Microscopy Mary Ann Seagren

CHAPTER 2 Microscopy Mary Ann Seagren

OUTLINE IDENTIFICATION OF PARTS OF THE Condenser USE OF THE COMPOUND MICROSCOPE Aperture (Iris) Diaphragm MICROSCOPE Oculars (Eyepieces) Coarse and Fine Adjustment Procedure: Use the Microscope to Objective Lenses Stage and Stage Brackets or Clips View the Letter “e” Degree of Magnifi cation CARE OF THE MICROSCOPE Procedure: Calculate Field Size Parfocal Putting the Microscope Away Procedure: Estimate Size of Resolving Power (Resolution) EXERCISE 1: LABEL THE PARTS OF THE Objects Using a Prepared Working Distance MICROSCOPE Blood Film Arm and Base EXERCISE 2: MATCH THE PARTS OF REVIEW QUESTIONS Light Source THE MICROSCOPE WITH THE Rheostat for Light Source CORRESPONDING DEFINITION INTRODUCTION In veterinary practices and laboratories, the compound light microscope is used by veterinarians and veterinary technicians daily in the course of diagnosing and treating animal diseases. A series of lenses is used to form an image from light passing through the specimen. In this way, the image can be magnifi ed R as much as 1000 times. In veterinary practices, compound light microscopes are used for a wide variety of reasons such as: 1. To examine the morphology and numbers of blood cells W (Figure 2-1), 2. To check for the presence of intestinal parasites in feces, P 3. To examine the contents of urine sediment, and 4. To examine an ear swab for the presence of infectious agents such as bacteria, mites, and fungi. The ability to properly use a microscope is essential to ensure the accuracy of these important laboratory tests. In a standard compound light microscope, light from an incandescent source is aimed towards a lens, called the condenser, which is located beneath the stage (Figure 2-2). The Figure 2-1 Blood smear. This is a photomicrograph (a condenser concentrates the light before it passes through a photograph of a microscopic fi eld) of a smear of stained hole in the stage where it then penetrates the specimen. From canine blood. Note the presence of two white blood cells. here the light passes through an objective lens, before being Each has a purple multilobed nucleus. They are surrounded magnifi ed a second time by the ocular or eyepiece. Finally, the by donut-like red blood cells, which have no nuclei. Platelets light reaches the eye so that what was too small to be seen is are seen interspersed among the red blood cells and they now made clearly visible. Some microscopes have a built-in appear as pale cell fragments or small dots. W, White blood illuminator as shown in Figure 2-2, A, whereas others use a cells; R, red blood cells; P, platelets. (Photo courtesy Manor mirror to refl ect light from an external source (see Figure 2-2, College.) B).

24 Standard compound Monocular light microscope light microscope

Ocular Ocular

Objective Objective

Slide Slide

Stage Stage

Condenser Condenser

Internal Mirror light source

A B External light source

Figure 2-2 A, Standard compound light microscope. Some microscopes have a built-in illuminator. B, Monocular light microscope. Others use a mirror to reﬂ ect light from an external source.

LEARNING OBJECTIVES In this laboratory, students will learn: 2. To use the microscope to view the letter “e.” 1. The location, name, and function of each part of the com- 3. To calculate the fi eld size of the various magnifi cations. pound microscope, and will learn to use, adjust, and main- 4. To estimate sizes of objects viewed using a stained blood tain these parts. fi lm. MATERIALS NEEDED • Compound light microscope • Immersion oil • Prepared microscope slide of newsprint letter “e” • Millimeter ruler • Prepared microscope slide of a stained blood fi lm • Lens paper • Prepared slide of a grid, ruled in millimeters (grid slide) • Lens cleaner TO BE IDENTIFIED Eyepiece or ocular (10× or 5× each) Illuminator or light source Arm Light on or off switch Stage Rheostat for light source Opening of the stage Iris diaphragm Fine adjustment knob Iris diaphragm lever Coarse adjustment knob Condenser Base Condenser knob Stage clips High-power or high dry objective (40×, 43×, or 45×) Control knob of mechanical stage clips Oil immersion (97× or 100×) Objective lenses Nosepiece Scanning objective (3.2×, 3.5×, or 4×) Body tube Low-power objective (10×) CLINICAL SIGNIFICANCE OF LIGHT MICROSCOPY Light microscopy is best suited to viewing very thin, stained specimens, such as sections of tissue, blood smears, and drop- lets of liquid containing bacteria. It is also useful for the examination of living, unstained specimens, such as mites, parasitic eggs from fecal fl otations, and cellular and noncellular compo- nents of urine sediments. Below is a list of specimens that might be observed in practice using light microscopy. • Stained bacteria from culture (1000×) (Figure 2-3) • Stained tissue sections (100×, 400×), thin sections with condensed chromosomes or specially stained organelles (1000×) • Stained blood smears—numbers and morphology of blood cells, presence of blood parasites (400×, 1000×) • Wet mounts of feces (fl otation or direct smear)—large pro- Yeast tists—(100×), protozoa oocysts (100×, 400× occasionally), nematode eggs (100×) • Wet mounts of ear debris or skin scrapings—mites and yeast (100×) (Figure 2-4) Figure 2-4 Yeast. This photomicrograph shows yeast cells • Stained smears from wounds and infections—fungal elements (oval and peanut-shaped) along with squamous epithelium and bacteria (400×, 1000×) from the ear canal. This is a common fi nding in a smear • Urine sediment, stained or unstained—presence of crystals, from a dog with a yeast ear infection. blood cells, casts, or bacteria (100×, 400×) • Stained vaginal smear—stage of estrus is determined by maturation of epithelial cells (100×, 400×) (Figure 2-5)

