MATERIALS

o Compound microscopeo Millimeter ruler

o Prepared slides of the letter e ornewsprint

o Immersion oil

o Lens paper

o Prepared slide of grid ruled in millimeters(grid slide)

o Prepared slide of three crossed coloredthreads

o Clean microscope slide and coverslip

o Toothpicks (flat-tipped)

o Physiologic saline in a dropper bottle

o Iodine or methylene blue stain (dilute) ina dropper bottle

o Filter paper or paper towels

o Beaker containing fresh 10% householdbleach solution for wet mount disposal

o Disposable autoclave bag

o Prepared slide of cheek epithelial cells

~ote to the Instructor: The slides and cov-erslips used for viewing cheek cells are to besoaked for 2 hours (or longer) in 10% bleachsolution and then drained. The slides and dis-posable autoclave bag (containing coverslips,lens paper, and used toothpicks) are to be au-toclaved for 15 min at 121°C and 15 poundspressure to ensure sterility. After autoclaving,the disposable autoclave bag may be dis-carded in any disposal facility, and the slidesand glassware washed with laboratory deter-gent and reprepared for use. These instruc-tions apply as well to any bloodstainedglassware or disposable items used in otherexperirnentru procedures.

.~ '"astenngA&P~

-ceess practice quizzes and more in the5.t..v~Area at www.masteringaandp.com.

The Microscope

OBJECTIVES

1. To identify the parts of the microscope and list the function of each.2. Todescribe and demonstrate the proper techniques for care of the

microscope.3. Todefine total magnification and resolution.

4. Todemonstrate proper focusing technique.5. To define parfocal, field, and depth of field.6. Toestimate the size of objects in a field.

PRE-LAB QUI Z

1. The microscope slide rests on the while being viewed.a. base c. irisb. condenser d. stage

2. Your lab microscope is parfocal. This means that:a. The specimen is clearly in focus at this depth.b. The slide should be in focus at higher magnifications once it is

properly focused at lower magnifications.c. You can easily discriminate two close objects as separate.

3. If the ocular lens magnifies a specimen 10x, and the objective lensused magnifies the specimen 35X, what is the total magnification beingused to observe the specimen? _

4. How do you clean the lenses of your microscope?a. with a paper towelb. with soap and waterc. with special lens paper and cleaner

5. Circle True or False.You should always start your observation ofspecimens with the oil-immersion lens.

6. Circle True or False. It is always necessary to use a coverslip with a wetmount to prevent soiling or damaging the objective lens.

With the inventi.on of the microscope, biologists gained a valuable toolto observe and study structures (like cells) that are too small to beseen by the unaided eye. The information gained helped in establish-

ing many of the theories basic to the understanding of biological sciences. Thisexercise will familiarize you with the workhorse of microscopes-the com-pound microscope-and provide you with the necessary instructions for itsproper use.

27

28 Exercise3

Care and Structure of theCompound MicroscopeThe compound microscope is a precision instrument andshould always be handled with care. At all times you must ob-serve the following rules for its transport, cleaning, use, andstorage:

• When transporting the microscope, hold it in an uprightposition with one hand on its arm and the other supporting itsbase. Avoid swinging the instrument during its transport andjarring the instrument when setting it down.

• Use only special grit-free lens paper to clean the lenses.Use a circular motion to wipe the lenses, and clean all lensesbefore and after use.

• Always begin the focusing process with the lowest-power objective lens in position, changing to the higher-power lenses as necessary.

• Use the coarse adjustment knob only with the lowest-power lens.

• Always use a coverslip with temporary (wet mount)preparations.

• Before putting the microscope in the storage cabinet, re-move the slide from the stage. rotate the lowest-power objec-tive lens into position, wrap the cord neatly around the base,and replace the dust cover or return the microscope to the ap-propriate storage area.

• Never remove any parts from the microscope; informyour instructor of any mechanical problems that arise.

ACTIVITY 1

Identifying the Parts of a Microscope1. Obtain a microscope and bring it to the laboratory bench.(Use the proper transport technique!)

• Record the number of your microscope in the summarychart on page 30.

Compare your microscope with the illustration in Figure3.1 and identify the following microscope parts:

Base: SUPPOlts the microscope. (Note: Some micro-scopes are provided with an inclination joint, which al-lows the instrument to be tilted backward for viewing drypreparations. )Substage light or mirror: Located in the base. In micro-scopes with a substage light source, the light passes di-rectly upward through the microscope: light controls arelocated on the microscope base. If a mirror is used, lightmust be reflected from a separate free-standing lamp.

