Contents

6: Aquaria

Living organisms are the most entertaining things to watch with the microscope. You may find it hard to believe that some jars of slimy water could become treasured possessions, but here is a rule of thumb: the more rotten and disgusting a biological material is macroscopically, the more interesting and beautiful it will be microscopically. Like any rule of thumb, this one has exceptions; but it is a useful guide to finding interesting specimens.

WARNING: Do not keep cultures near areas where food is prepared or eaten. Do not touch your eyes, nose, or mouth while handling cultures. After handling cultures, your hands should be thoroughly washed. It is also dangerous to breath certain cultures and to puncture your skin with sharp objects that have come into contact with cultures.

Jars and other glass containers of many sizes can be used for aquarium cultures. Preserve jars are commonly used. Cultures can be started in many ways. The classic method, known as a hay infusion, is to put a handful of dried hay or grass into a water filled jar. Loosely cover the jar and place it in indirect sunlight. Too much light damages some organisms. In a few days the aquarium will be populated with bacteria and, in a few more days, many other kinds of organisms will appear.

Fresh grass or parsley will result in different cultures than dry hay. Manure from farm animals can be put into water, and many interesting organisms will develop from cysts -- protective capsules formed by organisms which allow them to withstand unfavorable conditions. The water from ponds, streams, and quiet rivers teems with microorganisms. Skim dead leaves and scum from the top of the water, and mud, which will contain cysts, from the bed.

As the original water evaporates from the aquarium, it is best not to add tap water. Collect a few extra containers of water from the site for this purpose. If tap water must be used, let it set in an open jar for a day or two, allowing any chlorine in it to evaporate before use. Also try to find water that has not flowed through copper pipes. Copper is toxic to some organisms. Slides and cover slips used for viewing the organisms can also become toxic. Be sure that old slides and cover slips have no traces of iodine, bleach, or other chemicals adhering to them. Slides can be washed with soap and water, then rinsed twice in xylene. If organisms are still dying on slides that have been treated in this way, put the slides aside for another use.

The pH of a solution is a logarithmic measure of its acidity. A solution is acidic if its pH is below 7, alkaline if higher. A better selection of organisms will be available for observation if several aquaria are kept, each maintained at a different pH level. Use pH paper to measure the acidity of the aquarium. The pH should be kept between 6.5 and 8.5 for most organisms, although there are certainly exceptions. Leeuwenhoek made some of his earliest discoveries by observing cultures growing in pepper water. Acidity can be controlled by adding a bit of weak sodium hydroxide solution to make the aquarium more alkaline, or lemon juice to make the aquarium more acidic. If you have access to more sophisticated chemicals, buffers are sold that control the acidity of the aquarium automatically.

To maintain cultures that have been collected from streams and ponds, try to keep the pH as it was when the culture was collected. Even with this precaution, the cultures begin to change almost immediately. Aquaria that are years old constantly develop new organisms; bacteria, cysts, and spores are floating in the air, always ready to start new colonies in a friendly environment.

Microscopic plants can be encouraged to grow by adding a few drops of liquid plant food to the aquarium. Other organisms often thrive on a small amount of brewer's yeast or beef bullion added to the water. Water acidity is likely to increase after feeding, and should be checked. Specific organisms can be separated to new aquaria, where they may survive better than when competing with many other organisms. However, this procedure often fails; the organism may be dependent on others that were not transferred. It may become necessary to go to a library to collect information about the specific organism's feeding requirements.

To separate organisms, use a turkey baster to suck up a bit of the culture from any part of the aquarium and transfer the culture to a petri dish. Bring the petri dish onto the microscope stage and work while observing at low power. Use a fine medicine dropper to pick up organisms and move them to the new water.

Taxonomy

Taxonomy is the science of classification, particularly of living organisms. Before microscopes, living things were classified as either plant or animal; a five kingdom division is now favored. The five kingdom classification arranges organisms in order of complexity and evolution. Members of all five kingdoms will populate most aquaria, and will be available for examination in the living state. The figure illustrates the newer taxonomy, placing kingdoms of more complex organisms above kingdoms of simpler organisms.