Figure 2-3 Stained bacteria. This photomicrograph is of a Figure 2-5 Vaginal smear. This photomicrograph shows smear of Staphylococcus aureus bacteria stained with Gram epithelial cells that have sloughed from the lining of the stain. The bacteria that absorb the purple Gram stain are vagina. The appearance of the cells is used to gauge the called “Gram positive.” They are a common infectious agent stage of the dog’s heat cycle. (From Bassert J: Images for in mastitis and skin infections of all species. veterinary technician educators, Summer 2001.) Chapter 2 Microscopy 27

Oculars or eyepieces

Body tube

ARM Objective lenses Stage brackets Control knob Lever for iris of mechanical diaphragm stage clip

Light source Base

Condenser knob

Coarse focus Nosepiece adjustment knob

Opening of the stage Fine focus adjustment knob Condenser

Rheostat

Light switch B Figure 2-6 A and B, The most common microscope used in light microscopy is the compound light microscope. The compound light microscope has at least two magnifying lenses. One is in the eyepiece (ocular), and one is in the objective.

IDENTIFICATION OF PARTS OF Binocular vision offers greater clarity, detail, and THE MICROSCOPE a wider fi eld of view than does monocular vision, but A labeled photograph of a compound microscope is some adjustments to the microscope will need to be shown in Figure 2-6. Observe the labeled parts as you made to reap the benefi ts of binocular vision. First, read the description and function of each part. the distance between pupils (in the eye) varies from person to person, so the distance between the oculars OCULARS (EYEPIECES) will need to be adjusted to match your interpupillary A compound light microscope can either be binocu- distance. Second, one or both of the eyepieces may lar (containing two eyepieces) or monocular (con- be “a telescoping ocular,” which allows it to be taining one eyepiece). The usual magnifi cation of the focused. Most people see better in one eye than the eyepiece is 10×. It is important to periodically remove other. So the focusing of one or both eyepieces allows the eyepieces and clean them with lens cleaner and the visual acuity in both eyes to be matched to one lens paper. another. Because our visual acuity is measured in 28 Chapter 2 Microscopy units called diopters, this focusing process is called that is, objects in the specimen can be closer together the diopter adjustment. and still be seen as distinct from one another.

