Introducing Students to Epifluorescence Microscopy Using the OPN
Scope to Examine Leek Root Tips Stained with Acridine Orange
Chris Stewart and John Giannini*
St. Olaf College, Biology Department, 1520 St. Olaf Avenue, Northfield, MN 55057
*Email: [email protected]
Introduction
In this manual, we provide a new twist on an old standard – namely, the biology teaching lab
that introduces students to the concept of mitosis (or cell division) by viewing slides of onion
root tips. However, instead of using a compound light microscope to view prepared slides or
examining live cells stained with Toluidine Blue, this manual describes a way to use the lab to
further introduce students to some of the techniques involved in epifluorescence microscopy.
In particular, we describe how to stain leek root tips (instead of onion root tips) with Acridine
Orange (a fluorescent dye that, when bound to DNA, glows bright green if excited by blue light).
We then explain how to view the slides using a relatively inexpensive, open source
epifluorescence microscope that we have developed called the OPN Scope (Fig. 1).
Figure 1. The 3D-printed version of the OPN Scope (mounted on
an Olympus CH microscope).
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In the process, students should not only receive the opportunity to learn the basics about cell
division (including identifying cells in the various stages of mitosis), but also get the chance to
become familiar with epifluorescence microscopy – a rather sophisticated technique that is
often not available in a lab course until college (if not, graduate school).
Plus, because the onion root tip lab is such a staple in the biology teaching curriculum, we
believe that most instructors should be generally familiar with the exercise. Indeed, numerous
papers and manuals have described variations of this educational activity over the years – both
in the literature (Woodard, 1948) and online (Marietta College, 2017; Nuffield Foundation,
2017; Carboni, 2010). Still other websites offer virtual labs, where students can review images
of dividing cells taken from previously prepared slides (The Biology Corner, 2017; The Biology
Project, 2017; Marr, 2017; Rossi, 2017; Rutgers University, 2017; Davidson, 2015).
Of course, unlike a virtual lab, the activity described here affords students the opportunity to
develop their own lab skills by preparing live cell cultures for epifluorescence viewing.
However, we have streamlined many of the underlying procedures, so that students can focus
their efforts on searching for dividing cells (which often proves to be the most engaging and
exciting part of the lab). Ultimately, we hope that this exercise will provide both an enjoyable
and educational experience to students (and, ideally, their teachers as well). We further
encourage instructors to experiment with different techniques, stains, and cells to improve
upon this activity, and we look forward to seeing some of the results.
How a Basic Epifluorescence Microscope Works
For those who are not familiar with how an epifluorescence microscope works, we explain the
basic principles behind the instrument in our OPN Scope article, which we wrote for The Journal
of Chemical Education in 2016 (Stewart and Giannini, 2016). However, for readers who may not
have access to that journal, we briefly summarize the key concepts here.
Specifically, in order to induce fluorescence, an epifluorescence microscope sends an intense
(but narrow) band of light down onto a given sample, which excites electrons in the specimen
(Stewart and Giannini, 2016). As those electrons fall back to their “ground” state, they emit
light at a longer (and, thus, lower energy) wavelength, which is known as “fluorescence”
(Stewart and Giannini, 2016).
By way of illustration, as explained above, DNA that has been stained with the fluorescent dye
Acridine Orange will typically glow bright green when struck by blue light. Thus, to view the
corresponding nuclei or chromosomes of cells stained with Acridine Orange, an epifluorescence
microscope first sends a powerful white light through an “excitation filter,” which allows only a
narrow band of wavelengths in the blue spectrum to pass (Fig. 2, left; Stewart and Giannini,
2016). That blue light then strikes a “dichroic mirror,” which is designed to reflect all but the
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anticipated green fluorescent light down onto the sample (Fig. 2, middle). Upon reaching the
specimen, the blue light excites electrons in the Acridine Orange molecules (Fig. 2, bottom),
which have bonded with the DNA (and other structures) in the cells. As those electrons fall
back down to their “ground” state, they emit a green light, which has a slightly longer
wavelength (and, thus, a lower energy) than the blue light that was used to excite the electrons
(Fig. 2, bottom; Stewart and Giannini, 2016). That emitted green light then travels up through
the microscope and passes through the dichroic mirror as well as an “emission filter” (Fig. 2,
right) – both of which let only wavelengths in a narrow band of the green spectrum to pass (Fig.
