Exploring viral genetics with electron microscopy
S . M o n ro e D u b o i s e ,
K a re n D. M o u l t o n ,
a n d J e n n i fe r L . J a m i s o n
W
hen university scientists and high school
teachers share knowledge and resources,
there are mutual benefits. For example,
science teachers often excel in pedagogic
practice, but must teach with limited access to laboratory resources and specialized expertise unless they can
find willing partners at universities or other scientific
institutions. Collaboration with high school teachers and
students allows practicing scientists to do important outreach work—often required by their funding agencies—
and provide their graduate students with the opportunity
to increase communication and teaching skills. This article highlights one such partnership—a collaboration
of high school teachers and students with scientists and
graduate students at the University of Southern Maine
(USM)—and illustrates how access to a university research community’s advanced technological resources can
enrich science learning in high school classrooms.
32
The Science Teacher
Bacteriophages.
Bacteriophages, often simply called “phages,” are viruses that
infect bacteria. Phages are estimated to be the most diverse
and widely distributed genetic entities in the biosphere
(McGrath and van Sinderen 2007). They are found wherever
bacterial hosts are present, such as in soil, sea water, and
the intestines of animals. Like all viruses, phages consist essentially of a core of RNA or DNA surrounded by a protein
coat; since they are unable to replicate without a host cell,
they typically are not considered living organisms. Because
they only infect bacteria and cannot replicate except within
a host bacterial cell, bacteriophages are not capable of infecting humans or any other nonbacterial host. Therefore,
they are exceedingly safe as a tool for introducing students
to concepts of virology and molecular biology. In fact, bacteriophages have been used as an alternative to antibiotics
in the former Soviet Union and Eastern Europe, and have
been considered as a possible therapy against multiple drugresistant strains of bacteria (BBC Horizon 1997).
Head containing
DNA
Collar
Sheath
Base plate
Tail fibers
Protein capsid containing virus
DNA or RNA
Viral genome is injected into the cell
Bacterial cell
Bacteriophage genome
©2009 diagrams by Graham Colm
This partnership, the Maine ScienceCorps, is a project
sponsored by the National Science Foundation (NSF)’s
Graduate Teaching Fellows in K–12 Education (GK–
12) program. Through this program, USM’s virology
and transmission electron microscopy (TEM) research
group provides high school teachers and students in rural areas with access to the nanoscale world of viruses.
Graduate student fellows work with teachers to enrich
classroom content in microbiology and molecular biology.
This type of collaboration is especially important because
microbiology and virology concepts are fundamental to
understanding molecular biology and biotechnology, yet
laboratory experimentation and inquiry in secondary
classrooms are often constrained by safety considerations
(see “Keys to microbiological safety,” p. 37) and available
laboratory resources.
In the project described in this article, fellows bring safe
laboratory strains of bacteria—such as Escherichia coli B
(E. coli B) obtained from biological supply companies—
into high school classrooms and assist students and teachers in microbiology studies. They demonstrate necessary
safety precautions and facilitate classroom investigations
through the use of university resources, ranging from basic
tools to sophisticated instruments such as electron microscopes. It is unusual for electron microscopy to be available
for secondary science education, but this is just one example of how community partnerships can provide broad
educational benefits.
A micro- and nanoscale education
imperative
The unseen world, both living and nonliving, at the
micro- and nanoscale has immense importance for
health, disease, and the entire biosphere—and yet is
only visible in detail through microscopy. The biological world at these scales is richly complex in an ecological sense. It includes agents of infectious diseases
and other biological entities that contribute to the biosphere in numerous ways by
u driving evolution throughout the tree of life,
u influencing nutrition and developmental biology
of multicellular organisms,
u supporting food chains, and
u exerting controlling influences on Earth’s biogeochemical cycles (Fuhrman 1999; Hendrix
2005; Suttle 2005; Wommack and Colwell 2000;
Breitbart, Rohwer, and Abedon 2005; Schaechter, Ingraham, and Neidhardt 2006).
