BIOL 1020 Fall 2022
Lab 4 – Microscope & Lab Skills
Lab 4: Microscopy & Lab Skills
This laboratory session and associated activities are designed to help you develop skills involved
in working in a biological laboratory. Microscopy is an essential skill for any biology student.
Key Learning Objectives:
1.
2.
3.
4.
5.
6.
Identify the main parts of the compound microscope
Compare the uses of compound and stereo microscopes.
Draw specimens viewed with a compound microscope including a scale bar.
Calculate the magnification of the illustrations of microscopic specimens.
Apply statistical testing to cellular cultures
Calculate t-test statistic for hypothesis testing
For over 400 years, biologists have used microscopes as basic tools of discovery. Exploring the
microscopic world has led and continues to lead to ground-breaking findings in many areas of the
biological sciences. In this lab, you will be introduced to quality teaching-grade microscopes. These
instruments are expensive and must be treated with respect. Effective use of a microscope, like
any other skill, requires practice. In the following exercises, you will go through some of the basics.
You will not become an expert before you leave, but you will have taken an important first step.
Types of microscopes
In the introduction to this lab exercise, your TA will demonstrate the two most common types of
microscopes—the stereo or dissecting and compound microscopes. Although both magnify
specimens, these two instruments have very different uses.
Stereo or dissecting microscopes (Figure 4.1) typically have low power with a magnification range
of 4x to 40x. There main purpose is to manipulate larger specimens (i.e., dissections) in three
dimensions. Unfortunately, we don’t have enough for every student to use their own. Your TA will
demonstrate how this microscope works.
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Figure 4.1. The basic stereo or dissecting microscope highlights its major controls. The
microscope that you will most commonly be using in your biology courses will be the compound
microscope (Figure 4.2). While not in this exercise, these microscopes have enough magnification
to view bacteria (1000x). These microscopes shine light through a specimen on a slide which is
then captures by a series (hence compound) of magnifying lenses.
Figure 4.2. The major features of the compound microscope.
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General tips on using the binocular compound microscope
•
•
Unless you have astigmatism, you don’t need to wear glasses while using the scope.
o The microscope can correct for most prescriptions.
o You still can wear your glasses, but it makes viewing a little harder.
Use both eyes!
o Note that one or both ocular lenses are adjustable.
o To get the best image, first focus with one eye closed (the eye with the adjustable
ocular). Then switch which eye is closed and adjust the ocular lens until the image
is crisp. After that, using both eyes will help reduce headaches that can occur.
The specimens one views with a compound microscope can be prepared slides or fresh mounts.
Prepared slides are of processed or ‘fixed’ material for long-term preservation. They can be made
of whole organisms or thin (~5 µm) sections of organs or tissues. Fresh mounts can be made using
whole (and living) organisms or sections of organs and tissues without preservation. In the following
exercises, you will make two fresh mounts and view several prepared slides.
Microscope Exercise #1
This short exercise is intended to familiarize you with the basic function of the microscope on stage
movement focusing. Initially, navigating around your slides is challenging. With practice, however,
it will become more intuitive.
1. Take one of the ‘F’ slides in the small blue slides boxes
2. Place the slide on the microscope stage so that the label is still readable from your
perspective.
3. Focus on the ‘F’ with the lowest objective and draw what you see in the circle below.
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Adjusting light levels
If the light is too bright, it’s brightness can be controlled. You can also the diaphragm and
condenser under the stage. The diaphragm adjusts the contrast ratio, and the condenser adjusts
the lights focus.
Using the stage control knobs, move the stage up (i.e., away from you). Which way does
the image move? #ANALYZE
Move the stage the right. Which way does the image move? #ANALYZE
Microscope Exercise #2
Now that you are familiar with the microscope, you will use the prepared mixed protist cultures to
gain experience preparing your own wet mount slide. To prepare a fresh mount of a culture in the
lab you will first place a small droplet of the culture medium on the centre of a clean glass slide
then follow the steps outlined in Figure 4.3.
Figure 4.3. The proper technique for preparing a fresh mount of a biological culture.
