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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Never change slides while the oil immersion lens (100x) is in position.
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Apply immersion oil only to the 100x lens. Do not use oil with any other objective.
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Follow all steps to achieve good focus and prevent damage to the lenses.
👉Before returning the microscope to its cabinet:
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Clean all lenses and the stage using 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 so that the clips are not protruding.
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Coil the power cord neatly and secure it with a fastener.
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.
👉Cleaning 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.
It is helpful to practice moving the slide at low magnification to become familiar with scanning the entire sample area. This ensures that your observations are based on representative sections of the slide rather than areas that may be contaminated, overly dense, or poorly stained.
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
Above the stage is a rotating nosepiece that holds four objective lenses: 4x, 10x, 40x, and 100x. These provide the initial level of magnification. The image is then further magnified 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
Each objective lens must be at a specific distance from the slide 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
👉Technique tip: Place a small pen mark on the same side of the slide as your sample. Focus on this mark at 4x, then move through the objectives while maintaining focus on the mark. Once the 100x lens is in focus on the pen mark, you can move the slide to locate your sample—this ensures the correct focus plane.
The 100x objective lens requires immersion oil (a clear substance with the same refractive index as glass) to reduce light scatter and increase image clarity. Use this oil only with the 100x lens and clean it thoroughly with lens paper after use. Never allow oil to contact the other lenses.
MATERIALS (Per Group of 4)
-
Assigned microscope – each student uses their assigned scope slide with ink pen mark
- 1 Saccharomyces cerevisiae slide
- 1 Amoeba spp slide
- 1 Mixed bacteria slide
- 1 Black Sharpie medium point pen
METHODS/PROCEDURES
STEPS TO FOCUS
- Ensure all lenses are clean and free from dust, smudges, or oil before beginning.
- Rotate the lowest power objective (4x) into position directly above the slide.
- Begin by focusing on a pen mark drawn on the top surface of the slide. This landmark helps you identify the correct focal plane. If you experience difficulty focusing at higher magnifications, return to the 4x objective and refocus on the pen mark.
- Without looking through the oculars, observe the microscope from the side and use the coarse focus knob to raise the stage, bringing the 4x objective lens as close as possible to the slide—without touching it.
- Confirm the condenser is fully raised and that the iris diaphragm attached to it is completely open.
- Adjust the light intensity by opening the light source diaphragm and setting the rheostat (brightness dial) to high.
- 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.
- Increase magnification by rotating to the 10x objective then use the fine focus to sharpen the image. Once in focus rotate to the 40x objective and again use only the fine focus knob. You may see large bacteria or clumps at this level. However, to clearly observe individual bacterial cells, you must move to the highest power lens—the oil immersion objective.
Using the Oil immersion Objective: - Once the pen mark is sharply in focus at 40x, rotate the 40x objective away, place a small drop of immersion oil directly on the slide over your area of interest, and carefully rotate the 100x oil immersion lens into position. Using only the fine focus knob, adjust the image until it is sharp.
👉 Never use the coarse focus knob when the 100x lens is in place. The lens may crack.
👉 Although it may appear that the 100x objective will touch the slide, as long as the object is in focus under the 40x the 100x lens will slide into place
without hitting the slide. The 100x objective is designed to sit very close.
👉 You should never need to rotate the fine focus knob more than one full turn in either direction while using the oil immersion lens. If you cannot find the
object you are looking for under the 100x magnification it is typically faster and easier to start and try again. BE SURE TO REMOVE All OIL FROM THE
SLIDE AND LENSE BEFORE STARTING OVER. The lower objectives are not sealed. If oil touches any objective other than the 100x the objective will no
longer be able to give a clear image.
Lower magnification requires lower light.
Always use all the steps outlined above for each slide.
Focus each objective as you work your way up to the 100x objective, however, only draw the images you see 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. Label all your drawings with the genus, species (or name of the object) and the final magnification underneath the circle representing the field of view. Remember, You can abbreviate the genus but not the species. 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 compared to a
RESULTS
CONCLUSIONS:
1. When switching from 4x to 40x, do 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)?