Nematode Learning and Behavior: Part 1
In this first procedure of a two-session experiment, students will observe the morphology and behavior of
individual Caenorhabditis elegans from two strains (one wild type and one mutant) and begin an
experiment on the genetic basis of learning and behavior.*
CONTENTS
1
2
Objectives ............................................................................................................................................. 2
1.1
Experimental Goal ........................................................................................................................ 2
1.2
Prerequisite skills and Knowledge ................................................................................................ 2
1.3
Research Skills .............................................................................................................................. 2
1.4
Learning Objectives ...................................................................................................................... 2
Pre-Experiment ..................................................................................................................................... 2
2.1
OP50-pBAD Bacteria and Ampicillin Resistance ......................................................................... 2
2.2
Multicellular Model Organisms .................................................................................................. 2
2.3
C. elegans: An Introduction ......................................................................................................... 3
2.3.1
C. elegans: Reproduction .................................................................................................... 3
2.3.2
C. elegans: Lifecycle ........................................................................................................... 4
2.3.3
Thawing of C. elegans ......................................................................................................... 5
2.3.4
Growth after Thaw ................................................................................................................ 5
2.4
C. elegans Subculture................................................................................................................... 6
2.4.1
2.5
3
Standard Microbiological Practices ...................................................................................... 6
Prepare Laboratory Notebook ....................................................................................................... 7
Laboratory Manual................................................................................................................................ 8
3.1
Materials Check Off List............................................................................................................... 8
3.2
Safety and Waste Disposal Protocols............................................................................................ 8
3.3
Sterile Technique .......................................................................................................................... 8
3.4
Experimental Procedure ................................................................................................................ 9
3.4.1
C. elegans observation ......................................................................................................... 9
3.4.2
C. elegans subculture ........................................................................................................... 9
3.4.3
Create a Figure Showing C. elegans Larval Stages ............................................................ 10
*
Portions of this exercise derived from Bio-Rad Biotechnology Explorer C. elegans Behavior Kit (Catalog #1665120EDU) Instruction Manual, Planning Guide, and Neurology Supplement.
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2|Nematode Learning: Part 1
1
1.1
OBJECTIVES
EXPERIMENTAL GOAL
Students will subculture wild type and mutant C. elegans and create a figure showing the larval stages of
C. elegans development.
1.2
PREREQUISITE SKILLS AND KNOWLEDGE
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1.3
Students should be familiar with how to make solutions from a concentrated stock.
Students should know standard microbiological procedures.
Students should know how to use ImageJ to make basic length and area measurements.
RESEARCH SKILLS
After this lab, students will have had practice in:
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1.4
Standard microbiological procedures
Preparing solutions
Using a stereo dissecting microscope
Acquiring and calibrating digital microscope images
Using ImageJ
Subculturing C. elegans
LEARNING OBJECTIVES
After this lab, students will be able to:
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Explain the rationale for using ampicillin-resistant E. coli OP50-pBAD.
Explain the benefits of model organisms for biomedical research.
Describe the life cycle of C. elegans.
Culture C. elegans.
2
2.1
PRE-EXPERIMENT
OP50-PBAD BACTERIA AND AMPICILLIN RESISTANCE
You will be using culture dishes that have been inoculated with the OP50-pBAD strain of E. coli,
which is genetically modified to have resistance to the antibiotic ampicillin. Recall that the NGM Lite
agar in the culture dishes contains ampicillin. This prevents most species of bacteria from growing on
the plate, but still lets the E. coli OP50-pBAD thrive, which reduces the chance of contamination by
unwanted bacteria during the experiment. You will learn how to genetically transform bacteria later
in X-Lab 2.
2.2
MULTICELLULAR MODEL ORGANISMS
Scientists use model organisms in basic research because model organisms offer simplified cellular systems
that reproduce quickly, are easy to maintain, and are cost efficient. For example, if a DNA mutation is known
to result in a neurological disorder, more data can be generated using a model organism such as C. elegans,
which reproduces and matures every 2–3 days, rather than waiting for a human child to mature and show
symptoms. Commonly used basic model organisms include C. elegans, Saccharomyces cerevisiae (yeast),
Drosophila melanogaster (fruit fly), Danio rerio (zebrafish), and Mus musculus (mouse).
