2: Microscopy Experiment
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- 123862
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)We use a microscope to visualize objects that are too small to see with the naked eye. This is especially critical in Biology as all cells are quite small and require a microscope for visualization.
All living organisms are composed of cells. Most organisms are so small that they are one cell while some eukaryotic organism are much larger and are composed of somewhere between 2 and millions of cells. In this experiment you will learn to use a compound microscope and will measure and characterize single and multicellular eukaryotic samples as well as bacterial cells.
At the completion of this lab, students will be able to:
Content:
- Define magnification, resolving power, contrast, and field of view
- Recognize and give the function of the parts of a compound microscope
- Define cell, prokaryotic, eukaryotic, nucleus, and organelle
- Describe the similarities and difference between prokaryotic and eukaryotic cells
- Identify cell parts and describe their function
- Distinguish between plant and animal cells and know that they are both eukaryotic cells
Physical Skills:
- Properly care for a compound microscope
- Correctly use a compound microscope
- Make a wet mount
- Perform a simple stain
Scientific Communication Skills:
- Correctly label a microscopy image
- Format a figure legend
Introduction to Microscopy
The science of microbiology originated with the invention of the microscope in the late 16th century and its use by Robert Hooke and Antoni van Leeuwenhoek in the 17th century to view and describe microscopic living organisms. Microscopes remain one of the most important tools for modern microbiologists.
The microscopes we will use in class are compound bright field light microscopes (Figure 1). A light source in the base of the microscope is focused by the condenser and then passes through an experimental sample placed in the stage. The image is then magnified by the objective and ocular lenses (Figure 2). Our microscopes contain 10x ocular lenses, meaning this lens magnifies an image 10‐fold. They are stereomicroscopes, so the two ocular lenses need not focus on the same focal plane. They also contain 4 objectives: a 4x (scanning), 10x (low power), 40x (high dry) and 100x (oil immersion).
Parts of the Compound Light Microscope
Use Figure 1 to identify the various parts of your microscope. Read each description below, find them on your microscope and on the lab handout and manipulate the parts only where indicated. Before you start, make sure that the shortest objective is in the light path.
Figure 1. Parts of a compound light microscope
- Light source. The compound microscope uses transmitted light to illuminate a transparent (or super thin) sample mounted on a glass slide. Find the light source/illuminator, the off/on switch, and the rheostat which can vary the light intensity so that it is not too bright.
- Condenser. A condenser is composed of a condenser lens and iris diaphragm to focus the light on the specimen so that it is equally and fully illuminated. The lever for the iris diaphragm of the condenser is used to open and close the condenser to increase or decrease the area illuminated and thereby light intensity.
- Stage. Your slide containing a sample is placed on a mechanical stage over a central hole. Position a slide on the stage by releasing the tension in the spring loaded moveable arm and using this slide clip to hold your slide in place. There are two knobs to the right or left of the stage: one to move the specimen forward or backward and one to move it side to side.
- Focusing knobs. The coarse focus knob is for use with the low power objectives ONLY whereas the fine focus knob is critical for focusing especially with higher power objectives.
- Objectives. Our microscopes have four different objectives 4 objectives: a 4x (scanning), 10x (low power), 40x (high dry) and 100x (oil immersion). Note that they are labeled and color coded for ease of use. The other number written on the objective is the numerical aperture (NA). The higher the numerical aperture, the greater the resolving power.
- Ocular. The magnifying lens that you look into is called the ocular. Our microscopes have two 10x oculars (and are therefore stereomicroscopes). Since each objective has a different magnifying power, the total magnification is calculated by multiplying the magnifying power of the ocular by that of the objective.
Importantly, the objectives are parfocal meaning that once one objective has been focus, you can rotate another one into place without losing the focus. You will likely need to very finely adjust the fine focus, but it specimen will be very close to being in focus. Also, the center of the field of view will stay the same as you move from one objective to the next.
These features will allow you to generally focus at the low power (4x) where focusing is MUCH easier and then move to a higher power and only have minimal adjustments.
Magnification and Resolution
Magnification is the amount an image in increased in size; resolution is the ability to distinguish two distinct objects. Increasing the magnification does not help us see greater detail if the resolution is not sufficient to differentiate objects we are viewing at this high magnification. To be useful, an image must be clear, not just magnified.
