1. Culture epiphytic microorganisms from the surface of healthy plant tissues and become familiar with the morphology of typical plant-associated fungi and bacteria.
2. View nitrogen-fixing root nodules formed on bean plants by Rhizobium bacteria and observe the bacteria living within the nodules.

From the time a seed is placed in the ground until the final remnants of a dead plant are decomposed in the soil, the plant serves as home to a vast array of microorganisms. Many of these microbes are beneficial, helping the plant obtain water and nutrients and protecting the plant from pests and pathogens. Other microbes, including some fungi, bacteria, viruses, and nematodes, cause destructive diseases of plants. In this exercise, students will observe some of the beneficial microbes associated with bean plants.

A wide variety of saprophytic microorganisms grow on the surface of healthy plant tissues both above and below the ground. These microbes live on the nutrients (sugars, amino acids, etc.) that leach out of the plant cells and onto the plant surface. Although unseen with the naked eye, the leaf surface is covered with numerous bacteria and fungi. This will become clear to students as they make "leaf prints" and watch the colonies of microorganisms that develop when healthy leaves are touched briefly to the surface of a nutrient medium in a petri dish. The volume of soil immediately surrounding the root, referred to as the rhizosphere, is home to a wide variety of bacteria and fungi that help protect plants from the many pathogenic microbes found in the soil. Students can make "root prints" to demonstrate the presence of these rhizosphere microorganisms.

An important feature of the roots of beans and other legumes is the presence of nitrogen-fixing root nodules. These nodules develop following infection by bacteria in the genus Rhizobium. Once inside the root, the bacterium stimulates the root cells to divide forming the nodules. Within the nodules the bacteria produce enzymes that are capable of fixing atmospheric nitrogen, in essence making nitrogen fertilizer out of the air. In this exercise, students will observe the development of root nodules on bean plants and observe the bacteria that grow within the cells of the root nodule. Students can also attempt to isolate Rhizobium from the nodules.

Bean plants	Phaseolus vulgaris (string beans, snap beans, pinto beans, etc.) of any variety can be used for this exercise.
Rhizobium inoculant	Can be obtained from any garden center or nursery.
Potato dextrose agar (PDA)	Can be obtained in dehydrated form, as sterile media, or as poured culture plates from Carolina Biological Supply. Other media such as nutrient agar, plate count agar, etc., will also work well.
Congo red	1 g congo red stain dissolved in 50 ml water
Acid alcohol	3 drops conc. HCl in 50 ml 95% ethanol

 

Bean seeds can be dipped in water then shaken in the Rhizobium inoculant and planted in soil. In order to have a diverse array of microorganisms in the rhizosphere (the volume of soil immediately surrounding the root) it is best to use non-sterile garden soil or a mixture of non-sterile garden soil and potting mix. The plants should be grown with as much light as possible. Water the plants as needed, but do not fertilize. The plants should have good nodule development about four weeks after planting.

Making Leaf Prints: Using forceps carefully remove a leaflet from a bean plant and gently press either the top or bottom surface onto the surface of the nutrient medium in a petri dish. Take care not to touch the medium with your fingers. Remove the leaflet, cover the dish and incubate at room temperature. Colonies should start appearing in a few days.

Root Prints: Gently remove a bean seedling from the pot and shake or rub as much soil as possible off of the root system. Cut a 3-4 cm section of a root and use forceps to carefully remove most of the soil from the root segment so that only a thin layer remains. Using forceps, gently press the root segment onto the surface of the nutrient medium then remove the root, cover the dish, and incubate at root temperature. Again, fungal and bacterial colonies should begin appearing in a few days.

Observing fungal and bacterial colonies: When colonies are well developed on the leaf and root print plates students can view them with a dissecting microscope (or a hand lens). See if they can tell the difference between a typical bacterial colony (usually appearing as a droplet of slime on the culture plate) and a typical fungal colony (fuzzy looking). Have them describe the different types of bacterial and fungal colonies that they see.

If compound microscopes are available, have the students take a bit of a fungal or bacterial colony on a clean toothpick and transfer the tissue to a small drop of water on a microscope slide. They should put on a coverslip and look at the specimen and describe what they see. What do the bacteria look like? What do the fungi look like? How are the fungi from different types of colonies similar and how are they different when viewed with the microscope? I find it is a good idea to have the students make drawings of what they have seen.

Observing Rhizobium nodules: The teacher should carefully remove the soil from the roots of a four- to five-week-old bean plant. Soil should be removed by gentle washing in a bucket of water. Students can then collect some roots with nodules and observe them under a dissecting microscope (or with a hand lens). (The nodules will be from one to three or four millimeters in diameter.) Have them cut through a few nodules and notice the color. Healthy nodules should be pink inside.

Observing Rhizobium bacteria: Nodule tissue can then be placed in a drop of congo red stain on a slide and chopped into tiny pieces. After a few minutes, students should take clean toothpicks and gently remove the tissue pieces from the slide. They should then use the toothpick to spread the congo red stain in a thin film over the surface of the microscope slide. The preparation should be allowed to air dry. A few drops of acid alcohol should then be added and this will change the color from red to blue. After the alcohol has dried students should add a small drop of immersion oil (or mineral oil), put on a coverslip, and observe the preparation with a compound microscope at high power (100 or 400X). The bacterial cells will appear as white rods against a blue background. (There will also be irregularly shaped cellular debris on the slide along with the bacteria.) Each of these tiny bacterial cells is about 1/300 of a millimeter long.

Isolation of Rhizobium: If desired, students can isolate Rhizobium from a nodule and use the cultured organisms to inoculate bean seeds to see if nodules form. The nodules should be carefully removed from the plant and surface sterilized in 10% household bleach for 10 minutes. A large drop of sterile water is placed in a sterile petri dish and the nodule is transferred to the water using flame-sterilized forceps. The nodule is then crushed using the forceps. A milky fluid should appear within the water. Use a flame sterilized transfer loop to spread a loopful of this fluid on the surface of the PDA in a petri dish. Cover and incubate at room temperature. Rhizobium colonies should appear in a few days. These can be suspended in water and used to inoculate bean seeds prior to planting in sterile potting soil. Seedlings can be checked for nodule formation in about 3 weeks.