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Despite the seeming lack of a relationship to human beings, these model organisms have helped
researchers understand the basic cellular machinery underlying a host of human pathologies such as
cancer, neurological disorders, and aging. Furthermore, research studies utilizing model organisms have
helped develop powerful tools used by scientists in applied research that include RNA interference
(RNAi), DNA microarrays, and genome sequencing.
Combined, the scientific knowledge and breakthroughs that have been gleaned through the use of model
organisms have helped to significantly advance the knowledge of basic biology as well as human function
and pathologies. Studies that utilize model organisms often provide the data to both justify and direct cuttingedge therapeutic technologies.
Research on model organisms from prokaryotic (bacteria) to single cell eukaryotic (yeast), multicellular
eukaryotic (fruit flies and worms), and mammalian models (mice and rats) have shown that basic cellular
operating principles are shared between all living things. A finding made in bacteria can shed light on a
biological principle in eukaryotes, while a finding made in C. elegans may shed light on a biologic process in
humans.
Generally, the underpinnings of a basic biologic principle are studied in the simplest organism possible, and
as scientific knowledge is generated, the principles learned are studied in increasingly complex organisms
until we finally examine these principles in humans. The scientific knowledge acquired by studying model
organisms often could not have been acquired by studying humans or other complex mammals due to ethical
considerations, long lifespan, high cost, or, in the case of humans, the lack of a uniform genetic background
and environmental conditions.
2.3
C. ELEGANS: AN INTRODUCTION
C. elegans was selected as an experimental model organism in the early 1960s by the Nobel Prize–winning
researcher Sydney Brenner. C. elegans is a multicellular eukaryotic organism that, like humans, is made up of
specialized cells and complex systems such as neurons, muscle cells, a digestive and excretory system, and a
reproductive system. C. elegans was the first multicellular organism to have its genome completely
sequenced. As a result, its genetics are well understood. And since the human genome has also been
sequenced, we now know that C. elegans and humans share a large number of conserved genes and cellular
mechanisms.
C. elegans is a barely visible, free-living (not parasitic), microscopic roundworm (nematode) that reaches
approximately 1 mm in size when fully grown. This species of nematode has been found and isolated in
temperate soil environments worldwide. In nature, they feed on the bacteria that help to decay decomposing
plant matter. In the laboratory, however, C. elegans is typically fed lab strains of E. coli, and most commonly
E. coli OP50. C. elegans is not disease-causing and is not known to cause harm to any living organisms other
than its prey, bacteria.
C. elegans is a transparent nematode, thus facilitating the study of the cellular differentiation and
developmental fate of each of its cells. An adult C. elegans has about 1,000 somatic cells. The
developmental precursors of every C. elegans cell have been fully mapped by researchers. This deep
knowledge of the cellular developmental fate of the organism facilitated a detailed study of the genetics of
organ development and programmed cell death, for which Sydney Brenner, H. Robert Horvitz, and John
Sulston were awarded the Nobel Prize in Physiology or Medicine in 2002.
2.3.1 C. elegans: Reproduction
There are two C. elegans sexes: hermaphrodite (XX) and male (XO), as shown in Fig. 1. The
hermaphrodites are self-fertilizing. C. elegans hermaphrodites contain both male and female sex organs and
are capable of self-fertilizing to produce viable progeny even in the absence of other C. elegans. When selffertilized, C. elegans will typically lay 300–400 eggs. About 99.9% of the progeny from hermaphrodite
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4|Nematode Learning: Part 1
self-fertilizations become hermaphrodites, but about 0.1% become male. Hermaphroditic C. elegans are
convenient for laboratory use because no husbandry is required to maintain the laboratory strains.
Hermaphrodite
Male
Figure 1: Anatomy of C. elegans
On the other hand, C. elegans males contain only male sex organs. Males can inseminate a hermaphrodite,
which could then go on to lay as many as 1,000 eggs, of which about 50% become males. The ability to
mate males with hermaphrodites is important to researchers studying the function of specific genes and
genetic interactions as they are able to crossbreed C. elegans strains. Males can be visually distinguished
from hermaphrodites by the presence of a specialized tail that is used during mating.
