Download Exploring the Infectious Nature of Viruses

SA
Pl M
w ea PL
eb se E
lin re L
I
k fe TE
fo r
R
r c to A
T
i
or n U
re clu RE
ct d
ve ed
rs
io
n.
Edvo-Kit #209
209
Exploring the Infectious
Nature of Viruses
Experiment Objective:
This experiment explores the life cycle of viruses using classroom-safe
bacterial viruses. Students will use the viral plaque assay to visualize
the infection of E.coli by Bacteriophage T4 and determine the viral titer.
See page 3 for storage instructions.
209.150304
EXPLORING
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EDVO-Kit
EDVO-Kit 209
209
Table of Contents
Page
Experiment Components
3
Experiment Requirements
3
Background Information
4
Experiment Procedures
Experiment Overview
Using the Plaque Assay to Determine Viral Titer
Experimental Results and Analysis
Study Questions
8
9
11
12
Instructor's Guidelines
Pre-Lab Preparations
Experiment Results and Analysis
Study Questions and Answers
13
14
16
17
Appendices
18
Safety Data Sheets can be found on our website:
www.edvotek.com/safety-data-sheets
EDVOTEK and The Biotechnology Education Company are registered trademarks of
EDVOTEK, Inc. ReadyPour, COLORTOP, BactoBeads, and ViroBeads are trademarks of
EDVOTEK, Inc.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
2
EDVO-Kit 209
209
EDVO-Kit
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EXPLORING
Experiment Components
REAGENTS & SUPPLIES
Storage
•
•
•
•
•
•
•
•
•
•
•
4°C with dessicant
4°C with dessicant
Room Temp.
Room Temp.
Room Temp.
Room Temp.
Room Temp.
Room Temp.
Room Temp.
Room Temp.
Room Temp.
E.coli BactoBeads™
Bacteriophage T4 ViroBeads™
ReadyPour™ Luria Broth Agar
COLORTOP™ Agar
1X Phosphate Buffered Saline (PBS)
Luria Broth
15 ml screw-cap conical tubes
1.5 ml snap-top microcentrifuge tubes
Sterile loops
Petri Plates (large)
Wrapped 10 ml pipet (sterile)
Requirements
•
•
•
•
•
Check (√ )
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
Experiment #209 is
designed for 10 lab
groups.
(not included with this kit)
37° C incubation oven
Waterbath
Automatic micropipets (5-50 μl, 100-1000 μl) and tips
Pipet pump
White light box (optional)
All experiment components
are intended for educational
research only. They are not
to be used for diagnostic or
drug purposes, nor administered to or consumed by
humans or animals.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
3
EXPLORING
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EDVO-Kit
EDVO-Kit 209
209
Background Information
WHAT IS A VIRUS?
Table 1: Viruses that Affect Human Health
Class
Genome Envelope?
Example of Disease
In the late 1800’s, scientists were working to characterize the means through which microbes could cause
No
Adenovirus dsDNA
Respiratory Infection
disease. While some diseases like cholera, tuberculosis
Yes
Herpesvirus dsDNA
Chicken Pox, Shingles
and anthrax could be explained by the presence of
No
Parvovirus ssDNA
Rashes
specific bacteria, other diseases, like rabies, could not.
As such, these agents were considered to be a “bioNo
Diarrhea
Reovirus
dsRNA
logical chemical”; accordingly, they were named virus,
Yes
West Nile Virus
ssRNA
Flavivirus
from the Latin word that refers to poison or other
Yes
Hemorrhagic fever
ssRNA
Filovirus
venomous compounds. At this time, scientist MartiYes
Human Immunodeficiency Virus
Retrovirus ssRNA
nus Beijerinck was studying the infectious agent that
caused tobacco mosaic disease, a plant disease that
causes widespread crop damage. Through his studies,
Beijerinck proposed that the virus that caused this disease must be much smaller than bacteria, as it could not be
visualized by light microscopy. Furthermore, since viruses could not be cultured in nutrient media, they must only
be able to replicate in their host organism. The theory that viruses were in fact infectious particles was confirmed
in 1935, when Wendell Stanley visualized the tobacco mosaic virus using an electron microscope.