Despite the undeniable importance of microbes, even
college bioscience majors rarely experience substantial
microbiology education until late in their undergraduate
training, if at all. This accounts for a significant deficiency in the general public’s scientific literacy and in the
preparation of biologists, biomedical scientists, and health
April/May 2009
33
professionals (Schaechter, Kolter, and Buckley 2004;
BSCS 2006). Another unfortunate consequence is that
K–12 educators often lack any formal microbiology training and are thus limited in their capacity to convey this
foundational knowledge of the biological and biomedical
sciences to students.
The tools
The tools of microscopy are essential for making the usually invisible world of microbes and viruses less abstract
for learners of any age—teachers and students alike. For
the nanoscale dimensions of viruses, this requires electron microscopy, which uses electrons to create an image
of a specimen, has a much greater resolving power than
light microscopes, and can therefore obtain much higher
magnifications. Figure 1 shows an electron micrograph
that can be used to enhance students’ understanding of
the scientific process used to discover the function and
significance of DNA, which required distinct radioactive labeling of the proteins and DNA of bacteriophage
(virus)-infecting E. coli (Hershey and Chase 1952). (Note:
See “Bacteriophages,” p. 33, for further explanation of
how these viruses work.) The micrograph reveals several
intact virus particles (virions) with characteristic icosohedral heads that contain DNA and slender tail structures
that attach to host bacterial cells. One virus particle in the micrograph illustrates the
relationship of “form and function”—a unifying theme of
the National Science Education Standards (NRC 1996)—at
the molecular level. The structure of the virus is altered
when tail proteins contract to expel the viral DNA, which
is tightly packed in the virion head, into the E. coli. In
Figure 2
Revealing form, function, and history in
an electron beam.
Inquiry into the definitions of life.
All photos courtesy of Karen D. Moulton and Jennifer L . Jamison
Figure 1
The foundations of our knowledge of molecular biology, biochemistry, and many of the molecular tools that fuel progress
in modern biomedical sciences and biology are derived from
the study of viruses and microbes and their genetics (Cairn,
Stent, and Watson 1992; Summers 1999). This image of a bacteriophage isolated by a USM student from the Fore River in
Portland, Maine, can provide visual insight into the structural
basis for the classic Hershey and Chase (1952) experiment
demonstrating that DNA is the genetic material.
34
The Science Teacher
Concepts of viruses in particular remain abstract in many
biology classrooms; they are seen as important because of
the diseases some cause but are paradoxically described as
not really living. Like the zombies of classic horror films, viruses are “the undead” of the biosphere—biological entities
that lack defining characteristics of life and yet nevertheless
permeate its tree. “Using logic and evidence,” student inquiry
into whether viruses are nonliving or alive can lead to lively
opportunities to “communicate and defend a scientific argument,” as is recommended in the Standards (NRC 1996). These
bacterial viruses were isolated by high school students in
northern Maine with the help of GK–12 fellows.
C o l l a b o rat i o n at t h e N a n o s c a l e
understanding the classic Hershey and Chase experiment (1952), this image can provide visual insight into the
virus’s structural basis and mechanism: The radioactive
protein is left outside of infected cells while the distinctly
labeled DNA is injected into the cell.
The tree of life
The study of viruses has been the source of many important discoveries during the past century (Oldstone and
Levine 2000). But oftentimes virology concepts conveyed
in classrooms at all educational levels are biased toward
understanding the direct impacts of viruses upon health
and neglect their broader ecological and evolutionary importance in the biosphere. The three-domain molecular
tree of life (for example, as presented in the Baumgartner
and Pace article in the October 2007 issue of The Science
Figure 3
Astronomical viral abundance
revealed at the nanoscale.
Teacher) does not explicitly include viruses in any way—
despite their interplay within and across the Archaea,
Bacteria, and Eukarya domains. They are excluded because viruses are often considered nonliving, as they lack
their own metabolism and are dependent upon host cells
for their replication. However, they are genetic entities
with a powerful impact on the survival and evolution of
their hosts (Figure 2).
The bacterial viruses (bacteriophages)—molecular
parasites with elegant architecture—shown in Figure 2
were isolated by high school students in northern Maine
with the help of GK–12 fellows. Such micrographs,
showing the relative molecular simplicity of viruses in
comparison to cellular life, can stimulate lively discussion of that most fundamental question: “What is life?”
