The Compound Light MICROSCOPE
The basic tool of the microbiology laboratory is the bright field compound microscope. In this type of microscope, the field of view is “bright” or “light.” Familiarity with the proper use of the microscope will greatly assist you in the laboratory section of this course.To understand the proper use of the microscope, it is necessary to know the parts and how they function. You should refer to an actual microscope along with the picture in this manual or one in the lecture text.
The ocular lenses (eyepieces) are a lens system located at the top of the microscope. On our microscopes, the oculars provide a magnification of 10x for each eye. Bright field microscopes may be either monocular or binocular. Binocular microscopes have several adjustments which can be made to make your viewing more comfortable. The eye width is adjustable and the oculars are separately focusable. With the binocular microscopes, it is important to get comfortable viewing with both eyes at the same time so as to see one field of view (circle of light containing specimen). The objectives are a second lens system located just above the stage. They are found on a structure which rotates called the nosepiece (turret). These microscopes will have two objectives.
The scanning objective is the objective with the lowest magnification (4x-5x depending on microscope model). This is the objective which is always used first. Scanning power is used to examine the overall slide to determine the best area for viewing. This lens is shorter which allows for safe focusing with the coarse adjustment knob. Some microscopes lack this objective and therefore the a “low” power objective must be used first when viewing a specimen.
The highest power of magnification available on this microscope is the oil immersion objective. This objective provides a magnification of 100x. This amount of magnification is usually only necessary when viewing very small microorganisms such as bacteria. In order to eliminate light refraction, it is necessary to place
immersion oil between the slide and the lens. After the specimen has been focused on scan, oil is placed on the slide over the specimen. The oil immersion lens is then rotated into position in the oil. Use of this objective requires careful technique to prevent damage to the objective, the specimen, and the microscope. Please be sure to pay attention and take good notes as your instructor covers the procedure.
A third lens system is the condenser, which is found directly beneath the circular opening in the stage. The condenser focuses light from the illuminator onto the specimen. Some condensers may have their position changed by use of an adjustment knob. In this lab, it is usually best to start with the condenser raised to just beneath the specimen. In general, this will yield a sharper image.
Just beneath the condenser, there is a sliding lever to adjust the iris diaphragm. The iris diaphragm controls the width of the cone of light entering the condenser. Correct adjustment of the iris diaphragm with the lever can greatly sharpen the image of the specimen. One of the most worthwhile things for a student beginning to use a microscope, is to practice adjusting the iris diaphragm each time an objective is set into position.
All of the lens systems of your microscope must be kept clean. The only thing that is to touch the surface of a lens is lens paper. Never touch a lens surface with a finger, paper towel, bibulous paper, or anything other than lens paper. Lens paper may be used to gently wipe away dirt, smudges, and oil.
The illuminator (light source) is found in the base of the microscope. The base forms the foundation of the microscope while the connecting arm supports the the rest of the upper portion. When carrying the microscope always use two hands. Grab the arm of the microscope with one hand and support the base with the other hand.
The mechanical stage is the platform on which the specimen is placed. Please be sure you understand how to correctly place your slide into the slide assembly holder brackets. The slide assembly holder includes two knobs (silver) which allow controlled movement of the slide (left and right, up and down).
The coarse adjustment and fine adjustment are control knobs located near the junction of the arm and the base. These allow focusing of the specimen by bringing the objective lens either closer to the slide or further away. The coarse adjustment allows large adjustments in focusing and is used initially to find the specimen when viewing it through the oculars. It should only be used with the scanning lens (because these lenses are short enough to allow large adjustments of this knob). Never use coarse adjustment with oil immersion lenses (you may damage the lens).
The fine adjustment allows slight movement of the lens in order to focus and sharpen the detail of the image. It may be used with any lens because it only slightly adjusts the distance of lens from the slide so there is less danger of a collision between the lens and slide.
Follow this outline for all microscope work. Failure to do so may result in penalties.
