Exploring the Infectious - EDVOTEK · Exploring the Infectious ... the infection of E.coli by...

209.150304

209Edvo-Kit #209

Exploring the Infectious Nature of VirusesExperiment Objective:

This experiment explores the life cycle of viruses using classroom-safebacterial viruses. Students will use the viral plaque assay to visualizethe infection of E.coli by Bacteriophage T4 and determine the viral titer.

See page 3 for storage instructions.

SAMPLE LITERATURE

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Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures Experiment Overview 8 Using the Plaque Assay to Determine Viral Titer 9 Experimental Results and Analysis 11 Study Questions 12 Instructor's Guidelines 13 Pre-Lab Preparations 14 Experiment Results and Analysis 16 Study Questions and Answers 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.

Table of Contents

EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209

1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com

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Duplication of any part of this document is permitted for non-profi t educational purposes only. Copyright © 2015 EDVOTEK, Inc., all rights reserved. 209.150304


Experiment Components

REAGENTS & SUPPLIES Storage Check (√ )

• E.coli BactoBeads™ 4°C with dessicant ❑• Bacteriophage T4 ViroBeads™ 4°C with dessicant ❑• ReadyPour™ Luria Broth Agar Room Temp. ❑• COLORTOP™ Agar Room Temp. ❑• 1X Phosphate Buffered Saline (PBS) Room Temp. ❑• Luria Broth Room Temp. ❑• 15 ml screw-cap conical tubes Room Temp. ❑• 1.5 ml snap-top microcentrifuge tubes Room Temp. ❑• Sterile loops Room Temp. ❑ • Petri Plates (large) Room Temp. ❑• Wrapped 10 ml pipet (sterile) Room Temp. ❑

Experiment #209 is designed for 10 lab groups.

• 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 admin-istered to or consumed by humans or animals.

Requirements (not included with this kit)

EXPLORING THE INFECTIOUS NATURE OF VIRUSESEDVO-Kit 209

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WHAT IS A VIRUS?

In the late 1800’s, scientists were working to charac-terize the means through which microbes could cause disease. While some diseases like cholera, tuberculosis and anthrax could be explained by the presence of specifi c bacteria, other diseases, like rabies, could not. As such, these agents were considered to be a “bio-logical chemical”; accordingly, they were named virus, from the Latin word that refers to poison or other venomous compounds. At this time, scientist Marti-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 confi rmed 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 sur-rounded by a protective protein coat. As such, viruses rely entirely on a host organism for basic biological func-tions, 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 take over the translation machinery of its host organism. Early experiments characterizing the life cycle of viruses contributed to early discover-ies in molecular biology, lending valuable insights to the processes of DNA replication, transcription, 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 re-sponse 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 infl uence the behavior of the host cell, transforming a nor-mal cell into a cancer cell. Moreover, the effects of emerging viruses like Ebola or certain strains of Infl uenza on their human host can be deadly.

Background Information

Figure 1: Structure of Viral Capsids

Class Genome Envelope? Example of Disease

Adenovirus

Herpesvirus

Parvovirus

Reovirus

Flavivirus

Filovirus

Retrovirus

dsDNA

dsDNA

ssDNA

dsRNA

ssRNA

ssRNA

ssRNA

Table 1: Viruses that Affect Human Health

No

Yes

No

No

Yes

Yes

Yes

Respiratory Infection

Chicken Pox, Shingles

Rashes

Diarrhea

West Nile Virus

Hemorrhagic fever

Human Immunodeficiency Virus

Capsid

Helical(Tobacco Mosaic Virus) Icosahedral

(Bacilliform Virus)Complex virus

(T4 Bacteriophage)

Prolate(Adenovirus)

Envelope(HIV virus)



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Viruses are classifi ed by the nucleic acid composition of their genome and by whether the nucleic acid is single-stranded 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 fi ve 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 exam-ple, 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 differ-ent 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 fi ve steps: Attachment, Penetration, Replication, Assembly, and Release (summarized in Figure 2).

Attachment occurs when the T4 phage binds to specifi c 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 DNA. The phage recycles the nucleotides from the host DNA to replicate its own DNA. The phage also uses the host cell machinery to produce important proteins, in-cluding components of its protein coat. These newly 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 surround-ing environment. Release depends on the produc-tion of the viral protein holin, which creates pores in the bacterial cell mem-brane, and lysin, which degrades the bacterial cell wall. This damage encourages extracellular

Figure 2 - The Lytic Cycle of T4 Phage

1. Attachment

2. Penetration

3. Replication4. Assembly

5. Release Viral DNA released

T4 Phage

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Figure 3 - Timeline of Phage Infection

fl uid 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).