Once you have your slide prepared, you will place it on the stage of your microscope with the
lowest power objective (4x), and then you can start the process of getting the culture in focus
using your coarse and fine focus. Once the image is in focus, you can find a specimen and get
practice your stage control knob to “move” around the sample and follow microscopic
specimens! Once comfortable, switch to the 10x objective and re-focus your microscope.
REMEMBER to use ONLY the fine focus when not on the lowest power objective.
After some time getting familiar with looking at wet mounts under your microscope you switch to
the low power objective (4x) to remove the slide off the stage and place into the glass bowl
labelled “used slides”. This is a discard bowl for slides that you prepare yourself.
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Microscope Exercise #3
Continuing with wet mounts, your TA will now introduce the Anabaena cultures. Each culture has
been grown in two different environments. Anabaena are a genus of filamentous cyanobacteria
that can fix nitrogen. The nitrogen-fixation occurs in specialize cells with thickened cell walls
called heterocysts.
Figure 4.4. Microscope image of Anabaena cultures with heterocysts marked with the triangle
pointers. Image from: Zhang et al. 2006. doi:10.1111/j.1365-2958.2005.04979.x
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Your TA will assign your group to one of the cultures, you will then need to each prepare your
own slide with a single drop of the culture and begin viewing on the lowest objective. Once you
have a filament in focus, you can switch to higher objectives. You will then count one filament,
preferably the longest one you can find by scanning around the sample.
You will count all the cells that are between the heterocysts. For example, in Figure 4.4. there are
14 cells between the two heterocysts circled in black in panel B. If this was your samples your
recorded observation would be 14 cells. If you do not see heterocysts in your sample and instead
just long filaments of cells, you will count the entire filament of cells. Anabaena filaments break at
the heterocyst location, which would be why you may not see any.
Statistical Tests of the Two Cultures
One was grown in a controlled environment without excess nitrogen and the other was gown in a
controlled environment that contained nitrogen in the form of ammonia. Recall, inside the
heterocysts is where nitrogen fixation occurs. Therefore, heterocysts are important for Anabaena
that have limited nitrogen availability in their environment.
1. Do you think the two cultures will be different in terms of number of cells between
heterocysts?
Data Collection
Record the number of cells between the heterocysts of your sample here:
____________
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Table 4.1. Tallied numbers of cells between heterocysts in an Anabaena culture at your lab
bench. Your group will fill this in when your TA instructs you, to.
Number of Cells (#)
Sample 1
Sample 2
Sample 3
Sample 4
Group Mean (Average)
*If only 2 – 3 group members are present you can ignore sample 3, and sample 4. Each row
should be a single student’s observation. You will then average the data and complete the yellow
row.
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Table 4.2. Tallied numbers of cells between heterocysts in an Anabaena culture.
Number of cells between heterocysts
Red Culture
Blue Culture
Row to help with
Statistic Calculation
Group A
Group B
Group C
Group D
Group E
Sample Mean
1
Sample
Standard
Deviation
2
Sample Size
3
To fill in Table 4.2.
Step 1:
Calculate the mean of the red culture (Row A)
Step 2:
Calculate the mean of the blue culture (Row A)
Step 3:
Calculate the standard deviation of the red culture (Row B)
*TA will show you how to use excel for this
Step 4:
Calculate the standard deviation of the blue culture (Row B)
Step 5:
Count number of observations for each culture, this is your sample size (Row C)
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Recall from lab 1 that we will conduct statistical tests to determine if we support or reject a null
hypothesis.
1. What would the statistical null hypothesis (Ho) in this experiment be?
2. What is the statistical alternative hypothesis (HA)?
There are a variety of different statistical tests, and it is important to choose the correct one for
your experiment. In our experiment we are broadly asking if the two cultures are different, so in
other words, we are going to want to use a statistical test that will test the differences. In our case
that would be a t-test, which will compare the means of the two cultures so we can determine if
they are significantly different from one another, since we have two different cultures and we don’t
have previous knowledge of what the average number of cells should or would be we will be using
a two-tailed t-test. A two-tailed t-test will allow us to find significance in either direction of the
extreme, for our case this means we would consider a culture with more OR less cells from the
other culture, significant.
