using a compound light microscope - ms. smith's...

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4. W hy s h o u l dy o u u s e l e n s p a p e r o n l y o n c e ? (1) 3. W h y d os c i e n t i s t s u s e m i c r o s c o p e s ? (1) 2. W h y s h o u l da m i c r o s c o pe s l i d ea ndc o v e r s l i p b e h e l dby t h e i r e dg e s ? (1) L AB #:_ _ _ _ _ U S I N G A C O M P O UN D L I GHTM I C R O S C O P E I N T R O D U C T I O N : M a n y o b j e c t s a r e t o os m a l l t ob e s e e n b y t h e e y e a l on e . T h e y c a n b e s e e n , h o we v e r , wi t h t h e u s e o f a n i n s t r u m e n t t h a t m a g n i f i e s , o r v i s u a l l y e n l a r g e s , t h e o b j e c t . O n e s u c h i n s t r u m e n t , w h i c h i s o f g r ea t - i m p o r t a n c e t ob i o l og i s t s a ndo t h e r s c i e n t i s t s , i s t h e c o m po un dl i gh t m i c r o s c o p e . A c o m p o u ndl i g h t m i c r o s c op e c o n s i s t s o f a li g h t s ou r c e o r m i n o r t h a t i l l u m i n a t e s t h e o b j e c t t ob e o b s e r v e d , a n o b j e c t i v e l e n s t h a t m a g n i f i e s t h e i m a g eo f t h eo b j e c t , a nda n e y e pi e c e ( o c u l a r l e n s ) t h a t fu r t h e r m a gn i f i e s t h e i m a g e o f t h e o b j e c t a ndp r o j e c t s i t i n t ot h e v i e we r ' s e y e . O b j e c t s , o r s pe c i m e n s , t ob e ob s e r v e du n d e r a m i c r o s c o p e a r e g e n e r a l l y pr e p a r e di n o n e o f t wowa y s . P r e p ar e d o r pe r m a n e n t s l i d e s a r e m a d e t ol a s t a l o ng t i m e . T h e y a r e u s u a l l y pu r c h a s e df r o m b i o l o g i c a l s u pp l y h o u s e s . T e m p o r a r y o r we t -m o u n t s li d e s are m a d e t ol a s t on l y a s h o r t t i m e u s u a l l y o n e l a bo r a t o r y p e r i o d . T h e m i c r o s c ope i s a n e xp e n s i v e pr ec i s i o n i n s t r u m e n t t h a t r e q u i r e s s pe c i a l c a re a n dh a nd li n g . I n t h i s i n v e s t i g a t i o n , y o u will l e a r n t h e p a r t s o f a c o m po u n dl i g h t m i c r o s c o p e , t h e f u n c t i o n s o f t ho s e p a r t s , a n d t h e pr o p e r u s e a n dca r e o f t h e m i c r o s c o pe . Yo u wi l l a l s ol e a r n t h e t ec hn i q u e o f pr e pa r i n g we t - m o u n t s li d e s . A I M : T ol ea r n t h e p r o pe r u s e o f a c o m po u ndl i gh t m i c r o s c o p e . P R E -L AB D I S C U S S I O N : (5) R ea dt h e e n t i re i n v e s t i g a t i o n . T h e n a n s we r t h e fo ll o wi n g qu e s t i o n s . 1. W hy m i g h t it b e a g oo di d ea t ok ee p y o u r m i c r o s c o p e a t l e a s t 10 c m f r o m t h e e d g e o f t h e t a b l e ? (1)

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4. Why should you use lens paper only once? (1)

3. Why do scientists use microscopes? (1)

2. Why should a microscope slide and coverslip be held by their edges? (1)

LAB #:_____

USING A COMPOUND LIGHT MICROSCOPE

INTRODUCTION:Many objects are too small to be seen by the eye alone. They can be seen, however, with the use of aninstrument that magnifies, or visually enlarges, the object. One such instrument, which is of great-importance to biologists and other scientists, is the compound light microscope. A compound lightmicroscope consists of a light source or minor that illuminates the object to be observed, an objective lensthat magnifies the image of the object, and an eyepiece (ocular lens) that further magnifiesthe image of

the object and projects it into the viewer's eye.

