Title: Agar Overlays to Enumerate Bacterial Viruses
Grade: 9-12
Subject: Science
Introduction: Virus cells cannot live in their own, because they do not have any metabolic capacity of their own, therefore they can only be grown in other living cells. There are several methods to enumerate viruses. One of these methods is a plaque assay, also known as Plaque Agar Overlays. This method is used for bacterial viruses and is an effective method for doing such counts.
Objective: The students will be able to calculate the amount of viruses in a solution provided. The students will be able to grow a bacterial culture and correctly perform plaque assays of varying dilutions. ESOL students will be able to perform the same objectives.
Teacher Background Knowledge: Viruses are microscopic organisms that can infect bacterial, plants or animal cells. Viruses that infect bacteria are called Bacteriophages, which literally means "bacteria-eaters." They cannot live outside their host. In studying viruses, it is important to know how fast they reproduce and by how much their number increases. Because it is impossible to see them with the naked eye and the obligate requirement for a host, scientists must grow a culture of host cells, which in this case are bacterial cells. Once the culture has grown to a sufficient density, it is used to enumerate the viruses. This is done by diluting the viral stock, mixing it with the bacterial culture in molten (warm) agar (top agar), and pouring onto a sterile agar (bottom agar) plate. When an infected virus is present it will kill the host and the surrounding area of the host cell population, forming a "plaque." A plaque is a clearing formed by the virus on the bacterial "lawn". It is assumed that one viral cell caused one plaque on the plate. By seeing how many plaques there are on the plate, scientists can determine the amount of viruses in the original solution of the environmental sample. The lab exercise uses a marine bacterial host, Lisronella pelagia, and the bacteriophage HSIC (Williamson eral, 2001).
Student Prerequisite Knowledge: Students must have basic knowledge in biology, especially on bacteria and viruses. General knowledge in chemistry is needed as well as upper level math.
ESOL Vocabulary:
Lyse
Plaque
Enumerate/ Enumeration
Bacteriophage
Culture
Medium
Autoclave
Sterile/ Sterilizing
Plate Agar
Top Agar
Inoculate/ Inoculum
Dilute/Dilution
Host Cell
Phage
Teaching Strategies:
Discovery Learning
Chart
Lab Partners
Class Discussions
Independent Research
Assessment Strategies:
Plaques
Student Journals
Data Collection Chart
Connections to other Subjects:
Marine Microbiology, math, scientific notation, exponents, cheese formation, phage therapy
Standards / Benchmarks:
Sunshine State Standards:
-The student describes patterns of structure and function in living things, sc.f.1.3.2, sc.f.1.3.3, sc.f.1.3.5, sc.f.1.3.6, and sc.f.1.3.7
-The student understands the process and importance of genetic diversity, sc.f.2.3.3
-The student understands the competitive, interdependent, cyclic nature of living things in the environment, sc.g.1.3.1, sc.g.1.3.2.
-The student understands the consequences of using limited natural resources, sc.g.2.3.2, and sc.g.2.3.3
-The student uses the scientific processes and habits of mind to solve problems, sc.h.1.3.4, sc.h.1.3.5, and sc.h.1.3.7
The Nature of Science:
Scientific World View
-Scientists assume that the universe is a vast single system in which the basic rules are the same everywhere. The rules may range from very simple to extremely complex, but scientists operate on the belief that the rules can be discovered by careful, systematic study.
-From time to time, major shifts occur in the scientific view of how the world works. More often, however, the changes that take place in the body of scientific knowledge are small modifications of prior knowledge. Change and continuity are persistent features of science.
Scientific Inquiry:
-Investigations are conducted for different reasons, including exploring new phenomena, to check on precious results, to test how well a theory predicts, and to compare different theories.
-Sometimes, scientists can control conditions in order to obtain evidence. When that is not possible for practical or ethical reasons, they try to observe as wide a range of natural occurrences as possible to be able to discern patterns.
The Scientific Enterprise:
-Scientists can being information, insights, and analytical skills to bear on matters of public concern. Acting in their areas of expertise, scientists can help people understand the likely causes of events and estimate their possible effects. Outside their areas of expertise, however, scientists should enjoy no special credibility. And where their own personal, institutional, or community interests are at stake, scientists as a group can be expected to be no less biased than other groups are about their perceived interests.
-Funding influences the direction of science by virtue of the decisions that are made on which research to support. Research funding comes from carious federal government agencies, industry, and private foundations.
Resources/ Materials:
Heating Plate
Sterile Plates
Autoclave
Bacterial Host
1-Gallon Distilled Water
Pipettes
Glass Container
Zip-log Bags
1 Gal. Thermometer
Sterile 25mL pipettes
Phage stock
Shaker (if available)
Incubator (if available)
*Order the below from Fisher Scientific at 1-800-766-7000 or at Fishersci.com
To order online you must be registered.
Difco Marine Broth (Difco #279110), Fisher Catalog #DF0791-17-4. 500G.
