BIO 112.01 Lab 2

Monera is the former kingdom of the prokaryotes -- those single-celled organisms without a nucleus or other membrane-bound organelles. Recent research on bacterial systematics indicates that the bacteria actually belong in two distinct domains (a category higher than the kingdom): Archaea and Bacteria. The prokaryotes are the most ancient lineage of living organisms, with evidence of fossil bacteria being found in rocks 3.5 billion years old. The earth was ruled by bacteria and bacteria alone for at least 1.5 billion years! The bacteria are very diverse metabolically, but two main functional divisions can be made: some are heterotrophic, and others are autotrophic. The photosynthetic prokaryotes are the cyanobacteria, formerly called blue-green algae because they resemble the aquatic eukaryotic algae with which they occur. Due to the small size of bacteria and the lack of distinguishing characteristics visible under a light microscope, bacteria are often classified based on both the shape of their cells (e.g., sperical vs. rod-shaped) as well as their response to the Gram stain. All bacteria have cell walls, which stain differently depending on their chemical makeup.

All eukaryotes are classified in the domain Eukarya. The protists are single-celled eukaryotes, formerly all lumped into the kingdom Protista. Currently there are a number of classification schemes for the protists, reflecting the dynamic state of protist biology: some schemes even recognize multiple “candidate kingdoms.” This is a very diverse group because it includes both autotrophic and heterotrophic organisms. The autotrophs (“self-feeders”) are photosynthetic, manufacturing their own food by capturing the energy of sunlight in the chemical bonds of carbohydrates. Photosynthetic organisms can be readily recognized by the presence of pigments in their cells. Protists also include "mixotrophs," organisms such as Euglena that are both heterotrophic and autotrophic. Mode of locomotion is also diverse among the protists. They move via flagella (whip-like tails), cilia (short, hair-like structures that beat in unison), or pseudopods (“false foot” -- as in the oozing amoeba); some have no means of locomotion.

The members of the Kingdom Fungi were formerly lumped together in the same kingdom with the plants because both groups are sessile and have cell walls. In addition, both groups are eukaryotic, lack centrioles in their cells, and are mostly multicellular. The similarities that seem to unite these two groups of organisms disappear on closer inspection, however. Fungi have no motile cells at all in their life cycles (with one exception), whereas in plants the sperm are motile (except in the flowering plants, where this trait has been lost secondarily). Furthermore, although both have cell walls, they are composed of different kinds of polysaccharides: chitin in fungi (same as in arthropod exoskeletons!), cellulose in plants.

The fungi are heterotrophs that absorb their food after digesting it with extracellular enzymes. The structure of a fungus is essentially filamentous, rather than truly three dimensional as in the plants. Fungi are composed of threadlike structures called hyphae, which in aggregate form a mycelium. Some aspects of cellular structure in Fungi are quite unusual: the nuclear envelope does not break down in cell division, and the spindle is formed within the nucleus. Also, the cell walls between cells are often incomplete, allowing nuclei to move from cell to cell. Therefore, fungal cells can be multinucleate.

In this lab you will encounter representatives of all of these three groups -- bacteria, protists, and fungi -- and will learn about their basic biology and ecology.

Lab 2 Worksheet

Materials

Specimens at numbered stations arranged on the lab benches.

These consist of fresh materials and live specimens as well as preserved specimens, prepared microscope slides, and other written and illustrated materials.

Microscopes, slides, cover slips, forceps, lens paper.

Dissecting stereo microscopes.

Procedure

1. View the specimens of bacteria and protists, noting their key identifying features. Make a drawing of each specimen, labeling all of the characteristic structural features.

2. For each specimen, be sure to record its common name as well as its formal classification (e.g., domain, kingdom or candidate kingdom, phylum and species)

3. Be able to recognize all specimens as well as their distinguishing characteristics.

4. For the fungi, you may be asked to make a wet mount of a specimen, or examine a prepared slide, or look at an entire specimen, depending on the station.

5. In your lab notebook, create and fill in tables to summarize the key features of the different groups of fungi. The layout of these tables is specified in the text below.

Study Suggestions

1. Make detailed sketches and notes on specimens. This will help you in two ways: 1) when you attempt to draw a specimen, you are forced to look at it more closely, and 2) the drawings will help you study later.