Within each of the three domains of life, viruses are
capable of driving evolutionary change as powerful forces
of selective pressure and, sometimes, even by directly or
indirectly altering the DNA of the hosts they infect. The
widespread phenomenon of lateral gene transfer—in
which genetic material is shared across species boundaries—is sometimes mediated by viruses, enormously
complicating phylogenetic analyses that seek to understand evolutionary relationships among various groups
of organisms (Onishi, Kurokawa, and Hayashi 2001;
Daubin, Lerat, and Perrière 2003; Ochman, Lerat, and
Daubin 2005). In many cases, viral genomes are literally
inserted into the DNA of their hosts, adding genetic material and altering host gene activity. Such viral insertions
in bacteria drive bacterial evolution (Canchaya et al. 2003;
Hendrix 2005); their presence in human and other mammalian genomes is also well documented. In these genomes, retroelements, including retroviruses and their
remnants, have been reported to comprise large portions
of the total genetic material (Bannert and Kurth 2004)
and may have had a powerful impact on human evolution
and the evolution of mammals (Specter 2007).
“Dark matter”
The bacteriophage shown in this electron micrograph infects
E. coli C and was isolated from a local stream by students
at Caribou High School in northern Maine (with the assistance of GK–12 fellows). Using the 100 nm scale bar in the
micrograph, students can be asked to estimate the length of
a single virus particle (or virion) and then calculate how far
(in light years) a chain of 1031 virions would extend, as well as
how many bacteriophages would be needed to form a chain
length of 1 mm.
In recent years it has become clear that viruses are the
most abundant biological entities on Earth (Brüssow
and Hendrix 2002; Rohwer 2003; Filée et al. 2005; Suttle
2005). The serendipitous electron micrograph in Figure
3 shows a chain of five contiguous bacteriophages isolated
by our students that can be used to convey the scale and
abundance of tailed bacteriophages on Earth, estimated to
be 1031 by Hendrix (2005).
Great viral diversity and the fact that the abundance of
viruses infecting bacteria remain mostly unexplored have
led to the assertion that viruses are the “dark matter” of
biology (Filée et al. 2005). In partnership with teachers in
rural schools and GK–12 fellows, the virology and TEM
group at USM works to effectively reveal the powerful
ecological, biogeochemical, and evolutionary impacts of
this usually unseen biological dark matter.
April/May 2009
35
The collaboration
Since its inception in 2001, the Maine ScienceCorps
has brought laboratory-based bioscience enrichment to
underfunded rural high schools across Maine (Duboise
2004). Bioscience GK–12 fellow teams act as scientific
role models for precollege (K–12) students and provide
laboratory learning experiences that are aligned with the
Standards (NRC 1996).
In recent years, the program has provided authentic
research contexts for exploring biodiversity in natural or
model environments. Research experiences are increasingly part of student-driven, inquiry-based projects, particularly those focused on the mostly unexplored nature
of bacteriophage genomics (Brüssow and Hendrix 2002).
In the ecology classroom, a typical ScienceCorps project
considers the importance and human uses of particular
water sources and the microbes and viruses within these
environments. Known conditions, such as phosphorous
elevation—caused by phosphoric acid discharges from
a paper mill or the tidal influences that occur where a
river meets the ocean—may influence where the class
gathers water samples or the questions students ask.
the community to parents and school boards, and at a
university poster day presentation. Those students who
come to the university to present their work have the opportunity to visit university research facilities, including
the TEM laboratory.
Student learning
Students learn the fundamental concepts of virology,
viral ecology, and molecular biology through their study
of bacteriophages, and these concepts are reinforced
Essential steps for safely isolating
bacteriophages from the environment.
