Microscope lab Bio 120 PDF

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This is a lab we did in class that has notes and explains everything as well....

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Alexandria Frascone

1. USE OF THE MICROSCOPE OBJECTIVES: After completing this lab, you should be able to: a. identify the following parts of a microscope: ocular (eyepiece) arm base

nosepiece fine adjustment knob coarse adjustment knob

b. be able to define the following terms: compound microscope parfocal dissecting microscope total magnification

iris diaphragm stage clips objectives

stage

field diameter field of view

c. focus a specimen on ANY objective using proper microscope technique. d. determine what happens to the ORIENTATION of an image when viewed through the microscope. e. determine what happens to the SIZE of a specimen when viewed through the microscope f. detemine how the size of a specimen changes when you increase the size of the objective (go from low to high power) g. identify the relationship between magnification and field of view

One of the tenets of the Cell Theory developed by Schwann and Schleiden is that cells are the functional and structural unit of all living things. Many living things are multicellular and can easily be seen with the unaided eye. However, each individual cell that comprises these organisms cannot be viewed without the assistance of some magnifying instrument. In fact, there are many unicellular organisms that are invisible to the naked eye. In order to view these cells, we employ instruments called microscopes. The microscope is a device composed of a series of two or more lenses that serve two functions. First, they magnify, or enlarge objects. Second, they provide for greater resolution of those objects, which means that it allows you to distinguish two objects as separate entities. For example, the cells that make up the leaf of a maple tree cannot be seen individually. However, when the tissue is stained and viewed under a microscope, the individual cells and some of the organelles (e.g., nucleus, chloroplasts) can be distinguished. There are a number of different kinds of microscopes that vary with respect to their magnification and resolving power. For this lab, you will use two types of microscopes: the compound light microscope and the dissecting microscope. A microscope is considered compound if it has a minimum of two magnifying lenses. Under ideal conditions, compound light microscopes are used to view specimens as small as 0.25 μm (micrometers). Dissecting microscopes are typically used to distinguish macroscopic objects, such as organs and tissues in specimens. PARTS OF A MICROSCOPE In order to use a microscope effectively and efficiently, it is necessary to be familiar with

2 its parts. Obtain a compound light microscope from your instructor. When carrying a microscope, ALWAYS hold it by the arm and the base (see below). Microscopes are expensive instruments, so they should always be handled with care. NOTE: Not all microscopes have all of the parts listed below. arm – region of the microscope that suspends the magnifying lenses above the specimen. base – broad, heavy structure which supports the microscope stage – platform on which the specimen is placed. The stage on your microscope may be moving (through use of stage knobs) or stationary. stage clips – structures used to hold the slide in place. The stage clips can be opened and closed by pinching the ends together. stage knobs –structures used to control the movement of the slide on the stage. Note that each knob has a scale associated with. These scales allow you to determine the position of a specimen on the slide for future reference. diaphragm –structure that regulates the amount of light passing from the light source through the specimen. You may have an iris diaphragm (opens and closes similar to the iris of the eye) or a disc diaphragm with pre-determined openings. ocular –one of the magnification lenses, located in the eyepiece. It is found in the upper body of the microscope. objective lens – series of magnification lenses found on the nosepiece. There are a number of objective lenses on your microscope (either three or four). Each objective lens is labeled with the magnification produced by that lens. They are: low (scanning) power (typically labeled 4X) – this lens is the first one used when viewing a specimen. It is commonly called scanning power because it allows you to see a wider field of view and scan the slide for specific entities. medium power (typically labeled 10X) – this is an objective lens of intermediate magnification high power (may be labeled 40X or 45X) – this objective lens allows you to see greater detail on your specimen. oil immersion lens (commonly labeled 100X) – this objective lens is used with special oil (immersion oil) to see greater detail in your specimen and to

3 view very small specimens (e.g., bacteria). nosepiece – structure that holds the objective lenses focus knobs – structures used to move the objectives of the microscope coarse adjustment knob – typically the inner knob that moves the objectives large distances fine adjustment knob – outer knob used to move the objectives short distances. QUESTION 1: Using the above descriptions of microscope parts, label the following parts on Figure 1:

H ___H____ arm

A

___E____ base ___B____ objective lenses

B

___A____ ocular (eyepiece) __G_____ fine adjustment knob

C

___F____ coarse adjustment knob ___D____ iris diaphragm

D

___C____ stage

E F

G

Figure 1. Compound light microscope.