We will discuss a few of the many organisms in each of the kingdoms. This is only intended to be an orientation for beginners. As study progresses, more detailed works in biology, microbiology, botany, taxonomy, and animal biology will become useful.

Monera

Monera (sometimes referred to as Procaryotae) is the kingdom of organisms without a nucleus. That is, there are no internal membranes surrounding the genetic material. Such cells are called procaryotic. The kingdom is represented by the bacteria and the cyanobacteria. Tooth scrapings, buttermilk, and yogurt contain concentrations of bacteria in addition to those found in aquaria. The figure shows bacterial members of this kingdom likely to be encountered in an aquarium. It shows shapes, but no particular species. To identify bacteria by species requires special stains and microbiological analysis.

Some bacteria survive by absorbing nutrients from the environment through cell membranes. Others can use light to create carbohydrates from carbon dioxide in a process called photosynthesis. Some bacteria are capable of doing both.

The cyanobacteria often grow in filaments, either branched or unbranched. The figure illustrates Spirulina on the top and Oscillatoria on the bottom. Spirulina products are available in most health food stores. The filaments of Oscillatoria are capable of oscillating movement, and the novice may mistake it for a worm. The cyanobacteria were formally known as blue-green algae. They carry out photosynthesis as plants do, and they often grow in the same places as the true algae of the other kingdoms. However, the cyanobacteria are true procaryotes; the nuclear material within their cells is not surrounded by a membrane.

Some bacteria propel themselves with whiplike flagella. However, these structures are too small to be seen with a light microscope unless special flagella stains are used. These stains have mordants that build up around the flagella to a thickness that can be resolved by the microscope. Many other specialized staining techniques have been developed to allow visualization of bacteria.

Most procaryotes are so tiny that they are best observed with an oil immersion objective at a magnification of 1000X or more, although some will be visible at one third of that magnification. Bacteria will be present in all parts of the aquarium. They can be observed alive and moving about in a wet mount, but they are so transparent that another method is often used.

An aquarium will go through periods of dense bacterial colonization, during which the smear technique can be used with a drop of the aquarium water. Allow the smear to dry and then pass the slide over a small flame two or three times. This kills the bacteria and fixes them to the slide. Too much heat distorts the shape of the cells. Place a drop of stain on the smear and let it stand for a few minutes. Rinse the slide and let it dry. A drop of immersion oil (for a temporary mount) or resin is placed on the smear and a cover slip is added. Then place a drop of oil on the cover slip and view the bacteria with the oil immersion objective.

Protista

Protista is the kingdom of the simplest organisms with cells that have a nucleus and organelles. Both the nucleus and organelles are bounded by membranes, and the nucleus contains the genetic material for the cell. Such complex cells are said to be eucaryotic. Protists are usually unicellular, although they sometimes form colonies. Such multicellular colonies do not have cells that are differentiated into tissues. The protists evolved from procaryotes. The kingdom is represented in fresh water by amoebae, ciliates, flagellates, water molds, slime molds, and euglenoids.

Algae will inhabit almost every aquarium. Some algae are classified as protists, others as plants. The classification depends on structural makeup and pigmentation. An aquarium can be encouraged to grow all kinds of algae, as well as cyanobacteria, by adding two or three drops of liquid plant food. Often algae will be found on the surface or clinging to the sides of the jar as a thin green or red coating.

The flagella of Protists move in a whiplike motion instead of the rotating motion of the flagella of bacteria. The flagellated protists of the class Flagellata are algae. You may find the idea of swimming, green, photosynthetic organisms startling, but they are so common that one can expect to encounter them in most aquaria.

The figure illustrates Euglena. The longest flagellum bends back around the body so that the organism swims in the direction of the base of the flagellum. A shorter flagellum also originates from the same base, but it is short and difficult to make out with the light microscope. The body is not uniformly green. Instead, bright green organelles called chloroplasts are distributed throughout the cell. These contain chlorophyll, and carry out photosynthesis for the organism. Near the base of the flagella, a red eyespot is visible.The Dinoflagellate Ceratium has a covering armor called a pellicle. One of its flagella provides force for forward movement, while the other wraps around the body for a twisting movement.