The total magnification of your microscope is the product of the magnification of the objective and ocular lenses. For example, if you are looking at a slide through the 4x lowest power objective, the total magnification is (4)(10)=40x. If you switch to the 100x highest power objective, the total magnification is now (100)(10)=1000x. Remember you can select objectives of different magnification but on these microscopes your ocular lens is always 10x.
The resolution of a microscope is more difficult to control. Resolution depends on a built-in characteristic of the lens itself called the numerical aperture, as well as the wavelength of light. The theoretical maximum resolution of a compound light microscope is about 0.2 µm. Two objects (e.g. two bacteria or two subcellular structures) can only be distinguished as separate objects using a light microscope if they are at least 0.2 µm apart, no matter how great the magnification.
One way to maximize the resolution is to use an oil‐immersion lens. Since immersion oil has the same refractive index as glass, placing a small drop of oil between the coverslip and the objective reduces the refraction that would normally happen as the light travels through air. With less light scattered, the resolution of the lens is optimized.
Figure 2. Light path through the microscope and magnification of a specimen.
The 100x objective and ONLY the 100x on your microscope is an oil‐immersion lens. The three lower powered objectives are dry lenses – they cannot be used with immersion oil. Getting oil on a dry lens will severely damage it. If this happens, please notify your instructor ASAP so that the issue can be addressed and the objective thoroughly cleaned before it is damaged.
How to use the Microscope
Types of Cells
All cells have four basic cellular components: cell membrane, DNA genome, cytoplasm, and ribosomes. Beyond that, however, cell sizes and structures can differ greatly between different organisms.
Based on ribosomal sequencing work conducted by Carl Woese et al. in the 1990s, all life on earth can be subdivided into three domains of life. Eukarya have nuclear membranes surrounding their DNA genome and can be multicellular, while Bacteria and Archeae are single-celled organisms and do not have a nucleus. Bacteria and Archaea can be differentiated based on their ribosomal DNA sequences and the components of their cell membranes.
Archaea (shown in Figure 3 in red) are microscopic single cellular organisms including halophiles (live in high salt environments) and hyperthermophiles (live at extremely high salt conditions). They have unusual membranes with ether linkages which can help them withstand these extreme conditions.
Bacteria (shown in Figure 3 in blue) are also microscopic single cellular organisms with a phospholipid membrane and are incredibly diverse organisms. We'll be focusing on these in our research project.
Eukarya (shown in Figure 3 in brown) includes all macroscopic organisms including plants, animals, fungi and protists. Eukaryotic organisms can be either single celled or multicellular. All eukaryotic cells have a nucleus and membrane-bound organelles for example mitochondria and chloroplasts.
Figure 3: The universal tree of life. LUCA, at the base of the tree, refers to the Last Universal Common Ancestor (LUCA) - the original cell from which all cells and organisms have evolved. From this original cell, three domains of organisms derived - Bacteria (single celled organisms), Archaea (single celled organisms that are often found in harsh environments) and Eukarya (single and multicellular organisms that include plants and animals). by Chiswick Chap, CC BY-SA 4.0, via Wikimedia Commons
Types of Eukaryotic Cells
Eukaryotic organisms have a great diversity in cellular composition where some are single celled and some are multicellular. We will look at protists, freshwater plants and algae, onion cells, and human cheek cells. All of the eukaryotic cells that you will observe are alive and you will need to make your slides (called a wet mount) for observation of each cell type.
Protists: You may see protists in the isolated protist samples or a mixed pond water sample. These are small single-celled organisms that can sometimes move quite rapidly and prefer to avoid the bright light of the microscope. To image these, put one drop of sample on the slide and add one drop of methylcellulose to slow their movement. Cover with a coverslip and observe. You will need to eventually observe these organisms with the 40x objective (but remember to start at the 4x). Your instructor will tell you which samples we have available.
Possibilities include: Paramecium (single celled ciliated organisms which move very rapidly and have a large central vacuole to maintain water balance), Euglena (freshwater, photosynthetic, single celled organisms that move with a long flagella), Amoebae (translucent blobby shaped organisms that move by extending finger-like pseudopods).
