Biology

Chapter 2

Scientific Method/Microscope

Concepts/Ideas/Facts:

1.   science is a body of knowledge as well as a way of learning about the natural world.

Definitions:

1. Field Study – the observing and the collecting of data and sometimes specimens at a particular site. 
2. Scientific Method/Process – the posing of questions about nature and the discovering of facts that help answer those questions usually in an orderly fashion or sequence.

                A.   Observations– using one or more of the five senses, scientists “sees” something in nature that piques his interest.

a.       Collecting Data –gathering and recording of specific information based on observations.

b.      Measuring – process of determining dimensions of an object, the number of objects in a group, the duration of an event, or other characteristics in precise units.

c.       Organizing Data – placing observations and measurements in some kind of logical order, such as graphs, charts, tables, or maps.

d.      Classifying – process of grouping objects, organisms, or phenomena into an established scheme, or developing a new scheme

                 B.   Question – scientist asks a question about what is observed or studied

                 C.   Hypothesis – forming a testable statement about an observable phenomenon. A hypothesis cannot be proven beyond all doubt. It can only be supported by evidence.

D.      Prediction – stating in advance the result that will be obtained from testing a hypothesis. Often it is an if-then statement.

E.       Experimenting – process of testing a hypothesis or prediction by carrying out data-gathering procedures under controlled conditions.

a.       Controlled Experiment – is based on a comparison of a control group or phase with an experimental group or phase.  The control group and the experimental group are designed to be identical except for one factor, called the independent variable or manipulated variable.

b.      Dependent Variable (Responding Variable) – the measurement or observation of one main factor in both the control group and experimental group.  It is the “what happens after” measurement or observation.

3. Analyzing Data – process of determining whether data are reliable and whether they support or refute a given prediction or hypothesis.
4. Inferring – process of drawing conclusions on the basis of facts or premises instead of direct perceptions.
5. Modeling – involves constructing a representation of an object, a system, or a process that helps show relationships between data.  A model may be visual, verbal, or mathematical.
6. Communicating – sharing information which allows one scientist to build on the work of another.
1. Operational Definitions – definitions that are limited to repeatable and observable phenomena and make it clear to other scientists how something might be measured.
7. Law – general statement that describes a wide variety of phenomena.
8. Theory – most probable explanation for a set of data based on the best available evidence

The Microscope:                                                                                                                                   Notes:

1. the size of any feature of an object can be determined by measuring the size of that feature’s image and dividing it by the magnification.
2. eyepiece (ocular) magnification times objective magnification equals total magnification.
3. increasing magnification decreases the field of view size and may put image of specimen outside the field of view.
4. to go from millimeters to micrometers move the decimal point 3 places to the right of 3.  eg. 375 = 375, 000um
5. everything view through a microscope is upside a down and backwards
6. reducing light increases contrast
7. when switching from low to high power, the are in the field of view gets smaller and darker (you see a smaller area under slide under higher magnification)
8. the size of the objective lens aperture (opening) decreases with increasing magnification, allowing less light to enter objective lens, making it necessary to increase light intensity at higher magnifications

Definitions:

1.      Magnification – the apparent enlargement of an object under a lens.  The ratio of image size to actual size.

2.      Resolution – clarity and sharpness.  The ability of a microscope to show two very close points separately

3.      Field Size – the size of the field of vision under magnification.  The area of the slide you see when you look through a microscope.

4.      Total Magnification (TM) – equal to the power of the ocular lens multiplied by the power of the objective lens

5.      Resolving Power – determined by the amount and physical properties of the visible light entering the microscope.  The more light delivered, the greater the resolution

6.      Working Distance – the distance between the specimen (slide) and the bottom of the objective lens

7.      Parfocul – self-focusing lenses

8.      Depth of Field – the depth of a specimen clearly in focus  or relative depth.  Depth of field is lower at greater magnifications.

