The Microscope sffiffio€ . $e t . a5

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The microscope, developed more than three centuries ago, is the basic tool of the biologist. Amicroscope enables biologists io investigate living things and objects that are too small to be seen with theunaided eye. The microscope is able to magnifr these tiny specimens by means of lenses located in theeyepiece and objectives. T[e [gtrt microscope is also capable of revealing fine detail. This ability to reveal fineaitiit is known as resolving power. The type of microscope that you will be using throughout your study ofbiology is the compound light microscope.-

Specimens ihat are viewed under the microscope are mounted on one of two types of glass slides.Prepared slides are slides that are for permanent use. \ilet-mount slides are for temporary use. Most of theslides that you will use in biology will be wet mounts.

In ybur study ofbiology, it will be necessary for youto estimate the length and width of some olfg,urspecimens. To measure objecti under the microscope, a unit called the micrometer (pm), rygetimes calledtfre micton (p), is used. One micrometer equals 0.001 millimeter; one millimeter equals 1000 micrometers.

Give the purpose of the microscope:

Define resolving power:

Name the type of microscope used in high school biology plasses:

Contrast the use of prepared and wet-mount slides:

Name the preferred unit of measurement for microscope work:

Tell how many micrometers are in a millimeter:

also called the

Give the syrnbol for the micrometer:

Stage openingpermits light from light source topass uP through body tub€

Oiaphngmregulates amount ol l.lghtpassing through sPeclmen

Lamp or mirtordirects lighl through diaphragmand stage openrng

Carrying the Microscope: Take the microscope from the storage arca. Carry the microscope with one handunder the base and the other hand grasping the arm.

Place the microscope on the laboratory table. The microscope should be about 10 cm from the edge of thetable. Uncoil and plW the cord into an outlet at your lab station. The on(l)-otr(0) switch is located on theleft side ofthe base ofthe scope.

Parts of the Microscope: Look at the drawings on the previous page that is most like your microscope.Identifr the parts and functions of your microscope.

Cleaning the Microscope: Carefully clean the eyepiece and objectives lenses with lens paper. Locate thenosepiece and gently turn it so that the low-power scanning objective is in line with the body tube. Thenosepiece will click into place when the objective is in the proper position.

Field of View: Keeping both eyes opeo look through the eyepiece. You will see a circle of light. This iscalled the field of view. To make the circle of light as bright as possible, you may have to adjust thediaphragm.

Your Dominant Eye: If you are observing through a single lens (monocular) scope, learn to see with yourdominant eye while training the other eye to relax and not concentrate on anything. This technique is usefuland is easily learned with practice. It will come in handy when you draw your specimens as you can use yourweaker eye to help you see your drawing while observing with the dominant eye.

Right or Left? Which is your dominant eye?

In order to determine which eye is dominant (stronger), there is asimple test. Use your hands to form a circle at arm's length. Lookacross the roonl through the circle, both eyes open (relax), at someobject. Slowly bring the circle toward your face. Your "hand-circle" will probably go to one eye or the other-this is yourdominant eye. Use this eye for viewing.

Describe your hand-positionwhen carrying the microscope:

Give the rulme for the circle of light you see wlren looking through the eyepiece:

Name the microscope part used to adjust the amount of light in the field of view:

Contrast a monocular and binocular scope:

Which eye is your dominant eye?

There are three objective lenses on your microscope. The shortest lens is called the scanning lens-it has ared ring around it. The low-power objective is the medium-length lens and has a yellow ring around it. Thehigh-power objective is the longest lens and has a blue ring around it.

The lens you are using is the one that is "clicked" into place above the stage opening. You could also say itis the one below the body tube.

A4iust your micrqscope so the scanning lgns is in place for viewine a specimen.

People normally have a stronger(dominant) eye (the one that ismost used). It is often the oneused for microscopic observation,unless you are using a binocularstereo microscope. Then, youwould use both eyes to observeyour object or specimen.

