For this exercise, you will need:
Equipment:
- small jars with lids
- small glass or clear plastic dishes
- microscope slides, coverslips
- forceps, probes
- compound microscope, binocular dissecting microscope
Baits for oomycetes:
- whole or cut fruit such as apple or pear
- popped popcorn kernels (no butter or salt!)
- sesame seeds
- dead insects (flies, ants, etc.)
- spider web (wound on a toothpick)
- insect wings
- pollen
- thin pieces of plant tissue like onion skin, grass blades, etc.
- (Fresh materials used as baits, except for popcorn and pollen, should be boiled for a few minutes to reduce bacterial contamination.)
Water samples:
- water from a relatively clean, natural water source
Soil samples:
- boiled and cooled distilled water for baiting from soils
Fill a jar to half-full with water taken from a relatively clean natural water source, such as a pond or stream. Float several popcorn kernels in the uncovered jar. A white halo of mycelial growth will be observed in 2-4 days. Pollen baits should be examined after 12 hours.
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Figure 14. Mycelia of an oomycete growing from popcorn bait. Click image to see a larger view. |
The asexual sporangia and zoospores will appear first, usually as soon as some mycelium is visible in the floating popcorn (or other bait). A few days later on the oldest mycelium, the sexual antheridia and oogonia will begin to appear. For classroom demonstrations, it is recommended that several jars, started on consecutive days be available to see the various stages, e.g., one that is 3 days old, 4 days, and 5 days. The rate of development depends on the temperature of the room and will progress more quickly in hot weather.
A small amount of the mycelium can be transferred to a microscope slide and covered with a coverslip. It should be relatively easy to observe the nonseptate (coenocytic) mycelium and probably sporangia and oospores of oomycetes surrounding the bait. Fast-moving zoospores may also be seen zipping across the microscope field. These can usually be observed in a small glass or clear plastic dish using a binocular dissecting microscope.
Natural soils are also a source of oomycetes. Add about one teaspoon of soil to boiled and cooled distilled water in a jar. Add bait. Avoid adding too much organic matter, which will quickly contaminate the bait with bacteria. Observe fungal growth on baits in 2-4 days.
Fungi commonly trapped on various baits are Saprolegnia, Achlya, Aphanomyces, and Pythium. In the diagram section of this exercise we have included one page of line drawings of the asexual and sexual structures of a variety of oomycetes for you to copy and handout to your students to help them identify what they see. Also, most introductory mycology books and many general biology/botany texts have good diagrams of these common genera; two suggestions are listed under Supplemental Information. Also check out the websites listed under Supplemental Information to see additional photomicrographs of these organisms.
Note: This is not a pure culture system; students will probably see many bacteria and other common pond water organisms among the mycelia of the water molds.
Design your own experiment
Once students understand the basic procedure for baiting oomycetes, they can design their own experiments.
--Often when you first collect pond water it looks "clean". How do you know that the oomycetes you bait come from this water and not from the air?
Let students design experiments to test this. If you want to give hints probably the easiest way is to take two jars, fill one with pond water and one with sterile, distilled water. Float popcorn in both and see what happens. They might try a lid on one and not the other jar, but this introduces the problem of oxygen supply. They might also try boiling the pond water, allowing it to cool, and seeing if the oomycete still grows.
Students might also ask such biodiversity questions as the following:
-- Do different baits capture different oomycetes?
Students collect a variety of different things to try as baits (many possibilities are listed in Materials).
--Do different water or soil sources contain different kinds of oomycetes?
Students can collect water samples from different natural sources, e.g., pond, lake, marsh, flooded area, puddles, brook, river. Students can collect natural soil samples from different places, e.g., vegetable garden, lawn, wooded area, forest, fruit orchard, poorly drained area, well-drained area, sandy soil, clay soil, soils with different pHs, etc.
Students can also save clean jars to bring to class to use to set up their experiments, along with the assortment of baits, water samples, and soil samples they collect. Students working as lab teams decide what factor they want to test, plan their experiment, set up the jars appropriately, and decide how to record their data; at the appropriate time they examine the baits, record their data and draw conclusions about what they found. Different lab groups could test different factors, and at the end of the experiment, each group could share their results and conclusions with the rest of the class, perhaps by a short oral presentation. A class discussion could then follow.
