Course: Biology Agricultural Science & Technology

UUNIT: THE MICROSCOPE AND CELLULAR DIVERSITY

UState Standard:

UState Objectives:

UUnit Objectives: A. Learn how to use the compound microscope.

B. Learn the proper care and handling of the compound microscope.

C. Learn the names of the parts of a microscope and functions of each.

D. Learn how to prepare a wet mount slide for microscopic observation.

E. Learn to estimate the sizes of the field of view and objects observed in the microscope.

F. Learn to distinguish in the microscope prokaryotic and eukaryotic cells.

UMaterials Needed (Equipment): Computer and LCD Projector “Microscope” PowerPoint Presentation Copies of Worksheet: “The Microscope and Cellular Diversity Lab” (enough for each

student to have one)o Activity 1

Compound Microscope Stereomicroscope Lens Paper Lens Cleaner Newsprint Transparent Ruler Microscope Slides Cover Slips Dropper Bottle with Water

o Activity 2 Prepared Slide of Stained Bacteria Microscope Slides and Cover Slips Culture of Live Cyanobacteria (Anabena)

Culture of Live Cyanobacteria (Oscillatoria)o Activity 3

Microscope Slides and Cover Slips Protoslo (methyl cellulose) Culture of Amoeba (live) Culture of Paramecium (live) Mixed Culture of Protists Elodea Plants Onions Dropper Bottle of I2KI (Iodine Solution) Toothpicks Dropper Bottle of 0.9% NaCl Dropper Bottle of 0.5% Methylene Blue

Copies of Worksheet: “How to Measure Microscopic Objects” Lab (enough for each student to have one)

Supplies Needed: Microscopes for student groups Flat ruler with designated millimeter marks for students to look at under

microscope (rulers included in dissection kits work great) Prepared human blood smear for each microscope group Optional: Calculators for groups

Copies of Worksheet: “Microscope Use Experiment” (enough for each student to have one) Supplies Needed:

Newsprint so students may cut out a letter “e”. It also works fine to print a word document that is filled with “e”s

Microscopes, Slides, and Cover Slips for groups Two different colored thread spools so students may cut off threads to look at

Additional Activities to Increase Student Comprehension: “Cell Observation Exercise”“Microscope and Cell Observation Lab”

Facilities:Teaching ClassroomLaboratory Classroom (or space in teaching classroom to use microscopes in)

Interest Approach:Ask students how life forms are arranged. Compare the way that life forms are arranged as compared to how buildings are built. Stack building blocks to show students how plant cells are arranged as compared to how animal cells are arranged in a body (no certain way).

Objective A: Learn how to use the compound microscope.

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

0BLight MicroscopyThe light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments.

A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order,

obtaining sufficient contrast finding the focal plane obtaining good resolution recognizing the subject when one

sees it

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field microscopes, though. These pages will describe types of optics that are used to obtain contrast, suggestions for finding specimens and focusing on them, and advice on using measurement devices with a light microscope.

2BTypes of light microscopes

The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation

Demonstrate how to use a microscope for students in front of the class.

Activity:

“Microscope Use Experiment”

Interest Approach:

Objective B: Learn the proper care and handling of the compound microscope.

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

Use of the Microscope1. Carry with both hands.2. Set microscope in from edge of desk.3. Use lens paper to clean eyepiece and objectives.4. Start with lowest power (shortest objective) in place.5. Turn coarse adjustment so that stage is as close to low power as possible -- now while looking, turn coarse adjustment so stage starts to move away from objective.6. Clean up all spills and moisture from stage before putting away.7. Caution – Keep all water away from electrical parts8. Never use coarse adjustment with high power objective while trying to focus, because of danger of striking and breaking glass slide with objective.

14BCare of the microscope EVERYTHING on a good quality

microscope is unbelievably expensive, so be careful.

Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.

Hold the plug (not the cable) when unplugging the illuminator.

Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.

Always make sure the stage and lenses are clean before putting away the microscope.

NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents may separate or damage the lens elements or coatings.

Cover the instrument with a dust jacket when not in use.

Focus smoothly; don't try to speed through the focusing process or force anything. For example if you encounter increased resistance when focusing then

“Microscope” PowerPoint slides #12-15Have a microscope ready to demonstrate on

Go over these rules orally with students and have a microscope ready to demonstrate on.