Setting up the microscope:
1. Plug the cord into an outlet. Gently clean all lenses with lens paper.
2. Turn on illuminator (use the highest setting comfortable for your eyes- from 1-10).
3. Place a slide within the brackets of the slide assembly holder.
4. Position the scanning lens(red stripe) over the circular opening in the stage.
5. Use the coarse adjustment for rough focusing first, then the fine adjustment to
sharpen the image.
6. Adjust the lighting by using the iris diaphragm lever.Try not to bump the
condenser when adjusting the iris diaphragm.
7. Center the specimen within the field of view using the slide assembly control knobs.
Observe the specimen.
8.If low power is to be used: rotate nosepiece counterclockwise to position the low
power lens (yellow stripe) into place. Repeat steps 5, 6, and 7.
9. If high power is to be used: Rotate nosepiece counterclockwise to position the
high power lens (blue stripe) into place. Focus with the fine adjustment. Never use coarse adjustment with high power. Repeat step 6 and 7.
10. If oil immersion is to be used: Rotate the nosepiece to a position half way
between the oil immersion lens (white stripe) and the last lens you were using- either scan or high power. Place a small drop of oil on the slide over the specimen then rotate the oil immersion lens into place (it will be immersed in oil). Focus with the fine adjustment. Never use coarse adjustment with oil immersion. Repeat steps 6 and 7.
- When you are through, rotate the scanning lens into position before removing the slide. Raise the objectives all the way up using the coarse adjustment knob. Wipe oil off of permanent slides. Slides without a permanent coverslip will be disposed of in the sharps container.
Putting away the microscope:
1. The scanning lens should be positioned over the circular opening in the stage, and
the objectives should be separated the maximum distance from the stage using the coarse adjustment knob. The slide should be removed and cleaned with paper towels, stored, or disposed of.
2. Clean all microscope lenses with lens paper. Clean oil immersion lens last, clean-
ing until the lens is completely free of oil.
3. Clean oil off all non-lens surfaces of the microscope with a paper towel.
4. Turn off illuminator.
5. Gently unplug the cord and loosely wrap it around the oculars.
7. Make sure that the stage assembly is positioned so that its parts do not stick out
over the stage. This prevents possible breakage if it is bumped.
8. Carry microscope with both hands and return to the appropriate location.
Calculating Total Magnification:
OBJECTIVES times OCULARS = TOTAL MAGNIFICATION
Scanning Power 4X X 10X = 40 X
Low Power10X X 10X = 100X
High Power40X X 10X = 400X
Oil Immersion100X X 10X = 1000X
Troubleshooting Tips for Microscope Users:
Field of view is not circular:
*Make sure the lens has “clicked” into place.
*Condenser pins may need adjusting by instructor.
Two circles of light are visible when viewing through the oculars:
*To make one field of view (one circle):
-adjust distance of eyes from the microscope (about 1 inch)
-adjust width of oculars
Not enough light:
*Make sure light switch is on (-).
*Adjust iris diaphragm.
*Make sure illuminator intensity control is set to 10 or as bright as you can tolerate.
*Make sure black sliding illuminator switch located in front of the illuminator is opened.
*Make sure plug is working.
*Condenser position should be about 1 inch below the top of the stage initially for the scanning lens. It should not need further adjustment.
Specimen is blurry:
*Make sure focusing is done with the coarse and fine adjustment knobs (black), not the slide assembly holder knobs (silver). See diagram.
*Make sure the lenses are clean.
*Don’t forget to use the full range of focusing available in a systematic manner. If the focus knobs are turned too rapidly you may pass the focal point. Slow down when there is a change in the field of view because you are possibly approaching the focal point.
*If the field of view does not change (even as you are moving the slide) you need to reset the focal point. Rotate back to scan and reset the focal point. The oil need not be removed. Rotate back to oil immersion lens and try refocusing with fine adjustment.
Specimen was in focus on the scanning lens but cannot be found on oil immersion:
*Always make sure specimen is in the "bull’s eye" before moving from scan to oil.
*Sometimes it is necessary to return to scan, reset the focal point, then return to oil immersion, focusing with the fine adjustment.