Stage: The platform the slide rests on while beingviewed. The stage has a hole in it to permit light to passthrough both it and the specimen. Some microscopeshave a stage equipped with spring clips; others have aclamp-type mechanical stage as shown in Figure 3.1.Both hold the slide in position for viewing: in addition,the mechanical stage bas two adjustable knobs that con-trol precise movement of the specimen.

Condenser: Small substage lens that concentrateslight on the specimen. The condenser rna) have aand pinion knob that raises and lowers the c. -ndenservary light delivery. Generally, the best po iti••n forcondenser is close to the inferior surface of the stage

Iris diaphragm lever: Arm attached (0 the base ofcondenser that regulates the amount of light pthrough the condenser. The iris diaphragm perrnu thebest possible contrast when viewing the specimen.

Coarse adjustment knob: Used to focus on the specimen

Fine adjustment knob: Used for precise focusing oncecoarse focusing has been completed.

Head or body tube: Supports the objective lens syste(which is mounted on a movable nosepiece) and the <'(-

ular lens or lenses.Arm: Vertical portion of the microscope connecting thebase and head.

Ocular (or eyepiece): Depending on the microscope, then:are one or two lenses at the superior end of the head or bod:tube. Observations are made through the ocular(s). An oc-ular lens has a magnification of lOX. (It increases the ap-parent size of the object by ten times or ten diameters.) Ifyour microscope has a pointer (used to indicate a specificarea of the viewed specimen), it is attached to one ocularand can be positioned by rotating the ocular lens.

Nosepiece: Rotating mechanism at the base of the head.Generally carries three or four objective lenses and per-mits sequential positioning of these lenses over the lightbeam passing through the hole in the stage. Use the nose-piece to change the objective lenses. Do not directly grabthe lenses.Objective lenses: Adjustable lens system that permitsthe use of a scanning lens, a low-power lens, a high-power lens, or an oil immersion lens. The objectivelenses have different magnifying and resolving powers.

2. Examine the objective lenses carefully; note their rela-tive lengths and the numbers inscribed on their sides. Onmany microscopes, the scanning lens, with a magnificationbetween 4X and sx, is the shortest lens. If there is no scan-ning lens, tbe low-power objective lens is the shortest andtypically has a magnification of lOX. The high-power objec-tive lens is of intermediate length and has a magnificationrange from 40X to SOX, depending on the microscope. Theoil irrunersion objective lens is usually the longest of the ob-jective lenses and bas a magnifying power of 9SX to looX.Some microscopes lack the oil immersion lens.

• Record the magnification of each objecti ve lens of yourmicroscope in the first row of the summary chart on page 30.Also, cross out the column relating to a lens that your micro-scope does not have. Plan on using the same microscope forall microscopic studies.

3. Rotate the lowest-power objective lens until it clicks intoposition, and tum the coarse adjustment knob about 180 de-grees. Notice how far the stage (or objective lens) travels dur-ing this adjustment. Move the fine adjustment knob 180degrees. noting again the distance that the stage (or the ob-jective lens) moves. _

Condenserrack andpinion knob(regulatesheight ofcondenser)

FIG U RE 3. 1 Compound microscope and its parts.

Magnification and ResolutionThe microscope is an instrument of magnification. In thecompound microscope. magnification is achieved through theinterplay of two lenses-the ocular lens and the objectivelens. The objective lens magnifies the specimen to produce areal image that is projected to the ocular. This real image ismagnified by the ocular lens to produce the virtual imageseen by your eye (Figure 3.2).

The total magnification (TM) of any specimen beingviewed is equal to the power of the ocular lens multiplied bythe power of the objective lens used. For example, if the ocu-lar lens magnifies iox and the objective lens being usedmagnifies 45X, the total magnification is450X (or 10 X 45).

• Determine the total magnification you may achieve witheach of the objectives on your microscope, and record the fig-ures on the third row of the chart.

