Download Lab 9-Proeukaryote

9-1
Biology 1001
Laboratory 9
PROKARYOTES AND EUKARYOTES
PREPARATION
Read this exercise before you come to the laboratory.
TEXTBOOK READING
- Biology (Campbell et al.)- Chap. 27 (Bacteria), 28 (Protists) and 31 (Fungi)
- Biology 1001 Laboratory Manual: Appendix IV – Tables on page A-12
OBJECTIVES
- to correctly use the oil immersion objective
- to observe bacteria and understand cell types and Gram’s stain
- to prepare and stain wet mounts of yeast, mold and algae
- to discover and understand the differences between prokaryotes and eukaryotes
PREPARATION
Be able to answer the following questions:
1.
What are the differences between prokaryotic and eukaryotic cells?
2.
Why is immersion oil used?
3.
Why are bacteria heat fixed to the slide?
4.
Describe the differences between the cell walls of Gram positive and Gram
negative bacteria?
5.
Check the internet for images of bacteria (various forms of cocci, bacilli, spirilli) as well as
Anabaena, Zygnema, Rhizopus and Yeast.
LABORATORY ASSIGNMENTS
1.
After examining a slide of stained bacteria under oil immersion, prepare a
proper table indicating the cell types observed, cell sizes and their Gram
staining properties. Pass this in before you leave today.
2.
Prepare labeled drawings of fungal cells from wet mounts of Saccharomyces and Rhizopus.
3.
Prepare a labeled drawing of the prokaryotic Blue-green algae, Anabaena.
4.
Prepare a labeled drawing of the eukaryotic Green algae, Zygnema.
5.
Answer the questions on pages 9-9 and 9-10.
9-2
Prokaryotes and Eukaryotes - General Introduction
The major differences between prokaryotes and eukaryotes are listed below. Some, but not all
of these differences can be observed with light microscopy.
Prokaryotic Cells
Eukaryotic Cells
___________________________________________________________________________
Nucleus
not bounded by a nuclear
Membrane
DNA
single circular molecule
bounded by a double unit
Membrane
organized to form chromosomes
Complex organelles
Absent
Present
Microtubules
Absent
Present
Flagella
Simple
Complex (9+ 2)
Sexual reproduction
Absent
Present
___________________________________________________________________________
The basic living unit of all organisms is the cell. Although different cell types vary in size and
shape and although they perform widely different functions (e.g. muscle cells contract, red blood cells
carry oxygen, epidermal cells protect, etc...) their basic components are quite similar.
It is, however, possible to divide living organisms into two broad cellular groups: the
EUCARYOTES and the PROCARYOTES.
Prokaryotic cells belong to the smallest and simplest of all living organisms. Mycoplasms,
bacteria and blue-green algae are all prokaryotic cells and are considered primitive in evolutionary
terms. However, in terms of biomass and variety you should not think of them as unsuccessful
organisms. The oldest known fossils, approximately 3 billion years old, are of prokaryotes.
Prokaryotic cells do not have their chromosomal material organized into a membrane-bound nucleus,
they lack a nucleolus and membranous organelles such as mitochondria, Golgi, chloroplasts, etc...
A.
Bacteria
Introduction
Despite their minute size and simplicity of organization, bacteria are highly successful
organisms. As a group, they are adapted to occupy an extremely wide range of environments, and in
fact, they are more widely distributed than any other form of life.
In soil, where bacteria are especially abundant, some species play an important role in the
nitrogen cycle converting the inorganic nitrogen gas of the atmosphere into nitrogenous salts which
become available for plant growth. Bacteria are also responsible for much of the degradation of dead
9-3
organic material which releases carbon dioxide for reutilization in photosynthesis (the original
recycling factory).
Bacteria are important primary producers (many small organisms eat them) so they occupy an
vital place in world food chains. But, on the other hand, we have also been subjected to innumerable
diseases which can be traced to bacteria as causative agents: gonorrhea, pneumonia, scarlet fever,
tuberculosis, diphtheria and many more.
Bacterial cells, as we have already mentioned, are very small: we are talking about organisms
which are about one micron in diameter. If you consider that the limit of resolution of the light
microscope is 0.22 microns, then you can appreciate that until the development of the electron
microscope, not much was known about the internal structure of these organisms.
In fact during today’s lab session you should be able to get a first-hand appreciation of what
technical limitations mean in terms of the quality of information one can derive from light microscopy.
