4: MICROSCOPY
- Page ID
- 157063
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Students will be able to demonstrate proper care and handling of a compound light microscope, including cleaning, transport, and storage procedures.
- Students will accurately focus on a specimen at low and high magnification, including correct use of the oil immersion lens for observing bacterial cells.
BACKGROUND
One of the most essential tools in microbiology is the microscope (an instrument that uses lenses to magnify small objects). Before the invention of microscopes, the existence of microorganisms was completely unknown. Initially, it was difficult to persuade others—both scientists and the public—that such invisible life forms were real.
In this exercise, you will learn how to use a microscope effectively. Throughout this course, we will frequently examine bacteria which are extremely small. Proper care and precise use of your microscope will be necessary to observe them successfully.
A microscope functions similarly to a magnifying glass or eyeglasses by using glass lenses to enlarge objects. However, optimal performance depends on clean, undamaged lenses, sufficient lighting, proper alignment, and appropriate distance from the specimen. This exercise will teach you how to inspect and adjust these components to ensure accurate observations.
MICROSCOPE CARE
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Use only the microscope assigned to you.
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Always carry the microscope with two hands—one hand on the arm and the other supporting the base.
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Always lower the stage all the way down before removing a slide from the stage.
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Only use immersion oil with the 100x objective lens. Do not use oil with any other objective. If you accidently get oil on any objective other than the 100x let the instructor know right away so it can be cleaned properly and prevent permeant damage.
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Always start with the scanning (4x) objective.
๐Before returning the microscope to its cabinet:
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Wipe the oil immersion objective in one direction using only lens paper.
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Turn off the power and set the rheostat (brightness control) to the lowest setting.
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Rotate the 4x objective into place and lower the stage completely.
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Center the stage clip and holder so that they are balanced as much as possible over the stage.
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Coil the power cord neatly and secure it on the back of the scope.
MICROSCOPE FEATURES
OCULAR LENSES
The eyepieces, or ocular lenses, are located at the top of the microscope. Because your microscope has two, it is classified as binocular. Each ocular lens magnifies the image tenfold.
Adjust the distance between the oculars so that you can view a single, clear image with both eyes. If the image appears double or you can only see through one eye, the oculars need further adjustment.
You may remove your glasses while using the microscope. If your eyes require different prescriptions, one of the oculars can be adjusted to accommodate this difference.
๐Cleaning note: Use only lens paper to clean the oculars. Dust, oils from skin, and cosmetic products like mascara can easily soil the lenses. If you are unsure whether the dirt is on the ocular lens, gently rotate it—any dirt present will move with the lens.
STAGE
The stage is the flat platform on which the microscope slide is placed. Metal clips secure the slide, which can be moved forward/backward and left/right using the adjustment knobs located underneath the stage.
LIGHT SOURCE
The light source is located in the base of the microscope. Turn it on using the “O/I” switch and adjust brightness using the rheostat on the side. Lower intensity light may appear yellow, while higher intensity becomes blue-white. Because resolution (the ability to distinguish fine detail) improves with shorter wavelengths of light, full brightness should be used with high-power lenses. Lower brightness is appropriate for viewing samples under low magnification.
OBJECTIVE LENSES & MAGNIFICATION
Above the stage is a rotating objective ring (nosepiece) that holds four objective lenses: 4x, 10x, 40x, and 100x. These provide the initial level of magnification. The image is then further magnified 10x by the ocular lenses.
For example:
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4x objective × 10x ocular = 40x total magnification
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100x objective × 10x ocular = 1000x total magnification
The 100x objective lens requires immersion oil to reduce light scatter and increase image clarity. Use oil only with the 100x lens. Never allow oil to contact the other lenses. Oil will destroy the other objectives making it very difficult or impossible to focus.
Always label your drawings with the final magnification.
The final magnification for bacteria is always 1000x.
Each objective lens must be at a specific distance from the slide in order for the landmark or sample to be in focus. Your microscope is parfocal, meaning once a slide is in focus at low power, it should remain close to focus at higher powers. Once the sample is in focus, you should not need to rotate the fine focus knob more than two full turns in either direction. If after moving to a higher power and slowly rotating the fine focus two rotations in both directions you cannot find the landmark or sample, go back to the 4x and start over. Unless there is a cover slip on the sample, once there is oil on the slide you cannot start over or back to the 40x. The lower objectives are not sealed. If oil touches any objective other than the 100x the objective oil will get into the lens and destroy the objective.
๐Technique tip: Using a Sharpie pen, draw a circle (referred to as a landmark) on the slide around the sample. Be sure the circle and the sample are on the same side of the slide. Bacterial cells and the pen mark (circle) are on the same focus plane therefore when the circle is in focus the bacteria will also be in focus.