The compound light microscope has certain limitations.Although the level of magnification is almost limitless. the

TheMicroscope 29

Ocular lenses

-:----";~'-+- Rotatingnosepiece

~~~rr--Iris diaphragmlever

Substage light

resolution (or resolving power), that is, the ability to dis-criminate two close objects as separate, is not. The human eyecan resolve objects about 100 urn apart, but the compoundmicroscope has a resolution of 0.2 flm under ideal conditions.Objects closer than 0.2 urn are seen as a single fused image.

Resolving power is determined by the amount and physicalproperties of the visible light that enters the microscope. In gen-eral, the more light delivered to the objective lens, the greaterthe resolution. The size of the objective lens aperture (opening)decreases with increasing magnification, allowing less light toenter the objective. Thus, you will probably find it necessary toincrease the light intensity at the higher magnifications.

ACTIVITY 2

Viewing Objects Through the Microscope1. Obtain a millimeter ruler. a prepared slide of the letter eor newsprint, a dropper bottle of immersion oil, and some lenspaper. Adjust the conden er to its highest position and sw itch

30 Exercise 3

@Retlna

CD Real image

®Virtual image

FIG U R E 3.2 Image formation in light microscopy.Step (j)The objective lens magnifies the object, forming thereal image. Step ®The ocular lens magnifies the real image,forming the virtual image. Step @The virtual image passesthrough the lens of the eye and is focused on the retina.

on the light source of your microscope. (If the light source isnot built into the base, use the curved surface of the mirror toreflect the light up into the microscope.)

2. Secure the slide on the stage so that you can read the slidelabel and the letter e is centered over the light beam passingthrough the stage. If you are using a microscope with springclips, make sure the slide is secured at both ends. If your mi-croscope has a mechanical stage, open the jaws of its slide re-tainer (holder) by using the control lever, typically located atthe rear left corner of the mechanical stage. Insert the slidesquarely within the confines of the slide retainer. Check thatthe slide is resting on the stage (and not on the mechanicalstage frame) before releasing the control lever.

3. With your lowest-power (scanning or low-power) objec-tive lens in position over the stage, use the coarse adjustmentknob to bring the objective lens and stage as close together aspossible.

4. Look through the ocular lens and adjust the light for com-fort using the iris diaphragm. Now use the coarse adjustmentknob to focus slowly away from the e until it is as clearly fo-cused as possible. Complete the focusing with the fine ad-justment knob.

5. Sketch the letter e in the circle on the summary chart onpage 31 just as it appears in the field (the area you see throughthe micro cope).

How far is the bottom of the objective lens from the speci-men? In other words, what is the working distance? Use amillimeter ruler to make this measurement.

Record the working distance in the summary chart.

How has the apparent orientation of the e changed top to"tom, right to left, and so on?

6. Move the slide slowly away from you on the stage as yoview it through the ocular lens. In what direction does the im-age move?

Move the slide to the left. In what direction does the imagemove?

At first this change in orientation may confuse you, but withpractice you will learn to move the slide in the desired direc-tion with no problem.

7. Today most good laboratory microscopes are parfocal;that is, the slide should be in focus (or nearly so) at the highermagnifications once you have properly focused. Withouttouching the focusing knobs, increase the magnification byrotating the next higher magnification lens (low power orhigh power) into position over the stage. Make sure it clicksinto position. Using the fine adjustment only, sharpen the fo-cus. * Note the decrease in working distance. As you can see,focusing with the coarse adjustment knob could drive the ob-jective lens through the slide, breaking the slide and possiblydamaging the lens. Sketch the letter e in the summary chart.What new details become clear?

As best you can, measure the distance between the objectiveand the slide.

Record the working distance in the summary chart.

Is the image larger or smaller? _

Approximately how much of the letter e is visible now?

Is the field larger or smaller? _

Why is it necessary to cemer your object (or the portion of theslide you wish to view) before changing to a higher power?

*If you are unable to focus with a new lens, your microscope is notparfocal. Do not try to force the lens into position. Consult yourinstructor.

The Microscope 31

Summary Chart for Microscope # _

Scanning

Magnification ofobjective lens ____ x

Magnification ofocular lens __ 1.;_:0:..___ x

Total magnification ____ x

Working distance ____ mm

Detail observedLetter e

Field size (diameter) __ mm __ ~m

Low power

____ x

_----,-,1O:..___ x

