The Microscope for Aviculture Microbiology by
Linda Seger
I. SELECTION OF THE MICROSCOPE
II. FEATURES OF BIOLOGICAL AND STUDENT MICROSCOPES
The Compound Light Microscope
Illumination: Tungsten, Halogen, Tungsten-Halogen, Fluorescent
The Condenser
Diaphragm: Iris or Disk
The Specimen Stage
The Objective Lenses
The Arm and the Body Tube
The Ocular
Summary of Variables
III. SETTING UP THE MICROSCOPE
Adjusting the Microscope for First Time Use
Centering the Slide and Adjusting Focus
Adjusting Brightness
Adjusting Contrast Using the Iris Diaphragm
IV. USE AND CLEANING OF THE OIL IMMERSION LENS
V. CLEANING THE MICROSCOPE
VI. ERGONOMIC CONSIDERATIONS
VII. QUIZ ON THIS SECTION
I. SELECTION OF THE MICROSCOPE
The microscope needed for the study of Aviculture Microbiology is generally called a "biological" microscope. It is a compound light microscope with brightfield illumination. "Student" microscopes will have many of the same features as the biological scopes, and these too are appropriate for our use. The microscope to be used for Aviculture Microbiology must have a high-powered, oil-immersion lens which will be used to view Gram stains and other biological stains and smears.
The microscope is a relatively expensive piece of equipment and care should be taken in making your selection. Some features can be added after purchase to most microscopes, but it may save you money to include the features with your original purchase.
II. FEATURES OF THE BIOLOGICAL AND STUDENT MICROSCOPES
The Compound Light Microscope
The compound light microscope employs lenses through which light passes, going from the source, through the specimen, to the eye. Basically, it has a source of illumination, magnification and a focusing system.
The path of light goes from the microscope's light source, through the condenser system, the specimen itself, the objective lens and, finally, through the eyepiece, which further magnifies the image for the eye or camera.
Because the light rays from an object cross before reaching your eye, the image you see through most microscopes will be reversed and upside down. In describing the microscope, it might be easiest to follow the path of the light.
Illumination: Tungsten, Halogen, Tungsten-Halogen, Fluorescent
The intensity of illumination is controlled by the light source. A sub-stage mirror may be used to collect light, even from a candle, but most clinical microscopes have built-in light systems, controlled by an on and off switch or a rheostat.
Halogen provides very white light. It is commonly used in medical and research scopes. Tungsten light, also known as "incandescent light," provides an inexpensive, steady source of light. Tungsten light is yellowish.
WHAT IS TUNGSTEN-HALOGEN???
Both halogen and tungsten lighting generate heat, which can dry out slides or kill live specimens. Another lighting choice for a microscope, the fluorescent bulb, will yield a clearer image than the tungsten alternative and the bulbs are considerably cooler. The fluorescent light option is less common and more expensive to buy.
A microscope may have a simple on and off switch, or a rheostat which will increase and decrease both light and heat.
Your microscope may also have a field diaphragm just above the light source. The field diaphragm controls only the width of the bundle of light rays, not the intensity of the illumination. The narrower beam produced helps eliminate stray light. Better microscopes have a field iris diaphragm, which is a daisy diaphragm with interconnected "leaves." A 35mm camera also uses an iris diaphragm, so you are probably familiar with the mechanism.
The Condenser
The condenser is the small lens or set of lenses built into or under the stage with the purpose of collecting and focusing light. As the light passes into the bottom side of the condenser, it is condensed into a finer, focused stream of light, a parallel beam. A sub-stage, movable condenser is especially important with high magnification where light is scarce.
The Abbe type condenser consists of two lenses, constructed such that the upper lens element is fixed and the lower movable. The lower lens focuses the illumination between the elements so that light rays emerge from the upper stationary lens as a large diameter parallel bundle.
Light adjustment is made by raising and lowering the condenser, and opening and closing the aperture iris diaphragm or the disk diaphragm. Depending on the thickness of the slide and the subject, the location of the focal plane in relation to the light can vary. The condenser has a focusing knob used to move it up and down in order to focus the light on the specimen. The condenser may also house a holder for a filter, usually blue, to adjust the color of the light.