OBJECTIVE LENSES WORKING DISTANCE The objective lenses are attached to a revolving When a specimen is in sharp focus, the working dis- nosepiece. The power of each lens is engraved on the tance is the distance between the objective lens that side of the objective. The smallest power lens may is in use and the specimen. As stronger lenses are be 3.2×, 3.5×, or 4×, and is used when fi rst locating used, the working distance decreases. The specimen and viewing a specimen. Because some specimens and the glass slide on which it rests become closer can be quite minute and diffi cult to fi nd on a com- and closer to the objective as you move from scan- paratively large glass slide, this objective is some- ning to low power to high power. Caution should be times called the “scanning lens.” taken when using high dry and oil immersion objec- The next lens is called the low power (LP) objectives in particular, as there is risk of lowering these tive and is a 10× magnifi cation. It is used for initial, objectives too far when focusing and jamming the coarse focusing and for the examination of large objectives into the glass slide. Some carefully pre- specimens such as nematode eggs. pared slides have been broken this way, not to The high power (HP) objective or the high dry lens mention the potential for damaging the lens in the offers high magnifi cation without the use of oil. High objective. So, be very cautious when focusing with power lenses may magnify 40, 43, or 45 times, depending high powered objectives. Observe the working dis- upon the objective, and are used for small specimens, tance when using low power, high power, and oil such as protists, large yeast, and urine sediments. immersion. The distance should approximate 16 mm, The highest power lens is in the oil immersion objec- 4 mm, and 1.8 mm, respectively. tive. It has a magnifi cation of 97× or 100×. It is used with oil to improve resolution. This lens is used for viewing ARM AND BASE blood smears and bacterial smears, and to see details The arm connects the base, stage, and body tube. (such as cell organelles) in histologic samples. The base is the bottom platform of the microscope, which holds the illuminator. Always be sure to carry DEGREE OF MAGNIFICATION a microscope by its arm with one hand while using The degree of magnifi cation represents the total the other hand to support the base. amount of magnifi cation that is used to visualize a specimen. It is calculated by multiplying the ocular LIGHT SOURCE magnifi cation (usually 10×) with the objective magni- The light source is usually built into the base. A good fi cation. For example, if you are using the high power light source will have a wide dynamic range to provide objective (40×) to view a group of cells, the total high intensity illumination at high magnifi cations and degree of magnifi cation would be equal to10× × 40× lower intensities at low magnifi cations. The best micro- or 400×. scopes have controls that regulate the intensity and shape of the light beam. If your microscope requires an PARFOCAL external light source, make sure the light is positioned Most microscopes are parfocal, which means that so that it strikes the middle of the condenser. the image will remain focused as you increase mag- The apparent fi eld of an eyepiece is constant regard- nifi cation. This is a characteristic of a good quality less of the level of magnifi cation used. So it follows microscope. When the microscope is focused under that as magnifi cation increases, the area of visible low power, it will remain in focus as you switch to a specimen decreases. Because you are looking at a higher and higher power. It is good practice to begin smaller area, less light reaches the eye, and the image light microscopy in the lowest power and move up darkens. With a low power objective you may have to in magnifi cation incrementally, even if the micro- cut down on illumination intensity. When using a high scope is parfocal. This step-by-step approach allows power objective you will need all the light you can get, you the opportunity to center the specimen within especially when using a less expensive microscope. each new fi eld and to use the fi ne adjustment to perfect the image. RHEOSTAT FOR LIGHT SOURCE The rheostat regulates the intensity of light coming RESOLVING POWER (RESOLUTION) from the lamp or light source. To lengthen the life of Resolution is the ability of a microscope to produce the bulb, lower the light intensity before turning off a clear image. It is the ability to separate and distin- the light. guish fi ne details of a specimen. The resolving power is the minimum distance by which two points must CONDENSER be separated and still be perceived as two distinct The condenser is located directly above the light points. Better microscopes have higher resolution, source. It focuses the incoming light into a narrow Chapter 2 Microscopy 29

STAGE AND STAGE BRACKETS OR CLIPS The stage is the platform below the objectives upon which a glass or plastic specimen slide is placed. There is a hole in the stage through which the light from the condenser passes. A bracket holds a slide in place on a mechanical stage. Knobs, located below and to the right side of the stage, direct the brackets to move the slide to the right and left, and forward and backward. If a mechanical bracket is not present, stationary stage clips are used and the slide must be moved manually. Stage clips are found on older microscopes without mechanical stages. They are ﬂ exible pieces of metal that rest on the slide. Slides must be moved manually to change ﬁ elds.