2, top). Along the way, of course, the objective and ocular lenses in the microscope facilitate
the process by focusing the blue excitation light onto the sample and the emitted green light
for viewing (Fig. 2; Stewart and Giannini, 2016).
Figure 2. Examining cells stained with Acridine Orange, using an epifluorescence
microscope.
With respect to the mechanics of an epifluorescence microscope, typically, these instruments
house their excitation filter, dichroic mirror, and emission filter in a small plastic or metal
container called a “fluorescence filter cube.” Usually, these cubes are specifically designed to
fit a particular type of microscope and view a particular type of fluorescent stain, dye, or tag.
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Also, the microscopes that house these filter cubes normally have a way to slide or rotate each
cube into (and out of) place for epifluorescence or bright-field viewing. Of course, to learn
more about these instruments, readers can visit any number of websites, such as the Leica
Microsystems Fluorescence Microscopy web page, Nikon Microscopy U web page on
Fluorescence Microscopy, Olympus Fluorescence Microscopy Resource Center, and
ThermoFisher Scientific web page on Epifluorescence Microscope Basics (to name a few).
We have also included with this manual some of the Supporting Information from our OPN
Scope article – specifically, the supplement on how to 3D print one version of the instrument
(S1) along with the necessary CAD and STL files (S2 and S3, respectively) and the supplement on
how to build an alternate version of the microscope using mainly PVC parts (S4). We hope that
the background information provided in these supplements will help readers become more
familiar with how a simple epifluorescence microscope can be designed, built, and used.
Materials and Methods
At the outset, readers should know that, over the years, several papers and manuals have
described variations of the onion root tip lab with varying degrees of sophistication. For
example, Woodard (1948) sets forth a number of detailed procedures for preparing cells using
the Feulgen technique (see also Marietta College, 2017) while other online sources describe
staining methods using Toluidine Blue or other dyes (Nuffield Foundation, 2017; Carboni, 2010).
Although our protocols are similar to some of the procedures set forth in those publications, we
have tried to streamline the process for students and teachers in order to reduce the amount of
time that it takes to prepare the cells for epifluorescence viewing, the amount of equipment
needed to prepare these cells, and some of the safety risks presented by these other
techniques. Most notably, we (and others) have found that it is not absolutely necessary to
incubate the root tips in a hot water bath (60° C) as they sit in a container of hydrochloric acid
(HCl) in order to later see chromosomes in the dividing cells (Carolina Biological Staff, 2016).
Consequently, we do not include this step in our procedures, which not only eliminates the
need for the hot water bath itself, but also reduces the safety risks associated with the lab since
the HCl remains at room temperature throughout the exercise.
Nevertheless, readers should still make sure to review the “Hazards” section below since HCl
and Acridine Orange are dangerous chemicals that can cause serious injuries if not used or
handled properly. Also, before lab, instructors and their students should read the Material
Safety Data Sheets for these compounds in order to understand the risks posed by them.
Anyone in lab should further wear the appropriate protective equipment (e.g., gloves, goggles,
lab coats, etc.) when working with HCl and Acridine Orange since exposure to these substances
can cause severe injury or irritation (as well as other harmful effects).
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With that background in mind, the protocols for conducting this lab activity are fairly
straightforward, and we describe them below.
First, instructors should have the following items on hand for students before they begin the
exercise:
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1 to 2 organic leeks (prepared roughly one week in advance as described below);
100 mL of 1-M HCl in an appropriate container (e.g., a glass bottle with a tight cap);
100 mL of tap water also in an appropriate container (e.g., a glass bottle);
100 mL of Acridine Orange solution (50 mg/L) in a glass bottle wrapped in aluminum foil
with a tight cap (or other appropriate container); and
1 to 2 boxes of standard glass slides and corresponding cover slips.
We recommend wrapping the bottle containing the Acridine Orange solution in aluminum foil
in order to prevent any bleaching of the fluorophore by the surrounding light in the room
before the exercise. Otherwise, the nuclei and chromosomes of the cells may not appear as
bright when viewed with the OPN Scope. We also use deionized water to make our stock
Acridine Orange solution, but readers can use distilled water instead.
In addition to the items described above, we list in Table 1 below the supplies that each lab
group will need in order to conduct this educational activity. Ideally, each group should have
their own OPN Scope, which should be mounted on a conventional compound light microscope
with a removable head (S1). Alternatively, readers can make a version of the OPN Scope using
mostly PVC parts (S4), although making all of the necessary pieces and assembling the
instrument will likely require a woodworking or other similar tool shop. In addition, while not
absolutely necessary, we suggest that instructors also provide their students with digital
microscope cameras and corresponding computers, so that students can take photographs of
their fluorescent cells. Students can further use these images to create a slide show or figure
depicting the various stages of mitosis as part of a final lab report, poster, or paper for the
project.