Objects, or specimens, to be observed under a microscope are generally prepared in oneof two ways.Prepared or permanent slides are made to last a long time. They are usually purchased from biologicalsupply houses. Temporary or wet-mount slides are made to last only a short time—usually one laboratory

period.

The microscope is an expensive precision instrument that requires special care and handling. In thisinvestigation, you will learn the parts of a compound light microscope, the functions of those parts, andthe proper use and care of the microscope. You will also learn the technique of preparing wet-mount

slides.

AIM:To learn the proper use of a compound light microscope.

PRE-LAB DISCUSSION: (5)Read the entire investigation. Then answer the following questions.

1. Why might it be a good idea to keep your microscope at least 10 cm from the edge of the table? (1)

magnification of objective X magnification of eyepiece = total magnification

10XX10X= 100X

Figure 2

PROCEDURE:

Part A. Care of the Compound Light Microscope:

1.Figure 1 shows the proper way to carry a microscope.Always carry the microscope with both hands. Graspthe arm of the microscope with one hand and placeyour other hand under the base. Always hold themicroscope in an upright position so that the eyepiececannot fall out. Place a microscope on your worktable

or desk at least 10 cm from the edge. Position themicroscope with the arm facing you.

2.Notice the numbers etched on the objectives and onthe eyepiece. Each number is followed by an "X" that means"times." For example, the low-power objective may havethe number "10X" on its side, as shown in Figure 2. Thatobjective magnifies an object 10 times its normal size. Recordthe magnifications of your microscope in the Data Table. The

total magnification of a microscope is calculated by multiplyingthe magnification of the objective by the magnification of theeyepiece.For example:

J?ut on a laboratory apron. Always- handle Hie ^icroscope wiA extreme care. You are responsible for itsproper care and use. Use caution when handling microscope slides, as they can break easily and cut you.Never use direct sunlight as a light source for a compound light microscope. The sunlight reflectingthrough the microscope could damage your eye. Be careful when handling sharp instruments. Observeproper laboratory procedures when using electrical-equipment. Note all safety alert symbols next to thesteps in the Procedure and review the meanings of each-symbol by referring to Safety Symbols on page 8.

scissorsnewspaper

dropper pipettesoft cloth (or cheesecloth)dissecting probelens paper

SAFETY: 8-^^181

coverslipprepared slidemicroscope slidecompound light microscope

MATERIALS:

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1.Look at the microscope from the side. Locate the coarse adjustment knob that moves the objectives upand down. Practice moving the coarse adjustment knob to see how it moves the objectives with each

turn.

2.Turn the coarse adjustment so that the low-powerobjective is positioned about 3 cm from the stage.Locate the revolving nosepiece. Turn thenosepiece until you hear the high-powerobjective click into position. See Figure 3.When an objective clicks into position, itis in the proper alignment for light topass from the light source through theobjective into the viewer's eye. Now turnthe nosepiece until the low-power objectiveclicks back into position. Note: Always lookat the microscope from the side when moving an

objective so that the microscope does not hit ordamage the slide.

3.If your microscope has an electric light source, plug in the cord and turn on the light. If yourmicroscope has a mirror, turn the mirror toward a light source such as a desk lamp or window.

CAUTION: Never use the sun as a direct source of light. Look through the eyepiece. Adjust thediaphragm to permit sufficient light to enter the microscope. The white circle of light you see is thefield of view. If your microscope has a mirror, move the mirror until the field of view is evenlyilluminated.

4.Place a prepared slide on the stage so that it is centered over the stage opening. Use the stage clips tohold the slide in position. Turn the low-power objective into place. Look at the microscope from theside and turn the coarse adjustment so that the low-power objective is as close as possible to the stagewithout touching it

High-power,objective

4. Before you use the microscope, clean the lenses of the objectives and eyepiece with-lens papsr. Note:To avoid scratching the lenses, never clean or wipe them with anything other than lens paper. Use anew piece of lens papertm each lens you clean. Never touch a lens with your finger. The oils on yourskin may attract dust or lint that could scratch the lens.