Figure 2-7 Urine sediment. This photomicrograph of dog CARE OF THE MICROSCOPE urine shows crystals that are typically present when the dog Every part of a good quality microscope is expensive, has a bacterial infection of the urinary bladder. An unstained so be careful when moving, handling, and using one. specimen such as this is most easily viewed using high Carrying out the following recommendations will contrast (low condenser position). The illumination may need help ensure the health of your microscope. to be increased when the condenser is lowered. (From Bassert 1. Always carry the microscope in an upright posi- J: Images for veterinary technician educators, Summer 2001.) tion with two hands: one under the base and the other on the arm. Never grab a microscope beam that passes through the specimen and then by any part other than by the arm! enters the objective. Although some condensers are 2. When placing the microscope on a table top, fi xed in position and cannot be adjusted, adjustable be sure that the entire base is in contact with ones can be moved up and down to improve the the counter surface. The edge of the base should resolution and contrast of the image. Lowering the be no less than 10 cm from the edge of the condenser away from the stage increases contrast, table. which is helpful when viewing unstained specimens 3. Make sure the stage and objectives are as far such as urine sediments (Figure 2-7). Raising the con- apart from one another as possible and that the denser, on the other hand, decreases contrast and is lowest power objective is in position. helpful when viewing thin, stained specimens. 4. Unwind the lamp cord carefully, avoiding damage to any parts of the microscope. Hold APERTURE (IRIS) DIAPHRAGM the plug, not the cable, when plugging in and Most condensers contain an iris (aperture) dia- unplugging the illuminator. phragm, a device that controls the diameter of the 5. When using the focus adjustment knobs, focus light beam coming up through the condenser. When carefully. Don’t speed through the focusing the diaphragm is stopped down (nearly closed), the process, particularly when using the coarse light comes straight up through the center of the focus adjustment. condenser lens and contrast on the specimen is high, 6. Apply immersion oil only to the oil immersion but you lose resolution. When the diaphragm is wide objective. Never apply oil to the high dry or any open, resolution improves, but the light may glare other objective. The oil immersion lens has a and contrast is decreased. The optimum position of protective seal that prevents oil from leaking the iris diaphragm is an intermediate position. into the objective; the other objectives lack this protective seal. COARSE AND FINE ADJUSTMENT 7. Remove immersion oil immediately after use. The adjustment knobs located on the arm of the micro- Don’t let it dry on the lens. The oil can degrade scope change the distance between the stage and the the glue that holds the lens in place. objectives. The coarse adjustment knob is used during 8. Always use good quality lens paper on all optical low power and moves the stage quickly up or down to surfaces. Never use paper towels or other tissue bring the specimen into view. To fi ne tune this image paper products to clean the lenses. Only use and to focus specimens under high power and oil appropriate lens cleaner or distilled water to immersion objectives, the fi ne adjustment knob is used clean the lenses. Organic solvents may separate to carefully move the stage minute distances. Never or damage the lens elements or coatings. use the coarse adjustment knob when working under 9. Because bulbs are expensive, and have a limited high magnifi cation as there is risk of breaking the slide life, turn the illuminator off when you are not and scratching the objective. using the microscope. 30 Chapter 2 Microscopy

PUTTING THE MICROSCOPE AWAY 3. Unplug the illuminator cord and wrap it care- 1. Move the scanning objective into position and fully around the base of the microscope, being lower the stage as far as possible. careful of the illuminator. 2. Always make sure the stage and all of the objec- 4. Cover the instrument with a dust jacket when tives are thoroughly cleaned before putting away not in use. the microscope. Remember that oil can damage the lenses.

Exercise 1: Label the Parts of the Microscope

Below is an illustration of a compound microscope (Figure 2-8). Match the terms with the corresponding parts on the drawing.

Oculars Light source Coarse adjustment knob Nosepiece Condenser Fine adjustment knob Objective lenses Iris diaphragm Stage Base Light switch Stage adjustment knobs Arm Rheostat Chapter 2 Microscopy 31

Exercise 2: Match the Parts of the Microscope With the Corresponding Deﬁ nition

_____ Oculars a. Focuses the image. Can move the stage quickly a large distance. _____ Objective lenses b. Focuses the light into the objectives. Adjustment _____ Arm can change contrast. c. Controls the intensity of the illumination. _____ Condenser d. Magnifying lenses that are closest to the speci- _____ Iris diaphragm men. Can be changed to increase magniﬁ cation. e. Platform for holding the slide. _____ Rheostat f. You carry the microscope by this part. _____ Coarse adjustment knob g. Focuses the image. Moves the stage in very small increments. _____ Fine adjustment knob h. Controls the diameter of the light beam. _____ Stage i. Magnifying lenses that are closest to your eye. Usual magniﬁ cation is 10×.