The relationship between magnification and field of view is inversely proportional. As

4 magnification increases by a given factor, field of view decreases by the same factor, and vice versa. For example, if the magnification of an image increases by a factor of four, the field of view will decrease by a factor of four. When you look through the ocular of a microscope, you will see a circle of light. That circle is the field of view. As you move from low to high power, the field of view (what you are able to see in the circle of light) decreases - you are able to see less and less of the total specimen (see Figure 2).

Figure 2. Field of view using different objectives.

The field diameter is the diameter of the circle of light that you are able to see. When you move from low to higher objectives, you are still able to see a circle of light, but the diameter of that circle is reduced. field diameter

Every microscope has a pre-determined field diameter for each objective lens. For our labs, we will use the following field diameters.

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OBJECTIVE

FIELD DIAMETER

scanning (low)

____4____ X

______5.0______ mm

low (medium)

____10____ X

______2.0______ mm

high

_____40___ X

______0.2_____ mm

PROPER MICROSCOPE TECHNIQUE The steps used to properly focus a specimen on high power are listed below. Following the steps will enable you to quickly find a specimen under the microscope. Obtain a prepared slide from you instructor and focus a specimen on high power using these steps. 1. Plug in microscope and turn on light source. 2. Make sure the lowest power objective (4X) or the blank is in the light path. 3. Using the stage clips, place slide onto stage. Make sure that the specimen is near the center of the light path. 4. Using the coarse adjustment knob, bring the specimen into focus. If your specimen is not centered, center it at this time using the stage control knobs. The microscopes you use in this lab are parfocal, which means that once you get a specimen into focus on low power, it will be partially focused on higher powers. 5. Turn the nosepiece so that the medium (10X) power objective is in the light path. 6. Using the fine adjustment (outer) knob ONLY, focus the specimen. 7. Repeat steps 5 and 6 using the high (40X) power objective.

TO TAKE A SLIDE OFF THE MICROSCOPE: 1. Turn the nosepiece so that the low power objective or blank is in the light path. 2. Remove the slide. ALWAYS CARRY YOUR MICROSCOPE BY THE ARM AND THE BASE. WHEN FINISHED USING YOUR MICROSCOPE, WRAP CORD AROUND BASE.

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GUIDELINES FOR MAKING DRAWINGS IN BIOLOGY LABS In biology, it is often helpful to make sketches or drawings of the specimens you observe, either macroscopically (those that can be seen with the naked eye) or microscopically. When making drawings, you want your picture to closely reflect what you see. Your drawings should be as large or small as the specimen appears in the field of view. Granted, not everyone is an artist, and your drawings do not have to be true to life, but they should reflect the general features of the specimen you see. All good drawings have the following characteristics: ● ● ● ● ●

Well-defined lines Shading to reflect different colors / features Identification of the specimen drawn Properly labeled structures Drawing magnification

As an example, you may be observing human cheek cells under a microscope using the 40 X objective lens.

nucleus cytoplasm plasma membrane

This is an example of a poor drawing.

cytoplasmic inclusions ______human cheek cells______ This is an example of a good drawing.

Note that when making drawings, your specimen should be identified (human cheek cells) and labeled appropriately.

MICROSCOPE USE The microscopes commonly used for viewing specimens is the compound light microscope. One of the features of microscopes in general is that specimens viewed in the microscope are

7 inverted (upside down) and reversed (backwards) from how they look to the naked eye. Similarly, the way specimens "move" (if you move a prepared slide around on the stage, or you have a live specimen that is moving on the slide) is reversed and 'inverted'. So, if you are viewing a specimen through the ocular, and you physically move your slide a little to the left and up (away from you), in the microscope, the specimen will appear to move a little to the right and towards you. Often times, a total magnification of specimen is calculated. This is just the magnification of the ocular multiplied by the magnification of the objective. For instance, if you are viewing a specimen through a microscope with an ocular that is 10X, and you are using the 4X objective lens, then the total magnification would be 10 * 4, or 40X total magnification. This value is different from the 40X objective. LETTER "E" The letter "e" appears this way with the naked eye:

e When viewing a prepared slide of the letter "e" under the microscope, it will appear this way using the three objectives (low, or scanning (4X) power, medium (10X) power, and high (40X) power, respectively):

4X

10X

40X

The image is inverted and reversed

QUESTION 2: If what you view in the microscope is While looking at your specimen through the ocular, move the slide of the letter “e” up and to the right. In which direction do you think the specimen will move when looking into the microscope? __The specimen will move down and to the left because movements are all inverted or reversed._____