Part B. Use of a Compound Light Microscope:

Totalmagnification

Magnificationof Eyepiece

Magnification

of Objective

Other

High power

Low power

Objective

3. Use the formula to complete the Data Table.

DATA TABLE: (3)

What happens to the image as you move the slide (up)away from you?

What happens to the image as you move the slide to the left?

9. As you look in the microscope use your fingers to move the slide to the right and then to the left.

What happens to the image as you move the slide to the right?

8. As you look through the eyepiece, slowly adjust the diaphragm to obtain the appropriate light for

viewing.What happens as you adjust the diaphragm?

from the stage until the object pomes into focus. To avoid eyestrain, keep both eyes open wnue

looking through a microscope.CAUTION: To avoid moving the objective into the slide, never lower the objective toward the stage

while looking through the eyepiece.6.Turn the fine adjustment to bring the object into sharp focus. You may wish to adjust the diaphragm

so that you can see the object more clearly. In the appropriate space below, draw what you see through

the microscope. Record the magnification.

7.Look at the microscope from the side and rotate the nosepiece until the high-power objective clicksinto position. Look through the eyepiece. Turn the fine adjustment to bring the object on the slide into

focus.

CAUTION: Never use the coarse adjustment when focusing the high-power objective lens. This,could break your slide ordaraage the lens. In the appropriate space below, draw whatyou-see through

the microscope. Record the magnification.

PREPARED SLIDE: (6)

Low power magnificationHigh power magnification

High power magnificationLETTER "e": (6) Low power magnification

Figure 4

3.Hold a clean coverslip in your fingers as shown in Figure 4C. Make sure the bottom edge of thecoverslip is in the drop of water. Use a dissecting probe to slowly lower the coverslip onto the wetnewspaper. Slowly lowering the coverslip prevents air bubbles from being trapped between the slideand the coverslip. The type of slide you have just made is called a wet mount. Practice making a wetmount until you can do so without trapping air bubbles on the slide.

4.Center the wet mount on the stage with the letter "e" in its normal upright position. Note: Make surethe bottom of the slide is dry before you place it on the stage. Turn the low-power objective intoposition and bring the "e" into focus. In the appropriate place below, draw the letter "e" as seenthrough the microscope. Record the magnification.

5.While looking through the eyepiece, move the slide to the left.Notice the way the letter seems to move. Now move the slide to the right. Again notice the/way theletter seems to move. Move the slide up and down and observe the direction the letter moves.

6.Turn the high-power objective into position and bring the letter "e" into focus. In the appropriate placebelow, draw the letter "e" as seen through the microscope. Record the magnification.

Part C. Preparing a Wet Mount:

1.Use a pair of scissors to cut a letter "e" from a piece of newspaper.Cut out the smallest letter "e" you can find. Position the "e" on the center of a clean glass slide.

2.Use a dropper pipette to place one drop of water on the cut piece of newspaper. See Figure 4B.

10. Remove the slide. Move the low-power objective into position.

7. Inferring: Explain why a specimen to be viewed under the microscope must be thin. (1)

6. Observing: If you placed a letter "h" under the microscope, how would the image look in the field of

view? (2)

5. Observing: How does the letter "e" as seen through the microscope differ from the way an "e"

normally appears?(2)

4. Observing: How is the image of an object seen through the high power objective different from theimage seen through the low-power objective? (1)

3. Observing: Why must the specimen be centered before switching to high power? ^

2. Inferring: Why should you always begin to use a microscope with the lew-power objective? (1)

and coverslip with paper towels and return them to their boxes.

8. Rotate the low-power objective into position and use the coarse adjustment to place it as close to thestage as possible without touching. Carefully pick up the microscope and return it to its storage area.

ANALYSIS AND CONCLUSIONS: (42)

1. Inferring: Why do you place one hand under the base of the microscope as you carry it? (1)

1.Controls the amount of light passing up through the specimen and is located under the stage.

2.The lens nearest the eye and used to "look through".