2. Plan to come view the specimens once or twice more before the lab test. Test yourself by attempting to identify the specimens without first looking at their labels.

PART I. BACTERIA

Bacterial diversity includes the domains Archaea and Bacteria, both of which are characterized by having prokaryotic cells. Only specimens of domain Bacteria are presented in this lab exercise; however, pictures of typical archaean habitats and bacteria are provided.

The specimens of domain Bacteria in today's laboratory were selected to illustrate the diversity in bacterial shape, response to Gram stain, and mode of nutrition. The so-called “gram-positive” bacteria retain a Gram stain in the thick layers of peptidoglycan in their cell walls and therefore will appear purple/violet. In contrast, the “gram-negative” bacteria have less peptidoglycan (and it is beneath an outer membrane of their cell wall) and therefore do not take up the stain; they appear red. The Gram stain is an important way to differentiate among different types of bacteria that are otherwise difficult to distinguish under a microscope. View the slides of Bacillus cereus var. mycoides and E. coli and note the color of each: which species is Gram + and which is

Gram -?

Broadly speaking, bacterial cells may be classified into three groups based on their morphology: rod-shaped (bacilli; singular = bacillus), spiral (i.e., spirilli/helices) and spherical (cocci, sing. = coccus). View again the slides of Bacillus cereus var. mycoides and E. coli (see above) as well as the prepared slides of Spirochete and Streptococus, and classify each species based on its shape. Be sure to enter your conclusions in your lab notebook.

All of the above bacteria are heterotrophic, but the next specimen is photosynthetic and therefore belongs to the group of bacteria known as cyanobacteria. View the specimen of Oscillatoria sp., noting the presence of photosynthetic pigments that make the cells appear green. How would you tell Oscillatoria from a green alga (Chlorophyta)?

PART II. DIVERSITY OF THE PROTISTS

All protists are in Domain Eukarya, meaning that their cells are eukaryotic. The vast majority of protists are single-celled, though there are some exceptions, especially among the "algae." Protists are an extremely diverse group of organisms, and their classification seems to be in a constant state of flux. Just as an example of protistan diversity, their modes of nutrition range from ingesting food (the "protozoans") to absorbing food (fungus-like protists) to photosynthesis ("algae"). As was pointed out earlier, there are also "mixotrophs" than can both ingest food and carry out photosynthesis. Unfortunately, protistan systematics is not a straightforward affair: the various algae, for example, are not always closely related (i.e., monophyletic). In today's lab we will survey representatives from many of the "candidate kingdoms" of protists.

Candidate Kingdom Diplomonadida

The diplomonads (and their relatives the parabasalids) represent a group of eukaryotes that probably diverged very early from the rest of Domain Eukarya. They have multiple flagella, two nuclei, and cytoskeltons that are simple compared to those of most eukaryotes. View the prepared slide showing cysts of Giardia lamblia, the parasitic protist that causes giardiasis, which is characterized by severe diarrhea and cramping. Giardia is transmitted via these cysts in infected waterways, even in wilderness areas; therefore, campers and backpackers should always boil their water or treat it chemically with iodine to ensure that they don't pick up this parasite. Although probably not visible in this specimen, a key feature of this species and other members of its candidate kingdom is that they lack mitochondria.

Candidate Kingdom Euglenozoa

All members of Euglenozoa are single-celled protozoans with flagella. Their mode of nutrition varies: some species are heterotrophic, some are photosynthetic, and some are mixotrophic. Furthermore, some euglenozoans are free-living and others are parasitic.

In addition to the Euglenoids (next paragraph), candidate kingdom Euglenozoa also includes Typanosoma (a kinetoplastid), the parasitic organism that causes sleeping sickness.

Prepare a slide with live specimens of Euglena -- you may wish to add a drop of "Protoslo" on the slide to slow down the Euglena so you can view them closely. Euglena has an unusual flexible pellicle of protein embedded within its cell membrane -- this is quite unlike the solid cell walls of some protists. Can you detect changes in the shape of Euglena as it moves around? The two flagella -- one long, one short -- are located at the anterior end of the cell. Look for the light-sensitive stigma, also located near the anterior end. What color is Euglena, and what does this tell you about its mode of nutrition?

