Kingdoms Archaea, Bacteria, and Protista

Materials

1. Plates of gram-positive and gram-negative bacteria 2. Bacterial plates showing a variety of colonies 3. Bunsen burners

4. Live and/or preserved Anabaena or Nostoc colonies 5. Live cultures of Ulothrix, Spirogyra, Oedogonium,

Volvox, Scenedesmus, Euglena, or Phacus

6. Slides of stained and preserved Ulothrix, Spirogyra,

and Oedogonium

7. Diatomaceous earth

8. Herbarium specimens of seaweeds such as Gelidium, Porphyra, Gigartina, Ulva, Codium, Postelsia, Laminaria, Costaria, Nereocystis, or Desmarestia

9. Live or preserved dinoflagellates

10. Loaf of sliced white bread that contains no preservatives

11. Small petri dishes

12. Pond water

13. Dropper bottles of gentian (crystal) violet 14. Dropper bottles of safranin 0 dye 15. Dropper bottles of 95% ethyl alcohol

16. Live slime mold plasmodia

17. Non-expendable and expendable slime mold sporangia 18. Preserved, dried, or living specimens of slime molds

 

Some Sugqested Learning Goals

1. Know how to distinguish gram-positive bacteria from gram-negative bacteria, and all bacterial cells from those of Kingdom Protista.

2. Understand distinctions between heterocysts and akinetes.

3. Learn the differences among Ulothrix, Spirogyra, and Oedogonium with respect to reproduction and chloroplasts.

4. Understand how a diatom is constructed and how it moves.

5. Know the parts and structure of a dinoflagellate and of a kelp or other large seaweed.

6. Know how a slime mold plasmodium moves and the structure'of slime mold sporangia.

Note;; Slime molds (myxomycetes) are believed to be mem­bers of Kingdom Protista because they have several features commonly found in other members of this kingdom, includ­ing reproductive cells with flagella, that are not generally found in members of Kingdom Fungi. Nevertheless, because

they also have certain funguslike features, they have in the past been treated as fungi and have traditionally been studied along with members of Kingdom Fungi, discussed in the next exercise (Exercise 15). Your instructor may or may not choose to defer examining these organisms until you study Kingdom Fungi.

Introduction

 

Kingdoms Archaea and Bacteria

All the members of Kingdoms Archaea and Bacteria have prokaryotic cells. Prokaryotic cells have no nuclei or other organelles bounded by membranes. Bacteria occur in three basic forms: cocci, which are more or less spherical; bacilli, which tend to be rod-shaped; and spirilli, whose cells are twisted like corkscrews. The kingdom previously known as Monera is divided into two kingdoms, based on some fundamental differences in the chemistry, metabo­lism, and RNA molecules of the cells. Kingdom Archaea includes anaerobic methane bacteria, salt bacteria that carry on a simple form of photosynthesis and live in water saturated with salt; and sulfolobus bacteria, which live ex­clusively in hot springs. Kingdom Bacteria includes the majority of the better known bacteria, most of which are saprobes that depend on nonliving organic matter for their energy sources; parasites that use other living organisms as their energy sources; and autotrophic bacteria that syn­thesize organic compounds from inorganic compounds by photosynthesis or other means.

Nearly all bacterial cells are considerably smaller than those of complex plants and animals, and are best examined with the highest power of a compound microscope. We will examine representative bacteria that have no pigments within their cells, and cyanobacteria that have chlorophyll and other pigments in membranes within the cells.

Kingdom Protista

Members of Kingdom Protista all have eukaryotic cells with nuclei and various organelles discussed in earlier exer­cises. With the exception of protozoans, sponges, water molds, and slime molds (slime molds are the only group of these organisms that will be discussed here), virtually all members of Kingdom Protista possess chlorophyll and other pigments confined to chloroplasts.

The active state of slime molds is called a plasmodium. Unlike a true mold, which consists of delicate threads that in most fungi are compartmentalized into individual cells, a plasmodium consists of a multinucleate mass of cytoplasm without cell walls. Plasmodia move over dead leaves and debris in a "crawling-flowing" motion. As the plasmodia move, they engulf bacteria and other food materials. Slime molds, like fungi, have glycogen as a primary food reserve, and other fungus like features such as stationary reproduc­tive bodies (sporangia). They differ sharply from true fungi, however, in their flagellated reproductive cells-a feature that suggests they originated from other members of Kingdom Protista.