____ x

____ mm

High power Oil immersion

____ x ____ x

10 x---'--- __ 1:....:0:..____ x

____ x ____ x

____ mm ____ mm

__ mm __ ~m __ mm __ ~m __ mm __ ~m

Move the iris diaphragm lever while observing the field.What happens?

Is it more desirable to increase or decrease the light whenchanging to a higher magnification?

_______ Why? _

8. If you have just been using the low-power objective, re-peat the steps given in direction 7 using the high-power ob-jective lens. What new details become clear?

Record the working distance in the summary chart.

9. Without touching the focusing knob, rotate the high-power lens out of position so that the area of the slide over theopening in the stage is unobstructed. Place a drop of immer-sion oil over the e on the slide and rotate the oil immersionlens into position. Set the condenser at its highest point (clos-est to the stage), and open the diaphragm fully. Adjust the finefocus and fine-tune the light for the best possible resolution.

Note: If for some reason the specimen does not come into\ iew after adjusting the fine focus, do not go back to the 40Xlens to recenter. You do not want oil from the oil immersion

lens to cloud the 40X Jens. Turn the revolving nosepiece inthe other direction to the low-power lens and recenter and re-focus the object. Then move the immersion lens back into po-sition, again avoiding the 40x lens. Sketch the Jetter e in thesummary chart, What new details become clear?

Is the field again decreased in size? _

As best you can, estimate the working distance, and record itin the summary chart. Is the working distance less or greaterthan it was when the high-power lens was focused?

Compare your observations on the relative working distancesof the objective lenses with the illustration in Figure 3.3. Ex-plain why it is desirable to begin the focusing process at thelowest power.

10. Rotate the oil immersion lens slightly to the side and re-move the slide. Clean the oil immersion lens carefully with

32 Exercise 3

10x 4Sx 100x

c

"- /.........Stage

FIG U R E 3.3 Relative working distances of the 1Ox,45x, and 100x objectives.

lens paper, and then clean the slide in the same manner witha fresh piece of lens paper. _

The Microscope FieldBy this time you should know that the size of the micro copefield decreases with increasing magnification. For future mi-croscope work, it will be useful to determine the diameter ofeach of the microscope fields. This information will allowyou to make a fairly accurate estimate of the size of the ob-jects you view in any field. For example, if you have calcu-lated the field diameter to be 4 mm and the object beingobserved extends across half this diameter, you can estimatethat the length of the object is approximately 2 nun.

Microscopic specimens are usually measured in mi-crometers and millimeters, both units of the metric system.You can get an idea of the relationship and meaning of theseunits from Table 3.1. A more detailed treatment appears inAppendix A.

ACTIVITY 3

Estimating the Diameterof the Microscope FieldJ. Obtain a grid slide (a slide prepared with graph paperruled in millimeters). Each of the squares in the grid is 1 mmon each side. Use your lowest-power objective to bring thegrid lines into focus.

2. Move the slide so that one grid line touches the edge ofthe field on one side, and then count the Dumber of squaresyou can see across the diameter of the field. If you can seeonly part of a square, as in the accompanying diagram, esti-mate the part of a millimeter that the partial square represents.

_k....--~

-2.sml,\-<' (

\ )T 'r-r-1 i.t.: --r-/

TABLE 3.1 omparison of Metricnits of Length

Metric unit Abbreviation Equivalent

Meter m (about 39.3 in.)

Centimeter cm 1O-2m

Millimeter rom 103m

Micrometer (or micron) 11m(11) 10-6m

Nanometer (or nm (mil) 10-9mmillimicrometerormillimicron)

Angstrom A 10 10m

Refer to the "Getting Started" exercise (page xv) for tips on metricconversions.

Record this figure in the appropriate space marked "fieldsize" on the summary chart (page 31). (If you have beeD us-ing the scanning lens, repeat the procedure with the low-power objective lens.)

Complete the chart by computing the approximate diam-eter of the high-power and oil immersion fields. The generalformula for calculating the unknown field diameter is:

Diameter of field A X total magnification of field A =diameter of field B X total magnification of field B

where A represents the known or measured field and B repre-sents the unknown field. This can be simplified to

Diameter of field B =

diameter of field A X total magnification of field Atotal magnification of field B

For example, if the diameter of the low-power field (field A)is 2 mm and the total magnification is sox, you would com-pute the diameter of the high-power field (field B) with a to-tal magnification of 100 X as follows:

Field diameter B = (2mm X 50)/100Field diameter B = lmm

3. Estimate the length (longest dimension) of the followingmicroscopic objects. Base your calculations on the field sizesYOII have determined/or your microscope.