3.Causes a small amount of movement of the lenses; the smaller focus knob.

4. A flat platform on which the slide is placed.

7

Stage ClipsFine AdjustmentCoarse AdjustmentLight SourceDiaphragmStageObjective lensEyepiece or Ocular Lens

HGFEDCBA

12. Matching: Match the names of the parts of the microscope to the function. You will need to use the

names more than once. (11)

11. Drawing Conclusions: Suppose you were observing an organism through the microscope andnoticed that it moved toward the bottom of the slide and then it moved to the right. What does this tell

you about the actual movement of the organism? (2)

10. Inferring: If you are looking through a microscope at a freshly prepared wet mount and you seeseveral perfect circles that are completely clear surrounding you specimen, what is the most likely

explanation? (1)

b. high po-wer?

9. Inferring: If a microscope has an ocular with a 5x power, and has objectives with powers of lOx and

50x, what is the total magnification of: (Show your math for full credit!) (4)

a. low power?

'8. Inferring: Why should you never use coarse adjustment when focusing the high-power objective

lens? (1)

13. Label tbe.parts of the microscope below. (14)

6.Holds the slide in place.

7.There is only one of these lenses and usually magnifies lOx.

8 There are usually 2 or 3 of these lenses located the closest to the specimen.

9.The only focus knob you should use with high power objective lens.

10.The focus knob used only under low power (up to lOOx).

11.The focus knob used to sharpen the image.

illustrates the cell (object) in the center of the low-power field of view:

FIELD OF VIEW:• This is the diagram that represents where the cell should be located in the low-power field

of view to be sure the entire cell will be visible after switching to high power. This diagram

DLA.PHRAGM:•The diaphragm and fine adjustment are the parts of the microscope that should be used to

correct for the dark appearance . It is most probably the result of too little light passingthrough the specimen from the understage light source; adjusting the diaphragm to allowmore light to be directed through the specimen will brighten the field of view. The fuzzyappearance of the image most likely indicates that the microscope is slightly out of focus;using the fine adjustment knob will probably bring the image into sharper focus. When thehigh-power objective is in place, only the fine adjustment should be used because using thecoarse adjustment could smash the coverslip and slide and potentially damage the high-power lens.

•adjusting the diaphragm to obtain more light under high power is a proper techniques to

employ when using a microscope to view a specimen on a slide.

COVER SLIP:Reducing the size of air bubbles under the coverslip involves holding the slide at a slightangle and gently tapping the slide so that the bubbles escape from under the coverslip at itsupper edge.

•Placing a coverslip involves holding it over the specimen and touching one edge to the slide,then lowering the coverslip in a manner that allows the majority of air bubbles to escapefrom under it.

•Removing a coverslip involves moving it to the edge of the slide and using a fingertip orforceps to lift it off the specimen.

•keeping the organism from moving around is most easily attained by using a slide and

coverslip.

COARSE ADJUSTMENT: Using the coarse adjustment to focus the specimen under high power isthe activity that might lead to damage of a microscope and specimen. This action can easily crush acoverslip and specimen and even break a slide. Only the fine adjustment should be used when the

high-power objective lens is in place.

BROMTHYMOLE BLUE STAIN: One of the most commonly used stains, Lugol's iodine andbromthymol blue, provide a monochromatic (single color) stain for fresh microscopic specimensthat are absorbed differentially by different cell organelles. These and similar stains make studyingmicroscopic specimens easier.

INTRODUCTION TO THE MICROSCOPE REVIEW SHET;

INVERSION: Images are not reversed by compound microscopes, only inverted. Image inversionis characteristic of compound microscopes but only when comparing an actual specimen to itsmagnified image in the field of view. No image inversion (or reversal) occurs when switching from

low power to high power.

HIGH POWER:•high power: you see a few very LARGE cells. This best describes the relative number and

appearance of any cells observed using these two objectives. Using low power (likely lOOx),the student observes a field of view that encompasses perhaps 100 cells whose individualsizes appear small because of the relatively low magnification used. Switching to high power(likely 200x), the student observes a field of view only 25% (by area) that of the low-powerfield, encompassing only 25 cells, for example, whose individual sizes appear two timeslarger than under low-power magnification. {Low power: you see MANY small cells)

•focusing with low power first before moving the high power into position is a proper

techniques to employ when using a microscope to view a specimen on a slide.