In contrast to the lytic cycle, viruses that replicate via the Lysogenic Cycle survive in the host cell without causing any immedi-ate harm. Instead, temperate phages like bacteriophage lambda (or phage λ) inte-grate their genome into that of the host 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 quies-cent 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 initi-ated (Figure 4).

Virus Quantification Using the Plaque Assay

Viral quantifi cation tech-niques 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 infec-tions on the host organ-ism. The multiplicity of infection (MOI) is calculat-ed before performing the experiment to ensure that enough virus is added to the experiment to infect the entire sample. In industry, viruses are used

Figure 4 - The Lysogenic Cycle of T4 Phage

100

10

1

0.1

10 20 30 40

Total phage

Extracellularphage

Time (minutes)

Average burst size

Eclipse

Latent period Rise period

Bact

erio

phag

e pe

r in

fect

ed b

acte

rium

Virus attaches to cell walland injects viral DNA.

Viral DNAforms a circle.

Occasionally, the viralDNA separates from thebacterial chromosome,initiating a Lytic cycle.

Viral DNA inserts itself into the bacterial chromosome.

Bacterium reproduces normally,replicating viral DNA at eachcell division.

LYTIC CYCLELYSOGENIC CYCLE

New viral DNAand proteins areproduced & assembled.Viruses are released.

Cell and viral DNA arereproduced many times.

Under Normal Conditions

Under AdverseConditions


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Figure 5 - Plaque Assay

to introduce recombinant DNA into mammalian cells to pro-duce 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.

If a virus is lytic, virologists may quantify the number of infec-tious particles present in a sample using the plaque assay (outlined in Figure 5). A monolayer of host cells (pro-karyotic 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 bacte-ria/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 solidifi ed, 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 pre-pared. 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.

10 µl 100 µl100 µl 100 µl 100 µl 100 µl

Virus Stock 10-2 10-3 10-4 10-5 10-6 10-7

990µl 900µlSerial Dilution

Adsorb virus dilutions to cell monolayer.

Plaques develop in the cell monolayer

Fix and stain the cells, count the plaques

Agarose overlay

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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 conjunc-tion 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.

LABORATORY NOTEBOOKS:

Scientists document everything that happens during an experiment, including ex-perimental 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 hy-

pothesis to refl ect this change.

Experiment Overview

Wear gloves and safety goggles



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Using the Plaque Assay to Determine Viral Titer

1. LABEL four 1.5 ml microcentrifuge tubes as follows: 1:10, 1:100, and 1:1000. 2. ADD 180 μl of PBS to each of the labeled microcentrifuge tubes.3. 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.

4. LABEL fi ve agar base plates as follows: CONTROL, 1:10, 1:100, and 1:1000.

Wear gloves and safety goggles

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 Dilutions1:10 1:100 1:1000

180µl PBS 180µl PBS

Virus solution

180µl PBS

20µl 20µl 20µl

Control 1:10 1:100 1:1000

LabelPlates

Serial Dilution

5. PREPARE the control sample. ADD 100 μl of bacterial growth to the COLORTOP™ Agar tube labeled CONTROL.6. ROLL agar tube between your palms for 2-3 seconds to mix the bacteria with the agar. 7. Quickly POUR the mixture onto the pre-warmed plate labeled CONTROL. 8. 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.

Control

Roll 2-3 sec.

100µlBacterial growth

COLORTOP™ Control Tube

5. 6. 7. 8.Circular motionQuickly

Pour

WARNING!Bacteria and Phage must be added, mixed, and poured quickly to prevent hardening of the COLORTOP™ Agar.


9




Using the Plaque Assay to Determine Viral Titer

9. 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.

10. ROLL agar tube between your palms to mix the agar for 2-3 seconds to mix the bacteria and the virus with the agar.

11. Quickly POUR the mixture onto pre-warmed agar plate labeled 1:10. 12. 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..

13. 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 specifi cs.)

14. ALLOW the soft agar to harden for at least 10 minutes, keeping the plates in an upright position.

15. Once the agar has solidifi ed, STACK the plates on top of one another and TAPE them together. LABEL the plates with your initials or group number.

16. PLACE the plates in an inverted position (agar side on top) in a 37° C incuba-tor overnight (24 hours).

17. 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.

1:10

Roll 2-3 sec.

100µlBacterial growth

COLORTOP™ 1:10 Tube

9. 10.Quickly

Pour

11. 12.Circular motion

100µl

1:10Virus Dilution

13. 14.REPEAT Invert & Incubate

Steps 9-12 for 1:100

1:1000

15. 16.10min.

24hrs.

37°C


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Experimental Results and Analysis

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:

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.


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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 COLOR-TOP™ 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).



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Instructor's Guide

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INSTRUCTOR'S GUIDEEDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES

ADVANCE PREPARATION:

What to do: Time Required: When?

