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Secondary 2 · Life Sciences
Living Things · The cell

Observing Cells Under the Microscope

In Life Sciences class, your teacher hands you an onion and tells you that inside it there are invisible "little bricks." You can't see them with the naked eye: cells are too small. To see them you need an instrument that magnifies them hundreds of times. The optical microscope is the basic tool of the school lab, and using it well takes certain steps and tricks. By the end of this page you will know why a poorly prepared sample shows only smudges.

Parts of the microscope

To see the transparent skin of an onion you need to hold the glass, light it, and focus it. Which parts make all of that possible, and how do they work together?

Optical microscope seen from the side. Its optical parts are the eyepiece and the objectives. Its mechanical parts are the arm, the base, the stage with its clips, and the coarse and fine focus knobs. The light source illuminates the sample from below. eyepiece objectives clips stage light source arm coarse focus knob fine focus knob base optical parts mechanical parts

The two parts with lenses are the eyepiece (where you look, at the top) and the objectives (on the nosepiece, right above the sample). The sample is placed on the stage, held with the clips, and the light source illuminates it from below.

Because the image appears inverted (up and down, left and right), if you push the slide to the right, the image will appear to move to the left. If you are following a microorganism that is swimming, you will have to move the slide in the opposite direction from where you see it going.

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In the light microscope diagram, there are three parts labelled with "?". Write the name of each one and explain its function.

In the light microscope diagram, there are three parts labelled with "?". Write the name of each one and explain its function.

? body tube objective lens ? fine focus knob arm ? light source base
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Name the two parts of the microscope that contain lenses and explain where you look through and where the specimen is placed.

Name the two parts of the microscope that contain lenses and explain where you look through and where the specimen is placed.

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The microscope image is inverted. If you gently push the specimen to the right while looking, reason in which direction the image will seem to move in the eyepiece and…

The microscope image is inverted. If you gently push the specimen to the right while looking, reason in which direction the image will seem to move in the eyepiece and what practical consequence this has when you are following a moving microorganism.

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Total magnification

Optical microscope with the 10× eyepiece on top and the 40× objective over the sample; the image seen in the eyepiece is magnified 400×. 10× eyepiece 40× objective total magnification 400× 10× · 40× = 400×

When you switch to a stronger objective, three things happen: the image looks larger, the field of view (the area of the sample you can see) gets smaller, and less light gets through, so it looks darker. That is why, when going from $10\times$ to $40\times$, you should open the diaphragm or turn up the light.

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A microscope has an eyepiece lens of $10\times$ and objective lenses of $4\times$, $10\times$, and $40\times$. Calculate the total magnification achieved with each…

A microscope has an eyepiece lens of $10\times$ and objective lenses of $4\times$, $10\times$, and $40\times$. Calculate the total magnification achieved with each objective lens, and explain which one you should always start observing with and why.

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A microscope has an eyepiece lens of $10\times$ and an objective lens of $20\times$. Calculate the total magnification. If you switch to a $40\times$ objective lens,…

A microscope has an eyepiece lens of $10\times$ and an objective lens of $20\times$. Calculate the total magnification. If you switch to a $40\times$ objective lens, explain what happens to the size of the image, to the area of the specimen you manage to see, and to the amount of light.

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Real size and field of view

Total magnification tells you how many times the image is enlarged, not how big the cell is. An onion cell is not eight centimeters long just because you see it at $400\times$: that size is the apparent size. The real size is much smaller.

To estimate the real size you use the field of view, the circle you see through the eyepiece. If you know its diameter, you compare how many cells fit across it:

$$\text{real size} \approx \frac{\text{field diameter}}{\text{number of cells in a row}}$$
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With the $10\times$ objective lens (total magnification $100\times$), the field of view is about $1.5\ \text{mm}$ in diameter. If an onion cell is about…

With the $10\times$ objective lens (total magnification $100\times$), the field of view is about $1.5\ \text{mm}$ in diameter. If an onion cell is about $0.25\ \text{mm}$ long, estimate how many cells in a row would fit across the field of view, and explain what will happen to the diameter of the field of view when switching to the $40\times$ objective lens.

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A classmate observes a slide under a microscope at $400\times$. The field of view with that objective lens measures $0.4\ \text{mm}$ in diameter, and the cell they are…

A classmate observes a slide under a microscope at $400\times$. The field of view with that objective lens measures $0.4\ \text{mm}$ in diameter, and the cell they are observing occupies approximately half of that diameter. Your classmate claims: "Since the microscope magnifies $400$ times and the cell occupies $0.2\ \text{mm}$, its actual size must be $0.2 \times 400 = 80\ \text{mm}$, it's almost eight centimeters long!".\na) Identify the conceptual error in their reasoning and explain the difference between actual size and magnified apparent size.\nb) Calculate the correct actual size of the cell in millimeters and micrometers ($1\ \text{mm} = 1\,000\ \mu\text{m}$).\nc) If you placed identical cells one after another in a row, how many would fit along one centimeter of a ruler?

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Preparing a sample

Two pieces of glass are key: the slide is the rectangular plate on which the sample is placed, and the coverslip is the thin little piece of glass that covers it, flattens it, and protects it.