USE OF THE COMPOUND MICROSCOPE microscope slide. If the condenser has select- able options, set it to bright fi eld. Start with PROCEDURE: USE THE MICROSCOPE TO the iris (aperture) diaphragm in a closed posi- VIEW THE LETTER “e” tion. Slowly move the diaphragm lever so that 1. Carefully unwind the cord from around the the aperture slowly opens. You will see increas- base, making sure not to damage any parts of ing amounts of light coming through the spec- the microscope. Plug in cord. imen as you move the aperture diaphragm 2. Make sure the stage and objectives are as far lever to a more open position. apart from one another as possible and that 11. Take a moment to adjust the oculars. Make a the lowest power objective (scanning objec- diopter adjustment by adjusting the movable tive) is in position. eyepiece(s) so that both of your eyes see 3. Clean the oculars and objective with lens through the oculars equally well. Look at the paper, if needed. image with each eye individually and then 4. Turn on the light source by pushing the light together to confi rm balanced vision. switch to the “on” position. 12. Calculate the degree of magnifi cation by multi- 5. Adjust the oculars to match your interpupil- plying the magnifi cation of the ocular by the lary distance (distance between your eyes). magnifi cation of the objective. Enter this fi gure 6. Carefully place the prepared slide of the news- into the table below. Use the millimeter ruler print letter “e” on the stage and secure it in to measure the working distance from the top place with stage clips or mechanical brackets, of the slide to the bottom of the objective and if present. enter this measurement in millimeters (mm) in 7. Move the slide so that the letter “e” is centered the table below (Table 2-1). over the stage’s hole. 8. While looking from the side, use the coarse adjustment knob to carefully lower the lowest powered objective as close to the slide as pos- TABLE 2-1 Measurements and Calculations sible. Do not allow the objective lens to touch for Each Magnifi cation the slide. 9. Look through the oculars and use the coarse Objective Working Field focus knob to slowly raise the objective away Power Magnifi cation Distance Diameter from the slide. When the image becomes clear, 4× switch to the fi ne adjustment knob to perfect 10× the image. 40× 10. Using the condenser knob, raise the condenser 100× (oil) so that it is almost touching the bottom of the 32 Chapter 2 Microscopy

13. Turn the 10× objective until it is over the the bottom of the objective in millimeters and microscope slide. Turn the fi ne adjustment enter the fi gure in Table 2-1. knob to sharpen the image. Sketch the image 17. Rotate the 100× objective (oil immersion lens) you see in the circle provided. slightly, but do not click it into position. At this point, no objective should be in position. Care- fully place one drop of immersion oil on the portion of the slide that is directly over the condenser and stage hole. Do not get oil on your hands or on any part of the microscope. Rotate the 100× objective into position, being careful not to rotate any other lens into the oil. While looking into the oculars, use only the fi ne adjustment to focus the 100× (oil immersion) objective. Using the coarse adjustment could break the slide and damage the lens. Adjust illumination and contrast as needed. 10× Sketch the image that you see in the corresponding circle below.

14. Calculate the degree of magnifi cation and enter the fi gure in Table 2-1. Using the millimeter ruler, measure the working distance from the top of the slide to the bottom of the objective and enter this number in millimeters (mm) in Table 2-1. 15. Next, move the 40× objective into position. Turn the fi ne adjustment knob to sharpen the image. Remember, never use the coarse adjustment to focus when using high magnifi cation. Scan the slide by using the mechanical stage knobs to move the slide left and right and backward and forward while looking through Oil immersion (100×) the oculars. If you do not have a mechanical stage, you will need to move the slide on the stage by hand. Sketch the image in the corre- 18. Calculate the degree of magnifi cation and sponding circle below. You may need to adjust enter it into the table below. Using the milli- the lighting and contrast to generate the best meter ruler, measure the working distance possible image. from the top of the slide to the bottom of the objective and enter this fi gure into the table as well. 19. Rotate the 100× objective to the side and rotate the 4× objective into place (not the 40× objective because it would touch the oil). Carefully remove the slide without getting oil on your fi ngers or on any part of the microscope. Clean the slide with lens paper. Discard the oily paper right away. If you have oil on your fi ngers, you will need to wash your hands. Next, clean the 100× objective with fresh lens paper and lens cleaner to remove all traces 40× of oil. 20. Move the lowest power objective into position. Separate the objectives from the stage so 16. While using the 40× objective, calculate the that they are as far apart from one another as degree of magnifi cation and enter it in Table possible 2-1. Using the millimeter ruler, measure the 21. Re-examine the stage and lenses to ensure that working distance from the top of the slide to they are thoroughly cleaned. Chapter 2 Microscopy 33