Plants: To observe the freshwater plant Elodea, make a wet mount from a small piece of a single thin leaf. These leaves are composed of only a few layers of cells, you can adjust your focus to see each of them as you scan through the depth of the leaf. For Elodea, notice how all of the cells are rectangular. This regular shape is a result of the rigid cellulose cell wall which provides structure for these cells. If you look carefully and are in a hypertonic environment, you can see the area of the plasma membrane immediately inside the cell wall were it sometimes pulls away a little. Additional structural features that you will notice include the chloroplasts (small and green organelles floating in the cytoplasm), the central vacuole (a section of the central portion of the cell without chloroplasts) and sometimes the nucleus (clear and egg-shaped near the periphery of the cell).
To observe the onion cell, make a wet mount of the onion epithelium. To do this, cut a small wedge-shaped piece of the onion. Notice how the onion curves, break the piece against this curve to pull off a thin layer of cells that lie along the inner surface of the curve. The layer is only a few cells thick and can look like plastic wrap. Use this thin epithelial layer for your wet mount. Try to lay this as flat as possible to prevent overlapping cells. Add one drop of stain to increase the contrast and cover with a coverslip. You will likely see a circular nucleus and possibly tiny mitochondria around the edges of the cell.
Animals: To make a wet mount of your cheek cells, gently scrape the lining of your cheek with a clean toothpick. Carefully wipe the end of this toothpick on a slide and add one drop of iodine or methylene blue dye. These dyes will increase the contrast and allow you to identify the nucleus, cytoplasm, and the cell membrane. You might be able to see additional organelles inside the cell too.
Viewing of Prokaryotic Cells (Bacteria and Archaea)
There are three domains of living organisms: Bacteria, Archaea, Eukarya. All three of these domains are composed of cells. Bacteria and Archaea are single cells and using the microscopy techniques available to us, Bacteria and Archaea are almost indistinguishable and together are categorized as prokaryotic.
Prokaryotes lack a nucleus (including nuclear membranes and histone proteins) and membrane bound structures that perform specialized functions called organelles. The genetic material in prokaryotes is found in one central area of the cell called a nucleoid. Although single celled and generally quite small, prokaryotic cells come in a variety of cellular morphologies (shapes).
The three most common shapes of bacteria are coccus, bacillus, and spirillum. Coccus, cocci for plural, refers to round, spherical bacteria that might be arranged singularly or in larger groups. Bacillus, bacilli for plural, indicates longer rod-shaped bacteria which sometimes appear as a long chain of linked rods. Spirrilum are much less common in nature and look like corkscrews. Look at these slides carefully as often the bacteria are clumped together. Each fixed slide has all three types of bacteria. We'll make and fix some slides ourself as well.
NOTE: you will not be able to see any subcellular structures as they are too small for the resolution of our microscopes. You will need an electron microscope to see details including ribosomes, the plasma membrane, and the nucleoid.
Figure 4: Examples of the most common bacterial cell morphologies. Bacillus are rod-shaped, cocci are shaped as spheres, and spirillum are corkscrew shaped. The average bacterial size is ~1 micron and are only visible under a microscope.
Protocols
Protocol 1: Using the Microscope
Initial set up
- Carry your microscope to and from your lab bench by grasping the arm with your dominant hand and supporting the base with your other hand. Make sure to always keep the microscope upright and do not try to carry anything else at the same time.
- Remove the dust cover and, if needed, clean the lenses with a piece of lens paper. Never use anything else to clean your microscope as it could scratch and damage your microscope.
- Unwind and plug in power cord
- Turn on the light source – switch on right side of microscope
- Dial or Slider on right side of microscope will adjust the light intensity, depending on the sample and magnification used.
- Adjust the ocular focus:
- With your left eye closed and a slide on the stage (see below), focus your right eye on something on the slide using the normal focus knobs.
- Then close your right eye and adjust the focus of the left eye using the focus ring on the ocular lens.
- Adjust the spacing of the oculars so that you can look with both eyes simultaneously.
Viewing a Sample
- Select lowest power (4x) objective by turning the black ring just above where the objectives attach to the body of the microscope.
- Important: Never rotate by grabbing or putting pressure on an objective lens itself.