To complete our t-test we will use the following formula:
**You will not need to memorize this formula** We will show you how to calculate the values
inside the coloured boxes in the lab*
The components of this formula are defined on the next page.
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X1 = the mean of the red culture
X2 = the mean of the blue culture
S2= the standard deviation
Red box = the standard deviation formula of the red culture
Lab 4 – Microscope & Lab Skills
These values are in Row 1
of Table 4.2
These values are in Row 2
of Table 4.2
Blue box = the standard deviation formula of the blue culture
n1 = the sample size of the red culture
n2 = the sample size of the blue culture
These values are in Row 3
of Table 4.2
To Complete the t-test
Step 1:
Calculate s2 using the values you have recorded in Table 4.2 as the numerical value to represent
the red and blue boxed area (standard deviation)
Step 2:
Plug in the values for s2 (calculated in step 1) and the values from Table 4.2 into the t-test equation.
Record your calculated t-statistic here: ___________________
*Your TA will show you how to do calculations like this in Excel. You will not need to do it excel
yourself for this lab. When you do calculations like this, we will give you the steps so you can
complete the calculations in excel in future labs. For this lab, you can watch the TA demo*
Step 3:
Finding df (degrees of freedom)
df = Number of observations (n) – 1
Our degrees of freedom (df) = (n1 – 1) + (n2 – 1) *because we have two different sets of samples*
Step 4:
Determine significance. Recall in lab 1, we discussed how in biology we often use  =0.05. We will
be doing so again with this test.
Now, we must compare our t-statistic to a t-table that contains a variety of t-scores at different
levels of significance, we will look at the column of 0.05.
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Last step, compare our t-statistic value to that found in table 4.4.
If our t score is LESS than the value in table, we do not reject the null hypothesis
If our t score is GREATER than the value in the table, we reject the null hypothesis, and accept the
alternative.
Table 4.4. Two tailed T Distribution Table
Significance levels ()
df
0.2
0.10
0.05
0.02
0.01
0.002
0.001
1
3.078
6.314
12.706
31.821
63.656
318.289
636.578
2
1.886
2.920
4.303
6.965
9.925
22.328
31.600
3
1.638
2.353
3.182
4.541
5.841
10.214
12.924
4
1.533
2.132
2.776
3.747
4.604
7.173
8.610
5
1.476
2.015
2.571
3.365
4.032
5.894
6.869
6
1.440
1.943
2.447
3.143
3.707
5.208
5.959
7
1.415
1.895
2.365
2.998
3.499
4.785
5.408
8
1.397
1.860
2.306
2.896
3.355
4.501
5.041
9
1.383
1.833
2.262
2.821
3.250
4.297
4.781
10
1.372
1.812
2.228
2.764
3.169
4.144
4.587
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What can you conclude about the two cultures?
Do the results support the null hypothesis? Why or why not?
Can you predict which culture was in the nitrogen free environment? Explain your reasoning.
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Microscope Exercise #4
In this short exercise, you will gain an appreciation for the magnification of your microscope and
how it changes as you switch objective lenses. The total magnification of the image is the product
of the two magnifying lenses—the ocular lens and the objective lens. That is to say:
𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑡𝑜𝑡𝑎𝑙 = 𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑜𝑐𝑢𝑙𝑎𝑟 × 𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒
The ocular lenses of microscopes you will use have a magnification of 10x and your set of objectives
lenses will include 4x, 10x, and 40x magnifications. For this exercise, you will focus on producing
a clear, crisp image with each of the objective lenses (pun absolutely intended).
When you want to look at any specimen under the compound scope, you should always start with
the 4x scanning objective. Finding your specimen in the field of view is much easier at this low
power.
1. Get the stage micrometer scale (a tiny ruler) slide from your slide box and place it on the
microscope stage.
2. With the 4x scanning objective, find and focus on the ruler.
a. Calculate the total magnification and record it in Table 4.5.
3. Measure and record the diameter of your field of view using the 4x objective.
a. Move the ruler so that one tick is at the very edge of your field of view.
b. The distance between two small ticks on the stage micrometer scale equals 0.1 mm.