2.3.2 C. elegans: Lifecycle
One of the greatest advantages of working with C. elegans is its short life span of 2–3 weeks. After hatching
from an egg, C. elegans pass through four distinct juvenile developmental stages (L1–L4) before reaching a
fully mature adult stage (Fig. 2). Between developmental stages, C. elegans undergo a molting process in
which the old cuticle exoskeleton is shed, revealing a newly synthesized cuticle. Additionally, C. elegans
increase in size between each developmental stage, getting progressively larger in a stepwise fashion as the
worms develop from L1 to L4 larvae and finally to adults. Growth progresses from approximately 250 µm to
approximately 1,100 µm.
Figure 2: Life cycle of
C. elegans
When they are crowded or don’t have enough food, L1 and L2 C. elegans larvae can enter an alternative
specialized larval stage called dauer. Dauer larvae can be morphologically distinguished from other
developmental stages as they are thinner than normal and their oral orifice is plugged, preventing the
organism from feeding. The dauer larvae are highly stress-resistant and live much longer than regular C.
elegans. Dauer larvae allow C. elegans to be stored on plates for over a month, as long as the plates are sealed
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to prevent moisture loss. Once favorable conditions are encountered, such as a rich feeding environment,
dauer larvae reenter development in the L4 stage, where they continue to develop normally and can live for 2
more weeks.
2.3.3 Thawing of C. elegans
In the previous laboratory session, you received a sample of previously frozen C. elegans a. The sample was
a mixed culture containing all stages of C. elegans development (L1–L4, adults, and eggs) along with E. coli
OP50-pBAD. Typically, adult C. elegans do not survive the freezing process as well as L1–L2 larvae. Thus,
the majority of surviving C. elegans would been mostly larvae.
Figure 3. Image of wild-type C.
elegans after thawing.
2.3.4 Growth after Thaw
C. elegans grow well at room temperature, but E. coli grow best greatest at 37°C. However, even at room
temperature, E. coli will still grow at a faster rate than C. elegans. Therefore, in the days following the initial
thaw of C. elegans, the E. coli lawn will grow thick, potentially obscuring easy visibility of the C. elegans.
A very thick lawn of E. coli is desirable in the days after the thaw to support the nutritional needs of the C.
elegans population as it expands. Since C. elegans will be covered in E. coli OP50-pBAD during this period,
and C. elegans may explore the plate, they will tend to spread E. coli OP50-pBAD throughout the plate as
they move. This seeding of the plate with bacteria is normal and desirable since the growth of E. coli OP50pBAD will provide additional food for the C. elegans on the plate. As the C. elegans consume the E. coli,
the E. coli lawn will become thin and the growing C. elegans population will become apparent.
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Fig. 4. Image of wild-type C.
elegans. The nematodes are
seen in a thick E. coli OP50pBAD lawn 3 days after thaw.
2.4
C.
ELEGANS
SUBCULTURE
C. elegans will be overcrowded and may potentially exhaust their food supply after one week in culture. In
this procedure you will need to subculture the C. elegans to ensure that the C. elegans population is not
overcrowded and has an optimal feeding environment with fresh E. coli OP50-pBAD.
The day before this lab, NGM Lite culture plates were inoculated with E. coli OP50-pBAD and these
plates were placed in a 37 °C incubator. The lawn may be quite thin and difficult to see. Thin lawns are
preferred by researchers studying C. elegans as they facilitate visualization.
Fig. 5. Image of wild-type C. elegans
after subculture. As C. elegans move
through the thin E. coli lawns, sshaped tracks are left on the plate.
2.4.1 Standard Microbiological Practices
The E. coli OOP50-pBAD strain you will be working with in the next laboratory exercise is not
pathogenic; that is, it will not cause disease. In addition, this strain has been genetically modified to grow
only on a specially enriched medium. However, when handling E. coli of any kind, standard
microbiological practices must be followed. Even though you will not begin working with bacteria until
the next session, you are expected to adhere to these practices while preparing the culture plates.
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Standard microbiological practices are basic safe laboratory procedures for working with non-pathogenic
biological materials. These practices are outlined below.
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2.5
Wear a lab coat and tie back long hair.
Remove gloves before leaving the lab, touching the face, or keyboards.