In addition to photosynthesis, the flagellates absorb some nutrients through their cellular membranes. A few eat with a mouth as well. The flagellates multiply by longitudinal cell division. Studies with electron microscopes have shown that the structure of flagella in the sperm of higher animals, including humans, has the same internal structure as the flagella of the Protists.

Diatoms are armored algae that do not have flagella. Their outer covering is called a frustule, and it is made of silica. Diatoms are generally brown, even though they are photosynthetic. Some diatoms with a central groove on the frustule are able to move, but the details of how this movement are carried out are under contention. Diatom fossils are available from garden suppliers as diatomaceous earth. Huge deposits, some of which are thousands of feet thick, exist. (The fine ridges on the frustules have long been used in evaluating the resolution of microscope optics. Ialways take along a permanent slide preparation of diatoms when evaluating used microscopes.)

The ciliates are also common inhabitants of aquaria. The cilia, which give the ciliates their name, have the same structure as flagella, but are shorter. (Higher animals also posses cilia lining their bronchial and alimentary tracts. These have the same structure as the cilia of the Protists.) Cilia often cover a large part of the ciliate, as in Paramecium, below .

Other ciliates, such as Vorticella... and Stentor... have cilia near the mouth only. The mouth is called a cytosome. The cilia around the cytosome set up currents that bring in food. Often the movements of these currents are clearly visible because bacteria and other materials are present in the water. The cytosome is connected to a food vacuole within the organism. A food vacuole is an organelle that surrounds food within a membrane. You can put grains of carmine into the water with the ciliates and watch the grains be ingested. The brightly colored food vacuole will eventually break away from the cytosome and travel throughout the cell as the food is digested.

Some ciliates like Euplotes have cilia that are fused into leglike structures called cirri. Euplotes uses the cirri with surprising skill, moving about with the facility of a scurrying insect.

The ciliates do not photosynthesize. Some ciliates multiply not only by cell division (usually transverse), but also by sexual conjugation. One can occasionally observe this in an aquarium. The conjugating pair will perform a dance around one another which often lasts for many hours. The object of this activity is to trade genetic material. Concepts of male and female make little sense with some ciliates like Paramecium; each individual has characteristics associated with both sexes. Such ciliates are, however, divided into mating types. An individual of one mating type will only conjugate with members of certain other mating types within its species. Other ciliates, like Vorticella, have clearly distinguished male and female individuals.

If several aquaria are kept, conjugation can sometimes be induced by mixing ciliates from two of the aquaria together and starving the resulting population. If this fails to work, suppliers can provide cultures of different mating types. Suppliers also carry amphibians, roaches and termites which contain ciliates in their digestive tracts. A butcher can supply the rumen from a farm animal, which will also contain interesting ciliates.

The Sarcodines (or Rhizopods) include Amoeba and its close relatives. Everyone is familiar with movies in which giant amoebae engulf unwary humans. Real amoebae are microscopic but, in the case of one species, equally dangerous; it is the agent of amoebic dysentery.

Amoebae change shape to walk and to trap prey. The shape change is effected when the amoeba extends one or more pseudopodia from the main cell mass. The figure depicts an amoeba extending two pseudopodia in order to engulf a Paramecium. As the food is ingulfed, a food vacuole forms around it and the food is digested. Pseudopodia of other amoebae take different forms, such as latticed networks, thin sheets, or slender streams. The white blood cells of higher animals use the same method to engulf bacteria and foreign matter.

The testate amoebae live in a protective shell. This can either be secreted as in Arcella... or built up of cemented sand grains and foreign matter as in Difflugia.

Actinophrys is a member of the amoeboid order called Heliozoa or Sun Animalcules. The rays radiating from the cell are thin pseudopodia surrounding filaments anchored inside the cell. When a prey becomes entangled in these pseudopodia it is carried into the cell.