Object seen in low-power field:

approximate length:

____ mm

Object seen in high-power field:

approximate length:

____ mm

or f..lm

Object seen in oil immersion field:

approximate length:

The Microscope 33

____ Jlm

4. If an object viewed with the oil immersion lens looked asit does in the field depicted just below, could you determineits approximate size from this view?

If not, then how could you determine it? _

------------------------_-------111

Perceiving DepthAny microscopic specimen has depth as well as length andwidth; it is rare indeed to view a tissue slide with just onelayer of cells. Normally you can see two or three cell thick-nesses. Therefore, it is important to learn how to determinerelative depth with your microscope. In microscope work thedepth of field (the depth of the specimen clearly in focus) isgreater at lower magnifications.

ACTIVITY 4

Perceiving Depth1. Obtain a slide with colored crossed threads. Focusing atlow magnification, locate the point where the three threadscross each other.

2. Use the iris diaphragm lever to greatly reduce the light,thus increasing the contrast. Focus down with the coarse ad-justment until the threads are out of focus, then slowly focusupward again, noting which thread comes into clear focusfirst. (You will see two or even all three threads, so you mustbe very careful in determining which one first comes intoclear focus.) Observe: As you rotate the adjustment knob for-ward (away from you), does the stage rise or fall? If the stagerises. then the first clearly focused thread is the top one; thelast clearly focused thread is the bottom one.

Ii the stage descends. how is the order affected? __

Record your observations. relative to which color of thread isuppermost. midclJe. or lowest:

Topthread _

MidclJe thread _

Bottom thread III

Viewing Cells Underthe MicroscopeThere are various ways to prepare cells for viewing undera microscope. Cells and tissues can look very different withdifferent stains and preparation techniques. One method ofpreparation is to mix the cells in physiologic saline (called awet mount) and stain them with methylene blue stain.

If you are not instructed to prepare your own wet mount,obtain a prepared slide of epithelial ceUs to make the obser-vations in step 10 of Activity 5.

ACTIVITY 5

Preparing and Observing a Wet MountJ. Obtain the following: a clean microscope slide and cov-erslip, two flat-tipped toothpicks, a dropper bottle of physio-logic saline, a dropper bottle of iodine or methylene bluestain. and filter paper (or paper towels). HanclJe only yourown slides throughout the procedure.

2. Place a drop of physiologic saline in the center of the slide.Using the flat end of the toothpick, gently scrape the inner liningof your cheek. Transfer your cheek scrapings to the slide by ag-itating the end of the toothpick in the drop of saline (Figure 3.4a).

(b)

(c)

FIG U R E 3.4 Procedure for preparation of a WIllmount. (a) The object is placed in a drop of water .....on a clean slide, (b) a coverslip IS held at a 45 anglethe fingertips, and (c) it is lowered carefully ~ _ _.and the object.

34 Exercise 3

FIG URE 3. 5 Epithelial cells of the cheek cavity(surface view, 750 x).

, Immediately discard the used toothpick in the dispos-• able autoclave bag provided at the supplies area.

3. Add a tiny drop of the iodine or methylene blue stain tothe preparation. (These epithelial cells are nearly transparentand thus difficult to see without the stain, which colors the nu-clei of the cells and makes them look much darker than thecytoplasm.) Stir again.

Immediately discard the used toothpick in the dispos-able autoclave bag provided at the supplies area.

,•

4. Hold the coverslip with your fingertips so that its bottomedge touches one side of the fluid drop (Figure 3.4b), thencarefully lower the coverslip onto the preparation (Figure3.4c). Do not just drop the coverslip, or you will trap large airbubbles under it, which will obscure the cells. A coverslipshould always be used with a wet mount to prevent soiling thelens if you should misfocus.

5. Examine your preparation carefully. The coverslipshould be closely apposed to the slide. If there is excess fluidaround its edges, you will need to remove it. Obtain a pieceof filter paper, fold it in half, and use the folded edge to ab-sorb the excess fluid. (You may use a twist of paper towel asan alternative.)

, Before continuing, discard the filter paper in the dis-• posable autoclave bag.

6. Place the slide on the stage, and locate the cells at thelowest power. You will probably want to dim the light withthe iris diaphragm to provide more contrast for viewing thelightly stained cells. Furthermore, a wet mount will dry outquickly in bright light because a bright light source is hot.

7. Cheek epithelial cells are very thin, six-sided cells. In thecheek, they provide a smooth. tilelike lining, as shown in Figure3.5. Move to high power to examine the cells more closely.

8. Make a sketch of the epithelial cells that you observe.