Today, virology is a specialization of microbiology that explores viruses and the diseases that they cause. Studies
have shown that viruses are very simple infectious agents, comprising little more than a DNA or RNA genome surrounded by a protective protein coat. As such, viruses rely entirely on a host organism for basic biological functions, including DNA replication, transcription, and basic metabolism. For example, viruses lack ribosomes, which
are responsible for protein synthesis. Therefore,
for viral RNA to be translated into protein, it must
Helical
take over the translation machinery of its host
Icosahedral
Complex virus
(Tobacco Mosaic Virus)
organism. Early experiments characterizing the
(Bacilliform Virus) (T4 Bacteriophage)
life cycle of viruses contributed to early discoveries in molecular biology, lending valuable insights
to the processes of DNA replication, transcription,
Capsid
and translation.
Furthermore, virology addresses public health
concerns since viral infections impact the health
of their host (Table 1). Some viral infections, like
the common cold, are relatively harmless. The
majority of cold symptoms (fatigue, fever and
body aches) result from the body’s innate response to the infection, not the virus itself. Other
viruses, like Human Papillomavirus or Hepatitis B,
integrate their genes into the host genome. The
proteins coded for by these genes will influence
the behavior of the host cell, transforming a normal cell into a cancer cell. Moreover, the effects
of emerging viruses like Ebola or certain strains of
Influenza on their human host can be deadly.
Prolate
(Adenovirus)
Envelope
(HIV virus)
Figure 1: Structure of Viral Capsids
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
4
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
Viruses are classified by the nucleic acid composition of their genome and by whether the nucleic acid is singlestranded or double-stranded. A special layer of protein called the capsid surrounds and protects the viral genome
(summarized in Figure 1). This protective shell is built from protein subunits called capsomeres that assemble
into three major structural forms: helical, icosahedral and prolate. Some capsids are further surrounded by a lipid
bilayer envelope, which assists with viral entry into the host cell. Other viruses display a combination of two or
more structural forms and are thus categorized as complex. Viral capsids range in size from five to three hundred
nanometers in diameter, making it impossible to visualize most viruses by light microscopy.
Viral Replication Takes Place in a Host Organism
Viruses are not considered to be free-living organisms – all viruses require a host organism to replicate. For example, the viruses known as bacteriophages infect and replicate within bacteria. A typical bacteriophage comprises
an elongated capsid head that encloses the DNA and a protein tailpiece that facilitates attachment of the virus to
the bacterial host. After the bacteriophage injects its DNA into its host, the virus replicates using one of two different methods: the Lytic Cycle or the Lysogenic Cycle.
In the Lytic Cycle, the bacteriophage takes over the cell’s metabolic machinery to reproduce. After building new
phage particles, viral enzymes cause the bacterial cell to break down, or lyse, releasing the virus particles. The life
cycle of a lytic phage like T4 phage progresses through a series of five steps: Attachment, Penetration, Replication,
Assembly, and Release (summarized in Figure 2).
Attachment occurs when the T4 phage binds to specific receptors on the surface of the E. coli bacterium. The
phage then penetrates through the bacterial cell wall and injects its genome, leaving the phage coat behind. At
this time, viral enzymes
break down the host
1. Attachment
DNA. The phage recycles
T4 Phage
the nucleotides from the
host DNA to replicate its
own DNA. The phage
also uses the host cell
machinery to produce
important proteins, including components of its
protein coat. These newly
Viral DNA released
2. Penetration
5. Release
synthesized capsomeres
self-assemble around the
viral genome, resulting in
new viruses.