Diaphragm: Iris or Disk
An iris diaphragm or a disk diaphragm, housed within the condenser, serves to adjust contrast.
An iris diaphragm, as mentioned in the illumination section, has interconnected "leaves." The iris will give you a vast array of settings and will be easier to adjust while using the scope. If an "iris" diaphragm is not specified, the microscope may have a disk type.
A "disk diaphragm" is sometimes used in student scopes. The diaphragm is mounted beneath the stage. It will have six to eight apertures of progressively smaller sizes. The largest hole is wide open and transfers the most light. Adjustment is made by turning the disk. With the disk diaphragm the lens goes black between settings and fine tuning is not available.
The Specimen Stage
The prepared slide, or other specimen, sits on stage which holds it in place with a spring-loaded device. With the simple stage the slide must be moved manually.
A mechanical stage allows for simple, accurate movement of the slide. It has two control knobs: one moves the slide up and back, the other moves it from left to right. The mechanical controls allow you to move the slide slowly and precisely. This is especially useful when viewing at 1000X where a slight movement can move the subject area of the slide out of range.
A mechanical stage can be added to most scopes with a thumbscrew to hold it in place, although not all scopes are pre-drilled to accept mechanical stages. Mounting holes are fairly standard, but your microscope manufacturer should be able to guarantee the best fit.
The Objective Lenses
The objective lenses create a magnified image of the specimen. The more lenses you have, the more expensive the microscope will be and that difference represents a significant increase in the price of the microscope, perhaps by 50% or more for each additional objective. Typical configurations for the lenses on the turret, generally called the nosepiece, are 10X, 40X, 100X or 4X, 10X, 40X, 100X. A 4X lens is used to find areas for detailed observation. A 10X lens is useful for identifying tissues. A 40X lens can be used to see the details of cell and tissue organization. The 100X lens, with the use of immersion oil, can be used to see subcellular detail.
Your microscope should have achromatic lenses. To be called "achromatic," the lenses must be color corrected. If not constructed properly, the multiple layers of glass in a lens will cause certain colors to reflect outside the "focal plane," the area in focus under the lens, and any of those colors on your slides will not be visible. The standard for an achromatic lens also specifies that the center 60% of the field of view must be in focus.
The threading for the lenses should be "DIN" or "Deutsche Industrie Norm," which is an international standard for microscope objective lenses. This will insure that your objective can be easily replaced.
The Arm and the Tube Body
The arm is the structure that supports the magnification system. It is also the handle by which the microscope can be safely carried. The arm supports the tube body, which supports the objective lenses.
The Ocular
The eyepiece, or ocular, magnifies the power of the lens being used, such that a 10X ocular combined with the 100X lens creates a 1000X magnification. The eyepiece will be either "pinhole," commonly used on older microscopes, or "wide field." The wide field eyepiece lets you see more of the slide at one time, which means you will not have to move the slide as much. Once the eyepiece is in place on the microscope, you should not remove it, as it is a place for dust to enter.
The microscope can be monocular with a single eyepiece; binocular, for both eyes; and trinocular, with a set of binocular eyepieces plus a monocular one which allows for a camera or for a second person to look through the scope at the same time. If you are not going to be using the microscope for several hours running, you may be satisfied with a monocular objective. People who have been using binocular microscopes will probably prefer the binocular option, but it is an option you will want to evaluate as a purchase option.
Adjustments to oculars are made using the focus adjustment ring on the ocular itself.
Considerations in Buying a Microscope
Summary of Variables:
Lighting source and lighting control
Diaphragm Type
Lens quality and Type
Eyepiece type
III. SETTING UP THE MICROSCOPE
To begin using the microscope, you will need these items:
Lens cleaning paper
Slides
Cleaning fluids
Adjusting the Microscope for First Time Use
Turn on the light source. For optimal viewing of a slide, the intensity of the light source, the position of the condenser and the size of the viewing field must first be correctly adjusted.
You will need to remember these factors:
Brightness - adjusted with the illumination control and the condenser focusing knob
Light scattering - Extraneous light from an under- or over-corrected objective may cause glare which will mask detail.
