Description

In this laboratory activity, learners use a simple procedure to bait oomycetes from water and/ or soil and then examine these fungus-like organisms with the microscope to see how they look. They will see large hyphae, sporangia and swimming asexual spores (zoospores), the structures involved in sexual reproduction (oogonia and antheridia), and how the shapes and sizes of these structures may vary among different kinds of oomycetes. Learners may then design their own biodiversity experiments to see if the use of different natural water sources, different kinds of soils, or different baits affect the variety of oomycetes baited.
Quick Guide

Preparation Time: 1 to 2 hours Learning Time: 1 to 7 days Estimated materials cost: $1 - $5 per group of students Age Range: Ages 11 - 18 Resource Types: o Activity o Demonstration o Experiment/Lab Activity o Lesson/Lesson Plan Language: English

Materials List (per group of students)

small jars with lids small glass or clear plastic dishes microscope slides, coverslips forceps, probes compound microscope, binocular dissecting microscope 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 (would on a toothpick) insect wings pollen thin pieces of plant tissue like onion skin, grass blades, etc. water from a relatively clean, natural water source boiled and cooled distilled water for baiting from soils Natural soils

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.

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.

Learning objectives:
To learn about a group of fungus-like organisms; the oomycetes, by baiting them from natural sources (water and soil) and observing them.

Exercise description:
Students use a simple procedure to bait oomycetes from water and/ or soil and then examine these organisms with the microscope to see how they look. They will see large hyphae, sporangia and swimming asexual spores (zoospores), the structures involved in sexual reproduction (oogonia and antheridia), and how the shapes and sizes of these structures may vary among different kinds of oomycetes. Students may then design their own biodiversity experiments to see if the use of different natural water sources, different kinds of soils, or different baits affect the variety of oomycetes baited.

Time frame:

Oomycetes will be observable on the baits after 2-4 days (12 hours for pollen baits), and should remain viable for at least several days.

Study questions:
1. What characteristics make oomycetes different from the true fungi? 2. Do different baits capture different oomycetes? 3. Do different water or soil sources contain different kinds of oomycetes?

Answers to Study Questions:

1. What characteristics make oomycetes different from the true fungi? The oomycetes, commonly called water molds, are more closely related to other Protists than to true fungi. True fungi are more closely related to animals, than to plants and protists. There are some important characteristics of oomycetes that are very different from true fungi: 1. The mycelium cell wall is primarily cellulose, rather than chitin as in true fungi. Also, there are generally no septa or crosswalls dividing the cytoplasm into "sections". (Some do form in injured and old hyphae and sometimes at the base of reproductive structures.) In species studied, the vegetative nuclei are diploid. In true fungi, nuclei are generally haploid or dikaryotic (pairs of genetically different haploid nuclei that divide in tandem). 2. The sexual spores are oospores (or oospheres) produced from the pairing of antheridia and oogonia. These are an important source of genetic variation from generation to generation. In addition, the thick-walled spores are important survival structures during adverse conditions (drought, winter). 3. Oomycetes have a motile zoospore stage, which true fungi do not. These fast moving, swimming spores are one reason why they are commonly found in water and wet soils. 2. Do different baits capture different oomycetes? 3. Do different water or soil sources contain different kinds of oomycetes? The oomycetes trapped on various baits will be saprophytes or the saprophytic stage of a parasitic pseudofungus. There are a number of genera and species commonly found in soils and various water sources. They can be differentiated by the structure of the sporangia, the oogonia, and the antheridia which are illustrated in many biology and mycology texts. These structures usually form in less than a week and are easily seen with a compound microscope, so they make a good observational exercise in which students can see what kinds of variation in form exist. Some factors that will affect what species may be trapped include: 1. bait

2. water: still pond vs. moving stream, for example 3. time of year (although found even in water collected in mid-winter) 4. soil: drainage, depth, soil type, pH