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​Laboratory Exercise Instructor Notes
​Cytology of Fungal Infection


INSTRUCTOR'S NOTE #1

This is the second fungal lab of the semester in my introductory course. This laboratory gives students an opportunity to see the actual microscopic events in the infection process. Understanding these normally unseen and somewhat abstract events gives them a framework for fully comprehending many management issues, including those presented in the questions in Table 3.

My laboratories commonly have 6-8 pairs of students, most of whom have limited experience using a microscope. With that number, I find that having an assistant during the microscopy part is valuable to help students understand what they are seeing.

INSTRUCTOR'S NOTE #2

Large quantities of conidia of P. oryzae are produced by spreading  conidia onto the surface of 1/10-strength oatmeal agar; the addition of 2-3 pieces of autoclaved leaf of perennial ryegrass to a molten medium enhances sporulation. Cultures are maintained at room temperature under 15-25 µE m-2 sec-1 from fluorescent lights; 8-12 days after transfer, conidia are harvested into deionized water and quantified using a hemocytometer. Short-term culture maintenance can be on 1/5 or 1/10-strength Difco oatmeal agar. For storage, isolates are grown on sterile discs of Whatman #1 filter paper overlaid on 1/10-strength oatmeal agar, air-dried, and stored aseptically at -20ºC.

Boiling leaves in the glacial acetic acid/ethanol mixture kills the host tissue and eliminates any induced defense responses. As a consequence, results are more predictable between experiments than they might be with a live host.

Pyricularia oryzae works well for us in Kentucky, because of its economic importance and its readily recognized conidium. However, it should be possible to use any number of host-parasite combinations for this lab. Instructors may wish to experiment with other pathosystems available to them. For those interested in using P. oryzae, I am willing to provide a culture to instructors within the U.S. who obtain the appropriate APHIS permit.

Slides can be made several days in advance and sealed with clear nail polish, which greatly enhances the convenience of this lab to the instructor.

INSTRUCTOR'S NOTE #3

In my laboratory, I assign lab groups to work on either the 1 or 2-hr incubation period, the 7-8 hr period, and either the 14-16 or 24-hr incubation period. This is because time is limited in our lab session to 1 hr 50 min, and this assures that each group will have a good look at all the cytological events in the sequence. For P. oryzae, it is important to have data for all five incubation periods--1 hr, 2 hr, 7-8 hr, 14-16 hr, and 24 hr– each time the lab is conducted, to allow for the possibility of slight variation in fungal development between experiments.

INSTRUCTOR'S NOTE #4

Instructors may view an example data set in the password-protected area of the Instructor Communication and Scholarship section of this site, which can be accessed at www.apsnet.org/edcenter/instcomm/restricted/

INSTRUCTOR'S NOTE #5

Instructors may read additional comments from the author and suggested answers to discussion questions in the password-protected area of the Instructor Communication and Scholarship section of this site, which can be accessed at www.apsnet.org/edcenter/instcomm/restricted/

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