22. Turn off the microscope light and unplug the length of the diameter of the fi eld is 3.5 mm. illuminator cord. Wrap it carefully around the Calculate the length of the diameter of your fi eld base of the microscope. using the 4× objective and enter your result into 23. Cover the microscope with a dust cover. Table 2-1. 3. Move the 10× objective into position and again PROCEDURE: CALCULATE FIELD SIZE calculate the length of the diameter of the fi eld It is useful to be able to estimate the size of objects in millimeters. Enter your result in Table 2-1. viewed under the microscope. For instance, the 4. We could keep going and directly measure the oocyst of Isospora canis looks similar to the nema- length of the high power fi eld (HPF, 40× objec- tode egg Toxascaris leonina. The major difference tive) and oil immersion fi eld (100× objective) as between the two is that I. canis is half the size of you have been doing. But you can also calculate T. leonina. Being able to estimate the size of both these fi elds by using the measurements you I. canis and T. leonina, therefore, is important in already have of the low power fi eld (LPF, 10× making a correct identifi cation. But how is the size objective). Calculating these fi elds is easier and of a specimen calculated? The answer is not calcu- faster than measuring them directly. Use the lated directly, but indirectly by fi rst calculating the following formula to calculate the diameter of fi eld size. Field size refers to the amount of a speci- the fi elds seen under 40× and 100× objectives. men that is visible through the microscope. As the Enter your results in Table 2-1. magnifi cation of a specimen increases, the fi eld size decreases. By knowing the total fi eld size, you can diameter of LPF× degree of estimate the size of the specimen inside the fi eld. magnification of the LP objective Diameter of HPF = Degree of magnification of Here Is How It Is Done. . . . the HP objectiive The diameter of the microscope fi eld and the objects in it are measured in millimeters (abbreviation: mm, 1/1000th of a meter) and micrometers (abbreviation: Example: μm, 1/1,000,000th of a meter). More importantly (and What would be the diameter of the HPF if the more relevant to microscopy), a micrometer (μm) is diameter of the LPF is equal to 2 mm? The degree 1/1000th of a millimeter (mm) or 0.001 mm. To of magnifi cation of the low power objective is 100 measure the diameter of a microscopic fi eld, you will (10× objective × 10× ocular) and the degree of need a grid slide. A grid slide is a special microscope magnifi cation of the high power objective is 400 slide that contains ruled millimeter squares. Each (40× objective × 10× ocular). Therefore: square is 1 mm by 1 mm. Once a grid slide is acquired, complete the following steps: 2 mm× 100 1. Center the grid slide on the stage of the micro- Diameter of HPF = ==0. 5mm 500 μ m scope. Using the 4× objective, adjust focus, 400 brightness, and contrast until the image is clear and sharp. 2. Move the grid slide until one line of the grid is PROCEDURE: ESTIMATE SIZE OF OBJECTS lined up along the left hand side of the fi eld. USING A PREPARED BLOOD FILM Count the number of squares that extend across To estimate the size of objects in the fi eld, simply the diameter of the microscopic fi eld. If the last estimate the percentage of the diameter of the square is only partially in the fi eld, estimate the fi eld that is occupied by the object. For example, if a fraction of the square that is visible. For fi eld is 1 mm in diameter and an oocyst takes up half example, in Figure 2-9 there are three squares of the fi eld size, then the object is approximately in the diameter of the fi eld and approximately 0.5 mm in diameter. Refer to Figure 2-10 for another half of the fourth square is showing. So the example.

Set up for Viewing Clean the oculars and objective with lens paper as needed. Move the 4× objective into position. Adjust the oculars to your interpupillary distance (distance between your eyes). Carefully place a slide of a blood smear on the stage. If there is no cover slip, be sure that the blood Figure 2-9 3.5-mm microscope ﬁ eld. ﬁ lm is facing upward. 34 Chapter 2 Microscopy

Adjust Eyepiece Focus Look with the appropriate eye into the fi xed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope focus knob. Move the 10× objective into position over the microscope slide. Turn the fi ne adjustment to sharpen the image. Locate a specifi c red blood cell in the fi eld. Refer- ring to Table 2-1, fi nd the fi eld size for the 10× objective that you calculated earlier. Using this fi gure, estimate the diameter of the red blood cell. Draw the cell and write the diameter in the space provided below.