Candidate Kingdom Alveolata

This is a diverse candidate kingdom including both autotrophs and heterotrophs. All are single-celled and are characterized by "alveoli" (sub-surface, membrane-bound cavities), but their mode of nutrition and locomotion varies from group to group. There are three distinctive groups of alveolates:

Dinoflagellates

Dinoflagellates are mostly photosynthetic marine phytoplankton with two flagella; they also have distinctive internal armor of cellulose plates. See the prepared slide of Ceratium. Note the armored appearance and look for the flagella -- can you see the transverse groove that houses one of the flagella? (The other flagellum would be sticking out from the body of the dinoflagellate, perpendicular to the first flagellum.)

Ciliates

The ciliates are unicellular heterotrophs with cilia, fine hairlike projections used for locomotion. Prepare a slide from the culture of live Paramecium -- as with Euglena, you may need to use "Protoslo" to slow them down enough to observe them carefully. Paramecium, like most ciliates, is a freshwater organism. Try to locate the following structures on Paramecium: cilia, oral groove, macronucleus and micronucleus, and contractile vacuole. Observe the contractile vacuole closely under the oil immersion lens on your microscope -- what does it appear to be doing? What is its function?

Apicomplexans

The organism that causes malaria (Plasmodium) is an apicomplexan, one of the three sub-groups within Alveolata. This group is characterized by the presence of an "apical complex" of organelles that is used to penetrate the tissues of the host organism. (No specimen today in lab.)

Candidate Kingdom Stramenopila

The stramenopiles are a diverse group of algae and mold-like protists united by common structural features of their flagella (some of which bear fine hairlike projections) and their chloroplasts, if present (these are derived from endosymbiotic eukaryotes). In addition to the three groups mentioned below, stramenopiles also include the golden algae (chrysophytes).

Diatoms (bacillariophytes)

Diatoms are single-celled photosynthetic organisms with silica shells. Diatoms are found in both freshwater and saltwater. They lack flagella. Like their relatives the brown algae, diatoms store food in the form of the carbohydrate laminarin. Prepare a slide of water from Foster Lake and search for live diatoms -- their geometric siliceous shells are distinctive. See also the slides of preserved diatoms.

Brown algae (phaeophytes)

The brown algae are all multicellular "seaweeds," but range in size from microscopic to enormous (e.g., Macrocystis, the giant Pacific kelp, may be 100 meters long!). Brown algae are largely marine organisms, and are particularly common in the cold waters of the temperate zone. Their characteristic brown or olive-green color comes from the presence of the pigment fucoxanthin (a kind of xanthophyll); brown algae also have chlorophylls a and c. Phaeophytes store food as laminarin (a carbohydrate), in contrast to the storage molecules of the green and red algae.

Examine the preserved specimens of brown algae. Laminaria has a flattened leaf-like structure as well as a stalk and a "holdfast" which helps to secure it to its substrate (usually rocks). This differentiation into specialized structures is characteristic of many brown algae. Both Sargassum (of Sargasso Sea fame) and Fucus have air bladders that help to keep them afloat; see the plastomount of Fucus to get a good sense of the three-dimensional nature of these bladders.

Water molds (oomycotes)

The oomycotes are single-celled fungus-like protists Their thread-like hyphae are similar to fungal hyphae, but have cellulosic cell walls (not chitinous, as in kingdom Fungi); furthermore, the diploid condition dominates in oomycote life cycles, whereas the haploid condition dominates in the fungi. View the prepared slide of the water mold Achyla, noting the hyphae -- what advantage does the thread-like shape of the hyphae confer? (Hint: most oomycotes are either parasites or saprobes.)

Candidate Kingdom Rhodophyta

Rhodophyta includes the red algae, so-called because of their red color, imparted by the pigment phycoerythrin (they also have chlorophyll a). Red algae may be differentiated from brown and green algae not only by their color, but also because they store food as floridean starch (another kind of carbohydrate) and because they have no flagellated cells at any point in their life cycle. Like phaeophytes, red algae are primarily multicellular and mostly marine organisms, but unlike the brown algae, they are most abundant in the warm waters of tropical regions.