Objective C: Learn the names of the parts of a microscope and functions of each.

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

The MicroscopeAgricultural Biology

Two major types of microscopes based on energy used by device:– Light microscope • Uses visible light that is magnified as it passes through glass lenses• Source: Light• Magnifies up to 2000 times by using glass lenses• Materials can be living or dead– Electron microscope • Electron beam passes through magnetic lenses before magnified image is projected on photographic film or a fluorescent screen similar to television.• Source: Electrons• Magnifies up to 2 million times by using magnets• Materials should be dead

You will use the compound microscope– Why is it “compound”? • Two lenses collect and focus the light from the source as it is transmitted through the sample.• For light to pass through the specimen, the specimen must be very thin.• The image of the specimen seen in the microscope is not only magnified but the resolution is improved.

“Microsocope” PowerPoint slides # 1-11, and 16-34

Question Box: What are the parts of the microscope? What is the function of each part?

Microscope UnitThe Microscope• Resolution : Ability to distinguish and separate fine details in a specimen, such as two objects that are close to each other as being two rather than one– Our eyes can “resolve” two objects that are at least 0.1 millimeters (mm) apart– A good light microscope has a resolution up to 1000 times closer or 0.1 micrometers (µm)The Microscope

• Contrast : Ability to distinguish detail; different absorption of light by parts of the object being viewed– Can be increased by decreasing light intensity or by staining of the specimen• Proper selection of stains also increases our ability to identify specific structures within specimenMicroscopic Terms• Slide : Piece of glass upon which specimen is placed for viewing.

• Cover Slip : Thin square piece of glass or plastic used to cover specimen.

• Field of View : Area of specimen which is seen through lens system; bright circle you see when looking into a microscope.Microscopic Terms• Depth of Field : Ability of lens to furnish distinct image above and below focal plane. Decreases with increase of magnification.

• Parfocal : When objective is in focus, it can be switched from one power to another, with only small fine focus adjustments necessaryMicroscopic Terms

• Magnification : Number of times an object’s apparent size is increased by lens system– Determined by multiplying power of objective by the power of eyepiece (usually 10x).

Power of objectivex Power of eyepiece (usually 10x)

Total magnificationMicroscopic Terms

• High Power : High power objective on nosepiece (usually 40x).

• Medium Power : Middle power objective on nosepiece (usually 10x).

• Low Power : Lowest power objective on nosepiece (usually 4x).

Functions of Microscope Parts

• Ocular Lenses : Upper lenses nearest to eyes; magnify image already magnified by objective; brings light rays to a focus at the eye

• Rotating Nose Piece : Revolving part that supports three objectives with different magnifications

• Objective Lens: Lens closest to specimen; magnifies image of specimen

• Stage : Flat table-like area where slide or specimen is placed.

• Iris Diaphragm Lever : Disc-shaped device, located beneath stage, controls amount of light passing through specimen

• Condenser

• Substage Light : Source of light which passes through specimen.

• Base : Bottom stand, which houses the light.

• Body : Contains mirrors to reflect light to eyepiece.• Head

• Arm : Basic frame, used to carry microscope.

• Thumb Clip : Hold slide on stage, to prevent it from slipping around.• Mechanical Stage

• Coarse Adjustment Knob : Roughly focuses image by bringing the specimen and objective lenses closer together or farther apart.

• Fine Adjustment Knob : Sharpens image of specimen by moving specimen and objective lenses very small distances closer together or farther apart.• Mechanical Stage Control • Light Control

Objective D: Learn how to prepare a wet mount slide for microscopic observation.

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

How do you prepare a wet mount slide? IntroductionA wet mount slide is the most common type of slide preparation for HmicroscopeH work. Wet mount slides are used to view living organisms, as well as liquid substances of all kinds. They are also used for any sort of specimen that needs to be kept moist. If you work with microscopes or are planning to start, you should learn how to make a wet mount microscope slide.

InstructionsDifficulty: Moderately Easy

Steps

Step OneUse a flat glass slide to prepare a wet mount. The slide should be clean and free of dust or other fine particles.

2Step Two

Suck up a few drops of your liquid specimen into a medicine dropper.

3Step Three

Pick up a flat glass slide with one hand, grasping it by the outer edges.