Figure 2-10 Fecal fl otation. This photomicrograph of a fecal fl otation specimen shows an egg from the nematode Trichuris. The fi eld size is 140 μm, and the egg takes up half of the fi eld diameter. So we estimate the egg length to be 70 μm. (From Bassert J: Images for veterinary technician educators, Summer 2001.)

Optimize the Lighting 10× Turn on the illuminator and adjust it so that the ﬁ eld is bright without hurting your eyes. The higher the Diameter of RBC = ______magniﬁ cation, the more light you will need.

Adjust the Condenser Locate a speciﬁ c white blood cell in the smear and Adjust the condenser using the condenser knob so estimate its diameter. Draw the cell and write the that the condenser is almost touching the bottom of diameter in the space provided below. the microscope slide. This is the ideal setting for viewing stained smears. Start with the iris (aperture) diaphragm stopped down (high contrast). You should see the light that comes up through the specimen increase in brightness as you move the aperture diaphragm lever to a more open position. Adjust the aperture diaphragm to optimize viewing.

Focus, Locate, and Center the Specimen Start with the lowest magnifi cation objective lens (4×), to fi nd the specimen and/or the part of the specimen you wish to examine. Start with the specimen out of focus so that the stage and objective must be brought closer together. Use the coarse focus 10× knob to bring the stage and objective closer together. Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide Diameter of WBC = ______around on the stage until you have a good area for viewing. Find an area where the cells are close together but not touching. Use the fi ne focus knob to Turn the 40× objective into position above the micro- sharpen the image. scope slide. Use the fi ne adjustment to sharpen the Chapter 2 Microscopy 35 image. Scan the slide using the mechanical Locate a specifi c red blood cell in the smear and, stage knobs to move the slide on the stage, while referring to the fi eld size for the 100× objective that looking through the oculars. Adjust illumination if you previously calculated, estimate the diameter of necessary. the red blood cell. Draw the cell and write its diam- Locate a red blood cell in the smear and estimate eter in the space provided below. its diameter using the fi eld size for the 40× objective that you previously calculated. Draw the cell and record its diameter in the space provided below.

Oil immersion

= 40× Diameter of RBC ______

Diameter of RBC = ______Locate a speciﬁ c white blood cell in the smear and estimate its diameter. Draw the cell and write its diameter in the space provided below. Locate a white blood cell in the smear and estimate its diameter. Draw the cell and write its diameter in the space provided below.

Oil immersion

40×

Diameter of WBC = ______Diameter of WBC = ______

Move the 4× objective into position (not the 40× Rotate the 100× objective (oil immersion lens) to the objective because it would touch the oil). Carefully side slightly (so that no objective is in position). remove the slide, without getting oil on your fi ngers Place one drop of immersion oil on the portion of the or on any part of the microscope. Clean the slide slide that is directly over the condenser. Rotate the with lens paper. Discard the oily paper right away. If 100× objective into position, being careful not to you have oil on your fi ngers, you will need to wash rotate any other lens into the oil. While looking into your hands. Next, clean the 100× objective with fresh the oculars, use only the fi ne adjustment to focus the lens paper and lens cleaner to remove all traces of 100× (oil immersion) objective. Using the coarse oil. adjustment could break the slide or damage the lens. Separate the objectives from the stage so that they Adjust illumination if necessary. are as far apart from one another as possible. 36 Chapter 2 Microscopy

Re-examine the stage and lenses to ensure that minator cord. Wrap it carefully around the base of the they are thoroughly cleaned. microscope. Turn off the microscope light and unplug the illu- Cover the microscope with a dust cover.

REVIEW QUESTIONS

1. Why must oil be cleaned off the oil immersion lens before it dries? ______2. Why can’t you use oil with any objective other than the oil immersion objective? ______3. How do you calculate the degree of magnifi cation? ______4. If you are viewing cells through a 40× objective, what is the degree of magnifi cation at which you are seeing the cells? ______5. Which focus knob can you use with oil immersion? (fi ne, coarse, both) ______6. How does the apparent orientation of the letter “e” change when viewed through the microscope? ______7. When you move the slide away from you, in which direction does the letter “e” move? ______8. As you change to a higher degree of magnifi cation, do you have to decrease or increase the illumination? ______9. As you move to a higher power objective, does the object become larger or smaller? ______10. Does the fi eld size become larger or smaller? ______