Step 1: Enrichment of bacteriophages from aquatic
samples:
u
The project in action
Collaborating teachers ensure that personal safety issues are considered in field work and that students wear
laboratory gloves and chemical-splash goggles during
the collection of samples. (Safety note: Before field
trips, teachers should review and adhere to school
field trip policies and procedures.) Typically the
high school students, teachers, and GK–12 fellows work
together to collect water samples from nearby streams,
lakes, or ponds. While soils, sediments, and water samples are all likely to contain many bacteriophages, water
samples are most easily and safely used because filtration of unwanted microbes prior to the bacteriophage
isolation process is readily accomplished. Once students
are back in the classroom, they test for the presence of
bacteriophages using procedures described in “Essential
steps for safely isolating bacteriophages from the environment.” When students have successfully isolated a
bacteriophage, electron micrographs can be prepared at
the university to reveal the viral morphology (i.e., form,
shape, and structure).
In the high school classroom, students proceed to isolate the viral genomic material and explore the genetics
of the virus further using basic tools of molecular biology,
such as restriction endonucleases, with assistance from
the fellows. In some cases, university DNA sequencing
resources are used to provide students with some genomic
sequence from the isolated bacteriophage, allowing them
to see and understand the basics of comparing new sequences with public genetic databases.
The culmination of the project is the presentation
of research findings by students within the school, in
36
The Science Teacher
u
u
u
u
Collect local water samples from a lake, river, pond, or
beach site in clean (preferably sterile) plastic containers,
such as 50 mL plastic centrifuge tubes. In addition to any
safety precautions needed at particular field sites, nitrile
laboratory gloves and chemical-splash goggles should be
worn during water collection for personal safety and to
avoid contaminating the samples collected.
Filter water sample through a sterile non–protein binding
membrane filter (0.22 m or 0.45 m pore size) to remove
potentially hazardous bacteria but not most bacteriophages. Filters can then be disinfected with household
bleach diluted 1 part bleach to about 10 parts water prior
to disposal.
Add filtered water sample to culture of safe laboratory
strains of host bacterium (e.g., E. coli B or E. coli C).
Incubate and watch for clearing of culture, suggesting viral
lysis of host bacteria.
Remove remaining bacterial cells and cellular debris by
centrifugation or filtration of the enrichment culture.
Step 2: Detection and isolation of bacteriophages from
the enrichment culture:
u
u
u
Thoroughly spread a layer of actively growing host bacteria
(safe strains obtained commercially) on a petri plate of
nutrient agar medium with a sterile cotton swab; then place
small drops (about 10 m L) of the bacteriophage enrichment
(or dilutions of that enrichment) onto the agar surface.
As a bacterial “lawn” appears, observe any clearings in the
bacterial growth that could be “plaques” of virus-induced
bacterial cell lysis.
Individual viral plaques can then be used for virus purification and grown further for transport to the university
laboratory, where negative staining with heavy metals is
done prior to electron microscopic examination.
C o l l a b o rat i o n at t h e N a n o s c a l e
Keys to microbiological safety.
u
u
u
u
u
u
u
u
u
u
u
u
Review school board and local health department
policies relative to biotechnology experimentation
in the school science laboratory.
Send a letter home to parents and guardians advising them
of the activity.
Learn basic sterile culture methods well or seek expert assistance. Techniques for Microbiology by John M. Lammert
(2007) is a helpful guide.
Cultivating unknown microbes from the environment
poses unacceptable risks in the secondary school
classroom—and therefore should not be attempted. Cultivate only known safe laboratory strains (such as those
available from major educational supply companies). See
www.science-projects.com/safemicrobes.htm for further
discussion of microbes for educational use.
Use personal safety equipment, including lab coats, disposable nitrile gloves, and eye protection (chemical-splash
goggles).
Do not eat, drink, or apply cosmetics in the laboratory.
Keep fingers and writing instruments away from face and
mouth.
Wash hands with soap and water before and after handling
microorganisms and before leaving the laboratory, regardless of materials used. When handling microorganisms or
other living materials, cover cuts on hands before putting
on gloves to protect against infection.
Use only mechanical pipetting devices for transferring any
material. Avoid mouth pipetting.
Effect procedures carefully to reduce the formation of
aerosols. Keep pipette tips away from the face to avoid
inhaling any aerosol that may be formed.
Effectively disinfect surfaces using a freshly made solution of household bleach diluted with water to a 1–10%
concentration.
Sterilize used cultures and contaminated glassware in an
autoclave or pressure cooker or get assistance from a
laboratory that has this equipment.