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Table 1. Necessary Supplies for Each Lab Group to Examine Leek Root Tips Stained with
Acridine Orange Solution (50 mg/L) Using the OPN Scope.
Qty
Item/Description
Qty
Item/Description
1
Compound light microscope with a
5 mL
1.0-M HCl
removable head (or the PVC version
of the OPN Scope)
1
Set of 3D-printed parts for the OPN
5 mL
Acridine Orange solution (50 mg/L)
Scope (not necessary if using a PVC
version of the OPN Scope)
1
Fluorescence filter cube that
5 mL
Tap water
generates blue excitation light and
can view green emitted light (e.g., a
GFP filter cube will suffice)
1
High-intensity (e.g., 1,000-lumens or
3
Small glass bottles or beakers (e.g.,
higher) tactical LED flashlight that
10 mL) to hold the HCl, tap water,
will fit into the OPN Scope
and Acridine Orange solution
1
Digital microscope camera and
1
Pair of small scissors (to cut the leek
related software (optional)
roots)
1
Laptop or desktop computer for
1
Pair of small tweezers
viewing images from the digital
microscope camera (optional)
5 to 10 Glass slides and corresponding cover
1
Squirt bottle or plastic transfer
slips
pipette
1 to 2 Fresh (organic) leeks whose roots
1
Single-edged razor blade
were cut at least one week earlier
and have been re-growing in tap
water
1
Short length (e.g., 4 to 6 inches) of a
wooden dowel (3/4-inch diameter)
or a new pencil with a fresh eraser
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Step 1. Purchase fresh (organic) leeks from a local grocery store, super market, or coop (Fig. 3).
We recommend using leeks because we have experienced more success finding dividing cells in
leek root tips than in onion root tips. In addition, we believe that fresh organic leeks are less
likely to have been sprayed with chemicals that could interfere with the growth of any roots
(compared to other commercial leeks that may have been treated with different herbicides or
pesticides).
Figure 3. A fresh organic leek purchased from a local supermarket.
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Step 2. Cut off the roots of the leek (Fig. 4), making sure to leave roughly 0.5 cm of each
existing root intact (Fig. 4C) since this should help expedite the growing process.
Figure 4. Cutting the roots of a fresh leek to initiate the growing process (and, thus, cell
division). Make sure to leave roughly 0.5 cm of each existing root intact to facilitate new
growth.
Step 3. Then, place the leek in a beaker filled with tap water, so that new roots can begin to
grow (Fig. 5A). If necessary, stick three or four toothpicks into the leek to suspend it in the
beaker (and, thus, keep its roots from pressing up against the glass bottom, which could
interfere with the growing process). Also, in our experience, it typically takes about one week
for new roots to grow long enough for the lab. In addition, we have found that these new roots
tend to grow off to the side, often at an odd angle compared to the stalks of the roots that
were previously cut (Fig. 5B). While the new roots are growing, readers should further make
sure to keep the beaker filled with fresh tap water, so that the leek does not dry out.
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Figure 5. Growing new leek roots. (A) Placing a fresh leek into a beaker
of tap water after cutting the roots. (B) New leek roots growing after
roughly 1 to 2 weeks.
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Step 4. After about seven days, cut off the ends of some of the new leek roots and prepare
them for staining. Specifically, cut off a new root (which should be fairly white in color) roughly
1 cm from its pointed end. Then, use a tweezers to place the root into a small glass bottle or
beaker of HCl (1.0 M), and let it soak for 8 to 12 minutes (Fig. 6, left), depending upon the
results of earlier pilot tests. In the interests of expediency, we further suggest cutting off and
processing several new leek roots at once, so that students can prepare a series of slides in
advance (since some slides may not contain cells in every phase of mitosis). Relatedly, we have
found that roots which have turned a dull brown or appear somewhat fuzzy are likely too old to
be used in this lab activity, and they may not contain many (or any) mitotic cells.
Figure 6. Preparing a leek root for Acridine Orange staining by first letting it soak in 1.0M HCl for 8 to 12 minutes (left), then placing it in tap water for 10 minutes (middle), and
finally letting it soak in Acridine Orange stain (50 mg/L) for 30 seconds to 2 minutes.