Four steps to prepare an onion sample: place a thin layer from the inner surface of the scale on the slide, add a drop of iodine solution, lower the coverslip at a 45-degree angle, and remove the excess liquid with filter paper. 1 Place the sample onion scale thin sample slide 2 Stain with iodine drop of iodine 3 Lower the coverslip coverslip 45° slowly 4 Remove the excess filter paper excess liquid

In the school lab, methylene blue is also used, for example to stain cells from the lining of the mouth. A homemade wet preparation (a hair or a fiber from your sweater in a drop of water, with its coverslip) works for practicing, but it dries out and gets thrown away. A permanent sample comes fixed, sliced very thin, stained, and sealed, and it lasts for years.

Go back to the beginning: the onion shows its "little bricks" only if the layer is thin, stained, and well covered; a thick sample or one with bubbles gives only smudges.

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Put the steps for preparing an onion cell sample in order: (a) cover with the coverslip taking care to avoid air bubbles, (b) place a thin layer of onion on the…

Put the steps for preparing an onion cell sample in order: (a) cover with the coverslip taking care to avoid air bubbles, (b) place a thin layer of onion on the microscope slide, (c) add a drop of iodine solution (lugol) to stain, (d) remove excess liquid with filter paper.

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Why are many samples stained before observing them under a microscope? Give an example of a stain used in the school laboratory.

Why are many samples stained before observing them under a microscope? Give an example of a stain used in the school laboratory.

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Explain what the microscope slide and the coverslip are and what each one is used for when preparing a specimen.

Explain what the microscope slide and the coverslip are and what each one is used for when preparing a specimen.

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To observe something under the light microscope it must be cut into very thin sections. Explain why the specimen must let light through and what happens if it is too…

To observe something under the light microscope it must be cut into very thin sections. Explain why the specimen must let light through and what happens if it is too thick.

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Describe how you would prepare a wet mount of a hair or of a fiber from your jumper to view it under the microscope: what materials you would use and in what order you…

Describe how you would prepare a wet mount of a hair or of a fiber from your jumper to view it under the microscope: what materials you would use and in what order you would act. What is the difference between that homemade preparation and a permanent laboratory slide?

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During a practical activity observing cheek cell specimens stained with methylene blue, a group can only see shapeless blue smudges. Suggest at least three errors they…

During a practical activity observing cheek cell specimens stained with methylene blue, a group can only see shapeless blue smudges. Suggest at least three errors they might have made when preparing the sample and explain how you would repeat the experiment to get good results.

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Focusing correctly

Focus knobs of the microscope: the coarse knob moves the lowest-magnification objective a large distance and the fine knob moves the 40× objective only a tiny amount, as it sits almost touching the slide. Coarse focus knob Fine focus knob big turn coarse slide 4× big turn fine slide 40× big turn → big movement rough focusing with the lowest-magnification objective big turn → tiny movement sharpen the focus with the higher-magnification objectives

If the image looks very dark, the causes may be a weak light source or a closed diaphragm; you fix it by turning up the light or opening the diaphragm. If you see only a fragment of the sample, it may be poorly centered or you may be using a high-magnification objective; you fix it by centering it and going back to a lower objective.

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When observing a sample, the image looks very dark and only a fragment of the specimen can be seen. State two possible causes for each problem and what you would do to…

When observing a sample, the image looks very dark and only a fragment of the specimen can be seen. State two possible causes for each problem and what you would do to correct them without changing the objective lens.

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Why must you focus first using the lowest power objective lens and reserve the fine focus knob for higher power objective lenses? Explain what might happen if you use…

Why must you focus first using the lowest power objective lens and reserve the fine focus knob for higher power objective lenses? Explain what might happen if you use the coarse focus knob with the $40\times$ objective lens.

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You have focused a cell with the $10\times$ objective lens and switch to the $40\times$ one: the image is left slightly blurry and darker. Explain which focus knob you…

You have focused a cell with the $10\times$ objective lens and switch to the $40\times$ one: the image is left slightly blurry and darker. Explain which focus knob you must use to refocus (and which one you must not touch) and which lighting adjustment can improve the image.

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Care and safety in the lab

The microscope is delicate and the sample can be a hazard. These rules protect both the equipment and you.

When carrying it: take it out of the cabinet with both hands, one holding the arm and the other supporting the base. Never grab it by the eyepiece, the tube, or the stage.

When you finish the lab activity, in this order:

  1. Turn the nosepiece to the lowest-magnification objective and lower the stage.

  2. Turn off the light.

  3. Remove the slide and dispose of it where your teacher indicates.

  4. Clean the lenses only with special lens paper.

  5. Put the microscope away with its cover.

  6. Wash your hands.

Rules for samples and stains: wear a lab coat and gloves, because stains (Lugol's iodine, methylene blue) leave marks and can irritate the skin and eyes. Do not taste or smell the samples or the stains, because they can be toxic or contain microorganisms. Dispose of broken glass and samples in the designated container, to avoid cuts and contamination.

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Describe how a microscope should be carried from the cupboard to the work table without damaging it: which parts you hold and with how many hands.