A contractile vacuole is used by many protists as a pump to remove water that has been absorbed through the cell membrane. Watch a small ciliate for a few minutes and you will see this vacuole contract and apparently disappear. The vacuole can then be seen slowly growing as it refills. Most ciliates and amoebas, and some flagellates have these organelles. Nigrosine is a good stain to use in visualizing contractile vacuoles.

Phase microscopes and interference microscopes are the ideal instruments for studying living protists, but with a bit of extra work a standard microscope is an adequate visualization tool. Because many of the organisms are almost transparent, vital stains are useful, as are darkfield, oblique, and Rheinberg illumination.

Fungi

Fungi is the kingdom of organisms composed of eucaryotic cells which, lacking chlorophyll, live mostly on dead organic matter produced by other organisms. The kingdom evolved from protista and is represented by the sac fungi, conjugation fungi, club fungi (mushrooms), and yeasts. Except for the yeasts, fungi are multicellular, composed of structures called hyphae. When conditions are right, the hyphae can form a larger mass called a mycelium, which can sometimes be seen without magnification.

Members of this kingdom can often be found in aquaria, usually floating on the surface. Many fungi grow well in acidic conditions. This is why one is liable to find a culture growing in coffee or tea cups that have been left out overnight. Fungi can also be found on rotting food. The figure shows the very common Aspergillus. The large spheres at the top are composed of smaller spheres that are spores. Fungi reproduce by means of these spores. The next figure shows spores of the Penicillium fungus, from which we derive the drug penicillin.

Plantae

Plantae is the kingdom of (usually) multicellular organisms that photosynthesize food. The cells are eucaryotic. The kingdom evolved from Protista and includes green algae and all common plants. (Some biologists disagree with this classification of green algae and classify them, instead, in a kingdom similar to Protista. ) The first figure depicts the filamentous green algae, Zygnema on the top and Spirogyra on the bottom. Desmids, also green algae, are similar in appearance to diatoms, but have no shell, are green, and usually consist of a single cell that is constricted along the median. Chlamydomonas are green algae with an eyespot and two flagella. Individual cells of Volvox look very much like Chlamydomonas, but they form into colonies. (Surely Buckminster Fuller must have been aware of 19th century illustrations of these plants before he invented the geodesic dome!) Reproduction can be either sexual or asexual. Sperm masses, eggs, zygotes, or daughter colonies may form within a mother colony. The colony is surrounded by a gelatinous sheath, called a capsule, which is easily visualized if the surrounding water is darkened with India ink, nigrosine, or even food coloring. Many other algae also have a capsule that can be seen using the same stains. The individual cells of the Volvox colony are flagellated, and these flagella extend outward, beyond the sheath; hence the colony is motile.

Many larger organisms that we all recognize as plants can be found growing in streams. These can be dissected and sectioned to make slides. Of equal interest are the organisms that cling to them. On collecting trips water plants should be closely examined with a hand lens or field microscope.

Animalia

Animalia is the kingdom of multicellular animals that use a mouth to ingest organic matter. The cells are eucaryotic. The kingdom evolved from protista and includes insects, worms, and humans.

The first figure depicts a water flea (Daphnia), and the second depicts a member of Copepoda. Both are small crustaceans commonly found in fresh water aquaria. The third figure depicts Tardigrada. The tiny animal is shown circling a single filament of green algae. The movements of the unjointed legs are slow and lumbering, which is why the animals are commonly called water bears. Hydra is also common. It lies in wait for prey, waving its tentacles in the water. The tentacles and body are covered with capsules which, when they contact prey, discharge filaments that pierce the victim and release poison. The prey is then brought to the mouth at the base of the tentacles. Relatively large prey can be ingested by this tiny animal. Members of Rotifera (last figure) are relatives of the canine heartworm. Many species exist, and their shapes differ considerably. The mouthparts can be seen fanning the water for food and then contracting into the body. In some species the waving cilia make the mouthparts appear to be turning disks, which accounts for the class name.

Contents

7: Other Specimens

Arthropods

Arthropods (such as insects, spiders, scorpions, crustaceans, centipedes, millipedes, ticks, and mites) are uncommonly strange and beautiful things when inspected microscopically.