Step 5. After 8 to 12 minutes have passed, use a tweezers to remove the leek root from the HCl
and then place it in a small glass bottle or beaker of tap water so that it can soak for 10 more
minutes (Fig. 6, middle).
Step 6. Once the leek root has finished soaking in the tap water, use a tweezers to remove the
root and place it in a small glass bottle or beaker of Acridine Orange (50 mg/L), where it should
soak for 30 seconds to 2 minutes (Fig. 6, right), again depending upon the results of earlier pilot
tests. Readers can further wrap the container of Acridine Orange in aluminum foil to prevent
any bleaching of the fluorophore from the overhead lights. This step, however, is not
absolutely necessary in order to view (or obtain useful photographs of) dividing cells.
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Note: If simply seeking to view the cells using only bright-field microscopy, readers can let the
leek root sit in a small well or other container filled with 0.5% Toluidine Blue for 4 minutes
(Nuffield Foundation, 2017; Carboni, 2010). Then, rinse the root by successively placing it in at
least two other wells or containers filled with tap water for at least 1 minute each.
Step 7. After the leek root has finished soaking in Acridine Orange (or been rinsed following its
Toluidine Blue staining), use a tweezers to place the root onto a standard glass slide. Then, use
the single-edged razor blade to cut off the root tip, roughly 2 or 3 mm from its pointed end (the
rest of the root can be discarded as chemical waste). Readers can further use the single-edged
razor blade to gently tenderize the root tip (as if dicing it without actually cutting it into pieces),
which should make the tip easier to squash. Next, add a small amount of water on top of the
root tip using a squirt bottle or plastic transfer pipette. Then, place a standard cover slip over
the tip, and crush or squash it by pressing straight down on the cover slip with a short wooden
dowel or the eraser end of a new pencil. Flattening the root tip in this manner will make it
easier to view the cells under the microscope.
Step 8. After the leek root tip has been squashed, place the slide under a microscope, which
has the OPN Scope mounted on top (Fig. 1). Then, turn on the LED flashlight to the brightest
setting with its telescopic head fully extended, slide the drawer containing the filter cube into
place, and look through the eyepiece to view the stained cells (Fig. 7). Any nuclei and
chromosomes should appear bright green in color (Fig. 7). Students can further connect a
digital microscope camera to a laptop or other computer and take photographs of their
fluorescent cells (Fig. 7). As part of this process, students can also search for cells in the
different phases of mitosis to put together a gallery of images as part of the lab project (Fig. 7).
Step 9. Once finished, make sure to clean up the surrounding work space and properly dispose
of both the HCl and Acridine Orange (or Toluidine Blue) solution as chemical waste. None of
these items should simply be poured down any drains.
Results
Using the above protocols, it is possible to obtain sharp and vivid images of leek root tip cells in
the various stages of mitosis (Fig. 7), and these results are similar to those that have be
previously depicted in the literature or online using the Feulgen technique (Woodard, 1948) or
Toluidine Blue staining (Marr, 2017; Carboni, 2010).
Specifically, in our experiments, we have consistently seen cells undergoing prophase, where
the chromosomes start to condense in the nucleus (Figs. 7A and 7B); metaphase, where the
chromosomes begin to align themselves along the center of the mitotic spindle (Figs. 7C – 7F);
anaphase, where the chromosomes separate and move to opposite sides of the cell (Figs. 7G
and 7H); and telophase as well as cytokinesis, where the cell starts to divide (Figs. 7I and 7J).
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Figure 7. The stages of mitosis, as demonstrated by various leek root tip cells stained
with Acridine Orange (50 mg/L) and viewed at 400x total magnification using the 3Dprinted version of the OPN Scope (with a Nikon B-2A filter cube) mounted on an
Olympus CH-2 microscope. (A and B) Prophase. (C – F) Metaphase. (G – H) Anaphase.
(I – J) Telophase and Cytokinesis. Images taken using an OMAX 14-MegaPixel digital
microscope camera and its related TOUP software.
Of course, not every slide that we have prepared has contained cells in each phase of mitosis.
Nevertheless, we have been able to find cells in several different mitotic phases on one slide.
Plus, we have found that preparing multiple (e.g., 3 or 4) slides at once affords more time to
search for cells under the microscope, and having several slides at the ready enables us to
quickly move onto the next slide once we have finished examining the current one. Usually, we
have been able to find cells in every phase of mitosis by examining between 2 and 4 slides, but
additional slides might be required depending upon the age and quality of the leek roots.