Prepare LBAgar Base Plates

One hour 2-7 days before use

Prepare BacteriaHost Culture

16-20 hours

Night beforethe lab

Aliquot PBS 15 minutesDay beforethe lab

DAY OF THE EXPERIMENT:

Equilibrate waterbath at 60° C and incubator at 37° C

10 minutes1.5 to 2 hoursbefore performingthe experiment

Warm LB agar platesin 37°C incubator 60 minutes

1 hour before performingthe experiment

Preparation ofCOLORTOP Agar

1.5 hours 1.5 hours before lab

What to do: Time Required: When?

Preparation of VirusStock Solution

15 minutes Day of the lab

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.

Wear Hot Gloves and Goggles during all steps

involving heating.

1. BREAK solid ReadyPour™ LB Agar into small chunks by vigorously squeezing and shaking the plastic bottle.

2. 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 heat-ing may cause the bottle to break or explode.

3. 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).

4. COOL the ReadyPour™ Agar to 60°C with careful swirling to promote even dissipation of heat.

5. POUR 10 ml of the cooled ReadyPour™ Agar into each of the large petri dishes using a 10 ml pipet and pipet pump.

6. COVER and WAIT at least twenty minutes for the LB-agar plates to solidify. For optimal results, leave plates at room temperature overnight.

7. STORE plates at room temperature for no more than two days. Plates should be in-verted and placed in a sealable plastic bag to ensure that they do not dry out.

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 experi-ment to work properly.

• 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 fl ame across the surface of the medium.

• Cover the petri plate and allow the medium to solidify.

Quick Reference: Pouring LB Agar Plates

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.

:601. 3. 4. 5.2.

Agar

Loosen

Agar 60°C10 ml

Large source plates


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INSTRUCTOR'S GUIDE EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209

Pre-Lab Preparations - Advance Preparation

Preparation of Bacterial Host Culture

1. Using a sterile loop, ADD two BactoBeads™ to 10 ml of LB in a 50 ml screw-cap tube.

2. INCUBATE for 16-20 hours in 37°C incubator. 3. DISPENSE 600 μl of the bacterial culture into ten 1.5 ml snap-top

tubes.

Pre-Lab Preparations - Day of the Experiment

COLORTOP™ Agar Preparation

1. 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).

2. 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. Using a sterile disposable loop, ADD 5 phage beads into 10 ml sterile PBS in a 15 ml conical tube.

2. VORTEX thoroughly. This is the 1X stock solution.3. ALIQUOT 100 μl for each group.

PBS Preparation

1. LABEL ten 1.5 mL snap-top microcentrifuge tubes with 1X PBS. 2. ALIQUOT 1 mL 1X PBS into the microcentrifuge tubes. Each group

will receive one.

Each Group Should Receive:

4 LB-agar Base Plates, pre-warmed at 37° C4 15 mL Conical tubes containing 5 mL

COLORTOP™ Agar3 1.5 mL snap-top microcentrifuge tubes1 1.5 mL snap-top microcentrifuge tube

containing 1 mL 1X PBS1 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

15



INSTRUCTOR'S GUIDEEDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES

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)

50 plaques

0.01 (equals 1:100 dilution) X 0.1 mL of the diluted virus

= PFU per mL

= 50,000 PFU per mL

Experiment Results and Analysis


16


INSTRUCTOR'S GUIDE EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209

Please refer to the kit insert for the Answers to

Study Questions

A EDVOTEK® Troubleshooting Guide

Appendices

Safety Data Sheets can be found on our website: www.edvotek.com/safety-data-sheets


18


APPENDICES EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209

Appendix AEDVOTEK® Troubleshooting Guides

PROBLEM: CAUSE: ANSWER:

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.

The COLORTOP™ agar remains liquid for a longer period of time when the base plate is heated, making it easer to spread the agar.

Bacterial lawn does not form.

Plaques do not form.

Excessive heat inactivated the virus or killed the bacteria.

The COLORTOP™ agar should be cooled to 60°C before adding the phage/bacteria mixture.

Plaques concentrated in one area of the plate. COLORTOP™ agar was not spread evenly

across the surface of the plate.

Plaques are all different shapes and sizes.

Natural VariationAlthough the majority of plaques should be round, they can take differnent shapes. Count all distinct regions of clearing regardless of the shape and size.

Lumpy, bubbly and/or uneven overlay

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 intocontact with a cold base plate. This makes it difficult to evenly spread the agar.

Base plate was not properly warmed before pouring the COLORTOP™ agar.

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APPENDICESEDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES

Exploring the Infectious - EDVOTEK · Exploring the Infectious ... the infection of E.coli by...

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