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Using proper microscope technique, you would first focus your specimen using the 4X objective. The total magnification would be 40X, Next, you would switch to the medium (10X) objective and focus the specimen. QUESTION 3: Which adjustment knob do you turn in trying to focus the specimen? __You would want to use the Fine Adjustment knob to focus on specimen.____

QUESTION 4: Refer to Figure 2 above. Describe what happens to the letter “e” when you move to the 10X objective (increase the magnification)? Does it get larger or smaller? Can you still see the whole letter? ____The 10x objective lens just brings you in closer to the letter so it makes one point of the letter larger. You can not see the whole letter anymore, only a portion of it. Can also see the details in the ink and paper._________

QUESTION 5: If the ocular magnification is 10X, what is the total magnification using the 10X objective? _____The total magnification of the specimen is then 100x._____________ QUESTION 6: Refer to Figure 2 above. Describe what happens to the letter ‘e’ when you view it under the microscope using the 40X objective. ______The letter E image becomes larger and you can see just one line of the letter. The field of view is the decreased the most it can go.________

As you increase the magnification (switch to higher number objective lenses) you decrease the field of view (how much of the specimen you can see through the circle of light). The relationship between magnification and field of view is inversely proportional, meaning that as one measure gets larger, the other gets smaller.

ESTIMATING ACTUAL SIZE OF A SPECIMEN It is important to be able to determine the actual size of a specimen you are viewing under a microscope. As you have seen, increasing the magnification of the microscope will decrease the field of view, making specimens appear larger. When comparing different specimens, it is important to know the size relationship between or among them. For example, if

9 you view a specimen using the 4X objective, it may appear the same size as a different specimen viewed under the 40X objective:

specimen using 4X objective

specimen using 40X objective

The specimen viewed under the 40X objective is much smaller than the specimen viewed under the 4X objective, and in fact, may be food for the specimen viewed under 4X. Knowing the actual size of the specimens allows you to discern these relationships between specimens. When sketching your specimen it is important to DRAW YOUR SPECIMEN TO SCALE. That is, your drawing should be as large or as small as it appears in the field of view. To determine the actual size of a specimen, you need to know the field diameter for the objective you are using. These values are provided for you (see above).. When you look through a microscope, the field diameter is the distance from one end of the lighted circle to the other:

field diameter

Figure 3. Field diameter When viewing the specimen, you will ESTIMATE how much of the field of view is taken up by the specimen (another way to phrase this is to ask “How many of these specimens, if lined up, would fit across the field of view?”) When making this estimate, view the specimen through the ocular of the microscope. To determine the ACTUAL SIZE of a specimen, you then DIVIDE by the field diameter by the

10 number of specimens that would fit across the diameter.

Determine the actual size of the specimen drawn in Figure 3.

QUESTION 9. If the objective lens used to view this specimen was the 10X lens, what is the field diameter (refer back to page 4)? 10X objective = ___2.0____ mm. field diameter

QUESTION 10. In Figure 3, how many specimens would fit across the field diameter. Remember, you are estimating the number, it does not have to be exact. number of specimens that fit across the field diameter = ____4____ QUESTION 11: So, the actual size would be Filed Diameter/ # of specimens that first across 2.0 / 4 = 0.5mm The cell depicted in the previous example (Figure 3) was relatively uniform in shape, so estimating the number of cells that would fit across the field of view was straight-forward. Some specimens may be elongated, having a definite length and width. In such specimens, it is important to indicate which dimension you are estimating when calculating actual size.

QUESTION 12: A specimen of Euglena is viewed under a microscope using the 40X objective. The specimen appears as depicted below. What is the actual size (length and width) of the specimen?

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Figure 4: Euglena

ACTUAL SIZE: LENGTH

ACTUAL SIZE: WIDTH:

field diameter: ____0.2______ mm

field diameter: ___0.2_______ mm

# that fit across (length): ___1.5_____

# that fit across (width): ___1.5_____

actual size (length): ____0.133______ mm

actual size (width): ___0.133_____ mm

Always remember to include your units when calculating actual sizes of specimens.

It is important to remember that when you draw your specimen, it represent what you actually see in the microscope. This becomes critical when you calculate drawing magnifications.

QUESTION 13. This image was taken through a microscope using a 40X objective lens. Determine the actual size of this specimen.

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__Paramecium caudatum______________________

ACTUAL SIZE CALCULATIONS: field diameter : ____0.2_____ # that fit across: ____2_____ actual size: ___0.1mm_____...