Also, for schools that seek to use only bright-field microscopes with their students, the above
protocols can generate similar images of dividing cells when the leek roots are stained with
Toluidine Blue (Fig. 8), and these results are again similar to other images that have appeared
online (Marr, 2017; Carboni, 2010) or in the literature (Woodard, 1948). In particular, following
the above procedures, we have repeatedly seen chromosomes that were stained a dark blue or
a deep purple in each of the various phases of mitosis (Fig. 8).
Of course, as with the leek root tips that had been stained with Acridine Orange, we were not
always able to find cells in every stage of mitosis on one slide. Instead, at times, we needed to
search multiple slides to find examples of cells in each one of the mitotic phases. Thus, as
suggested above, instructors may want to have their students prepare several leek root tips for
Toluidine Blue staining at the outset, so that more time can be dedicated to searching the slides
for dividing cells.
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Figure 8. The stages of mitosis, as demonstrated by various leek root cells stained with
Toluidine Blue and viewed at 400x total magnification using the 3D-printed version of
the OPN Scope mounted on an Olympus CH-2 microscope (without the filter cube in
place). (A - C) Prophase. (D – E) Metaphase. (F – H) Anaphase. (I – J) Telophase and
Cytokinesis. Images taken using an OMAX 14-MegaPixel digital microscope camera and
its related TOUP software.
Discussion
Given the ease with which the OPN Scope can be 3D-printed, assembled, and used as well as
the simplicity of the fluorescent staining technique described above, we hope that this
educational lab activity will be an enjoyable and informative experience for students and
teachers alike. Not only can the exercise introduce students to the idea of cell division (i.e.,
mitosis), in the process, students will have the chance to learn some of the basic concepts and
techniques involved in epifluorescence microscopy (a rather sophisticated field) in addition to
developing some of their own lab skills. Also, for more advanced courses, instructors could
expand upon this lab activity to grow new leek roots under different conditions (or treat the
roots with different chemicals), so that students can explore how these changes affect cell
division. Plus, if using digital cameras to take photographs of their leek root tip cells, students
could later prepare figures that show the various stages of mitosis (Figs. 7 and 8) or the effects
of different conditions or treatments on the cells, and these figures could further be included in
a poster, paper, or lab report that was assigned as homework for the project. Given the
possibilities, we hope that the lab exercise (as well as the OPN Scope itself) will help to create a
variety of new opportunities for both students and their teachers to conduct their own
scientific inquiries while also developing their technical skills.
As to cost, many schools should already have most of the basic equipment needed for this lab
exercise (e.g., glass beakers or bottles, slides and cover slips, scissors, tweezers, razor blades,
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etc.). In addition, fresh leeks are relatively inexpensive, costing around $2 to $4 each
(depending upon the size and current market price).
Also, for schools that already have a set of compound light microscopes with removable heads,
then the most expensive component of this lab activity will likely be the fluorescence filter
cube. For example, used ones typically cost around $100 to $500 each on eBay or other similar
websites, depending on the make and model (S1). Alternatively, for schools that do not have
these types of microscopes and cannot afford buying a set of new or used educational models,
teachers could build a PVC version of the OPN Scope as long as they had access to an adequate
wood or other tool shop (S4). In this case, the necessary raw materials would cost around $180
to $600 in total, depending upon the exact items used (S4). However, some of these supplies
(e.g., the PVC board and pipe) would make several PVC models.
In addition, for schools that already have a standard 3D printer, one spool of 1.75-mm diameter
filament (which typically costs $20 to $25 online) will print the parts for at least two OPN Scope
models that could fit on microscopes with removable heads. As a result, given a reasonably
priced LED flashlight ($15) and used fluorescence filter cube ($250), an instructor could equip a
teaching lab with OPN Scopes for under $300 per instrument (again, assuming that the school
already had compound light microscopes with removable heads).
Alternatively, for schools without a 3D printer, the website www.3dprintingpricecheck.com
estimates that it would cost approximately $85 to 3D print all of the components for the OPN
Scope using an online 3D-printing service like makexyz (at January 2017 prices, not including
any taxes or shipping), which increases the total cost to around $350 per instrument (once the
LED flashlight and used filter cube are included). However, in the long run, purchasing a
relatively inexpensive 3D printer (e.g., one under $500) may make the most economic sense.