In this exercise you will also be introduced to a few representatives of the thousands of species of pigmented algae. Algae vary in size from minute single-celled organ­isms to giant kelps that may attain lengths of 45 meters (nearly 150 feet). External water is essential to algae com­pleting their life cycles, and the great majority of them are aquatic. All possess chlorophyll a, but each phylum ex­hibits unique combinations of pigments, different food re­serves, and distinctive reproductive cells. They occur in na­ture as single cells, colonies, filaments, thalli (flattened bodies), or in mutually beneficial associations with fungi.

A. Non-Photosynthetic Bacteria

Examine the plates of bacterial colonies growing on agar, a gelatin like substance obtained from several red and a few brown seaweeds. Note the colors and textures of the colonies, which consist of many thousands of bacteria. Mount a small amount of bacteria in a drop of water on a slide by touching the tip of a probe to a colony and then vigorously rotating it in the drop of water. Cover with a coverslip and, after locating cells under low power, switch to high power. Note the shapes and sizes of the bacteria.

In the nineteenth century, Christian Gram discovered that some bacteria retain a stain he devised and others do not retain it. His stain became known as the Gram stain; those bacteria retaining the stain were called gram-positive and those not retaining the stain were called gram-negative. Variations of the Gram stain are now routinely used as a first step in identifying bacteria. If your instructor decides to have you check bacteria provided for their response to a gram stain, he/she will show you how to make bacterial smears. You should then be ready to proceed as follows:

On clean microscope slides, make smears of bacteria in the plates marked A and B, one smear to a slide. Dry the slides by passing them rapidly through a Bunsen burner flame four or five times. After the slides are dry, add a drop of gentian (crystal) violet dye to each slide. Tilt the slides `so that any excess dye drains away from the bacteria, and add a drop of Gram's iodine reagent. Allow the iodine to stand for 1 to 2 minutes and then add, one at a time, drops of 95% ethyl alcohol until the violet color is no longer ap­parent to the naked eye. Now add one drop of safranin 0 dye and wait for 30 seconds. Then wash gently with water,

add a coverslip, and examine with the microscope. Note that the bacteria on one slide are stained purple and those on the other are not. The gram-positive bacteria are stained purple; the gram-negative are not stained.

B. Cyanobacteria

Mount a small amount of the cyanobacteria Nostoc or An­abaena on a slide or, if fresh material is not available, exam­ine the prepared slides of these cyanobacteria. Note that the pigments are diffused throughout the cells and not located in plastids. Can you see any nuclei? Are there any colorless heteorocysts (cells that appear to have a slightly thicker wall) scattered throughout the filaments? Heterocysts are nitrogen-fixing cells at which cyanobacterial filaments may fragment (break). Are there any dense-looking cells that are somewhat oblong in outline (akinetes) present at the ends or within the filaments? Akinetes are resistant to freezing and desiccation, and are a means of ensuring the survival of the organisms over winter or when water is lacking.

 

C. Pond Water Organisms

Agitate the pond water and place a single drop on a clean slide; add a coverslip. Examine with the compound micro­scope. Make drawings or diagrams of at least three differ­ent organisms. To help you identify the organisms, there are pictures of some of the more common ones on the pages at the end of this exercise. Your instructor may help you identify other organisms not illustrated. Do you see any motile forms (i.e., forms that are moving)? Movement may be by means of whiplike tails called flagella or by means of numerous short, moving hairs called cilia. Diatoms have a rigid glasslike cell wall composed primarily of silica; their movement may be brought about by means of cytoplasm extending through pores and functioning somewhat like a Caterpillar tractor track. Both flagella and cilia are minute in diameter and may be difficult to see without special equipment or techniques. Notice the wide variety of chloro­plast types and the small, round, colorless pyrenoids (starch accumulation centers found in green algae) on some of the larger chloroplasts (they may or may not be present on your particular slide).

D. Spirogyra

Mount a small amount of Spirogyra in a drop of water on a slide. Locate a pair of conjugating filaments. If your fresh material is not conjugating, examine a prepared slide show­ing this. Observe the papillae that unite, forming conjuga­tion tubes (short cylindrical tubes between adjacent cells). Are any gametes migrating through the conjugation tubes to adjacent cells? If the cells of one filament are empty, note the relatively thick zygotes in the cells opposite those of the empty filament. No special asexual reproductive cells are produced by Spirogyra. Instead, new cells are added by mitosis after filaments break.

E. Ulothrix and Oedogonium

Examine the cultures of Ulothrix and Oedogonium avail­able, and study the prepared slides of these two green algae. How do these two algae differ from one another and from Spirogyra with respect to chloroplasts and reproduction?

