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: giannini@stolaf.edu

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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:

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

removable head (or the PVC version of the OPN Scope)

5 mL 1.0-M HCl

1 Set of 3D-printed parts for the OPN Scope (not necessary if using a PVC version of the OPN Scope)

5 mL Acridine Orange solution (50 mg/L)

1 Fluorescence filter cube that generates blue excitation light and can view green emitted light (e.g., a GFP filter cube will suffice)

5 mL Tap water

1 High-intensity (e.g., 1,000-lumens or higher) tactical LED flashlight that will fit into the OPN Scope

3 Small glass bottles or beakers (e.g., 10 mL) to hold the HCl, tap water, and Acridine Orange solution

1 Digital microscope camera and related software (optional)

1 Pair of small scissors (to cut the leek roots)

1 Laptop or desktop computer for viewing images from the digital microscope camera (optional)

1 Pair of small tweezers

5 to 10 Glass slides and corresponding cover slips

1 Squirt bottle or plastic transfer pipette

1 to 2 Fresh (organic) leeks whose roots were cut at least one week earlier and have been re-growing in tap water

1 Single-edged razor blade

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 1.0-

M 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 3D-

printed 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 3D-

printed 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).

http://pages.stolaf.edu/opn-lab/equipment/ 18 February 16, 2017

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

http://pages.stolaf.edu/opn-lab/equipment/ 19 February 16, 2017

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-onion-

and-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

http://pages.stolaf.edu/opn-lab/equipment/ 20 February 16, 2017

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-hazards-

safety-tips

The Nuffield Foundation. (2017). Investigating mitosis in allium root tip squash. Available

online at http://www.nuffieldfoundation.org/practical-biology/investigating-mitosis-allium-

root-tip-squash

OPN Lab Equipment Web Page. (2017). Available online at http://pages.stolaf.edu/opn-

lab/equipment/

Rossi, J.C. (2017). Onion Root Mitosis. Available online at http://www.microscopy-

uk.org.uk/mag/indexmag.html?http://www.microscopy-

uk.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.