The free living soil nematode, Caenorhabditis elegans, offers numerous advantages as a teaching organism, including its small size, ease of maintenance, ready availability, and number of mutant strains. Using C. elegans, we have devised a laboratory exercise using fluorescence microscopy to study endocytosis and phagocytosis in the digestive system. Instructors prepare the worms prior to class in each of three treatments. Some worms are fed FITC-conjugated microspheres, which can only be absorbed by phagocytosis. Others are fed RITC-conjugated dextran, which can be absorbed by pinocytosis. The last group of animals is suspended in acridine orange, a fluorochrome that concentrates in acidic organelles. Students examine worms from each treatment, as well as controls, to determine which cellular transport mechanism this nematode uses for food absorption and to relate the mechanism's location to acidic compartments, i.e. lysosomes. This exercise gives the students an opportunity to study organelles in a living metazoan, offers perspective on organelle size and distribution in cells; allows them to manipulate the widely used C. elegans; and it introduces them to epi-illumination fluorescence microscopy. Furthermore, the images obtained are aesthetically pleasing and can be spectacularly colorful.

Aside from being a powerful research tool, the free living soil nematode, Caenorhabditis elegans, offers numerous advantages as a model organism in instructional settings. Its small size (ca. 1 mm) and simple culture requirements allow it to be grown cheaply in large numbers, while taking up relatively little storage space. Adults have only about one thousand somatic cells, all of which are transparent, lending to straightforward descriptive studies of cell fate, morphogenesis, and physiology. With an average life span of only 2-3 weeks and many mutant strains readily available, students can demonstrate a variety of phenomena in Mendelian, population, and developmental genetics. In the exercise we describe, students used C. elegans in our "Introduction to Cell Biology" course to study vesicular transport across the plasma membrane of intestinal epithelium.

The University of Wisconsin-Whitewater is a premiere, comprehensive, undergraduate state university, enrolling approximately 10,000 students. The Biological Sciences programs emphasize laboratory and field instruction, and are striving to implement investigative learning approaches at all levels, from introductory general studies biology all the way to independent research programs for high achieving seniors in biology.

There are approximately 250 Biology majors, most of whom take our Introduction to Cell Biology course. It is offered every semester, with a maximum section size of twenty. Each section meets three times per week: twice for fifty-minute lectures, and once for a two-hour laboratory. We work with a limited budget. The lecturing professor also prepares and teaches the laboratories, with minor assistance only in purchasing supplies and in preparing stock solutions.

Introduction to Cell Biology is pivotal in the biology majors' curriculum, serving as a gateway to upper division courses in cell biology and physiology for about 65 of our students, and as the recommended option to fill a biology breadth requirement for the remaining 35. To register for the course, students must first complete a one year introductory biology sequence, and either previously completed or be concurrently enrolled in their second semester of introductory chemistry.

This exercise gives students an opportunity to study organelles in a living metazoan, allows them to manipulate C. elegans, and introduces them to epi-illumination fluorescence microscopy. We asked students to determine if C. elegans epithelial cells use phagocytosis or endocytosis to absorb ingested materials. We pulse fed adult C. elegans with various dyes and dye conjugates, then washed them in phosphate buffered saline (PBS). Students made wet mounts of living worms and viewed them using epi-illumination fluorescence microscopy. By comparing the location of fluorescently labeled lysosomes with RITC-dextran and FITC-labeled microspheres, student investigators can determine whether endocytosis or phagocytosis is the primary mechanism for epithelial absorption. The images obtained are aesthetically pleasing and can be spectacularly colorful, helping to maintain student interest and improve student retention of associated subject content.

Populations of C. elegans were maintained at 16^o on lawns of Escherichia coli (strain OP50) grown on nutrient agar. We used fine forceps or the bent tip of a hypodermic needle to transfer twenty-five to fifty worms at a time to 50 µl drops of four different PBS reagents. (1) 0.05 mg/ml acridine orange concentrates in acidic spaces, particularly lysosomes, due to acidic protonation. (2) Rhodamine B isothiocyanate (RITC) conjugates of dextran are absorbed by endocytosis. One of us made the conjugates we used (Clokey and Jacobson, 1986), but they are commercially available from Molecular Probes (Catalogue #D-1824, PO Box 22010 Eugene, OR 97402-0469, http://www.probes.com/). Working solutions of RITC-dextran were prepared by simply transferring a small crystal of the conjugate to the PBS for a few minutes, until the solution developed a light pink color. No further attempt was made to control RITC-dextran concentration. (3) Latex microspheres conjugated to fluorescein isothiocyanate (FITC) can enter the cell only by phagocytosis. Microspheres were purchased from Polysciences (catalogue #15700, 400 Valley Road, Warrington, PA 18976, http://www.polysciences.com/) as fluoresbrite YG carboxylate microspheres, 0.50µm, and diluted 1:10 or 1:20 in PBS before use. (4) Control animals were incubated in PBS alone.