For example, $85 would buy at least three spools of ABS filament, which would print the parts
for at least six OPN Scopes. Conversely, at $85 per instrument, it would cost roughly $510 to 3D
print six OPN Scopes using the makexyz website, which is roughly the cost of a reasonably
priced, basic 3D printer (at January 2017 prices, not including any taxes or shipping). Plus, with
a 3D printer, students could start designing their own instruments and equipment, which
should help to open up many new possibilities for them.
Finally, for schools that do not currently have a supply of Acridine Orange on hand, small
samples of the compound (e.g., 5 or 10 g) can be purchased online through chemical supply
companies like Sigma-Aldrich or Cayman Chemical for between roughly $7 and $8 per gram (or
approximately $40 to $80 total) at February 2017 prices, not including any taxes or shipping and
depending upon the exact quantity purchased. Similarly, for schools that plan to use Toluidine
Blue for this lab activity, small quantities of the stain (e.g., 5 or 10 g) can be purchased online
from chemical companies like Sigma-Aldrich , Fisher Scientific , or Flinn Scientific for roughly $3
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to $6 per gram (or about $30 total) – again at February 2017 prices, not including any taxes or
shipping and depending upon the quantity purchased.
Ultimately, we hope that the affordability and versatility of this educational lab activity will help
teachers provide their students with interesting and challenging projects as they progress in
their studies. In particular, by introducing students to both the concept of cell division and the
technique of epifluorescence microscopy, we hope that the lab exercise (and the OPN Scope
itself) will help to spark a curiosity and sense of exploration in students that continues to grow
over time. We also hope that the lab activity and all of the various OPN instruments and OPN
experiments that we have created can help encourage students (and their teachers) to think
creatively about making and using some of their own scientific equipment (not just for their
own purposes, but to help others as well), and we hope to see some of the results.
Helpful Hints
Although most aspects of this lab exercise are fairly straightforward, we provide a few helpful
hints below to assist readers with the educational activity.
First, as explained above, for anyone who is unfamiliar with epifluorescence microscopy in
general or the OPN Scope in particular, we suggest reading our OPN Scope paper (and related
supplements) to gain a better understanding of both the field and the instrument. In addition,
the supplemental materials contain a number of their own helpful hints, which readers may
find useful when assembling or using the device.
Second, as indicated above, we typically use an Olympus CH-style microscope with our 3Dprinted OPN Scopes since this model has both a removable head and removable eye pieces,
which make setting up the instrument relatively easy. However, we have designed mounting
adapters and eye piece tubes for other commercial microscopes as well – specifically, the Nikon
AlphaPhot-2 YSR, Nikon Eclipse E300, Zeiss AxioStar Plus, and Zeiss PrimoStar models (S2, S3).
Of course, readers can design parts to fit their own compound light microscopes (if they are not
one of the above models). Also, while we use the free version of DesignSpark Mechanical for
our Computer Aided Design (CAD) work, readers can use whatever CAD software they prefer.
Third, for the lab activity itself, we suggest that students prepare several (e.g., 3 or 4) leek root
tips for staining at the start of lab, so that more time can be dedicated to searching the slides
for mitotic cells (which is usually the more engaging part of the lab exercise). Also, in our
experience, finding dividing cells (especially ones in each phase of mitosis) can sometimes be a
lengthy process. For example, we have found that, sometimes, even a new leek root may not
have many (or any) mitotic cells. As a result, having multiple slides prepared at the start of
viewing should enable a lab group to quickly move onto the next slide if need be. Not only
should this approach save some time in the long run, by preparing all of the slides in advance,
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each student in a lab group should also be guaranteed the chance to make at least one slide on
their own (so no one will feel left out of the process).
Fourth, if students do prepare multiple slides at the start of lab, we suggest placing any slides
that are not being viewed at the time in a drawer and closing it or covering the slides in some
other way (e.g., with a small cardboard box that is turned upside down). This approach should
reduce any photo-bleaching of the Acridine Orange stain by the overhead lights, which should
help the chromosomes appear brighter when excited by the blue light of the filter cube.