FINE ADJUSTMENT: The diaphragm and fine adjustment are the parts of the microscope thatshould be used to correct for the dark appearance . It is most probably the result of too little lightpassing through the specimen from the understage light source; adjusting the diaphragm to allowmore light to be directed through the specimen will brighten the field of view. The fuzzyappearance of the image most likely indicates that the microscope is slightly out of focus; using thefine adjustment knob will probably bring the image into sharper focus. When the high-powerobjective is in place, only the fine adjustment should be used because using the coarse adjustment

could smash the coverslip and slide and potentially damage the high-power lens.

^ PowerHig^ Power

The diameter of the field of view decreases is the best explanation for the changes observedby the student when switching from low to high power. Most student laboratory microscopesare set up to provide a low-power to high-power ratio of 1:4 (100x:400x). Because the high-power objective is viewing the cell (object) 4 times more closely than is the low-powerobjective, the diameter of the high-power field of view is only about V* that of the low-powerfield of view.The high-power field of view is decreased compared with the low-power field of view, notincreased.

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• When the microscope turret is rotated carefully to position the high-power objective overthe specimen, the image of the cell will be centered in the high power field of view. Whenthe low-power image is not centered, it could look like this under high power:

STAINS:• The purpose of cell observation is to observe all cell organelles, not just some cell

organelles. Stains do not eliminate cell organelles.The laboratory procedure represented in the diagram is one in which a researcher is adding

SLIDE: keeping the organism from moving around is most easily attained by using a slide and

coverslip.

OCULAR LENS:•Cleaning the ocular and objectives with lens paper is a proper techniques to employ when

using a microscope to view a specimen on a slide.•The ocular lens is the lens closest to the observer's eye; using only this lens will not improve

the focus.

OBJECTIVE LENS:•The objective lens is already being used; using only this lens will not improve either

brightness or focus. However, using it with the fine adjustment knob will likely bring theimage into sharper focus.

•Cleaning the ocular and objectives with lens paper is a proper techniques to employ when

using a microscope to view a specimen on a slide.

NOSE PIECE: The nosepiece is a fixed part of the microscope that serves as a swivel mount forthe turret holding the objective lenses. Using the coarse adjustment with the high-power objectivein place would likely not improve the sharpness of the image and could smash the coverslip and

slide and potentially damage the high-power lens.

1 mm* 1000 tm

1 | I | I | 1 | 1 | 1 |Tffim

yl 1 [ 11! [u1 1 1 1 1 1 1 1 1 1 1 1 1 1Plaslic ruler

MICROMETERS: 1000 /xm is the average length of all 5 cells in micrometers (um) in thisdiagram. The diagram shows five cells end to end that together cover the distance of 1 millimeter.By definition, 1 millimeter (1 mm) equals 1,000 micrometers (1000 um). One-fifth (20%) of 1000|im is 200 urn, which is the average length of the cells illustrated.

LUGOL'S IODINE STAIN: One of the most commonly used stains, Lugol's iodine, provide amonochromatic (single color) stain for fresh microscopic specimens that are absorbed differentiallyby different cell organelles. These and similar stains make studying microscopic specimens easier.

LOW POWER:Low power: you see MANY small cells &. This best describes the relative number andappearance of any cells observed using these two objectives. Using low power (likely lOOx),the student observes a field of view that encompasses perhaps 100 cells whose individualsizes appear small because of the relatively low magnification used. Switching to high power(likely 200x), the student observes a field of view only 25% (by area) that of the low-powerfield, encompassing only 25 cells, for example, whose individual sizes appear two timeslarger than under low-power magnification, (high power: you see a few very LARGE cells)

• focusing with low power first before moving the high power into position is a proper

techniques to employ when using a microscope to view a specimen on a slide.