Difco Marine Agar (Difco #212185), Fisher Catalog #DF0979-17-8. 500G.
| Teacher | Students |
| Start the lesson by asking the students about the smallest organism there is, what can it be? Once the students have given ideas, introduce viruses to the students. Explain what a virus is and how they live. Then explain that viruses live everywhere, they are all around, including in the oceans. | Give the teacher some ideas about what they think is the smallest organism on the planet. Listen to the teacher explain about viruses and where they live. |
| Explain to the students that viruses cannot live without a host and therefore it is impossible to count viruses without putting them in a host culture. Then explain about plaque assays and what they are used for. | Listen to the teacher talk about plaque assays and viral counts and why they are needed. |
| Give the students the instructions for the experiment and explain them to the students. Review sterile techniques with them and go over the layout of the lab and where tools and materials are. | Go over the instructions of the lab with the teacher and pay attention to where all the tools are and where materials are. |
| Have the students break up into the lab groups and start the experiment. Walk around the classroom and help students who need it. | Do the experiments; each student does two pairs of plates for their experiments, three host plates and three infected plates. |
| When the experiment is done, show the students where to put their plates and explain to the students about their journal entries that they need to start. | Put their plates in the "incubator" and start the first journal entry for the experiment. |
| Make sure that all the students have done their 6 plates and have cleaned up their work areas and have completed their journal entries. Review the important points of the experiment and discuss with the class their thoughts on the experiment and what they think will happen. | Clean up work area and put plates away and finish journal entries. Discuss the experiment and hypothesize what will happen with the plates. |
| Explain to the students that they will be checking their plates the next day and will take notes in their journals on what is occurring and how many clearings there are on each plate. Then explain that once everyone has gotten their counts and have their graphs, the class will discuss what the results are. | Listen to the teacher and prepare for the next day's journal entries. |
Preparing Bottom Agar for Plates - Can be done either as a separate lab period for students or prepared by the teacher or teacher's assistant. Procedure is designed for 15 groups of 2.
- Use Zobell's 2216 Agar to make agar for the plates.
- Make a 1.5% concentration of 2216 Agar using distilled water, (or use ASWJP in place of 2216 Agar). Make up two liters of Agar, (should yield 135 plates).
- Make sure the Agar is completely dissolved, using a hot plate. When Agar is dissolved, separate the Agar into four 1 liter flasks, 500mL in each one.
- Autoclave these for 2 hours.
- When they are finished, put the flasks into a 50°C water bath for 20 minutes or till they are warm to touch.
- Pour warm agar into plates, enough agar to cover the bottom of the plate (15mL), let it sit and cool for about an hour. Each group of students should have 9 plates.
Top Agar
- Make a 1% concentration of top Agar using the 2216 Agar and distilled water, (or use ASJWP in place of 2216 Agar). Make up 400mL of top Agar in a 1 liter flask. This should make up 135 tubes.
- Dissolve the Agar completely using a hot plate.
- Autoclave the Agar for an hour and once it is finished, cool it in a 50°C water bath for 20 minutes.
- Pipette 3mL into sterile tubes, each group should have 9 tubes.
- Let the agar cool and harden in tubes.
Preparation of Listonella pelagia
- Inoculate Listonella pelagia host into 10mL Zobell's broth (or ASWJP) in a 50mL tube and leave in shaker at 100-200 RPMs at 28°C overnight.
- Transfer 10mL into 130mL Zobell's broth. Let shake at 28°C for 2-4 hours. Use immediately.
Viral Plaques
- Heat overlay tubes in a boiling water bath until the agar is melted. Once the agar is melted, put the tubes in a water bath at 50°C. This should be done about a half-hour before actual plate making to ensure the tubes are melted.
- Prepare the phage dilution series. To do this, take 100uL from your undiluted phage stock and put into 900uL sterile Zobell's broth. Mix (vortex) the tube; this is now your 10-1. Take 100uL from this tube and place in a new tube with 900uL sterile Zobell's broth; this is your 10-2 dilution tubes. Plates will be 10-5, 10-6, 10-7, 10-8, the plates will be an added dilution. Repeat, making sure to vortex between dilutions, until you have reached your endpoint for the series, which will be 10-7. Students will use 10-4, 10-5, 10-6, 10-7, (two plates each and one control plate).
- To make the overlay plates, take 1mL from your actively growing host, put in the agar tube, add 100uL from your appropriate phage dilution, and rub the tube between the palms of your hands quickly. Pour the contents of the tube onto the plate, close the lid of the plate, and make figure eights with the plate on the bench top until the overlay completely covers the base. This part must be done quickly as the agar will harden quickly once out of the water bath.
- Let the plates sit for 20-30 minutes before moving them to an incubated area at 28°C, (room temperature) overnight. Plates can be stored upside down.
- Check plates the next day for plaques and record the numbers in journals.
- Do calculation [(# of plaques/0.1mL] x dilutions= phage/mL
10-8
10-9
10-10
10-11
Assessment
The students will be assessed based on their experiments with the plaques, did they do them correctly, and if so, what occurred. Their journals will count toward their grade, as they will show how the students calculated the final viral abundance. The students will also be graded on their graphs of their calculations and participation in the experiment and class discussions.
ESOL students will be assessed in the same way, whether or not they could correctly perform a plaque assay and determine the viral counts. Participation in the experiment and in class discussions will also be assessed.
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