Tools and Measurement

 

       

 

 

Scientific Method

Organizing Data

Tools and Measurement

Laboratory Safety

Skills

     Vocabulary: arm, base, body tube, chromatography, compound microscope, course adjustment knob, diaphragm, dissection microscope, eyepiece, field of view, fine adjustment knob, gel electrophoresis, high power objective, inclination joint, inverted, light, low power objective, meniscus, mirror, nosepiece, ocular, opaque, restriction enzymes, reversed, ruler, solvent, stage clips, stereomicroscope, total magnification, translucent, triple beam balance

Instrumentation
Work in the living environment/biology laboratory requires some knowledge about and the ability to use several pieces of specific scientific apparatus.    Some of these tools include the light or compound microscope for looking at microscopic specimens and the dissection microscope for looking at three dimensional specimens larger than the naked eye. More specialized laboratory techniques include that of gel electrophoresis and paper chromatography.

Measurement
Measurements of mass, volume, and length are commonly required in lab.  The triple beam balance and graduated cylinder are other commonly used pieces of equipment for determining mass and volume respectively. Various types of rulers may be employed to provide measurements of length depending upon the specimen being studied or observation being made.

Stereo Microscopy

Light Microscopy

                               
 

 

 

 

 

 

 

 

 

 

 

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. Never use the course adjustment knob on high power. The lens is closer to the stage making it easier to break a slide or the lens itself. 

  4. Notify teacher if a slide or cover slip breaks. Students should not handle broken glass.
     
  5. If using a microscope with a light, turn off light when not in use

    6.    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 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 Electrophoresis 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

 

Measurement

Measurements of mass, volume, and length are commonly required in lab.  The triple beam balance and graduated cylinder are other commonly used pieces of equipment for determining mass and volume respectively.    Various types of rulers may be employed to provide measurements of length depending upon the specimen being studied or observation being made.

Volume Measurement
A commonly used instrument to measure liquid volume is the graduated cylinder
This instrument usually measures liquid volume in milliliters (ml).   

Using a Graduated Cylinder

It is important to remember to read to the bottom of the curved line or meniscus when measuring solutions involving water or most liquids.   The graduated cylinder at the left is divided into increments of 2 ml, so the volume in it is 12 ml. The graduated cylinder on the right is divided into increments of 1 ml, so the volume in it is 16 ml.

Mass Measurement
The triple beam balance is commonly used to measure mass in the biology lab. This device is named for its three long beams on which sliding bars called riders (or tares) are used to determine the mass of an object placed on its platform. It is very important that the riders on the rear beams are in the notch for the whole number of grams and not in between notches. The front beam is a sliding scale graduated in grams. The rider on this beam can be positioned anywhere on the scale. Masses on a triple-beam balance can be read to tenths of a gram and estimated to hundredths of a gram.

Using the Triple Beam Balance

The picture at the upper left shows two different models of triple beam balances commonly used in the biology laboratory.    The picture at the lower left shows the measurement of a mass in progress.    Without estimation, the mass of the object appears to be 373.3 grams (g).

Length Measurement  
Most measurements in biology will involve metric units of measurement.  When using a ruler, it is good to start at a whole number increment that isn't 0. Many times the end of a ruler will be worn away by student/teacher use or is inaccurate due to the manufacturing process. It is important to remember to take away the whole number increment one has moved in on the ruler (in the example below 1 cm) from the measurement obtained.

Using a Ruler to Measure Length

Problem:  How long is leaf A?

The tip of the leaf is at about 6.5 cm, but note the measurement started at 1 cm. Therefore, Leaf A is 5.5 cm or 55 mm. in length.

Microscopic Measurement
The magnifying power of most objectives and oculars is engraved on them. On the ocular, the marking can be found on the top edge or on the smooth cylinder that fits inside the body tube; on the objectives, magnification is on the side of the cylinder. For example, a marking "10x" means that the particular lens forms an image ten times larger than the object being viewed. The total magnification of a microscope is equal to the power of the eyepiece (ocular) X power of the objective used. For example, if a student is using a microscope with a 10 X ocular and a 43 X high power objective, the total magnification of the specimen the student is viewing is equal to 10 X 43 or 430 X (times).

Formula for Total Microscope Magnification

Total magnification

=

Power of the eyepiece

x

Power of
the
objective

Measuring the diameter of the field of view on a microscope can be made by placing a clear metric ruler on the stage of a microscope. The light microscope is used to look at cells or other similarly sized microscopic objects, so small units of measure such as millimeters or micrometers are used.  It is important to remember that there are 1,000 micrometers in 1 mm (millimeter) and 1000 millimeters in a meter.
 

Finding the Size of a Microscope Field of View

In the pictured field of view at the left, it can be observed that there are approximately 3 1/2 divisions equal to a length of 3.5 mm.   Therefore this field of view is equal to 3.5 mm
or 3,500 micrometers.

 

Finding the Size of Multiple Cells in a Field of View

The two cells in this field take up a field of view of one millimeter.  Therefore, the size of the specimen is equal to 1 mm/2 cells or 0.5 mm per cell.  There is 500 micrometers in 0.5 mm., so the average size of each cell is 500 micrometers.

 

Estimating Cell Size When the Field of View is Known

It is often difficult to approximate the approximate size of the field of view, but this ameba considered lengthwise appears to occupy approximately 1/3 of the field of view. The field of view in the left image is 3 mm. Given that the ameba in the image takes up about 1/3 of that field, we can find its approximate length by multiplying the 3 mm  X 1/3 = 1 mm length or 1,000 micrometers for the approximate length of this ameba.

The student is viewing the same ameba in the field of view at the right on a higher power. The field of view gets smaller which makes the ameba appear larger in this field.

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