4. Without adjusting anything else, rotate the revolving nosepiece to move the 10x low power
objective into place.
a. Always watch from the side when changing objectives to make sure there is room.
b. Calculate the total magnification and record it in Table 4.5.
5. Look through the eyepiece. Is the image crisp?
a. You will likely have to adjust the fine focus a bit. You should NEVER use the coarse
focus when using the 10x and 40x objectives!
6. Measure and record the diameter of your field of view using the 10x objective.
7. While looking from the side, switch to the 40x high power objective.
a. Calculate the total magnification and record it in Table 4.5
8. Adjust the fine focus ONLY.
9. Measure and record the diameter of your field of view using the 10x objective.
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Table 4.5. Magnification properties of your compound microscope.
Objective
Power
Scanning
4x
Low
10x
High
40x
Magnification total
Field of view diameter
(mm)
Field of view
diameter (µm)
Part B – Drawing from your Microscope
Since the beginning, illustrations have been essential for recording microscopic discoveries. Even
with modern, high-resolution microphotography, drawing what you see is an easy way to capture
the ‘moment’—especially since your microscope it not fitted with a trinocular camera mount. For
this exercise and your assignment for the lab, you will be illustrating what you see through the
microscope.
To be most meaningful, there are a few requirements for illustrations. First, they must only include
what you can see through the scope. You should never include an organelle or structure because
you know it should be there. Doing so would be an inaccurate representation defeating the purpose
of the illustration. Second, using the diameters you determined above, you need to determine and
include the actual size of the specimen on your illustration. And third, to show how big your drawing
is relative to the actual cell, you must include a scale bar and the magnification of the illustration.
Scale bars give a quick visual reference to the approximate size of the illustration (Figure 4.5). A
good scale bar is a line that represents a unit of length. Your scale bar should be easily workable
(i.e., 1 mm, 1 µm, or even 10 µm). That is to say, it should be a length that can easily be multiplied
to measure larger features like cell width or divided into smaller pieces to measure components
like an organelle. Typically, scale bars do not span the entire length of your drawing or be of an
irregular length. Just think, the counting the number of ‘bars’ long the specimen should be an easy
task. You want the multiples to be easy to count off in your head.
Figure 4.5. An illustration diagramming
an appropriate and inappropriate scale
bar.
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Using Figure 4.5, a viewer could easily ascertain that the cell is 7 µm long by counting how many
bars it would take to go the length, similar to the technique you used to measure your cells in the
field of view. Moreover, the viewer could also easily determine that the nucleus is about 2 µm by 1
µm.
In the context of illustrations, we use the term magnification in a different way than when referring
to the power of the microscope. Here, you are indicating the size of your illustration relative to the
actual size of the specimen using the following formula:
𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑙𝑙𝑢𝑠𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =
𝑆𝑖𝑧𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑙𝑙𝑢𝑠𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑠𝑖𝑧𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑝𝑒𝑐𝑖𝑚𝑒𝑛
When using this formula, it is essential that both sizes are in the same units. You cannot divide cm
by mm or µm. For example, if you measure your specimen to be 5 µm and your illustration is 10
cm, the magnification of your illustration is NOT 10 cm/5 µm = 2x. You must first convert the 10
cm to µm. Appendix A can help you with this.
Lab 4 Assignment information
The Lab 4 assignment outline will be available during your lab period. You will be drawing a
specimen as seen as from the microscope, all the information you will need will be presented to
you in your lab.
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Appendix A – Metric Conversions
Getting Smaller
Getting Bigger
1 metre (m) = 100 centimetres (cm)
1 nm = 0.001 µm
1 cm = 10 millimetres (mm)
1 µm = 0.001 mm
1 mm = 1000 micrometres (µm)
1 mm = 0.1 cm
1 µm = 1000 nanometres (nm)
1 mm = 0.001 m
Therefore, 1 m =
Therefore, 1 nm =
100 cm
0.001 µm
1000 mm
0.000001 mm
1 000 000 µm
0.0000001 cm
1 000 000 000 nm
0.000000001 m
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