Remember: If at anytime (before adding oil to the slide) you experience difficulty focusing at higher magnifications, return to the 4x objective and refocus on the pen mark. Once there is oil on the slide DO NOT return to lower magnification. Ask your instructor for assistance.
MATERIALS (Per Group of 4)
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4 Microscopes (One per student - only use your assigned microscope)
- 1 Saccharomyces cerevisiae slide
- 1 Amoeba spp slide
- 1 Mixed bacteria slide
- 1 Black Sharpie medium point pen
METHODS/PROCEDURES
STEPS TO FOCUS
- Make sure the stage is in the lowest position by gently rotating the course focus knob (do not force it).
- Rotate the scanning objective (4x) into position directly above the stage.
- Place the slide on the stage and secure it in place with the stage clip.
- Adjust the slide so that either the pen mark (landmark) is directly over the light source.
- Use the course focus knob to raise the stage all the way up.
- Use both eyes to look through the oculars while slowly rotating the coarse focus knob to slowly bringing the stage down until the landmark (pen mark/circle) comes into focus.
- Use the fine focus to sharpen the landmark.
- Increase the magnification by rotating to the 10x objective then use the fine focus to sharpen the landmark.
- Center the landmark
- Rotate to the 40x objective. From now on use only the fine focus knob.
- Increase the light intensity.
- Focus on the landmark - you may start to see bacterial cells under 40x objective.
- Look through the oculars and slowly turn the coarse focus knob to lower the stage slightly, which will bring the pen mark into sharp focus. Once the image is clear at 4x, scan the slide to find a suitable area to observe. Make sure the region of interest is centered in the field of view. At higher magnifications, only a small portion of the slide is visible—any object not centered at low power will be lost from view at high power.
Using the Oil immersion Objective:
- Once the landmark is centered and as sharp as possible (some of the 40x objectives have gotten oil on them by previous students and therefore the sharpness is limited) DO NOT LOWER THE STAGE. Rotate the objective ring past the oil objective lens until the space between the 100x and 4x is over the sample. Place a drop of immersion oil directly on the sample. Remember - do not lower the stage - rotate the 100x oil immersion lens into position. Using only the fine focus knob, adjust the image until it is sharp.
๐ Never use the coarse focus knob when the 100x lens is in place. The lens and/or slide may crack.
๐ Although it may appear that the 100x objective is too close and will hit the slide, as long as the sample is in focus at 40x the 100x lens will be at the right height to move into place without hitting the slide. The oil must touch the 100x objective in order for the light to be directed into the lens properly - otherwise you will not be able to see the bacteria.
You must focus the sample/landmark under each objective as you work your way up to the 100x objective, however, only draw the sample at the magnifications listed in the table below.
|
Pen Mark |
X40 |
X100 |
X400 |
X1000 |
|
Amoeba |
X40 |
X100 |
X400 |
------- |
|
Saccharomyces |
*** |
*** |
X400 |
X1000 |
|
Mixed bacteria |
*** |
*** |
X400 |
X1000 |
*** view and focus but do not draw
--- do not view/focus or draw at that magnification
Drawings are to be done as you look through the microscope. You are welcome to take pictures if you want to, but drawings are to be completed in class while looking at the object through the microscope. All labels are to be placed directly under your drawing. Labels should include: genus, species (or name of the object) and the final magnification.
Remember, the genus can be abbreviated but not the species can not. Drawings should be done using colored pencils. Do not use ink pens as they will bleed through to the back of the page.
The following is to be completed during lab then turned in on Canvas as a PDF
*If you are using an iPad or tablet you will need to take screen shots of your competed work, save the screenshots as one PDF then submit them on Canvas by the due date designated on Canvas.
*You can also print out the entire exercise to bring to lab with you. If you choose to complete the lab on paper, take pictures of the completed results and conclusions sections only, save them as one PDF, then submit to Canvas by the due date designated on Canvas.
MICROSCOPY
NAME ______________________
EXPECTATIONS
If you prepare a bacterial smear without staining it, what do you anticipate the bacteria will look like at 100x?
RESULTS
CONCLUSIONS:
1. When switching from 40x to 10x, would you expect to see more of the specimen or less of the specimen in your field of view? Why?
2. Which magnification provided the clearest view of the amoeba? Why is it difficult to focus clearly on thicker specimens when using high-power magnification? Explain your answer.
3. Which objectives can the course focus (the large dial) be used with? ______________________
4. Which objectives can the course focus (large dial) NOT be used with? __________________
5. What objective(s) require the addition of immersion oil and what is the purpose of the oil?
6. What will happen if immersion oil is used with the wrong objective(s)?