Resolution - In microscopy, resolution is defined as the fineness of detail in an object revealed by an optical device. In practical terms, resolution refers to how small and how close objects can be and still be recognizable. In the brightfield microscope, resolution is affected by the numerical aperture (NA) of a lens and the aperture iris or disk diaphragm.
Contrast - adjusted by opening or closing the condenser diaphragm
Magnification - adjusted by selecting the appropriate lens
Focus - adjusted with the coarse and fine focus knobs
Oil immersion - Used only with specially designated lenses, immersion oil is specially formulated to adjust its refractive index, and which allows the oil immersion lens to achieve maximum resolution.
In the following steps you will adjust the brightness of the source light and close the iris diaphragm as far as possible to give maximum contrast.
Adjust Brightness
Your light must be powerful enough to give good illumination - especially for the higher magnification/reduced light needed for microscopic slide evaluation - and allow good contrast to see maximum detail.
If you have an external light source with a mirror, position the light about 10 inches in front of the microscope to shine directly on the mirror. Adjust the mirror position so that the bright light reflects into the center of the condenser.
If you have a rheostat, turn the light all the way up.
If your microscope has a field diaphragm, first center it in the optical path and then close it down until, looking through the objective, it just disappears from view. This does not affect optical resolution; however, as scattered light will degrade the quality of your image, a field diaphragm must be properly adjusted.
Adjust the Condenser System
The condenser position must be adjusted with each objective used to maximize light focus and resolving power.
The Iris Diaphragm
The iris diaphragm is often used incorrectly to control light intensity. While this might seem logical, its proper use is to control the size of the cone of light entering the objective lens. If this diaphragm is opened too wide, contrast will be lost because of glare. If closed too far, resolution through the lens will be lost.
Using the lever, fully open the diaphragm to allow maximum light. Using a 10X objective, reduce the aperture of the iris diaphragm until you can see at least part of its edge through the ocular.
Adjust the image of the diaphragm by raising or lowering the condenser using the condenser focus knob until the edge is in sharp focus. The image should also be in focus. If not, refocus the image using the coarse focus knob. Repeat until the image of the specimen and of the diaphragm are in focus at the same time.
Open the diaphragm until it just vanishes from the optical field. The height of the condenser lens is correct; however, when magnification is changed, the condenser system will need to be readjusted.
The Focal Plane
Both brightness and contrast are now properly adjusted for good microscopic work. The location of the focal plane in relation to the light can vary, depending on the thickness of the slide and of the subject.
Center a slide on the stage. Turn the nosepiece to the 10X lens. Looking directly at the stage - not through the ocular - turn the coarse adjustment until the objective is as close to the slide as possible. Stop turning the adjustment when the objective no longer moves.
Look through the ocular(s). Slowly back off the 10X lens using the coarse adjustment until the image on the slide is visible.
Use the fine adjustment to focus on the image.
Adjusting the Binocular Microscope
If you are using a binocular microscope, the distance between the oculars should be adjusted to fit the viewer's eyes. Use first coarse, then fine adjustments to focus on the image using the right eye. Now close the right eye and look through the left ocular. Use the focus ring on the left ocular to achieve sharp focus for the left eye. Because the right ocular was already in focus, you should not make the adjustments using coarse or fine focus controls which would raise or lower the objectives.
Centering the Slide and Adjusting Focus
Put a slide on the specimen stage and use the condenser centering screws to center the image of the field diaphragm, closing the diaphragm down further, as necessary, to center it accurately. Using the 4X power or other low power lens, adjust the focus with the coarse focus control (the inside, bigger wheel on the arm of the scope).
IV. USE OF THE IMMERSION OIL LENS
Some lenses require that oil be placed between the lens and the slide. Oil immersion lenses are indicated by having the word "oil" engraved on the outside of the lens. Don't allow any of your dry lenses to come in contact with this oil.
Immersion oil is a specially formulated oil whose refractive index has been adjusted. The oil reduces the light reflected back from the slide and bends the light rays inwards to focus on the specimen, such that there is more light and less interference, and therefore better detail
To use the 100X oil immersion lens:
Adjust the illumination and focus the specimen on a dry objective lens, such as the 40X.
