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 A 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.

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