E Diatomaceous Earth

In a drop of water on a clean slide, mount a small amount of diatomaceous earth, which consists of the "shells" of millions of marine diatoms. What are the most common shapes of marine diatoms? Is there variation in the patterns of pores present? Note that diatoms may appear to have one shape in valve (top or bottom) view and another shape in girdle (side) view.

G. Dinoflagellates

In a drop of water on a clean slide, mount a small amount of the dinoflagellate material provided, or alternatively, ex­amine the demonstration that has been set up. Note the "armor" plates. How are the grooves in which the flagella are located arranged?

H. Seaweeds

Examine the herbarium specimens of marine algae (sea­weeds) on display. Although some of the larger seaweeds have forms of food- or water-conducting tissues, none have true xylem or phloem, root, or leaves. Do any of them have holdfasts (rootlike structures that anchor them to rocks), bladders (bulblike swellings that enable seaweeds to float), stipes (stalks), or blades (flattened, leaflike bodies)?

I. Slime Molds

With the aid of your dissecting microscope, examine the petri dishes with living plasmodia of slime molds. Focus on the leading edge of a plasmodium and note the rapid flow­ing of the protoplasm. Does the direction of flow ever change? Can you see individual cells?

Examine the specimens of slime mold reproductive bodies available. If expendable materials are provided, mount a sporangium in a drop of water on a slide. Observe the numerous spherical spores and the capillitial threads interspersed among the spores. A few species of slime

molds lack capillitial threads, which are unknown in true fungi.

 

Drawings to Be Submitted

1. Draw a group of bacterial cells from those you mounted in water. Also draw one or two filaments of cyanobacteria such as NOSTOC or ANABAENA. Be sure to indicate the magnifications of your drawings.

2. Draw at least three different algae occurring in your pond water. If they are not illustrated on the pages at the end of this exercise, ask your instructor to identify them for you.

3. Label the drawings of SPIROGYRA and ULOTHRIX provided. Labels for SPIROGYRA should incude: VEGETATIVE FILAMENT, PAPILLAE, CONJUGATING FILAMENTS, CHLOROPLAST, PYRENOID, GAMETE, ZYGOTE, and GERMINATING ZYGOSPORE. Labels for ULOTHRIX should include: HOLDFAST, ZOOSPORES, NEW FILAMENT, GAMETES, FERTILIZATION, ZYGOTE, MEIOSIS, and MATURE FILAMENT. Show where MEIOSIS takes place in both organisms.

4. Draw a filament of OEDOGONIUM from a prepared slide. Label VEGETATIVE CELL, OOGONIUM, EGG, ANTHERIDIUM, and SPERM.

5. Draw a diatom, showing its markings.

6. Draw a dinoflagellate showing its grooves and "armor" plates.

7. Draw a marine alga (seaweed). Identify any HOLDFASTS, BLADDERS, STIPES, or BLADES present.

8. With the aid of your dissecting microscope, draw several slime mold sporangia. Label SPORANGIUM and CAPILLITIAL THREADS.

When you have completed your assignments for this exer­cise, break off a portion of bread small enough to fit within the petri dish provided. Then add no more than one drop of water to the bread (if you add more than one drop, yeasts are very likely to multiply and interfere with the growth of other fungi you will be trying to cultivate). Next, sprinkle dust from the corners of the floor in the laboratory or else­where, or comb your hair over the bread. Close the dish and print your name and section number on the outside, then set it aside until the next laboratory session. At that time we will examine any fungi that develop on the bread.

 

1. What is the difference between prokaryotic and eukaryotic cells?

2. If a bacterium is gram-positive, how does it respond to a Gram stain?

3. Which organisms are most likely to contain heterocysts?

4. Of what advantage to a cyanobacterium is an akinete?

5. What is the function of conjugation tubes?

6. Specifically where in a cell are pyrenoids located?___________________________________________

7. What substance gives rigidity to diatom cell walls?___________________________________________

8. How do diatoms move?

How do dinoflagellates move?

9. How does the plasmodium of slime mold differ from the mycelium of a true fungus?__________________

10. What is the equivalent of a root in a seaweed?

 

 

 

Kingdoms

1. What is a pyrenoid?__________________________________________________________________

2. In which alga would you expect to find conjugation tubes?

3. If you find a pair of algal filaments conjugated but the cells of one of them are empty, what are the dark objects in the cells of the other filament?__________________________________________________________

4. What are the colorless, slightly thicker-walled cells present in some cyanobacteria?__ 5. What is an akinete?__________________________________________________

6. In addition to differences in the way they reproduce, what should help you distinguish Spirogyra from Oedogonium?

7. What is a motile alga?_________________________________________________________________

8. What type of organism mentioned in this exercise may have bladders?

9. Where are the pigments of cyanobacteria located?

10. Which algae have rigid, glasslike walls?___________________________________________________

 

                     ALGAE LAB

AIM:  To make yourself familiar with specimens of algae.  As with all other groups of organisms, certain species of algae are found in certain habitats.  The purpose of this material is to demonstrate that you can identify some of the more common species and learn where some of these species are commonly found.