- Set slide on stage and hold in place with clip.
- Use the coarse and fine focus knobs to focus on the sample at lower magnification before rotating the collar (not touching the objectives themselves!) to switch to a higher objective.
- Refocus at the desired magnification, using the fine focus knobs.
- If you want to use the 100x oil objective, check with your instructor before using. Turn the collar so that the slide is centered between the 100x oil lens and the adjacent objective lens (neither one is clicked into place).
- Carefully put one small drop of immersion oil on the cover slip.
- Slowly rotate the collar so that the 100x objective clicks in place.
- The immersion oil should form a connection between the slide and the lens.
- Important: Use care never to get oil on any part of the microscope other than the 100x oil lens. Especially do not get oil on the other dry objectives – this can severely damage them.
- Adjust the level of illumination with the slider on the side of the microscope.
Tips For Focusing
- Always start by getting in focus at the lowest power objective (4x). Then increase objective strength one at a time, focusing before moving to a higher objective.
- If you have trouble finding the correct focal plane in which to view your sample, try focusing first on the edge of the cover slip.
- Then, without moving the focus knob, scroll the stage over so that the specimen is in the field of view.
- It now should be close to in focus, requiring only minor adjustment with the fine focus knob.
Putting the Microscope Away
- Clean the immersion oil off the 100x oil objective if used.
- Tear off a piece of lens paper and gently swipe it across the lens.
- Never use a Kimwipe or anything but lens paper on a lens; it could scratch the lens.
- Rotate the paper to a clean portion or get a fresh sheet and gently swipe again.
- Repeat until there is no visible oil coming off the lens onto the paper.
- Never “scrub” on the lens by pressing with your finger or rubbing back and forth – any tiny pieces of dirt can scratch the lens.
- If desired, clean the other lenses in the same way, with clean lens paper.
- They should not have any oil on them, but sometimes they will get a little dusty or dirty.
- Wipe up any oil that accidentally got on other parts of the microscope.
- The stage is most likely to become contaminated by stray smudges of oil.
- If necessary, moisten the corner of a Kimwipe with a little ethanol to help wipe up the oil. Remember, Kimwipes are never to be used on the lenses themselves.
- Turn the objectives so that either no objective points down toward the stage or the 4x objective points down (this helps to prevent damage to the objectives)
- Adjust the stage scroll knobs so that it doesn’t stick out.
- Wrap the cord around the holders.
- Carefully store it in the appropriate place in the microscope cabinet.
Protocol 2: Using the Microscope
During the semester, we will perform multiple experiments with the microscope, so it is critical to feel comfortable using one. This first activity will familiarize you with how to use a microscope. Please refer to the microscope guide in your lab manual.
Using some of the fixed samples provided, practice viewing and focusing on your samples. Remember to start with low power and then step-by-step move to higher power. This will help you avoid colliding the objective into the slide and make focusing easier. As you practice, answer the following questions:
Materials
- microscope
- ruler (or letter e) slide
Protocol
• Using the ruler slide, measure the length of the field of view at each magnification and record in the lab handout. Which objective has the largest field of view? Which has the smallest?
• Using the ruler slide, compare the direction of the image. Is this image right side up or upside down with respect to the actual ruler? Is the image backwards or forwards? As you move the slide from right to left which way does the image move? As you move from front to back, which way does the image move?
• As you focus on the slides, which objectives can you adjust the focus the most? This objective has the greatest depth of field. The one with the narrowest band in focus has the least depth of field.
Protocol 3: Making a Wet Mount
Materials
- microscope
- slides
- coverslips
- protist samples
- ProtoSlo
- onion sample
- Elodea sample
- toothpick
- stains (iodine and methylene blue)
- blotting paper.
Protocol
- Gently place your sample on a clean slide, if needed, add a drop of water, dye to improve visibility, or Protoslo to slow rushing protists.
- Lower the coverslip slowly from one edge to avoid creating bubbles.
- If using dye, place the slide between a few pieces of blotting paper and press down gently (don't rub) to soak up extra dye.
- View the suspension under the microscope starting at low power.
- When finished, place the slide and coverslip in the bucket of soapy water to be washed.