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Preparing and Observing a \ilet Mount

Obtain a glass slide and cover slip. Wipe both sides of the slide with a cloth to remove dust, etc. The cleanedslide and cover slip should be handled by their edges.

Cut out a small piece of newspaper that contains the letter "e." This should be a standard newsprint letter-NOT a headline-size letter-Avoid dark backgrounds on either side of the newsprint. Place the letter "e" in thecenter ofthe slide as it would appear in the newspaper-face-up. rieht side up.

Using a pipette, place a drop of tap water on top ofthe "e." IJse a probe to hold the "e" in place while you addthe drop of water, if necessary.

Hold a cover slip at about a45o ang,le over the drop of water. Gently lower the cover slip onto the slide. If airbubbles appear, gently tap the cover slip with the back end of the probe.

Dissecting needle

Place the wet mount of the letter "e" on the stage of the microscope rryith the letter facing yor+ as you wouldread it in print. Adjust the slide so that the letter is above the opening of the stage.

Look at the slide at eye level. Observe the space between theslide and the scanning objective. The scanning objective shouldbe in line with the body tube (over the stage opening). Slowlyturn the coarse-adjustment knob, raising the stage to its highestposition. You are now ready to view your specimen.

Look through the eyepiece and SLOWLY turn the coarse-adjustment knob until the letter "e" comes into focus. Inthecircle below (which represents a field of view), sketch what yousee.

Calculate the magnification (eyepiece x objective).Show your math in the space below.

Leave your slide on the stage and have your instructor veriffyour view. Your instructor will initial the blank below.

CAUTIONz Never raise the stagewhile looking through the eltepiece;you may hit and danrage the slide orohjective lens. Look to the side, raisethe stage to its nruxintum height, thenfocus u,hile the stage is movirtg AWAYfi'om the objective.

CompletedMagnification

Make a second slide of a letter o'e" but this time from print provided by your instructor. Once again, check theorientation of the letter (it should be in the same position as it appears in print).

Rotate the nosepiece to the actual low-power lens. This is the mid-length lens (with a yellow circle) and ismarked with a magnification of 10X.

Following proper technique, locate and focus on the letter.

Calculate the magnification below (show your math):

Sketch what you observe below.

Completed Magnification

Paradoxical Movement: The microscope exhibits an optical phenomenon called paradoxical movement.

Move the slide to the right......Which way does it "appear" to move in the field of view? Move it

to the left.. ..Which way does it o'appear" to move? Move the slide away from you, then toward

you. Which way does it "appear" to move each time? What does it mean

when an optical instrument exhibits paradoxical movement?

Practice centering an object in the field of view. For example, an object is on the edge of the field of view andyou want to center it before you ask your partner to take a look. Determine which way you would move theslide to center the specimen. Below are some sample problems. Use an arrow (or two arrows) to show whichway you would move the slide to center the object:

Y

To observe a specimen at high-power magnifrcation, turn the nosepiece until the high-power objective clicksinto place. (The high-power objective is the longest object, has a blue ring, and is marked 40X.)

You microscope is parfocal. This allows you to focus the scope at low power, switch to high power, and, withonly minor adjustment of the fine-adjustment knob, see the object at high power. For that reason, here isanother general rule of microscopy:

To view an object at highswitch to high power and

Observe the letter at high power. Draw what you observe

Calculate the magnification below (show your math):

Completed Magnification

Measuring an Object Under the Microscope

Turn the nosepiece so the scanning lens (4x, 40x total) is in place. Place a millimeter scale of a transparentplastic ruler over the center of the stage opening in the microscope.

Use the scanning objective to locate the millimeter lines of the ruler. Place these lines in the middle of the fieldof view and use the coarse-adjustment knob to bring them in to focus. The distance between two lines on theruler represents I mm.

power, always begin by focusing on it at low power. Then,use the fine-adjustment knob to bring the object into view.