The last step of the viral
reproduction cycle is the
release of the new phage
viruses into the surrounding environment. Release
depends on the production of the viral protein
holin, which creates pores
in the bacterial cell membrane, and lysin, which
degrades the bacterial
cell wall. This damage
encourages extracellular
4. Assembly
3. Replication
Figure 2 - The Lytic Cycle of T4 Phage
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
5
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
100
Bacteriophage per infected bacterium
fluid to enter the bacterium, swelling and
eventually bursting the cell. Once the
bacterial cell lyses, the new viruses can
infect nearby bacterial cells. The life cycle
of bacteriophage T4, from penetration of
the phage to the release of new phage
particles, takes about 40 minutes (Figure
3).
Total phage
Average
burst size
10
Extracellular
phage
1
10
20
30
40
Time (minutes)
In contrast to the lytic cycle, viruses that
0.1
replicate via the Lysogenic Cycle survive in
Eclipse
the host cell without causing any immediLatent period
Rise period
ate harm. Instead, temperate phages like
bacteriophage lambda (or phage λ) integrate their genome into that of the host
Figure 3 - Timeline of Phage Infection
cell. Similar to the lytic cycle, the lysogenic
cycle begins when the phage attaches to
the host cell and injects its DNA. Instead of immediately forcing the host cell to produce new virus particles, the
injected viral DNA integrates with the bacterium’s chromosome. Each time the host cell divides, the latent viral
DNA (known as a prophage) is also copied and passed into the daughter cell. The viral genome exists in a quiescent state until the cell is exposed to adverse conditions such as exposure to UV or mutagenic chemicals. At that
time, the viral genome
excises itself from the
host cell chromosome
and the lytic cycle is initiated (Figure 4).
Cell and viral DNA are
reproduced many times.
Virus Quantification
Using the Plaque
Assay
Virus attaches to cell wall
and injects viral DNA.
Viral quantification techniques are performed to
determine the number
of viruses in a given
sample. As such, this is
an essential technique in
the academic, industrial
and medical sectors. For
example, virologists in
academic labs study the
effects of the viral infections on the host organism. The multiplicity of
infection (MOI) is calculated before performing the
experiment to ensure that
enough virus is added to
the experiment to infect
the entire sample. In
industry, viruses are used
Occasionally, the viral
DNA separates from the
bacterial chromosome,
initiating a Lytic cycle.
Viral DNA
forms a circle.
Under Adverse
Conditions
Under Normal
Conditions
LYTIC CYCLE
Bacterium reproduces normally,
replicating viral DNA at each
cell division.
LYSOGENIC CYCLE
New viral DNA
and proteins are
produced & assembled.
Viruses are released.
Viral DNA inserts
itself into the
bacterial
chromosome.
Figure 4 - The Lysogenic Cycle of T4 Phage
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
6
EDVO-Kit 209
to introduce recombinant DNA
into mammalian cells to produce biological medications like
the hemophilia drug Factor VIII.
This method of gene transfer,
called transduction, creates
cell lines with the foreign DNA
permanently integrated into
the host cell’s genome. After
purifying the medication, the
pharmaceutical company must
ensure that it is free from viral
contamination. Furthermore,
clinicians will often determine
a patient’s viral load, or the
amount of virus in a patient’s
sample, to determine the
severity of the infection. For
example, the viral load of HIV
patients is regularly monitored
to ensure that the treatment is
effective.
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
10 µl
100 µl
100 µl
100 µl
990µl
Virus Stock
100 µl
100 µl
900µl
-2
10
Serial Dilution
10
-3
10
-4
10
-5
10
-6
-7
10
Adsorb virus dilutions to cell monolayer.
Agarose overlay
Plaques develop in the cell monolayer
Figure 5 - Plaque Assay
Fix and stain the cells, count the plaques
If a virus is lytic, virologists may
quantify the number of infectious particles present in a sample using the plaque assay (outlined in Figure 5). A monolayer of host cells (prokaryotic or eukaryotic) is plated on a solid nutrient medium and infected with the virus. The samples are incubated
for a period of time, during which the virus begins to reproduce within its host. Eventually the host cell will lyse,
releasing the virus from the infected cell into the environment, where they will infect the surrounding cells. This
creates a visible region of clearing called a plaque. The plaques are counted to calculate the initial concentration of
virus particles present within the sample. Each dilution is performed in triplicates to minimize error and to obtain a
more accurate concentration.
