf

Rf = distance the pigment travels from the original spot of solvent
distance to the wetting front of the solvent

Electrophoresis
Gel electrophoresis is a procedure used to separate charged molecules of different sizes by passing them through a gel in an electrical field.   The gel serves to act as a support for the separation of the molecules of different sizes.   The gel is usually composed of a jelly-like material called agarose which is made from seaweed.

Electrophoresis Setup

Molecules such as DNA fragments of different lengths and proteins of different sizes are often separated in the gel.   Holes are created in the gel which serve to hold the particular DNA mixtures to be separated.   The DNA fragments are then loaded into the wells in the gel.

 

  

Separation of DNA

The gel contains very small holes which act to regulate the speed which molecules can move through it based on the size of the molecules.   The smaller molecules will move much more easily through the small holes in the gel.   As a result, large fragments of DNA lag behind small fragments, thus allowing the experimenter to separate these molecules based on their size. 

Sometimes molecular weight markers are electrophoresed along with the specimen, so the experimenter may know the size of the DNA fragment which has been separated.  Different individuals or organisms form different banding patterns in the plate when their DNA has been separated.    DNA is cut into pieces for separation for electrophoresis by restriction enzymes.
These enzymes were originally discovered in bacteria and were used by the bacteria to defend themselves from invasion by other bacteria and viruses.

Some Uses for the Gel Electrophoresi DNA Separation

  1. It may be used to determine an individual's genetic relationship to his or her ancestors, as the more closely matched the banding pattern between two individuals, the more closely they will be genetically related.   In theory, no two individuals will form the same DNA banding pattern when the electrophoresis is completed.
     
  2. It may be used to identify an individual that have committed crimes based on the ability to match the suspects DNA to evidence which has been collected at a crime scene.
     
  3. It may be used to determine evolutionary relationships between organisms, as organisms with a closer genetic relationship will form more similar banding patterns.

 

Electrophoresis Setup with Power Supply

Web Resources
Gel Electrophoresis for Separating DNA Molecules
(Dr. Karen Hughes/University of Tennessee at Knoxville)

 

Created by James M. Buckley, Jr.
Copyright 1999-2003 Oswego City School District Regents Exam Prep Center
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