Bacteria occur in three typical shapes: round (the cocci, coccus, singular); rod-shaped
(bacilli; bacillus, singular) and curved-shaped (spirilli; spirillus, singular). See Figure 27.2 in your
text book. All of these can occur singly or in groups known as bacterial colonies.
Beyond the actual shape differences, bacteriologists observe certain clues which enable them to
further classify bacterial types.
(a)
Clustering – bacteria may exist as individual cells or as groups of two cells, clusters or chains of
individual cells. These characteristics are observable microscopically and these characteristics are
useful in identifying bacteria.
(b)
Colony Characteristics - bacteria organize themselves in large distinct groupings presenting
characteristic colours, shapes, margins and profiles. These characteristics are observable
macroscopically and used with microscopic evidence to identify bacteria.
(c)
Motility - whether a particular species of bacteria is capable or not of movement (i.e., is motile)
may aid in its identification.
(d)
Staining Reaction - The reaction of different species of bacteria to certain stains may be a
useful identifying feature.
Over the years, the Gram stain (named for its discoverer, Christian Gram) has proved to be a
very useful diagnostic tool. The reaction to the Gram stain correlates with differences in the cell wall
composition of bacteria. It is called a differential staining procedure because it can be used to divide
bacteria into two groups, called Gram positive (G+) and Gram negative (G-). After Gram staining, the
positive bacteria appear blue and the negative bacteria, appear red. Not all bacteria are either Gram
positive or Gram negative; a few display both staining reactions and are referred to as “intermediate”
or Gram variable. See Figure 27.3 in your text book.
Demonstration: Colony Types
On the demonstration bench, observe a number of Petri dishes containing bacterial cultures grown on
an agar medium. Note the variety of colours, colony shapes, surface characteristics and margins.
9-4
Macroscopic characteristics such as these are used to help identify what kind of bacteria constitute
these colonies. What are the most common colony shapes, colony margins and colony surface
characteristics found in the species you observed on the demonstration bench?
Microscopic observation of bacteria:
Examine slide 108 from your slide box. This is a bacteria smear showing a variety of bacterial
shapes and gram-staining properties. You will need to examine the slide using the 100X objective lens.
You must use the oil immersion technique to do this. Before you begin, thoroughly clean the slide by
breathing on it and polishing with a piece of lens tissue. This will remove dust and fingerprints.
This is a good time to clean the 40x and 100x lens on your microscope. Use lens cleaner from
the stain rack and lens tissue to do this. Smudgy lenses will make it hard to bring microscopic
organisms into sharp focus.
Procedure for using Oil Immersion
1. Centre the slide using the scanning (4X) lens. Bring the smear into focus.
2. Change to the low power (10X) lens and then the high power (40X) lens, centering and focusing the
slide each time. Be sure to use only the FINE FOCUS knob with the 40X lens.
3. While watching carefully from the side, swing the 40X lens out of the way and the oil immersion
(100X) lens toward the functional position. Before snapping it into place (aligned with the body tube),
place a small drop of immersion oil (found in your stain racks) on top of the circle of light from the
condenser, showing through the slide.
4. Still watching carefully from the side, snap the 100X lens into position and allow it to contact the oil
droplet.
5. Look through the ocular lens and carefully focus the microscope, using the FINE FOCUS KNOB
ONLY.
NOTE: you cannot go back to high power (40X) without removing the immersion oil from the 100X
lens and the slide.
6. You may need to increase the light intensity if the field of view seems too dark.
7. Examine the slide, using the mechanical stage to move the slide as you require. Use the ocular scale
to measure each cell type you observe. Refer to the calibration information from lab 4 to calculate the
length on one ocular unit at 1000X.
8. When you are finished examining your slide, you must clean all the immersion oil off your lens and
slide while being very careful not to get oil on the high power lens while doing so. While watching
carefully from the side, swing the 100X lens out of the way and the scanning (4X) lens into place.
Using lens tissue, clean the 100X lens of immersion oil. Repeat using a new piece of
lens tissue moistened with lens cleaner. Remove the slide and clean it thoroughly with
tissue and lens cleaner before replacing it in your slide box.
9-5
Assignment 1:
Carefully survey slide 108, locate and measure as many different kinds of bacteria as possible.
Note the size and Gram staining property of each. (Figure 27.2 and 27.3 in your text provides
information on Gram stain and bacterial cell shapes) Be sure to only measure individual cell size, NOT
the size of a cluster of cells.