In this experiment, we will use the T4 phage and its host organism E. coli to study the dynamics of a viral infection.
The assay is performed using double agar overlay method, which creates a thin, homogenous layer of the bacteria/phage mixture in soft agar on top of a hard agar base. By trapping the microbes in a layer of agar, the virus
is only able to spread to cells in its immediate vicinity. The soft overlay is prepared by melting the agar at 95°C,
then cooling it to 60°C. At this temperature, the agar is warm enough to remain liquid, but cool enough to add
the E.coli/phage suspension without killing the bacteria. The agar and bacterial suspension are mixed and evenly
poured over a hard agar base plate. Once the agar has solidified, the plates are incubated for 24 hours, during
which time the bacteria and the virus grow.
In order to determine the concentration of the virus, or the titer, serial dilutions of the viral stock sample are prepared. Bacterial samples are inoculated with a small volume of each dilution, and assayed using the plaque assay.
The concentration of a bacteriophage stock is determined in plaque-forming units (PFU) per milliliter. To calculate
the virus concentration, the plaques formed on the agar plate are counted and multiplied by the dilution factor. For
a valid phage count, the number of plaques per plate should be between 30 and 300.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
7
EXPLORING
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EDVO-Kit
EDVO-Kit 209
209
Experiment Overview
EXPERIMENT OBJECTIVE:
This experiment explores the life cycle of viruses using classroom-safe bacterial
viruses. Students will use the viral plaque assay to visualize the infection of E.coli by
Bacteriophage T4 and determine the viral titer.
LABORATORY SAFETY
1.
Gloves and goggles should be worn routinely as good laboratory practice.
2.
Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.
3.
DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4.
Exercise caution when using any electrical equipment in the laboratory.
5.
Always wash hands thoroughly with soap and water after handling reagents or
biological materials in the laboratory.
Wear gloves
and safety goggles
LABORATORY NOTEBOOKS:
Scientists document everything that happens during an experiment, including experimental conditions, thoughts and observations while conducting the experiment,
and, of course, any data collected. Today, you’ll be documenting your experiment in a
laboratory notebook or on a separate worksheet.
Before starting the Experiment:
•
•
Carefully read the introduction and the protocol. Use this information to form
a hypothesis for this experiment.
Predict the results of your experiment.
During the Experiment:
•
Record your observations.
After the Experiment:
•
•
Interpret the results – does your data support or contradict your hypothesis?
If you repeated this experiment, what would you change? Revise your hypothesis to reflect this change.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
8
EDVO-Kit 209
209
EDVO-Kit
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EXPLORING
Using the Plaque Assay to Determine Viral Titer
180µl PBS
Serial Dilution
20µl
Virus solution
180µl PBS
20µl
180µl PBS
20µl
1:10
1:100
1:10
1:100
1:1000
1:10
1:100
1:1000
20 µl of
virus
solution +
180 µl PBS
20 µl of
1:10 virus
solution +
180 µl PBS
20 µl of
1:100 virus
solution +
180 µl PBS
Table 2: Making Serial Dilutions
Label
Plates
Control
1.
2.
3.
4.
1:1000
LABEL four 1.5 ml microcentrifuge tubes as follows: 1:10, 1:100, and 1:1000.
ADD 180 μl of PBS to each of the labeled microcentrifuge tubes.
PREPARE serial dilutions of the virus solution using Table 2. For example, to
prepare the 1:10 dilution, add 20 μl of the virus solution to the tube labeled 1:10
containing 180 μl PBS.
LABEL five agar base plates as follows: CONTROL, 1:10, 1:100, and 1:1000.
Wear gloves
and safety goggles
100µl
Bacterial growth
5.
6.
Roll 2-3 sec.
7.
8.
Quickly
Pour
COLORTOP™
Control Tube
5.
6.
7.
8.
Circular motion
WARNING!