LENS ~PAPER:Cleaning the ocular and objectives with lens paper is a proper techniques to employ

when using a microscope to view a specimen on a slide.

WET-MOUNT:•A wet-mount slide preparation of a specimen is stained in order to make cell structures more

visible. Stains such as iodine and bromthymol blue are routinely used in wet-mountpreparations. They are absorbed by the organic compounds in cells at different rates,increasing visual contrast and making the cell structures visible under the objective lens of acompound microscope.

•Excess water in a wet-mount is removed from a slide by the use of a paper towel, not stain,

(we need the stain and shouldn't use too much of it)

Stains such as iodine and bromthymol blue are routinely used in wet-mount preparations.They are absorbed by the organic compounds in cells at different rates, increasing visualcontrast and making the cell structures visible under the objective lens of a compoundmicroscope.

The high-power lens can be used whether or not the cell is stained. Stains help the observerto differentiate among different structures of the cell.the most common reason for staining a specimen being observed is NOT to make the viewmore colorful. There are many momochromatic stains that contain no color, and we can learn

much from using these types of stains as well as the color ones.

Paper towel

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on the slide at one edge of the coverslip. The stain is drawn under the coverslip and aroundthe specimen by means of capillary action. A small piece of paper towel is used to absorbexcess moisture from under the opposite edge of the coverslip, speeding the rate of capillaryaction.

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S. Base your answer on the accompanying information and on your knowledge of biology.

A student observes a red onion cell with a compound light microscope using low, then high power. The two views are

represented in the accompanying diagrams.

The best ej^jlanation for the changes observed by the student is that, when switching from low to high power, the

4. When using a compound light microscope, the most common reason for staining a specimen being observed is to

1.keep the organism from moving around3. determine the effects of chemicals on the organism

2.make the view more colorful4. reveal details that are otherwise not easily seen

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3. A elearplastic ruler is placed across the middle of the field of view of a compound light microscope. A row of cells can be

seen under low-power magnification (100x). What is the average length of a single cell in micrometers (um)?

2> A wet-mount slide preparation of a specimen is stained in order to

1.eliminate some organelles3. use the high-power lens

2.make cell structures more visible4. remove water from the slide

3.adding stain to a slide without removing the

covers lip

4.reducing the size ofairbubbles undera coverslip

1.placing a coverslip over a specimen

2.removing a coverslip from a slide

Paper towel

sm-mcrscolabpqsOctober 20,2014

1. Which laboratory procedure is represented in the accompanying diagram?

8. A student prepared a slide of pollen grains from a flower. First the pollen was viewed through the low-power objective lens

and then, without moving the slide, viewed through the high-power objective lens of a compound light microscope.

Which statement best describes the relative number and appearance of the pollen grains observed using these two

objectives?1. low power: 25 small pollen grains3. low power: 25 large pollen grains

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7. A slide of human blood cells was observed in focus under the low-power objective of a compound light microscope that

had clean lenses. When the microscope was switched to high power, the image was dark and fuzzy. Which parts of the

microscope should be used to correct this situation?

1.nosepiece and coarse adjustment3. objective and fine adjustment

2.diaphragm and ocular4. diaphragm and fine adjustment

(2)(4)

6. Base your answer on the accompanying information and on your knowledge of biology.

A student observes a red onion cell with a compound light microscope using low, then high power. The two views are

represented in the accompanying diagrams.

Which diagram represents where the cell should be located in the low-power field of view to be sure the entire cell will be

visible after switching to high power?

Low PowerHig^ Power

1.diameter of the fold of view decreases3. diameter of the field of view increases

2.slide was accidentally moved4. image is inverted and reversed

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9. Which activity might lead to damage of a microscope and specimen?1.cleaning the ocular and objectives with lens paper 3. using the coarse adjustm^nt to focus the specimen

under high power2.focusing with low power first before moving the high 4. adjusting the diaphragm to obtain more light underpower into positionhigh power

high power 100 large pollen grainshigh power: 100 small pollen grains2. low power: 100 small pollen grains4. low power 100 large pollen grains

high power: 25 large pollen grainshigh power 25 small pollen grains