Label containers holding any remaining contaminated
solid and liquid materials that have come in contact with
microorganisms for appropriate disposal.
Additional information on biosafety can be found at:
u
u
ational Institutes of Health Biosafety in Microbiological
N
and Biomedical Laboratories: www.nih.gov/od/ors/ds/
pubs/bmbl
School Improvement in State of Maryland—Biotechnology and Recombinant DNA Research: http://mdk12.org/
instruction/curriculum/science/safety/hazards.html
(Editor's note: See also the Safer Science column by Ken Roy
on p. 12.)
with the TEM images provided through the university
laboratory. Bacteriophages that infect safe laboratory
strains of bacteria can be safely and readily cultivated
and studied in secondary school classrooms, as they only
infect the bacterial host and thus pose no human risk of
infection. The laboratory processes involved in collaborative bacteriophage research projects are
u adaptable to many educational levels and settings;
u relatively inexpensive and rapidly accomplished;
u linked to the historical roots of molecular biology;
u well-suited for introducing the diversity of microbes and viruses and the importance of their
interactions; and
u can be inquiry-based, especially if students are
encouraged to ask questions about environmental variables and contribute to the design
of the field investigation and laboratory experimental processes.
Bacteriophage projects and similar classroom investigations are readily aligned with state and national science education standards (NRC 1996) and benchmarks
(AAAS 1993) for secondary education. The use of
university electron microscopy resources complements
the bacteriophage projects, but a lack of access to these
specialized facilities should not prevent well-prepared
and confident teachers from conducting such projects in
their classrooms.
The impact
Bacteriophages have long been used as safe viral models
in research and in the classroom. TEMs, such as those
shown in Figures 1, 2, and 3 (pp. 34–35), have made the
unseen world of viruses a reality to students and teachers
in our project. While electron microscopy must be done
in the university laboratory, most other procedures are
part of the high school classroom investigations facilitated
by the teams of GK–12 fellows. Through the combination of isolating the virus from the environment, seeing
the virus in electron micrographs, and working with the
viral DNA, students learn a variety of fundamental lessons in basic microbiology, virology, and molecular biology. In addition to the interest and understanding gained
through hands-on field and classroom laboratory experiences, students’ science experiences are also enriched
through the interactions with the fellows and university
research staff.
The hands-on research context allows students to
“combine processes and scientific knowledge as they use
scientific reasoning and critical thinking to develop their
understanding of science” (NRC 1996, p. 105). In observations by teachers, fellows, and others, it is frequently
noted that even students who do not typically excel academically are highly engaged in the active research process. This is in keeping with results obtained through a
April/May 2009
37
widely used scientific attitude inventory (Moore and Foy
1997) that was taken by almost 200 students working
with Maine ScienceCorps fellows. While only 16% said
they would like to study science and even fewer (6%)
said they would like to be a scientist, almost 27% said
that working in a lab would be fun. Thus it seems likely
that opportunities to enjoy active laboratory-based scientific inquiry could contribute to some students becoming
more interested in science.
Evaluation and assessment
The professional external team evaluating the Maine
ScienceCorps project focuses much attention on the
partnership’s benefit to the communication and teaching
skills of the graduate fellows, as well as the classroom
laboratory activities and resources for participating
teachers. In twice yearly surveys, both fellows and teachers usually report benefits of student learning, engagement, and aspirations.
It is primarily the high school teachers who formally
assess student learning throughout the experimental process. Teachers integrate the laboratory research process
into their curriculum with careful alignment to state and
national standards and use a variety of assessment tools,
including student journals, pre- and posttesting, and student presentations.
A key opportunity for university faculty to view
student achievement is at a research poster presentation event that occurs on the USM campus every spring.
Student posters provide an opportunity for a summative
view of what was learned through the research experience, including misconceptions that may remain.