Figure 3. Making a wet mount. Note how the hand is placing the coverslip by dropping it at an angle. This helps prevent some air bubbles. Witia, CC BY 3.0, via Wikimedia Commons
Protocol 4: Observing Bacterial Cells
Theory
Before bacterial cells can be stained, they must first be attached to a glass slide in a thin film called a smear. A smear can be prepared from a broth culture or from a single colony on a plate. Put a tiny drop of water ~2µl in the circle on your slide. You can do this with either a pipette or using an innoculation loop. Then transfer a small amount of bacteria from a single colony to that drop of water, mix thoroughly, and spread it around into a thin film on the slide.
NOTE: Take care not to transfer too much bacteria or the cells will be clumped together and difficult to distinguish (this is a common challenge for beginning microbiologists). Allow the bacterial smear to air dry completely.
Now that you have prepared a smear of bacterial cells on the slide, you need to fix the cells. Fixing has two purposes. First, it kills the cells quickly and in a way that minimizes shrinkage and distortion so that you can accurately determine the cell morphology. Additionally, fixing sticks the cells to the slide so that they will not wash off during the staining process. We will be using heat fixation. To heat‐ fix the bacteria on your slides, pass the slide through the flame of a Bunsen burner. Be sure to hold the slide carefully with a slide clamp or clothespin so that you don’t burn your fingers. Your heat‐fixed smear is now ready for staining.
Simple Staining
Staining procedures that use only one type of stain are called simple stains. They increase the contrast between the bacterial cells and the slide background. Many different dyes can be used and we will use methylene blue. Methylene blue is a basic dye and binds to negatively charged cellular components, including the cell wall.
To stain a heat‐fixed smear, hold the slide over a beaker. Using a dropper, put several drops of the dye directly on top of the fixed bacteria. Let the stain sit for about a minute, then tilt the slide over the beaker and rinse the stain off using the distilled water wash bottle. Rinse for 10 seconds or more, until the water running off the slide is clear.
Gently blot the slide dry. Don’t wipe the slide or the fixed bacteria might wipe off. The simply stained bacteria are now ready to look at under the microscope. To view your sample, place a coverslip over the stained sample. The purpose of the coverslip is to help prevent contamination of the microscope with any of the bacterial sample.
Figure 6. Step-by-step image guide for simple staining a bacterial sample.
Materials
- microscope
- slides
- coverslips
- dH2O
- bacterial strains
- sterile sticks, micropipette and/or inoculating loop
- clothespin
- methylene blue stain
- beaker
- Kimwipes or blotting paper
Protocol
- Label the slides with the identification information for your microbe.
- Make a dime sized smear with a plate culture of the your bacteria.
- Put 2µl of water (or your bacterial sample if it is in broth) on a slide. You can use either an inoculating loop or a pipette
- Spread the bacteria in this small volume
- Set the slide aside and allow the smear to dry completely before fixation.
- Heat‐fix the smears by passing the slide through the flame of a Bunsen burner several times
- Stain the slide with 1-2 drops of methylene blue; let sit for 1 min
- Rinse off the stain with dH2O
- Carefully blot slide dry between Kimwipes or using blotting paper
- Observe the bacterial morphology using the 40x dry objective (start at 4x and work your way up).
- Draw what you see in the field of view or take a photo. Be sure to label your drawing with the name of the sample, the total magnification, and the cellular morphology of that species
- Repeat the simple stain procedure for each bacteria.
Simple Stain Video
When finished:
- Clean off your microscope
- Lower the stage of the microscope to the bottom
- Turn the 4x (lowest power objective) to the down position
- Have your instructor sign off on your microscope - and then cover the microscope and return it to the correctly numbered space
- Return the ruler slides and any previously prepared slides the appropriate box
- Place the onion and Elodea slides in the tray of soapy water
- Place any cheek (and toothpick) and bacterial slides in the red biohazard sharps container.
- Using the 70% Ethanol or Sanizide sprayer on your bench, spray your bench space to disinfect it
- Wipe the bench down with a paper towel.
- Wash your hands prior to leaving.
References
Biology 105 Lab Manual
Perry et al., Lab Manual for Majors General Biology, 1st edition. Brooks Cole. 2008
Powell and Sullivan. MyMicrobe Project – A Guided Research Project Academex. 2015