Question Box: How do you prepare a wet mount slide so that it can be viewed under a microscope?

4Step Four

Place one drop of the liquid specimen in the medicine dropper on top of the slide. Make sure the drop goes on the center portion of the slide.

5Step Five

Put down the medicine dropper. Do not tilt the slide in your other hand while you are doing this.

6Step Six

Use your free hand to carefully pick up a cover slip (they are extremely fragile). Grasp the cover slip by the outer edges.

7Step Seven

Place the cover slip on top of the slide, making sure the edges of the cover slip match up with the edges of the slide. Do not press down on the cover slip.

8Step Eight

Continue to grasp the slide/cover slip combination by the outer edges. You should keep the slide as horizontal and steady as possible, and place it on the viewing tray of your microscope.

1BHow to Prepare a Wet Mount (Vaseline mount)Living specimens do not survive long in the heat from an intense microscope illuminator

Question Box: What if the wet mount keeps drying out before we can look at it under the microscope?

bulb, usually because the specimen dries up. This problem is easily solved by preparing a Vaseline chamber. Simply take a single cover slip and hold it between thumb and forefinger by the edges. Pick up some Vaseline on the other forefinger and rub it over your thumb to make a film. Scrape your thumb carefully on each edge of the cover slip to make a continuous Vaseline ledge.

Place a drop or two of suspension on a clean slide, and turn the cover slip over on top of the drop. Press down the edges to seal the chamber against evaporation. When preparing a Vaseline mount, keep in mind that the image becomes degraded with thicker mounts, especially at high powers in dark field or phase contrast. Unless the specimen is large and fragile enough to be damaged by pressing down too hard on the cover slip, keep the chamber depth very shallow.

Activity:

Have students practice making a wet mount and pass it off to the teacher. Also, have students practice making a wet mount with Vaseline to ensure student comprehension.

Objective E: Learn to estimate the sizes of the field of view and objects observed in the microscope.

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

Microscopic MeasurementThe 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 ofthe

objective

The size of a microscopic field of view can be determined on low power using a device called an optical micrometer.   An economy version of this can be made by placing a clear metric ruler onthe stage of a microscope and using it to estimate the field of view.  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.

Question Box: How do you estimate how big objects under the microscope are?

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.

Activity:

“How to Measure Microscopic Objects” Lab Supplies Needed:

One lab sheet for each student Microscopes for student groups Flat ruler with designated millimeter marks for students to look at under

microscope (rulers included in dissection kits work great) Prepared human blood smear for each microscope group Optional: Calculators for groups

Objective F: Learn to distinguish in the microscope prokaryotic and eukaryotic cells.

Activity: “The Microscope and Cellular Diversity Lab”

o Activity 1 Compound Microscope Stereomicroscope Lens Paper Lens Cleaner Newsprint Transparent Ruler Microscope Slides Cover Slips Dropper Bottle with Water

o Activity 2 Prepared Slide of Stained Bacteria Microscope Slides and Cover Slips Culture of Live Cyanobacteria (Anabena) Culture of Live Cyanobacteria (Oscillatoria)

o Activity 3 Microscope Slides and Cover Slips Protoslo (methyl cellulose) Culture of Amoeba (live) Culture of Paramecium (live) Mixed Culture of Protists Elodea Plants Onions Dropper Bottle of I2KI (Iodine Solution) Toothpicks Dropper Bottle of 0.9% NaCl Dropper Bottle of 0.5% Methylene Blue

Curriculum (Content)(What to teach)

Instruction (Methodology)(How to teach)

“The Microscope and Cellular Diversity Lab” worksheet allows students to practice using the microscope while looking at different types of cells

Question Box: What is the difference between prokaryotic and eukaryotic cells?

Review and Evaluation:Objectives will be reviewed before examinations. Exams will be developed based on objectives as taught in class.

Instructional Support and References:

Bierer, Loretta Kett. 1991. Life Science: The Challenge of Discovery Program. D.C. Heath and Company. Lexington, Massachusetts.

Oram, Raymond F. 1994. Biology: Living Systems. Macmillan/McGraw-Hill Publishing Company. Westerville, Ohio.

Oswego City School District Regents Exam Prep CenterHhttp://regentsprep.org/Regents/biology/biology.cfm