Beyond students’ evident enthusiasm and the sense
of ownership they convey to the USM community, their
schools, and their own communities, the projects seem
to be particularly valuable to students from remote rural areas. These students often have limited exposure
to scientific role models and, in some cases, to cultural
expectations that higher education is important for their
future success. USM bioscience graduate students also benefit from
working with these students. They experience interdisciplinary research and education collaboration beyond
the focus of their graduate research and make significant
gains in both broad scientific knowledge and in communication skills. Faculty mentors of the GK–12 fellows
are stimulated to find more cross-disciplinary connections
with their colleagues and new opportunities for communication and collaboration with regional high school
science educators.
Connecting with the scientific community
While many university scientists work in demanding
research environments that consume much of their
time and effort, most realize that the preparation of
38
The Science Teacher
their students is directly linked to the quality of precollege education, and many are making efforts to
enrich secondary science education as a result (Goodman 2002). Scientific professional societies such as the
American Society for Microbiology (ASM), Microscopy Society of America (MSA), and American Chemical
Society (ACS) consistently work to make educational
resources in their disciplines widely available (see “On
the web”). To address the micro- and nanoscale education imperatives described in this article, MSA offers
online resources through Project Micro, and ASM
provides Microbe World and, at a modest cost, Microbe
Library (see “On the web”).
Research-funding agencies, most notably NSF, offer incentives for scientists to extend the educational
impact of their research efforts beyond the university
and their scientific communities. As part of building
the vitality of the national scientific workforce and
the professoriate of the future, NSF has created the
GK–12 and other programs that specifically seek to
encourage interdisciplinary interaction and enhance
the communication and teaching skills of graduate students in science, technology, engineering, and
mathematics through close collaboration with K – 12
educators. The collaboration outlined in this article
illustrates the experience of one GK–12 project from
among over 200 that NSF has funded across the nation
since the program began in 1999. Teachers who are
interested in incorporating similar programs in their
classrooms should visit the website for more information (see “On the web”).
Recently, the authors have extended the Maine ScienceCorps to also provide virology, microbiology, and microscopy summer experiences for teachers through support
provided by the National Institutes of Health Science Education Partnership Award (NIH SEPA) program (see “On
the web”). These and other projects supported by government agencies and other organizations provide many opportunities for collaboration and sharing of resources by
scientists and precollege educators.
Finding access to scientific resources and expertise at
universities and other research institutions can enhance the
pedagogical opportunities of secondary school teachers and
thus enrich the learning of precollege students. University
graduate students and faculty gain new insights into how
students learn scientific concepts through collaborations
with master science teachers. High school students who
have worked collaboratively with members of a university
scientific community are often better informed of future
opportunities and better prepared for successful transition
to the learning environments found in higher education. n
S. Monroe Duboise ([email protected] ) is an associate professor of applied medical sciences, Karen D. Moulton (kmoulton@
usm.maine.edu) is virology laboratory manager and TEM facility
C o l l a b o rat i o n at t h e N a n o s c a l e
director, and Jennifer L. Jamison ([email protected]) is a
research associate and electron microscopist, all at the University
of Southern Maine in Portland.
On the web
ASM: www.asm.org
ACS: https://portal.acs.org/portal/acs/corg/memberapp
GK–12 project: www.nsfgk12.org
MSA: www.microscopy.org
Microbe World: www.microbeworld.org
Microbe Library: www.microbelibrary.org
NIH SEPA: www.ncrrsepa.org
Project Micro: www.msa.microscopy.org/ProjectMicro/PMHomePage.html
Acknowledgments
The Maine ScienceCorps GK–12 project has been supported by
NSF grants DGE-0086341 and DGE-0440560. The integration of
molecular virology research projects into graduate education in the
USM Department of Applied Medical Sciences has been supported
by NSF CAREER award MCB-0093347. Summer experiences in
virology, microbiology, and light and electron microscopy for teachers are now included in an NIH SEPA project funded by National
Center for Research Resources (NCRR) grant 1R25RR024280-01.
Bacteriophage ecology research development at USM was also supported by a USM Faculty Senate Research Award to S.M. Duboise.
The TEM capabilities of the laboratory were established in March
2006 with combined NSF (CNS-0521262) and USM support. The
work of GK–12 fellows Jennifer Walker and Eric Hazelton in facilitating bacteriophage isolation in northern Maine high schools is
gratefully acknowledged.
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