All worms were incubated in a drop of solution in a humid dish for a minimum of four hours or a maximum of overnight at 16^o, washed in one ml of PBS, gently centrifuged, and transferred to clean PBS in 35 mm dishes.

Students worked in small groups, and each group was assigned one of the four treatments. They prepared wet mounts by placing a number of worms in a drop of PBS on a slide then covering with a cover slip. The worms must be alive to maintain the integrity of the lysosomes but immobilized for proper viewing. This can be done by using a dissecting microscope to monitor the animals, then removing enough PBS from the slide with a bit of paper tissue to immobilize the worms by coverslip pressure. Removing too much PBS can crush the animals, and students sometimes needed several attempts to get this right. The edges of the coverslip were sealed with a drop of low-fluorescence immersion oil to reduce evaporation.

The live mounted worms were viewed on an inverted Olympus microscope equipped with epifluorescence and a "B" filter set to view fluorescein, and a "G" filter set to view rhodamine. Either filter allowed observation of acridine orange labeling. (Table 1).

Table 1: Optimal excitation and emission filters for epi-illumination microscopy

Dye or Pigment	Excitation l	Emission l
Acridine orange	250 nm - 475 nm, peak 325 nm	> 478 nm
FITC- microspheres	320 nm - 490 nm, peak 405 nm	> 500 nm
RITC-dextran	542 nm - 550 nm	> 590 nm
Lipofuscins	300 nm - 400 nm, peak 370 nm	> 418 nm

Students were escorted in small groups to the fluorescence microscope, instructed in safe use of light path and filter controls, and allowed to observe their worms in phase contrast, with the rhodamine filter, and the fluorescein filter. They were encouraged to sketch their observations, or to draw the fluorescence pattern over a downloaded phase contrast photograph of C. elegans. Rhodamine-labeled conjugates (Fig. 1: ) and the acridine orange lysosomal stain (Fig. 2: ) appeared to co-distribute in the gut epithelium. Most students were able to identify the fluoresceinated beads in the gut lumen of the worms, but not intracellularly (Fig.3: ). Students submitted their results in the second of two formal lab reports required this semester. The lab reports demonstrated that students were able to identify goals, perform the experiment, and correctly interpret the results.

Although our lab is not so equipped, it would be useful to also check worms with a 365 nm exciter filter and 420 nm barrier filter to identify the autofluorescent secondary lysosomes in the epithelial cells. Students would compare the lipofuscin distribution in residual bodies with the rhodamine-labeled endosomes and acridine orange labeled lysosomes. They should find them co-distributed. If available, the use of a CDC image capture apparatus would allow students to incorporate their own images into lab reports, rather than depend on memory and hand drawn sketches over bright field images.

You may also order mutant strains with deficiencies in endosomal uptake for comparison to wild type strains. We recommend the constitutive dauer daf-4 mutant which shows endocytosis defects. Dauer is a halted developmental state found normally in response to low food conditions, crowding, or elevated temperatures. Double mutants of daf-4 with daf-3, daf-5, or daf-12, repress the dauer form, but show endocytosis defects nonetheless, and may also be of interest for this laboratory exercise. All these strains, as well as wild types, are available from the Caenorhabditis Genetics Center (CGC).

Click here to download sample lab instructions and an instructor's check list.

• for Caenorhabditis endosomal labeling:
  Clokey, G.V. and L.A. Jacobson (1986); Mech. Ageing Dev. 35: 79-94
  
• for worm culture:
  • Avery, L. (1999); http://elegans.swmed.edu/
  • Caenorhabditis Genetics Center (1999); gopher://elegans.cbs.umn.edu/