Fifth, as with other OPN instruments and OPN experiments that we have developed, we
recommend that instructors conduct a sufficient number of “test runs” with this lab activity in
advance to become familiar with the equipment and protocols used. Such pilot tests should
also help instructors better understand the types of issues that could arise during the exercise,
which should help with “trouble shooting” any student set-ups if the need arises. In addition,
by conducting a series of pilot tests, instructors should get a sense of the appropriate amount
of time that the leek roots should sit in HCl and Acridine Orange. For example, we have found
that letting the roots sit in HCl for between 8 and 12 minutes generally suffices (with 10
minutes usually providing an adequate amount of time to soften the roots). Similarly, letting
the leek roots sit in the Acridine Orange solution for between 30 seconds and 2 minutes also
tends to light up the nuclei and chromosomes of the root tip cells under the OPN Scope fairly
brightly. However, at times, the stain can appear too bright, so an incubation period around 30
seconds might be preferable to a longer one. Plus, all of these time periods could differ
depending upon the age and condition of the leek roots. For this reason, we also suggest using
relatively fresh leeks with new roots (i.e., no more than two weeks old) that appear fairly white
(as opposed to light brown) in color. Such leek roots may have a greater number of cells
undergoing mitosis, which will make the search for dividing cells easier for students.
Hazards
Readers should know that hydrochloric acid (HCl) is an extremely dangerous chemical, and
anyone using the acid should exercise a great deal of caution and care around it. In particular,
the acid is corrosive as well as toxic; and, in concentrated forms, it can release an acid mist that
is also very hazardous (MSDS Online, 2014; ScienceLab.com, 2013a). Specifically, both the acid
itself and any mists that it creates can severely – even irreversibly – damage the skin, eyes, and
internal organs on contact – including if swallowed or inhaled (MSDS Online, 2014). In some
cases, this damage can even prove fatal. In addition, prolonged exposure to HCl (including any
acid mists) can further have toxic effects on numerous organs and organ systems, including the
eyes, kidneys, liver, lungs, skin, and teeth as well as the circulatory and respiratory systems
(ScienceLab.com, 2013a).
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Also, as explained in the Supporting Information (S5) to our OPN Scope article, Acridine Orange
is a hazardous chemical as well, which can cause severe skin and eye irritation on contact.
Exposure to the compound may also cause genetic defects.
Likewise, Toluidine Blue is also a potential irritant of the skin and eyes as well as the digestive
and respiratory tracts. As such, the chemical may be harmful if absorbed through the skin,
ingested, or inhaled. Toluidine Blue has further displayed mutagenic effects on bacteria and
yeast, and it may adversely affect genetic material in humans as well (ScienceLab.com, 2013b).
In addition, if introduced intravenously, Toluidine Blue has been found to cause different blood
diseases (e.g., hemolytic anemia, leukopenia, methemoglobinemia) as well as negative effects
on the central nervous system (e.g., headaches and convulsions), the cardiovascular system
(e.g., hypertension and dysrhythmia), and the renal system (acute renal failure and hematuria)
(ScienceLab.com, 2013b).
As a result, anyone working with HCl, Acridine Orange, or Toluidine Blue should exercise great
care around these substances. This includes wearing the appropriate protective equipment
(e.g., gloves, goggles, lab coats, masks or respirators when necessary, etc.) and reading the
Material Safety Data Sheet for each one of these substances before working with them. In
addition, given some of the risks involved, we suggest that instructors prepare any stock
solutions of HCl, Acridine Orange, or Toluidine Blue themselves. Also, for safety reasons, we
recommend purchasing a container of 1-M HCl (instead of a more concentrated supply of the
acid).
In addition, as explained in our OPN Scope article and several of the papers on our OPN Lab
“Equipment” website, there are a number of hazards associated with 3D printing. For example,
the 3D printing bed can become quite hot when it is in use. As a result, readers should exercise
a great deal of caution when removing parts from a 3D printer. In addition, the process of 3D
printing can release harmful nanoparticles into the air, which can cause significant health
issues, such as asthma attacks, respiratory arrest, strokes, and even cardiac arrest. Therefore,
readers should keep their 3D printer in a well-ventilated space and wear the appropriate
protective equipment when operating the printer (e.g., masks or respirators to protect their
respiratory systems from any nanoparticles, goggles or safety glasses to protect their eyes from
the same, etc.). Also, to learn more about 3D printing safety, readers can review the following
helpful websites and PDFs (or search for their own information online): Carnegie Mellon
University PDF on 3D printing safety; University of Florida Website on 3D printing policies and
safety; and University of Vermont Website on 3D printing safety.
