Lesson 2: Isolating a Potato Killer
Funeral For Famine Victim
“Isolating a potato killer” is a lesson designed to have students use Koch’s postulates to demonstrate the causal relationship between microbe and disease by transmitting P. infestans from an infected potato tuber to a healthy potato specimen. In this lesson, students will use laboratory technique and inquiry to determine how the plant pathogen (the oomycete P. infestans) infects its host (the potato) as a part of the module “CSI Dublin: The Hunt for the Irish Potato Killer.” “CSI Dublin: The Hunt for the Irish Potato Killer” is an integrated project incorporating science concepts of earth science, biology, biotechnology and art to allow students to apply their understandings of epidemiology and plant pathology into the historical context of science and human health. This unit plan would be best used after previous units or discussions on DNA, biotechnology, taxonomy, bacteria, viruses, plants, protists, fungi and immunity.
- The learner will employ the scientific method to inquire how pathogens infect their hosts and disrupt their normal life plant processes.
- The learner will use Koch’s postulates to understand the interplay of infection and disease.
- The learner will isolate P. infestans from a plant sample using aseptic laboratory techniques.
Curriculum Alignment – National
- Content Standard A: “As a result of activities in grades 9-12, all students should develop Abilities necessary to do scientific inquiry, Understandings about scientific inquiry”
- Content Standard C: “As a result of activities in grades 9-12, all students should develop understanding of: The Cell, Interdependence of organisms, Behavior of organisms ”
- Content Standard E: “As a result of activities in grades 9-12, all students should develop Abilities of technological design, Understandings about science and technology”
- Content Standard F: “As a result of activities in grades 9-12, all students should develop understanding of: Population growth.”
Curriculum Alignment – State
- North Carolina Standard Course of Study for Biology, Goal 1
- North Carolina Standard Course of Study for Biology, Goal 3.02
- North Carolina Standard Course of Study for Biology, Goal 4 (all)
- North Carolina Standard Course of Study for Biology, Goal 5.01
Classroom Time Required
This lesson requires 40 minutes of class time. An additional 25 minutes are required 7 days later, Analysis questions may be completed at home or in class at the completion of the entire activity. Pre-activity Teacher prep: Obtain an infected potato tuber to procure infected samples. Pre-activity discussion: 5 minutes depending on student questions. Student Activity: 20 to 30 minutes depending on student ability levels. Post-activity discussion: 5 minutes depending on student questions. 7 days later Activity: 15 to 20 minutes depending on student ability levels. Post-activity discussion: 3 to 5 minutes depending on student questions and clean up.
Students should understand the steps of the scientific method, pathogen, host, plant cell structure (cell walls, guard cells, stomata), taxonomy, ecology, symbiotic relationships (parasitism,) and fungi. They do not need to know any information about P. infestans or Koch’s Postulates as they will learn this within this lesson. Most of this information will be conveyed in the PowerPoint teacher presentation.
- Infected late blight (P. infestans) potato tuber (available for purchase through supply companies or donated by local universities) (See Comments section)
- Sterile probes or needles (or a heat source for sterilization), microscope slide, cover slip(s), sterilized petri dishes, bleach, distilled water, parafilm, sharpie pen, dissecting microscope (or hand lens), compound light microscope, gloves, rubbing alcohol, plastic container with wire cage, paper towel, forceps/tweezers, timer, ruler, small healthy potatoes, sterilized pipette, Cotton Blu dye (or other type of available stain), Sharps container for used needles
- Lab Sheet
- Laminated (optional) student sheets for Day 1 and Day 7 activities.
- Disposal/Cleaning for Potatoes, containers & microscope slides (Bleach solution or autoclave)
- Incubator (optional for autoclave, see bullet item above)
- Students will need access to common laboratory equipment, dissecting microscopes, and compound light microscopes. PowerPoint presentation requires an overhead projector (LCD projector is preferred with internet access for external links to the internet, but not necessary.) Activities
- To open the activity, the teacher should brainstorm disease, and introduce the ideas surrounding Koch’s postulates and the Disease Triangle of plant pathology. Students should be able to relate how scientists determine that certain pathogens are the causative agent of disease. (Presentation is in PowerPoint.)
- The teacher will explain that in order to study a pathogen it must be isolated into a pure culture. Students should brainstorm why they cannot simply take an infected plant from the field and be able to extract one single type of microorganism.
- Using that information, students should create a hypothesis based upon the information from their observations (from lecture) as well as the pre-lab discussion. Make sure all students have recorded their own unique hypotheses before they begin their laboratory activity. (See comments section.)
- Before beginning the lab, the teacher should inform students of proper and safe laboratory techniques. (See Comments section.)
- Have students begin the laboratory protocol (complete with Day One Lab procedure sheets) while circulating around the room answer questions. (See Comments section.)
- Allow students 20 to 30 minutes to work independently on their data collection while monitoring their progress. (See Comments section.)