Wash hands after removing gloves and before leaving the lab.
Keep work areas clean and uncluttered.
Decontaminate your work surface with 70% ethanol at the beginning and end of the lab class, as
well as after any spill of viable material.
Decontaminate work surfaces before and after completion of day’s work, and after any spill.
Perform procedures in a manner that minimizes the creation of aerosols.
Eating, drinking, and applying cosmetics are not permitted in the work area
Wear protective eyewear and gloves.
PREPARE LABORATORY NOTEBOOK
Read ahead in the lab manual for this experiment, so that you can prepare your lab notebook.
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3
3.1
LABORATORY MANUAL
MATERIALS CHECK OFF LIST
Each small group of (2-3) students will have:
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Two NGM Lite agar plates with fresh (24 h) E. coli OP50-pBAD lawn
Three microcentrifuge tubes, 2 ml
NGM Lite agar plate with C. elegans wild type
NGM Lite agar plate with C. elegans mutant
M9 stock solution (prepared in previous session)
Two disposable plastic transfer pipet (DPTP)
P20 adjustable-volume micropipet and tips
P1000 adjustable-volume micropipet and tips
Marking pen
Scissors
Vernier calipers or ruler
Dissection microscope (10–30x magnification) with digital camera
Laptop with digital camera image acquisition software and ImageJ
Microscope calibration slide
70% ethanol in squirt bottle or spray bottle
The class will have access to:
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Sterile water
Scissors or single-edged razor blades
3.2
SAFETY AND WASTE DISPOSAL PROTOCOLS
Ampicillin or Penicillin Allergies - Ampicillin is a member of the penicillin family of antibiotics. If you
have allergies to penicillin or to any other member of the penicillin family of antibiotics, you should avoid
contact with the agar in the culture dishes containing NGM lite. If you have such an allergy, notify your
instructor.
Standard Microbiological Practices - You will be working with transformed E. coli OP50 bacteria.
Follow the standard microbiological practices, as outlined in the pre-experiment section above, including
the use of protective eyewear, gloves, and a lab coat.
Waste - Place all solutions and components (including pipet tips) that come into contact with bacteria and C.
elegans into in a fresh 10% bleach solution for at least 20 min for sterilization. A shallow pan of this solution
will be available at a common workstation. Sterilize petri dishes by covering the agar with 10% bleach
solution. Let the plate stand for 1 hour or more, and then pour excess plate liquid down the drain. Once
sterilized, the agar plates can be double bagged and treated as normal trash. Wear safety glasses and gloves
when using bleach solutions.
3.3
STERILE TECHNIQUE
With any type of microbiology technique (such as working with C. elegans), it is important not to
introduce contaminating bacteria (or mold and other fungi) into the experiment. Because contaminating
bacteria are ubiquitous and are found on fingertips, benchtops, etc., it is important to avoid these
contaminating surfaces. Follow standard microbiological practices throughout the procedures and when
you are working with the pipets and agar plates, the tip of the pipet and the surface of the agar plate
should not be touched or placed onto contaminating surfaces.
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3.4
EXPERIMENTAL PROCEDURE
3.4.1 C. elegans observation
1. Familiarize yourself with the operation of the stereo dissecting microscope. Note that it can
provide trans-illumination (light from the bottom), epi-illumination (light from the top), or both,
and that it has two magnification settings.
2. Place the calibration slide on the microscope stage, and adjust the oculars so that you can focus
easily on the scale bars on the slide.
3. Use the scale bars to estimate the area of the microscope’s field of view at each of the two
microscope magnifications.
4. Place the culture dish containing the wild type C. elegans on the microscope stage. Keep the lid
on the culture dish at all times.
5. Experiment with the microscope illumination, focus and magnification to find the settings that
allow you to best observe the C. elegans.
6. Now also observe the mutant C. elegans.
Q1. Describe the appearance of the E. coli remaining on each plate.
Q2. Do you see C. elegans at a variety of larval stages on each plate?
Q3. Can you find any eggs on either plate?
Q4. Do the mutant and wild-type C. elegans look the same?
Q5. Provide an estimate of the number of wild type and mutant C. elegans on each plate.
Q6. Explain the strategy you used to determine the preceding estimate.