View the preserved specimens of Polysiphonia, a red alga. See also the package of roasted seaweed -- the red alga Porphyra ("nori"), is used as a wrapper for sushi. Agar and carrageenan are two other products derived from red algae that are very valuable commercially. Agar is used in the lab to grow bacterial cultures, and carrageenan is found in a wide range of products, from paint to ice cream.

Candidate Kingdom Chlorophyta

The green algae, Chlorophyta, are the protistan group most closely related to the Kingdom Plantae (true plants). Like true plants, the color of green algae is imparted by chlorophyll a and b; also like plants, their food is stored as starch (how do these two features differ from those of red algae and brown algae?) Green algae may be single-celled, colonial, or multicellular; they are mostly freshwater, but some are marine or even terrestrial.

View the live and preserved specimens of Volvox, a freshwater, colonial green alga. Describe the shape and motility of Volvox. Can you see "daughter colonies" within a colony? See also the preserved specimens of Ulva (sea lettuce), an edible seaweed that is only two cell layers thick!

Candidate Kingdom Mycetozoa

This candidate kingdom includes two distinct groups of fungus-like organisms: the plasmodial slime molds (Myxogastrida) and the celular slime molds (Dictyostelida). Although superficially similar to true fungi, both groups of slime molds have amoeboid stages that are much more similar to amoebas than they are to fungi. Both plasmodial and cellular slime molds have complex life cycles that involve both feeding and reproductive stages. A key distinction is that the feeding stage (plasmodium) of the plasmodial slime molds is a massive multinucleate cell, whereas the feeding stage of the cellular slime molds is made up of many independent amoeboid cells. When the cellular slime mold cells start running low on food, they form an aggregate colony that looks like a plasmodium, but it is composed of many cells separated by plasma membranes.

Examine the prepared slide of the cellular slime mold Dictydium -- this is a whole mount of the sporangium, or spore-producing structure, of this organism.

Protists with Pseudopodia

A variety of similar protistan groups have pseudopodia ("false feet") for feeding and locomotion: the amoebas (rhizopods), radiolarians and heliozoans (actinopods), and the forams (foraminifera). The relationships among these three groups are uncertain at present. The actinopods have silica shells, forams have shells of calcium carbonate, and amoebas either lack shells or have proteinaceous shells. In the shelled (testate) forms, the pseudopodia extend through holes in the test.

Prepare a slide with a single live Amoeba. Use either a depression slide or support the coverslip or a regular flat slide with a few grains of sand so as not to crush the amoeba. Observe the locomotion of the amoeba. How do the pseudopods work? Try to locate food vacuoles within the cell. How does an amoeba ingest its food? Make a labeled diagram of an amoeba.

PART III: DIVERSITY OF THE FUNGI

There are five divisions in the kingdom fungi: Chytridiomycota, Ascomycota, Basidiomycota, Zygomycota, and Deuteromycota. (Note that use of the term “division,” rather than “phylum,” is a throwback to the days when fungi were lumped with plants; plant phyla are also called divisions.) Fungal divisions are distinguished by differences in their life cycles and in the structures associated with reproduction. Create a table in your lab manual summarizing the distinguishing features of each of the following five groups. Arrange the table as follows:

DIVISION	Characteristic Structural Features	Diagnostic Reproductive Features	Ecology	Examples of Species in This Group
Chytridiomycota
Ascomycota
Basidiomycota
Zygomycota
Deuteromycota

Chytridiomycota (chytrids)

The chytrids, such as Chytridium, were formerly lumped with the protists, but molecular and structural evidence places them in kingdom Fungi. They have chitinous cell walls, have an absorptive mode of nutrition, and some species form coenocytic hyphae. These species do, however, have spores with a single flagellum, unlike all other fungi. The chytrid lineage is believed to represent the most primitive group of fungi, and probably evolved from flagellated protists. The chytrids are mostly aquatic and are saprobes and parasites. View the prepared slide of Allomyces, which is a whole mount of the sporophyte.