- As students finish isolating their cultures, make sure the plastic containers are properly wrapped in parafilm and stored in a warm, undisturbed location. (See Comments Section)
- If students finish their data collection early, have them help other students to finish or clean up.
- In the last remaining minutes of class recap on what they have done and if there are any questions. Relate the process of the sandwich technique to how P. infestans infects its host, reproduces within the context of its complete life cycle. Since it takes 7 days for P. infestans to grow, students will not be able to complete their analysis and conclusion questions for homework until later in the week.
- In 7 days, have students retrieve their isolate samples and examine them under the dissecting microscope to prepare stained slides to observe under the light microscope. (Using the Day 7 Lab Procedure sheets.) Make sure students use alcohol swabs to wipe off the eye pieces of the microscopes to prevent any transmission of infectious, communicable eye diseases like pink-eye. (See Comments and Website section)
- Allow students 15 to 20 minutes to prepare slides and record their final data. Have students clean their slides and station before working on their analysis and conclusion questions. If students do not finish in class, they can finish these questions for homework. Monitor students sketches to ensure they are drawing mycelium and not other debris under the microscope. (See Modifications Section)
- In the post lab discussion, in 3 to 5 minutes answer student questions and have them clean and sanitize their lab stations.
- Assure students that only their data questions will be assessed upon accuracy whereas the rest of their score depends on the thought and quality of their responses. Ensure that all students are able to complete the assignment. (See Modifications Section)
- The teacher will score the student responses based upon completion of each step of the scientific method. The data questions should be assessed upon accuracy and quality of student responses. The analysis and conclusion questions should be graded based upon use of critical thinking skills. Appropriate drawings should be compared to actual specimens. (See Websites section)
- For students who have physical disabilities, have teacher prepared slides before class available for them to view underneath the microscope.
- For ESL and ELL students, they may use an online translator such as Babelfish (http://babelfish.yahoo.com/) to translate entire web pages into their preferred language for information about P. infestans as well as Oomycetes. (See Websites)
- Topics: Pathology, Taxonomy, Immunology.
- Pathology/Taxonomy: Students may wish to create a sampling of microorganisms around the school. They can isolate each of these in culture to repeat the experiment and create a dichotomous key. http://nerds.unl.edu/pages/preser/sec/skills/dkeys.html
- Immunology: Students may wish to investigate other pathogens that cause disease and investigate these in a separate experiment. http://pathmicro.med.sc.edu/book/welcome.htm Alternative Assignment
- If this lab is not appropriate for your school circumstances, the American Phytopathological Society (APS) website has an inquiry based lesson plan on Water Molds and other information about Oomycetes that can be used instead of P. infestans. http://www.apsnet.org/education/k- 12plantpathways/TeachersGuide/Activities/WaterMold/lessonplan.htm
- Because P. infestans must be procured before the start of this lab, it should be ordered in advance and may be stored in a moist, cool location wrapped in paraflim. Inoculate tuber samples by taking small cores of infected tissue before this activity to provide enough cultures for all lab groups. Another possibility is baiting with rhododendron leaves from pond water to grow oomycete cultures.
- It is important for students to understand the importance of observations in formulating a hypothesis. Teachers should foster the creative and unique thought process that the hypothesis requires and emphasize that there is no “right or wrong” answer, only supported or not supported.
- Since students are working with pathogens and needles, it is important that they follow proper laboratory safety rules. A proper sharps container should be supplied as well as strict adherence to emergency procedures as prescribed by the Centers for Disease Control and Prevention (CDC.) http://mylist.net/archives/mcoh-eh/2004-July/001156.html
- The idea behind creating an isolate is preventing cross contamination of opportunistic microorganisms. Make sure that students are wearing gloves and other safety gear. Remind students often to clean their implements (with a gas fire or alcohol wash) and/or to use sterilized equipment every time. They should clean their lab bench before, during and after with the 10% bleach solution to prevent contamination, including containers and wire mesh. Paper towels may be autoclaves by shortly microwaving them before the activity.
- The question may arise, that if the since the leaves are infected (due to stomata), then how does it spread to the tuber? Actually, that question still largely remains unsolved, but it is thought that the mycelium may move to infect the tuber or zoospores may spread. http://www.apsnet.org/online/feature/lateblit/chapter1/epidemic.htm
- Make sure the potatoes incubate in a cool (68 degrees is their optimal temperature) well lit environment where they won’t be disturbed. This is the optimal condition for P. infestans growth.
- When students are making their microscope slides, it may be a good opportunity to review proper microscope technique including how to properly place a cover slip on a slide as well as proper adjustment techniques. Failing to do so may cause some students to accidently crack their slides or ruin the lenses of the compound light microscopes.
- Chia-Hui “Julia” Hu, post-doctoral researcher in the department of Plant Pathology at North Carolina State University under the direction of Dr. Jean Beagle Ristaino, professor of Plant Pathology.