While looking through the eyepiece, move theruler so that one of the millimeter lines is justtouching the left side of the field of view. Youruler will look like the diagram but you will beable to see more millimeter lines since you areworking at a lower magnification.

To determine the diameter of the field of view forthe scanning lens, count the number if millimeterlines (actually spaces) that are visible. You willneed to estimate the diameter to the nearest tenthof a millimeter.

Field of view

2 millimeters

Since the micrometer (pm) is the preferred unit of measurement for use with the microscope, convert yourmeasurement in millimeters to micrometers by moving the decimal three places to the right (effectivelymultiplying by 1 000).

Scanning lens (4x, 40x total) field of view: mm or

Next rotate the nosepiece to the low-power objective (10x, 100 x total) and determine the field of view. Countthe number of millimeter lines that are visible. Since the millimeter marks themselves are now rather large, youwill need to move the edge of one of the marks to the left side of the field of view and count from the left edgeof one mark to the left edge of the next. Don't forget to estimate the nearest 0.1 mm.

Low-power objective (10x, 100x total) field of view: mm or pm

Because under high power (40x, 400x total) the thickness of one of the millimeter lines takes up practically theentire field of view, it is difficult to estimate the diameter of the field veiw under high power magnification.

The diameter under high power can be calculated on paper, however. Here are the steps:

l. Divide the magnification of the high-power objective by the magnification of the low power objective.Show your math in the space below:

2. Then divide the diameter of the low-power field size of view in micrometers by the answer to the stepone above. Again, show your math:

High-power objective (40x,400 total field of view: mm or pm

Sample problem: Here is what you see in your scope at 100x (using the 10x objective):

For low power (10x, l00x total):

Field of view Edge of ruler

For high power (40x, 400x total):

Mill imeter l ines

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2 millimeters

Record your group's field sizes for the scanning (4x, 40x total), low-power (10x, l00x total), and calculatedhigh-power (40x, 400x total) on the data table in class. The class will adopt a standard set of field sizes fromclass data. Do not write anything in the section below until all the class data has been recorded.

Class standards:

Prepare a wet-mount of a human hair. Observe and draw the hair at scanning, low, and high powers.

Low-power x High-power

To calculate the width of a human hair, estimate how many could be placed side-by-side across the field ofview. Divide the size of the field of view by this number. Show your math for each problem in the spacebelow:

7

Practice problems:

Human Hair l00x Human Hair 400x

/tPrepare a slide of two crossed threads of different colors.in the space below. You drawings should include color.

View the threads at low power and draw what you seeEstimate the width of the threads.

Crossed Threads

Magnification

Width of a thread pm

v<. qrhPl"[" o\

\ l iJ U\ c,*1"^\q{, ons :

Make a temporary wet mount of material from your'oPond inaJar" and view it using the microscope. Attemptto choose one microorganism and draw it in the space below.

Pond in a Jar

Magnification

Width or length _ pm\^ri A+h c.o.tc\^lslions :

Continue your work with the pond water by preparing additional slide(s) and locating at least two othermicroorganisms. Draw them in the spaces below and use the field guide to identi$z them.

Magnification x

Width or length

Cq[o.rt\.^li snr ..

Magnification

Width or length

C"o.l.-,rl q{'on; }

pm pm

1

Label the parts of the microscope in the figure below:

Looking through the microscope, in what direction does the letter "e" appear to move when you moved the slide

to the rieht? to the left? away from you? toward vou?

What is the above phenomenon called?

Calculate the total magnification of your microscope for the following objectives. (Show your math)

Scanning

Low-power

High-power

What happens to the focus of the letter'oe" as you change from low-power to high-power magnification?

When the above occurs, your microscope is said to be

How many times is the magnification increased when you change from low-power to high-power

masnification?

What happens to the size of the field of view when you change from low-power magnification to high-power

maenification?

How many micrometers are in a millimeter?

Give the alternate name for the micrometer:

Give the unit for the micrometer:

Give its unit:

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