Finally, as also explained in our OPN Scope article and some of the papers on our OPN Lab
“Equipment” website, we frequently use an Outlite WT03 tactical LED flashlight with the OPN
Scope. However, this flashlight can become rather warm – and, sometimes, even quite hot – if
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left on for long periods of time. Although we have not run into any problems due to this
overheating, readers may want to use a different LED flashlight with the OPN Scope for this lab
activity (especially if working with middle or high school students), and we have had success
with the UltraFire, SkyWolfeEye, and GeakLight brands. Instructors could also tell their
students to turn off their LED flashlights when they are not viewing fluorescent cells. This
approach would not only reduce the chances of an Outlite WT03 overheating, but also limit the
amount of any fluorescent bleaching that might occur (enabling students to view the cells on
their slide for a longer period of time).
Disclosures
The authors declare that they have no conflicts of interest related to any product, brand,
company, website, or other item discussed in this manual. In fact, as with other open source
equipment and experiments that we have created, http://pages.stolaf.edu/opn-lab/, we
encourage readers to improve upon this lab activity by experimenting with other materials and
by bringing their own insights and inspirations to the project.
References
The Biology Corner. (2017). Onion Root Tip. Available online at
https://www.biologycorner.com/projects/mitosis/onion_root.html
The Biology Project at the University of Arizona. (2017). Online Onion Root Tips. Available
online at http://www.biology.arizona.edu/cell_bio/activities/cell_cycle/cell_cycle.html
Carboni, G. (2010). Mitosis in the Root Tips of Garlic and Onion. Available online at
http://www.funsci.com/fun3_en/mitosis/garlic.htm
Carolina Biological Staff. (2016). Squash an Onion and Learn the True Age of Your Cells.
Available online at http://www.carolina.com/teacher-resources/Interactive/squash-an-onionand-learn-the-true-age-of-your-cells/tr10865.tr
Davidson, M.W. (2015). Mitosis in Onion Root Tips. Available online at
https://micro.magnet.fsu.edu/micro/gallery/mitosis/mitosis.html
Marietta College. (2017). Mitosis in Onion Root Tip Cells (PDF Lab Handout). Available online
at http://w3.marietta.edu/~biol/introlab/Onion%20root%20mitosis.pdf
Marr, K. (2017). Mitosis in Onion Root Tips. Available online at
http://www.instruction.greenriver.edu/kmarr/biology%20211/Labs%20and%20ALEs/B211%20L
abs/B211%20Labs/Onion%20and%20Whitefish%20Mitosis/Onion%20Root%20Tip%20Mitosis/
Onion%20Root%20tip%20Mitosis.html (adapting similar work from
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http://staff.jccc.net/pdecell/celldivision/oniontip.html and
http://district.bluegrass.kctcs.edu/billd.snyder/sharedfiles/biowebsite/BiologyLabs/BIO137/13
7Lab2/Lab2MitosisSlides.html, whose links are no longer current)
MSDS Online. (2014). Hydrochloric Acid Hazards & Safety Tips. Available online at
https://www.msdsonline.com/blog/health-safety/2014/09/10/hydrochloric-acid-hazardssafety-tips
The Nuffield Foundation. (2017). Investigating mitosis in allium root tip squash. Available
online at http://www.nuffieldfoundation.org/practical-biology/investigating-mitosis-alliumroot-tip-squash
OPN Lab Equipment Web Page. (2017). Available online at http://pages.stolaf.edu/opnlab/equipment/
Rossi, J.C. (2017). Onion Root Mitosis. Available online at http://www.microscopyuk.org.uk/mag/indexmag.html?http://www.microscopyuk.org.uk/mag/artnov04macro/jronionroot.html
Rutgers University. (2017). How Do Cells Reproduce? Available online at
http://bio.rutgers.edu/~gb101/lab2_mitosis/index2.html
Science Lab. (2013a). Material Safety Data Sheet for Hydrochloric Acid. Available online at
http://www.sciencelab.com/msds.php?msdsId=9924285
Science Lab. (2013b). Material Safety Data Sheet for Toluidine Blue O. Available online at
http://www.sciencelab.com/msds.php?msdsId=9927627
Stewart, C. and Giannini, J. (2016). Inexpensive, open source epifluorescence microscopes. J.
Chem. Educ. 93(7), 1310-1315. Available online at
http://pubs.acs.org/doi/abs/10.1021/acs.jchemed.5b00984.
Woodard, T.M. (1948). Some features of mitosis in the onion root tip. J. Tenn. Acad. Sci.
23(3), 191-199.
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