Nematodes, also known as roundworms, are a phylum of simple, unsegmented animals found in nearly every ecosystem on Earth, including soil, marine environments, and as parasites in plants and animals. Most free-living species measure less than a millimeter in length, making them invisible to the naked eye. Microscopy is the only way to observe their morphology, study their behavior, and understand their internal anatomy.
The Importance of Nematodes in Science
Scientific research relies heavily on nematodes due to their utility as biological models and their wide-ranging ecological impact. The species Caenorhabditis elegans is a primary model organism in genetics and developmental biology. Its transparent body, short life cycle, and mapped nervous system make it ideal for studying fundamental biological processes relevant to human health, such as aging and programmed cell death.
Nematodes are also important in agricultural and environmental science. Free-living species are integral to nutrient cycling in soil ecosystems, regulating microbial populations by feeding on bacteria and fungi and releasing nutrients for plants. Conversely, plant-parasitic nematodes, like the root-knot nematode, cause billions of dollars in crop damage annually by feeding on plant roots. Microscopic identification of these pests is necessary for global food security.
Selecting the Right Microscope
Observing nematodes effectively requires utilizing two distinct types of light microscopy. For initial work, such as isolating specimens from a soil sample, sorting different life stages, or observing general movement patterns, a stereomicroscope is the preferred tool. This low-magnification instrument typically provides 7x to 40x magnification, offering a large working distance and a three-dimensional view necessary for manipulating the small worms with a picking needle or micropipette.
For detailed analysis, observation shifts to a compound microscope, which provides the necessary high magnification. This microscope uses objectives ranging from 4x to 100x, allowing for total magnifications up to 1000x. The compound microscope is essential for visualizing internal structures, such as the pharynx, gonads, and specialized mouthparts, which are used for precise species identification.
Preparing Nematodes for Viewing
The nematode’s constant thrashing movement makes imaging difficult, so live specimens must be temporarily immobilized for high-magnification viewing. A common technique involves placing the worms on an agar pad, which provides a thin, flat surface for mounting and helps restrict movement. To achieve complete stillness, a paralytic agent is often incorporated into the mounting solution or applied directly to the slide.
Two common chemical immobilizing agents are levamisole and sodium azide. Levamisole acts as a cholinergic agonist, binding to acetylcholine receptors to induce spastic paralysis. Sodium azide is an inhibitor of mitochondrial respiration, which rapidly depletes the cellular energy required for movement. For temporary immobilization without chemicals, some researchers use cold shock, which is less disruptive to cellular processes and allows for recovery.
Identifying Key Features Under Magnification
Successful observation focuses on specific morphological markers that reveal the nematode’s species, feeding type, and sex. The outermost layer is the cuticle, a non-cellular, multilayered structure that can appear smooth or feature distinct rings or ridges. The anterior end contains the mouthparts, which indicate the worm’s feeding habits. For example, some species have a simple mouth opening for consuming bacteria, while plant-parasitic species possess a sharp, hollow stylet used to pierce plant cells.
Internally, the muscular pharynx, or esophagus, is the primary pumping organ that draws food into the digestive tract, and its shape is a key identification feature. The reproductive system is also highly visible, as most nematodes are sexually dimorphic. Males are generally smaller and possess copulatory spicules near a curved tail. Females are typically larger and contain one or two ovaries, with a vulva opening along the ventral side of the body.