Describe how a microscope should be carried from the cupboard to the work table without damaging it: which parts you hold and with how many hands.

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What must be done when finishing a microscopy practical? Put these tasks in order and explain them: switching off the light, cleaning the lenses with the special paper,…

What must be done when finishing a microscopy practical? Put these tasks in order and explain them: switching off the light, cleaning the lenses with the special paper, removing and disposing of the specimen, putting the microscope away, and washing your hands.

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Name three safety rules for working with biological samples and stains in the school laboratory and explain the reason for each one.

Name three safety rules for working with biological samples and stains in the school laboratory and explain the reason for each one.

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Beyond the optical microscope

The human eye can tell two points apart if they are separated by about $0.1\ \text{mm}$ ($100\ \mu\text{m}$). The optical microscope reaches about $0.2\ \mu\text{m}$, which is enough to see cells and bacteria. For anything smaller, such as a virus, you need the electron microscope (down to $0.0002\ \mu\text{m}$), which uses electrons instead of light.

Instrument Smallest visible detail What you can see well
Human eye $100\ \mu\text{m}$ A grain of sand
Optical microscope $0.2\ \mu\text{m}$ Cells, bacteria
Electron microscope $0.0002\ \mu\text{m}$ Viruses, membranes

The electron microscope works in a vacuum and with heavily treated samples, so it shows only dead cells. The optical microscope, on the other hand, lets you see living organisms, such as a protozoan swimming in a drop of water.

To compare the size of two types of cells (for example, those from the growth zone of an onion root and those from the bulb), prepare a sample of each, estimate the size using the field of view, record the data in a table (sample, cells in a row, estimated size), and measure several cells, for example 10 of each, and take the average so the result is reliable.

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A research team wants to film in real time how a unicellular protozoan swims and captures bacteria in a drop of stagnant water, and at the same time, photograph in full…

A research team wants to film in real time how a unicellular protozoan swims and captures bacteria in a drop of stagnant water, and at the same time, photograph in full detail the internal structure of the viruses that infect those bacteria. A student proposes using only a transmission electron microscope to achieve both goals. Evaluate their proposal with reasoned arguments: explain why it is not possible to observe the live protozoan with this device and determine which microscope should be used in each case and why.

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Analyze the following data on the limit of resolution (the minimum distance to distinguish two separate points) of three instruments:\n• Human eye: $0.1\ \text{mm}$…

Analyze the following data on the limit of resolution (the minimum distance to distinguish two separate points) of three instruments:\n• Human eye: $0.1\ \text{mm}$ ($100\ \mu\text{m}$).\n• Compound light microscope: $0.2\ \mu\text{m}$.\n• Electron microscope: $0.0002\ \mu\text{m}$.\n\nYou have four biological samples:\n1. An onion plant cell ($100\ \mu\text{m}$).\n2. A bacterium ($2\ \mu\text{m}$).\n3. A flu virus ($0.1\ \mu\text{m}$).\n4. The plasma membrane of a cell ($0.008\ \mu\text{m}$).\n\na) Deduce the minimum instrument you need to distinguish each of the four samples.\nb) Justify, using these data, why cells and bacteria could already be observed in the 17th and 18th centuries, but the existence and structure of viruses could not be demonstrated until the invention of the electron microscope in the 20th century.

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You want to check whether the cells in the growth zone of an onion root are smaller than those in the bulb. Design the practical activity: which two specimens you will…

You want to check whether the cells in the growth zone of an onion root are smaller than those in the bulb. Design the practical activity: which two specimens you will prepare, how you will estimate the size of the cells using the field of view, which data table you will use, and how many cells you will measure so that the comparison is reliable.

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The inverted image and the cell cycle

The microscope's lenses invert the image: a letter "e" looks rotated $180^\circ$ (upside down and flipped). That is why a "p" will look like a "d". You can check this by cutting out a letter from a newspaper and looking at it under the microscope. Then compare the result with your prediction.

The microscope also shows that every cell comes from another cell: the cell cycle is the sequence of stages in which a cell grows, duplicates its genetic material, and divides in two. Understanding it is key to growth, tissue repair, and diseases such as cancer; you will study it in depth later on.

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When observing the letter "e" cut out from a newspaper, the microscope shows it rotated 180° (upside down and back to front). Predict how the letter "p" will look before…

When observing the letter "e" cut out from a newspaper, the microscope shows it rotated 180° (upside down and back to front). Predict how the letter "p" will look before viewing it, explain why this inversion happens, and describe how you would test your prediction.

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An online store advertises a "School optical microscope with magnifications of up to $50\,000\times$ that allows you to observe viruses and molecules for only 30…

An online store advertises a "School optical microscope with magnifications of up to $50\,000\times$ that allows you to observe viruses and molecules for only 30 pesos".\na) Using the analogy of enlarging a low-resolution digital photograph (where you only get larger pixels, but no new details), refute why this advertisement is deceptive and predict what you would actually see when trying to force such high magnification using visible light.\nb) Explain what fundamental physical change the electron microscope introduces compared to the optical microscope to overcome this limit and sharply display structures measuring just a few nanometers.

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Summary

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