Wings of insects require no preparation except clearing before mounting in resin. Moths and butterflies are covered with scales, which are modified hairs; many other insects, such as mosquitoes, also have scales. Scales can be scraped off the insect and mounted without preparation. Very small insects, such as aphids, can be mounted whole after a short dehydration and clearing. Whole insects can be embedded and sectioned. Because of the hardness of the exoskeleton, a slicing instead of a pushing cut is often required with the microtome blade. Arthropods can be killed by being placed in a covered jar containing a cotton ball soaked with household ammonia. Similar killing jars are available from suppliers.

Mounts of the exoskeleton without the soft tissues are the most common insect preparation. The insect is dissected in alcohol, then dropped into potassium hydroxide or sodium hydroxide solution (25% or so) and boiled until the soft tissues are removed. This process is called maceration.

WARNING: Both potassium hydroxide and sodium hydroxide can easily injure you when heated; the chemical splatters out of its container. Incline the mouth of the container (which might be a test tube or the like) away from your face when applying heat. Wear eye protection, an apron, and gloves. Immediately wash with water any part of your body that comes into contact with these chemicals and seek medical attention. Younger microscopists should obtain permission and supervision before attempting to macerate insect parts.

After maceration, wash the parts of the exoskeleton in water, dehydrate, clear, and mount. Staining is normally unnecessary, since the chitin that composes the exoskeleton is colored. Chitin will, however, yield new information when viewed with polarized light. Reflected illumination is also often useful for the larger, more opaque parts of the exoskelton, as is a stereo or dissecting microscope.

All arthropods have interesting eyes. Spiders have a number of simple eyes, while insects and crustaceans have compound eyes, each of which has hundreds or even thousands of facets. These compound eyes can also be dissected from the head and macerated.

Small arthropods can be observed alive and moving around in a well slide. The beating heart, blood circulation, and other internal movements can be observed. Do not neglect to microscopically observe the larva, pupa, and habitat of the species if you can.

The habitat of spiders is of particular interest. One is likely to find within the web a tiny graveyard of prey that represents the insect population of the area. Wasps prey similarly on spiders, and representatives of the local spider population can be found in wasp nests.

Quick Botanical Specimens

For beginners who do not want to immediately tackle embedding and sectioning, onion membranes and moss leaves provide a perfect view of a tissue one cell thick without the need for elaborate preparation. To obtain an onion membrane, split an onion into quarters and remove one of the sections from the center of one of the quarters. Remove the outer membrane of this section. This will be an extremely thin, transparent sheet. Cut a small square from this sheet, and stain the square. Moss can be found growing close to the ground in many areas. It can be recognized as a velvety green ground cover in spots where there is no grass. Remove a bit of moss, wash off the dirt that clings to the bottom, and stain the leaves. Permanent preparations can easily be made of either onion membrane or moss leaves by fixing, staining, dehydrating, clearing, and mounting.

Textiles, Fibers, and Hair

The microscope can be used to identify most natural textile fibers. Synthetic fibers are more of a problem because they are often visually identical. Chemical and physical tests are required as a supplement to microscopic examination.

Fibers and hair can be mounted whole for a longitudinal view, and embedded and sectioned to obtain a cross sectional view. Since fibers require no dehydration or fixing, working with them is simpler than working with biological specimens. For a quick examination, the fibers can be clamped in a split carrot and sectioned with a razor blade. However, the resulting sections will be inferior to those that have been properly embedded.

You can quickly assemble a large collection of permanent slides that can be used for purposes of identification. Since extremely small amounts of fiber or hair are required for each slide, samples can be taken from virtually any item in your wardrobe.

Sometimes sections, instead of whole mounts, are useful for longitudinal views. This is especially true of the roots of animal hair, which are quite interesting in longitudinal cross sections that include a layer of the surrounding skin from which it was taken.

Fibers from different kinds of paper are distinctive. Boil a bit of the paper in a one or two percent sodium hydroxide solution, rinse, and tease the fibers apart with a couple of needles.

Fibers and hair can be studied with brightfield illumination with or without stains. Polarized illumination will often show differences between two fibers that are otherwise similar.

Contents