Ascomycota, or ascomycetes (sac fungi and yeasts)

A characteristic feature of the sac fungi is that the spores (ascospores) are borne inside a sac-like structure called the ascus (plural: asci). Also, the hyphae have perforated cross walls separating the cytoplasm of adjacent cells. Examples of ascomycetes are yeasts, mildew, Neurospora, morels, and truffles. The fruiting body of ascomycetes, which is often cup-shaped, is called an ascocarp.

View the prepared slide of Morchella with asci. This is the morel, a much-sought after edible mushroom often found beneath oak trees.

The yeasts are single-celled fungi of tremendous economic significance. Put a drop of live yeast cells on a slide, then view them under the microscope. You may be able to see yeast cells “budding” off --- this is how they reproduce. Yeasts undergo anaerobic respiration, producing carbon dioxide gas and alcohol as waste products. The bubbles in champagne, beer, and rising bread are made of carbon dioxide. Alcohol produced by yeasts in a closed container eventually builds up to toxic levels, killing the yeasts and putting a halt to ethanol production -- this is why wine naturally has an alcohol content of only about 12 percent.

Basidiomycota, or basidiomycetes (club fungi)

The characteristic feature of this group is the basidium (a club-like structure on a stalk), which contains the basidiospores within it. Often the basidia are borne on gills or within pores. Like the sac fungi, hyphae have perforated cross walls separating cells. Examples of basidiomycetes are the mushrooms and rusts.

Examine the specimens of the commercial mushroom (Agaricus bisporus): note the gills underneath the cap. The mushroom is the fruiting body (or basidiocarp) of the fungus; most of the mycelium is actually underground. Remove a segment of gill and mount it on a microscope slide. Can you see the basidia and basidiospores? Also slice a stem lengthwise and try to see the filamentous structure of the mushroom.

Next view a prepared slide of Coprinus. Here the basidia should be plainly visible.

Zygomycota, or zygomycetes (common molds)

In this group of fungi, the hyphae lack cross walls. An example is the black bread mold, Rhizopus nigricans. View the prepared slide of Rhizopus zygotes. Zygotes are produced sexually and are the only diploid stage of this fungus. The thick-walled structure around the zygote is the zygosporangium, which prevents desiccation of the zygote. Spores are produced asexually by sporangia borne on stalks.

Deuteromycota, or fungi imperfecti

This group includes species where sexual reproduction has not yet been observed, making precise classification impossible; rather, these species reproduce asexually by conidia.

Examples include Penicillium, which is used in cheeses and is an important antibiotic.

View a prepared slide of Penicillium conidia.

PART IV: ECOLOGY OF FUNGI

Fungi occupy diverse ecological roles in biological communities. Many fungi are decomposers, i.e., saprobes, subsisting on dead organic matter. Fungi are among the few organisms that can break down cellulose or lignin, the major components of fallen tree trunks. View the prepared slide of the woodrot fungus, which shows the hyphae penetrating the wood. The hyphae have lots of surface area for absorption of nutrients, and so are ideal structures for decomposers.

Many agricultural diseases are caused by parasitic or pathogenic fungi. For example, the bracket fungus on display can penetrate the living tissues of trees. The rusts are a very damaging group of parasites. View the slide of wheat rust (Puccinia graminis) showing the telia stage (one of a number of different stages of its very complex life cycle). Can you see the fungal spores? Also see the preserved wheat stems showing wheat rust stages. There is also a preserved specimen of Claviceps purpurea (ergot) on wheat. The economic costs of fungi destroying human foods are staggering.

Not all fungi have sinister lifestyles, however. Many fungi engage in mutualisms with other organisms. For example, lichens are mutualisms between a cyanobacterium or alga and a fungus, usually an ascomycete. Lichens can tolerate incredibly harsh conditions, and are often the dominant organism in extreme habitats such as Antarctica, where they grow on rocks. Examine the specimens of lichen.

Another important symbiosis (“living together”) is found in the mycorrhizae, which means “fungus root.” View the slide of endomycorrhizae in an orchid root -- can you distinguish between root and fungus? Can you see the hyphae penetrating the cells? Other fungi are ectomycorrhizae, meaning they form a sheath around the root but do not actually penetrate the cells. The mycorrhizae provide enhanced uptake of nutrients, water, and trace metals to the plants, and the plants provide carbohydrates from photosynthesis to the fungi.