9.      Stereomicroscope – used for magnifying large specimen.  It acts like a large magnifying glass.

Parts of a Microscope:

1. Objective Lenses – the lenses closest to the specimen, which can be rotated to increase or decrease magnification.
2. Nose Piece – rotating part of the microscope that holds the objective lenses
3. Ocular Lens (Eye Piece) – lens in the eye piece which magnifies 10X
4. Body tube – between the nose piece and ocular lens, it maintains the correct distance between the eye piece and objective lens.
5. Stage or Platform – area where the specimen is heal beneath the objective lenses.
6. Stage Clips – metal clips that hold the glass slide firmly in place beneath the lens
7. Coarse Adjustment Knob – Moves body tube or stage up or down for major adjustments
8. Fine Adjustment knob – Moves body tube or stage up or down very slightly for sharpening image
9. Arm – supports the body tube (eye piece)
10. Base – supports the microscope
11. Iris/Diaphram – similar to a camera shutter, it controls the amount of light let through the stage and through the slide sample
12. Illuminator (light source) – light source from beneath the stage that illuminates the specimen.  Less is best.

Focusing

1. Start with the lowest magnification objective lens closets to the specimen (slide) then focus by using the coarse adjustment knob to slowly Raise the lens until it is in focus.  If it doesn’t focus properly then use the fine focus adjustment knob.
2. Adjust the Diaphragm as you look through the Eyepiece, and you will see that MORE detail is visible when you allow in LESS light!  Too much light will give the specimen a washed-out appearance.
3. center your specimen before raising the magnification
4. Once you have it on High Power you only use the fine focus knob!
5. When using the oil immersion lens, the oil is needed to help gather enough light to actually see through the lens!  Do not use oil for any other lens!

Making a Wet Mount

1. gather a thin slice/piece of specimen.  If your specimen is too thick, the coverslip will wobble on top of the sample like a see-saw.
2. Place one drop of water directly over the specimen.  Too much water will cause the coverslip to float on top of the water and the specimens will float past the field of view.
3. with one end of the coverslip touching the water (the drop on the specimen), place the cover slip at a 45% (about) and gently lower it (prevents air bubbles).

How to Stain a Slide

1. wipe off the excess stain so the lens doesn’t get covered in blue   
2. Place one drop of Methylene Blue stain onto one edge of an already made wet mount slide  (coverslip) (coverslip) and a piece of paper towel on the other edge of the coverslip to draw water out from  under the  coverslip  This will draw the stain under the coverslip..
3. at the end of the experiment be sure to wash and dry both the slide and coverslip and return to their correct places.

Oil Immersion

1. The only reason to use an oil-immersion objective is to take advantage of the highest resolving power available to the microscopist.  
2. The drop of immersion oil becomes the only optical medium between the specimen and the frontlens of the objective.
3. Place the covered slide on the stage and locate the area of interest using the lowest magnification
4. With the specimen focused close the lamp diaphragm to a small aperture in the center of the field
5. Swing the lower magnification out of the way and add a drop of oil on the coverslip
6. Swing the oil lens into position and focus with the oil lens
7. Open the lamp diaphragm and focus the proper light
8. When done remove the oil with a piece of tissue paper, clean with lighter fluid not alcohol!

Calculations:

Total Magnification:

ocular magnification multiplied by the objective magnification equals total magnification.

Ocular magnification times objective magnification = total magnification

Example:      10x  X  50x  = 100x total magnification                                                                             

Calculating fields of view:

The diameter of field A multiplied by the total magnification of field A  =  diameter of field B multiplied by the total magnification of field B                          Or:

Diameter of Field B = diameter of field A x total magnification of A

                                                  total magnification of B

Example:

Field A:  diameter  =  2mm             total magnification = 50x

Field B    diameter =  ?                    total magnification = 100x

Field B ? =  (2mm x 50x)    =   1mm      

                         100x

The fraction of the area seen under high power is the same as the ratio of the low and high power magnification.  Example; if the low power objective is 20x and the high power objective is 40x, then under high power you’ll see 20/40 or ½ the area of the slide as seen under low power.

MICROSCOPY MEASUREMENTS:

Nanometer (nm) – 1/1,000,000,000 meter  (1 one billionth of a meter)                                                            Micrometer (um) - 1/1,000,000 meter  (1 one millionth of a meter)                                                                  Millimeter (mm) - 1/1,000 meter (1 one thousandth of a meter)                                                                      Centimeter (cm) – 1/100 of a meter (1 one hundredth of a meter)

                                                           SCIENTIFIC METHOD

Test supports hypothesis:                                                                                                                      Test does not support hypothesis:                                                                                  Make more predictions and test them.                                                                                                                           Revise hypothesis.