Use information on your summary chart (page 31 ) to estimatethe diameter of cheek epithelial cells.

____ mm

Why do your cheek cells look different than those illustratedin Figure 3.5? (Hint: What did you have to do to your cheekto obtain tbem?)

, 9. When you complete your observations of the wet• mount, dispose of your wet mount preparation in the

beaker of bleach solution, and put the coverslips in an auto-clave bag.

10. Obtain a prepared slide of cheek epithelial cells, andview them under the microscope.

Estimate the diameter of one of these cheek epithelial cellsusing information from the summary chart (page 31).

____ mm

Why are these cells more similar to those seen in Figure 3.5and easier to measure than those of the wet mount?

11. Before leaving the laboratory, make sure all other materialsare properly discarded or returned to the appropriate laboratorystation. Clean the microscope lenses and put the dust cover onthe microscope before you return it to the storage cabinet. _

The Microscope

~E _

LAB TIMEIDATE __

Care and Structure of the Compound Microscope1. Label all indicated parts of the microscope.

1. Explain the proper technique for transporting the microscope.

35

36 Review Sheet 3

3. The following statements are true or false. If true, write T on the answer blank. If false, correct the statement by writing onthe blank: the proper word or phrase to replace the one that is underlined.

I. The microscope lens may be cleaned with any soft tissue.

2. The microscope should be stored with the oil immersion lens in positionover the stage.

3. When beginning to focus, use the lowest-power lens.

4. When focusing, always focus toward the specimen.

5. A coverslip should always be used with wet mounts and the high-powerand oil lenses.

4. Match the microscope structures given in column B with the statements in column A that identify or describe them.

ColumnA Column B

1. platform on whicb the slide rests for viewing a. coarse adjustment knobb. condenserc. fine adjustment knobd. iris diaphragme. mecbanical stage or spring clipsf. movable nosepieceg. objective lensesh. oculari. stage

____ 2. used to increase the amount of light passing throughthe specimen

____ 3. secure(s) the slide to the stage

____ 4. delivers a concentrated beam of light to the specimen

____ 5. used for precise focusing once initial focusing hasbeen done

____ 6. carries the objective lenses; rotates so that the differ-ent objective lenses can be brought into position overthe specimen

5. Define the following terms.

virtual image: _

resolution: _

ReviewSheet3 37

Viewing Objects Through the Microscope6. Complete, or respond to, the following statements:

1. The distance from the bottom of the objective lens in use to the specimen is called the

2. Assume there is an object on the left side of the field that you want to bring to the center (thatis, toward the apparent right). In what direction would you move your slide?

3. The area of the specimen seen when looking through the microscope is the _

4. If a microscope has a lOx ocular and the total magnification at a particular time is 950x, theobjective lens is use at that time is x.

5. Why should the light be dimmed when looking at living (nearly transparent) cells?

6. If, after focusing in low power, only the fine adjustment need be used to focus the specimen atthe higher powers, the microscope is said to be _

7. If, when using a lOX ocular and a l5X objective, the field size is 1.5 mm, the approximatefield size with a 30X objective is mm.

8. If the size of the high-power field is 1.2 mm, an object that occupies approximately a third oftbat field bas an estimated diameter of mm.

7. You have been asked to prepare a slide with the letter k on it (as shown below). In the circle below, draw the k as seen in thelow-power field.

k

8. Figure out the magnification of fields I and 3, and the field size of 2. (Him: Use your ruler.) Note that the numbers for thefield sizes below are too large to represent the typical compound microscope lens system, but the relationships depicted areaccurate.

5 mrn1. ..-O.._

0.5 mm3. ..- •.._

mrn2. "-O.._

lOOX____ x ____ x

9. Say you are observing an object in the low-power field. When you switch to high-power, it is no longer in your field of view.

Why might this occur? _

What should be done initially to prevent this from happening? _

10. Do the following factors increase or decrease as one moves to higher magnifications with the microscope?

resolution: _ amount of light needed: _

working distance: _ depth offield: _

38 Review Sheet 3

11. A student has the high-dry lens in position and appears 10 be intently ob erving the pecimen. The in uuetor, nooo:ing distance of about I em, knows the student isn't actually seeing the specimen.

Howso? __

12. Describe the proper procedure for preparing a wet mount.

13. Indicate the probable cause of the following situations arising during use of a microscope.

a. Only half of the field is illuminated: _

b. Field does not change as mechanical stage is moved: _