Bacteria and Phage
must be added, mixed,
and poured quickly to
prevent hardening of
the COLORTOP™ Agar.
Control
PREPARE the control sample. ADD 100 μl of bacterial growth to the COLORTOP™ Agar tube labeled CONTROL.
ROLL agar tube between your palms for 2-3 seconds to mix the bacteria with the agar.
Quickly POUR the mixture onto the pre-warmed plate labeled CONTROL.
COVER the plate and lift it from the bench. Holding the plate in your hand, gently rock and rotate the plate in
a circular direction. This disperses the COLORTOP™ agar evenly across the surface of the agar plate.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
9
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
Using the Plaque Assay to Determine Viral Titer
100µl
Bacterial growth
9.
10.
100µl
Roll 2-3 sec.
1:10
Virus Dilution
13.
REPEAT
Steps 9-12 for
1:100
1:1000
12.
11.
Quickly
Pour
Circular motion
1:10
COLORTOP™
1:10 Tube
14.
15.
16.
Invert & Incubate
10
min.
37°C
24
hrs.
9.
10.
11.
12.
13.
14.
15.
16.
17.
PREPARE the experimental samples. ADD 100 μl of the 1:10 virus dilution
and 100 μl of bacterial growth to a fresh tube of COLORTOP™ Agar.
ROLL agar tube between your palms to mix the agar for 2-3 seconds to mix
the bacteria and the virus with the agar.
Quickly POUR the mixture onto pre-warmed agar plate labeled 1:10.
COVER the plate and lift it from the bench. Holding the plate in your hand,
gently rock and rotate the plate in a circular direction. This disperses the
COLORTOP™ agar evenly across the surface of the agar plate..
REPEAT steps 9-12 for the 1:100, and 1:1000 virus dilutions. Be sure to work
quickly to prevent the COLORTOP™ agar from solidifying. (Refer to Table 2 for
specifics.)
ALLOW the soft agar to harden for at least 10 minutes, keeping the plates in
an upright position.
Once the agar has solidified, STACK the plates on top of one another and
TAPE them together. LABEL the plates with your initials or group number.
PLACE the plates in an inverted position (agar side on top) in a 37° C incubator overnight (24 hours).
VISUALIZE the control and dilution plates. PROCEED to Experimental Results
and Analysis.
WARNING!
Bacteria and Phage
must be added, mixed,
and poured quickly to
prevent hardening of
the COLORTOP™ Agar.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
10
EDVO-Kit 209
209
EDVO-Kit
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EXPLORING
Experimental Results and Analysis
RECORD your observations in Table 3. Make note of size, color, and appearance of plaques. Be sure to count each
plaque on a dilution plates if possible. Calculate the viral titer using plates that contain between 30 – 300 colonies.
Table 3: Observations
Plate
Observations
Number of Plaques
Control Plate
Plate 1:10
Plate 1:100
Plate 1:1000
Use the appropriate dilution plate to calculate the concentration of viral particles (PFU) using the formula below:
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
11
EXPLORING
EXPLORING THE
THE INFECTIOUS
INFECTIOUS NATURE
NATURE OF
OF VIRUSES
VIRUSES
EDVO-Kit
EDVO-Kit 209
209
Study Questions
1.
What are viruses? How are they different from plants, animals and microbes?
2.
What are bacteriophages?
3.
Name and summarize the two reproduction pathways of bacteriophages.
4.
What is a plaque assay? Why do we need to quantify viruses?
5.
In order to determine the viral titer of a T4 phage solution, serial dilutions of the
sample were prepared. 100 μl of each dilution was mixed with 3 ml of COLORTOP™ agar containing E.coli and poured onto an agar base plate. After a 24-hour
incubation period at 37°C, 150 plaques were observed on the 1:1000 dilution
plate. Using this data, calculate the concentration of the bacteriophage (pfu/ml).
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
12
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
INSTRUCTOR'S GUIDE
Instructor's Guide
ADVANCE PREPARATION:
What to do:
Time Required:
When?