Rotate the nosepiece so no lenses are over the slide.
Place one drop of immersion oil on the slide at the place that was under the lens.
Swing the 100X lens into position, making sure that it makes contact with the drop of oil. If it does not, slowly lower the objective, while watching from the side rather than through the objective, until it just touches the drop.
Look through the objective and make fine adjustments to the focus - do not use the coarse adjustment. As necessary, to the iris diaphragm and condenser to obtain the best image. If you are using modern parfocal lens, which means that all objectives are approximately in focus at the same setting, the specimen should already be roughly in position. As the working distance with high magnification lens is very narrow, be very careful not to force the lens into the slide.
Adjust the contrast and illumination as necessary to produce a good image.
Cleaning the Oil Immersion Lens
When you are finished with the specimen rotate the nosepiece and clean the 100X lens. Remove the slide.
Remove excess oil from the slide with lens paper or another absorbent paper moistened with ether. Never use gauze to clean the optical parts of a microscope.
To use the oil lens on a new slide repeat the preceding steps.
When you are finished with the oil immersion lens, clean the tip of the oil immersion lens with lens paper. Moisten the paper with ether and draw the wet paper over the surface of the lens to float the oil off onto the paper. Repeat as necessary to remove all the oil.
V. CLEANING THE MICROSCOPE
In addition to regular cleaning of the immersion oil lens after each use, cleaning of your microscope should also be regularly performed.
Eye makeup, especially mascara, present a constant cleaning problem. It's easier to remove the makeup before using the microscope than it is to clean it off the eyepiece.
Lenses should be cleaned only with lens paper that has been moistened with water to remove general dirt or ethyl ether to remove oil.
Use the lens paper to apply a film of the fluid to the surface of the lens and another to remove the fluid. Never rub the lens with the paper.
Kimwipes or similar absorbent paper may be used to remove dirt and oil from the stage and other mechanical parts.
Stubborn dirt may require other solvents such as xylene or commercial lens cleaner applied to the lens paper. Xylene can damage the lens mounting if allowed to get beyond the front seal. Ask for advice from a knowledgeable person before trying this yourself.
ERGONOMICS
We should not put ourselves in a position of using a microscope for hours at a time without consideration for ergonomics. Pain and injury to the neck, shoulders, back, arms, wrists, hands, gingers, legs and feet, as well as eyestrain and headaches have been associated with microscope use. Poor posture and awkward positions are risk factors, particularly head and upper back inclinations. Some of the newer microscopes are designed with ergonomics in mind.
The Occupational Safety and Health Administration (OSHA) finds that "Microscope work is straining both to the visual system and the musculoskeletal system. Operators are forced into an unusual exacting position, with little possibility to move the head or the body. They are often forced to assume an awkward work posture such as the head bent over the eye tubes, the upper part of the body bent forward, the hand reaching high up for a focusing control, or with the wrists bent in an unnatural position."
In using the microscope infrequently and for not more than a few hours a day, the following recommendations may improve your comfort:
Keep a minimum of two inches between the thigh and desk or counter with the leg free from obstructions.
Keep the limbs close to the body with the forearm parallel to the floor and resting on the bench top. Use armrests for older microscopes having controls located in high positions.
Use padded edges for workstations or countertops to avoid contact stress on arms.
Reference:
Kathleen E. Carr and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
QUIZ ON THIS SECTION
What is the purpose of the condenser?
What liquid should you use on a microscope to remove oil?
What is the function of immersion oil?
What are the two types of eyepiece?
Why is a mechanical stage important for use with high magnification?
What is the disadvantage of both tungsten and halogen light sources?
What mechanism provides contrast?
By what part of the microscope should you pick it up?
What is magnification power of the typical oil-immersion lens?
References:
Linne, J.J. and Ringsrud, K. M. Clinical Laboratory Science, The Basics Mosby, Inc. 1999
Walters, N. J. Basic Medical Laboratory Techniques Delmar Publishers, 1996
New York Society Microscopical Society, Glossary of Microscopical Terms and Definitions, 1989, ASTM document E 175-82