3.4.2 C. elegans subculture
1. Label one NGM Lite agar subculture plate (containing E. coli OP50-pBAD lawn) “Wild Type” and
the other “Mutant.” (Remember to label the bottom of the plate.) Add your section number and
group ID to the label.
2. Label one microcentrifuge tube “Wild Type” and the other “Mutant.”
3. Record your observations of what the E. coli OP50-pBAD lawns look like. Observe qualities such
as whether the lawn is continuous, its color, whether it is transparent or translucent, and whether
the agar surface is fully covered.
4. Using a clean 2 mL centrifuge tube, your M9 stock, and sterile water, prepare 2 mL of 1X M9
buffer.
5. Sterilize the edge(s) of a clean single-edged razor blade or scissors with
70% alcohol, then cut the ends off two 20 µl pipette tips to make larger
openings, as shown at right.
Q7. Why should you avoid touching the ends of the pipette tips with your fingers?
What should you do if you happen to touch the tip with your finger?
6. Using the P1000 micropipette and a clean tip, transfer 1 ml of C. elegans M9
buffer (1x) to the NGM Lite agar plate containing wild-type C. elegans.
7. Rotate the plate to cover the entire surface with the buffer.
8. Incubate the plate at room temperature for 30 sec.
9. Tilt the plate containing the wild-type C. elegans at a 45° angle to allow the worms in solution to
collect on one side of the plate.
10. Transfer the wild-type C. elegans in solution into the microcentrifuge tube labeled “Wild Type.”
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11. Allow C. elegans in the microcentrifuge tube to settle for 2 min.
12. Using one of the 20 µL tips that you trimmed, transfer 10 µl of the C. elegans pellet to the
appropriately labeled plate containing a lawn of E. coli OP50-pBAD bacteria for food.
13. Cover the plate.
14. Using clean 1000 andn 10 µL tips, repeat the preceding steps to subculture the mutant C. elegans.
15. Place the two culture dishes in safe location on your benchtop.
3.4.3 Create a Figure Showing C. elegans Larval Stages
1. Place the calibration slide on the microscope stage and practice viewing the slide at each
magnification.
2. Record an image of the calibration slide at the microscope’s high magnification setting.
3. Using the wild type C. elegans culture dish, record an image of at least one C. elegans at each
larval stage. If you see a male, record an image of it, too.
4. When you are done recording images, place the culture dish back in a safe place.
5. In ImageJ, open the image file of the calibration slide. Save the image under a NEW file name.
This way, if you need to access the original image again, you can do so.
6. Select the straight line tool. Draw a horizontal line exactly over the appropriate part of the
calibration scale. Record the number of pixels that is equivalent to the length of scale bar
(displayed on the bottom of the small ImageJ toolbar).
a. Click Analyze > Set scale
b. Click “Known distance” and enter the value for the scale bar.
c. Set “unit of length” as appropriate.
d. Click “OK”.
7. Open one of your C. elegans images.
8. Select a segmented line tool (right-click to select the line type from the line selection button).
Note that information about the line is displayed on the status bar.
9. Determine the length of the worm of interest. You can select Analyze → Measure (or Ctrl+M) to
record a line length and Edit → Draw (or Ctrl+D) to make a line permanent.
Q8. How many segments should you use to obtain an accurate measurement? Explain your answer.
10. Repeat the preceding step with the freehand line tool.
Q9. Which do you believe is more accurate, the line segment tool or the freehand tool? Explain your
answer.
11. Use the rectangular area selection tool to surround an area on the image. After selection, use the
menu commands (or the standard Windows keyboard shortcuts) to copy the selection to a Word
or PowerPoint document.
12. Repeat the preceding steps to create a labeled figure that shows each of the larval stages, creating
a life cycle diagram of C. elegans. Be sure to include a scale bar in the figure (similar to the
figures you used in the Bite Force laboratory experiment).
13. In the figure legend, indicate the length of each worm the figure.
14. If you have time, repeat the preceding procedure to create a similar figure for the mutant C.
elegans (switch tasks with your group partner).
15. When you are done, place the two C. elegans culture dishes in the location designated for your
section, then submit your C. elegans life cycle figure(s) to your instructor.
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