Prepare LB
Agar Base Plates
One hour
2-7 days before use
Prepare Bacteria
Host Culture
16-20 hours
Night before
the lab
Aliquot PBS
15 minutes
Day before
the lab
DAY OF THE EXPERIMENT:
What to do:
Time Required:
When?
Equilibrate waterbath
at 60° C and
incubator at 37° C
10 minutes
1.5 to 2 hours
before performing
the experiment
Warm LB agar plates
in 37°C incubator
60 minutes
1 hour before
performing
the experiment
Preparation of Virus
Stock Solution
15 minutes
Day of the lab
Preparation of
COLORTOP Agar
1.5 hours
1.5 hours before lab
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK, Inc., all
rights reserved.
209.150304
13
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
INSTRUCTOR'S GUIDE
EDVO-Kit 209
Pre-Lab Preparations - Advance Preparation
POUR AGAR BASE PLATES
If plates are prepared 2-7 days before use, they must be stored in a sealed plastic bag at 4°C.
1.
2.
Loosen
3.
:60
Agar
Agar
5.
4.
60°C
10 ml
Large source plates
1.
2.
3.
4.
5.
6.
7.
BREAK solid ReadyPour™ LB Agar into small chunks by vigorously squeezing and
shaking the plastic bottle.
LOOSEN, but DO NOT REMOVE, the cap on the ReadyPour™ Agar bottle. This allows
the steam to vent during heating. CAUTION: Failure to loosen the cap prior to heating may cause the bottle to break or explode.
MICROWAVE the ReadyPour™ Agar on high for 60 seconds to melt the agar. Carefully
REMOVE the bottle from the microwave and MIX by swirling the bottle. Continue to
HEAT the solution in 30-second intervals until the agar is completely dissolved (the
amber-colored solution should be clear and free of small particles).
COOL the ReadyPour™ Agar to 60°C with careful swirling to promote even dissipation
of heat.
POUR 10 ml of the cooled ReadyPour™ Agar into each of the large petri dishes using a
10 ml pipet and pipet pump.
COVER and WAIT at least twenty minutes for the LB-agar plates to solidify. For optimal
results, leave plates at room temperature overnight.
STORE plates at room temperature for no more than two days. Plates should be inverted and placed in a sealable plastic bag to ensure that they do not dry out.
Wear Hot Gloves and
Goggles during all steps
involving heating.
NOTE for Step 3:
Use extra care and make
sure the agar does not
boil out of the bottle. Pay
close attention and stop
the heating if it starts to
bubble up.
NOTE: If plates are prepared more than two days before use, they should be stored inverted in a plastic bag in
the refrigerator (4°C). Remove the plates from the refrigerator and warm in a 37°C incubator for 60 minutes
before use.
On the day of the experiment, place
agar base plates for students in 37°C
incubator to warm for at least one hour
before performing the experiment. The
plates MUST be warm for the experiment to work properly.
Quick Reference: Pouring LB Agar Plates
•
Use a sterile 10 ml pipet with a pipet pump to transfer the
designated volume of medium to each petri plate. Pipet carefully to
avoid forming bubbles.
•
Rock the petri plate back and forth to obtain full coverage.
•
If the molten medium contains bubbles, they can be removed by
passing a flame across the surface of the medium.
•
Cover the petri plate and allow the medium to solidify.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
14
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
INSTRUCTOR'S GUIDE
Pre-Lab Preparations - Advance Preparation
Preparation of Bacterial Host Culture
1.
2.
3.
Using a sterile loop, ADD two BactoBeads™ to 10 ml of LB in a
50 ml screw-cap tube.
INCUBATE for 16-20 hours in 37°C incubator.
DISPENSE 600 μl of the bacterial culture into ten 1.5 ml snap-top
tubes.