MATERIALS:  Microscopes, algae samples, herbarium samples

PROCEDURE: Examine specimens of the different groups of algae noting the distinguishing characteristics of each.

 

DIVISION CHLOROPHYTA

    The Chlorophyta are the green algae.  They are an ancient group, possibly extending back to the origin of photosynthetic, cellular plants.  Complex green plants are considered to have arisen from green algae.

Distinguishing Characteristics:

    1.  Pigments---chlorophyll a and auxiliary pigments, chlorophyll b and carotinoids (yellow and orange pigments).

    2.  Food reserve--true starch.

    3.  Cell wall--cellulose..(exceptions)

    4.  Flagellation--when present, always two to four; always anterior.

 

I.  Examine the various specimens on demonstration.  Draw and Describe one macroscopic ON your paper.  Use the following terms to help with the description;

    Branching, cell shape, color, chloroplasts..(shape, size, location), filaments, presence of any reproductive structures, any outer sheath present and anything else you can describe it with!

 

DIVISION PHAEOPHYTA

  Most brown algae (Phaeophyta) grow in the intertidal zone.  Nearly all are marine.  There are no unicellular genera.  Forms vary from simple  branched filaments to giant seaweeds over 60 meters long.  Many of the giant species, especially the kelps, show a high degree of external differentiation into a root-like HOLDFAST, a short, stem like STIPE, and a long strap-like BLADE.  All larger species have air bladders of various sizes.

 

Distinguishing characteristics are:

    1. Pigments--chlorophyll a and auxiliary pigments, chlorophyll c, carotinoids and fucoxanthin (brown pigment).

    2.  Food reserve--laminarin and mannitol.

    3.  Cell wall of cellulose and algin (a commercially valuable compound).

    4.  Reproductive cells with two laterally placed flagella.

    5.  Well developed alternation of generations.

 

II.  Examine the various specimens on demonstration.  Draw and Describe one macroscopic sample on your paper.  Use the following terms to help with the description;

    Branching, cell shape, color, chloroplasts..(shape, size, location), presence of any reproductive structures, any outer sheath present and anything else you can describe it with!

 

 DIVISION RHODOPHYTA

  The red algae (Rhodophyta) are relatively small plants, most species being less than 0.7 meters long.  Their growth forms are simple filaments, highly branched filaments or sheet-like bodies.  They are abundant in warm marine waters. A few are fresh water.  They are capable of living at depths greater than those of any other algae.

 

Distinguishing characteristics:

    1.  Pigments--chlorophyll a and the auxiliary, pigments, chlorophyll d, phycoerythrin, and phycocyanin. 

    2.  Food reserve--floridean starch.

    3.  Cell wall of cellulose sometimes covered with gelatinous material commercially known as AGAR.

    4.  Absence of any kind of motile cells.

    5.  Complex life cycles in many.

 

III.  Examine the various specimens on demonstration.  Describe one macroscopic and one microscopic sample on your paper.  Use the following terms to help with the description;

    Branching, cell shape, color, chloroplasts..(shape, size, location), presence of any reproductive structures, any outer sheath present and anything else you can describe it with!

 

 

DIVISION CHRYSOPHYTA  (golden-brown algae (diatoms))

The golden-brown algae (Chrysophyta) possess evolutionary trends in size increase some of which are exhibited by filamentous and colonial forms.  We will examine diatoms, either filamentous or unicellular forms.  Diatoms are characterized by cell walls composed of two overlapping halves that fit together in a manner similar to the parts of a Petri dish.

ALGAE LAB       DRAWING SHEET   NAME______________________________ PD______

Label all parts ith holdfast, stipe, blade, name of speciman, collection point if included.

Chlorophyta (Green Algae) Macroscopic                                                            

 

 

 

 

 

 

 

 

 

 

 

 

Phaeophyta (Brown Algae) Macroscopic                                                           

 

 

 

 

 

 

 

 

 

 

 

 

 

Rhodophyta (Red Algae) Macroscopic                                                                   Rhodophyta (Red Algae) Microscopic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                Chrysophyta (Diatoms) Microscopic