Pre-Lab Preparations - Day of the Experiment
Each Group Should Receive:
4 LB-agar Base Plates, pre-warmed at 37° C
4 15 mL Conical tubes containing 5 mL
COLORTOP™ Agar
3 1.5 mL snap-top microcentrifuge tubes
1 1.5 mL snap-top microcentrifuge tube
containing 1 mL 1X PBS
1 1.5 mL snap-top microcentrifuge tube
containing 100 μL Bacteriophage Stock
Solution
1 1.5 mL snap-top microcentrifuge tube
containing 600 μL E. coli culture
• Adjustable volume micropipet
COLORTOP™ Agar Preparation
1.
2.
MICROWAVE the COLORTOP™ Agar on high for 60 seconds to
melt the agar. Carefully REMOVE the bottle from the microwave
and MIX by swirling the bottle. Continue to HEAT the solution in
30-second intervals until the agar is completely dissolved (the
red-colored solution should be clear and free of small particles).
Once melted, DISPENSE 5 ml of agar per 15 ml screw-top conical
tube (4 tubes per group, 40 tubes total).
PLACE tubes of melted agar in 60°C waterbath to keep the agar
from solidifying. The agar MUST be kept warm at all times.
Bacteriophage Stock Solution Preparation
1.
2.
3.
Using a sterile disposable loop, ADD 5 phage beads into 10 ml
sterile PBS in a 15 ml conical tube.
VORTEX thoroughly. This is the 1X stock solution.
ALIQUOT 100 μl for each group.
PBS Preparation
1.
2.
LABEL ten 1.5 mL snap-top microcentrifuge tubes with 1X PBS.
ALIQUOT 1 mL 1X PBS into the microcentrifuge tubes. Each group
will receive one.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
15
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
INSTRUCTOR'S GUIDE
EDVO-Kit 209
Experiment Results and Analysis
Sample Calculation:
The 1:100 dilution plate (above) has 50 plaques. 100 μL of the bacteriophage dilution was added to the E. coli culture.
Average # of plaques
Dilution factor X Volume of diluted virus (in mL)
= PFU per mL
50 plaques
0.01 (equals 1:100 dilution) X 0.1 mL of the diluted virus
= 50,000 PFU per mL
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK,
Inc., all rights reserved. 209.150304
16
Please refer to the kit
insert for the Answers to
Study Questions
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
APPENDICES
EDVO-Kit 209
Appendices
A
EDVOTEK® Troubleshooting Guide
Safety Data Sheets can be found on our website:
www.edvotek.com/safety-data-sheets
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK, Inc., all
rights reserved.
209.150304
18
EDVO-Kit 209
EXPLORING THE INFECTIOUS NATURE OF VIRUSES
APPENDICES
Appendix A
EDVOTEK® Troubleshooting Guides
PROBLEM:
Bacterial lawn does not
form.
Plaques do not form.
Plaques concentrated in
one area of the plate.
CAUSE:
Excessive heat inactivated the virus or
killed the bacteria.
The COLORTOP™ agar should be cooled to 60°C before
adding the phage/bacteria mixture.
Phage/bacteria solution not thoroughly
mixed into the COLORTOP™ agar before
the overlay was poured.
Be sure to spread the COLORTOP™ agar across the entire
surface of the agar base plate by moving the plate in a circular
direction.
COLORTOP™ agar was not spread evenly
across the surface of the plate.
Base plate was not properly warmed before
pouring the COLORTOP™ agar.
Plaques are all different
shapes and sizes.
Lumpy, bubbly and/or
uneven overlay
ANSWER:
The COLORTOP™ agar remains liquid for a longer period of
time when the base plate is heated, making it easer to spread
the agar.
Natural Variation
Although the majority of plaques should be round, they can
take differnent shapes. Count all distinct regions of clearing
regardless of the shape and size.
COLORTOP™ agar was below 60°C when
it was poured on the plate.
COLORTOP™ agar should be warm enough to avoid
hardening while adding to the plate to avoid lumps and
bubbles in agar.
Base plate was not properly warmed before
pouring the COLORTOP™ agar.
COLORTOP™ agar may solidify too quickly if it comes into
contact with a cold base plate. This makes it difficult to evenly
spread the agar.
1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com
Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 2015 EDVOTEK, Inc., all
rights reserved.
209.150304
19