Organize your notes and construct a table to summarize: a) the variety of cell types (cocci,
bacilli, spirilli), b) the length of each cell type and c) the Gram-staining properties of each type. Be sure
to refer to your lab manual, Appendix IV, page A-12 for instructions on preparing proper tables. Pass
this table in before you leave today.
B.
Fungi
Introduction
Say “Fungi” and people immediately think of awful, unpleasant things. Fungi are often
thought of as sprawling blobs which occupy a very low place in the evolutionary order of things. In
fact, the truth is nearly at the opposite extreme. Many fungi are exquisitely constructed and their life
cycles are among the most complex to be found any where in nature. There is great diversity in
anatomy and life cycles and for this reason the Kingdom Fungi is difficult to define as a taxonomic
unit. The current view is that the Kingdom encompasses all heterotrophic organisms with absorptive
nutrition. They are saprophytes (living on dead matter) or parasites.
The fungi are placed in division Eumycophyta. This division can be divided into four phyla,
we will only examine two, the Ascomycetes and the Zygomycetes.
The Ascomycetes, the sac fungi, have hyphae (singular: hypha) with crosswalls and each
resulting cell has a single nucleus. We will look at Saccharomyces (yeast, a unicellular variety) from this
class.
The Zygomycetes, the algal fungi, have multinucleated hyphae without crosswalls. From this
class we will examine Rhizopus, the black bread mold.
Procedure
YEAST:
1.
Obtain a clean dry slide. Use only a small drop of culture and a VERY SMALL drop of
methylene
blue. If you put the stain on the slide first, you can wipe away any excess before you
add the yeast. Add a small drop of yeast culture and a coverslip.
2.
Observe and draw a few typical cells. These cells are alive. They reproduce vegetatively by
budding. You will see buds of all sizes, some as big as the parent cell. (Usually only one bud
per cell). Dead cells will stain dark blue. Live cells will remain clear. Find an example of
budding and include it in your diagram. These cells have organelles inside, but you will not be
able to identify them. Refer to Figure 31.7 in your text.
3.
When you have completed the drawing of a few yeast cells, put the slide aside while you go on
with the exercise. After 20 or 30 minutes, recheck the slide. Do you notice air bubbles that
were not there before? Where did they come from?
9-6
Assignment 2:
Prepare a labeled drawing of Saccharomyces. Don’t forget to include title, magnification and scale bar.
You may wish to refer to Appendix III to review the instructions for preparing biological drawings.
Procedure
1.
you
Rhizopus has been grown on agar in Petri dishes. Examine the colony in the petri dish
under the dissecting scope on the demonstration bench. Observe patterns of
growth and formations. The hyphae are the network of clear tubular strands. Occasionally
will see large upright round expanded ends on some hyphal strands. These are sporangia
(sporangium = singular), the spore producing structures. Refer to Figure 31.12 in your text.
2.
Prepare a stained whole mount of Rhizopus. Use a clean dry slide. Bread mold is not easy to
spread on the slide. Put a SMALL DROP of methylene blue and a drop of water on the slide.
Then, using two dissecting needles or toothpicks, remove some of the bread mold from the
petri dish and spread the strands apart in the drop of liquid on the slide. Carefully tease the
clump in the liquid to immerse it and separate the hyphae. Add cover slip.
3.
View your slide under your microscope. Locate an intact hyphal tip and watch for 3-5
minutes, you may see the hyphae growing.
4.
Draw a portion of your Rhizopus slide, showing branching pattern and as many structural
Assignment 3:
Prepare a labeled drawing of Rhizopus. Don’t forget to include title, magnification and scale bar. You
may wish to refer to Appendix III to review the instructions for preparing biological drawings.
C.
ALGAE
Introduction
Although an extremely diverse and plentiful group, the algae are often overlooked or ignored.
They range from small unicellular and simple, to large, multicellular and complex. They come in a
variety of colours and can be found living alone, in colonies or in association with others (Paramecium,
seaweeds, lichens). Algae are food suppliers, oxygen producers and some are nitrogen fixers. These
interesting organisms can stabilize ice cream and toothpaste, kill fish or poison cattle.
Algae can be subdivided into several divisions, we will examine two, Cyanobacteria (the called
the blue-green algae) and Chlorophyta (green algae).
The prokaryotic Cyanobacteria (blue-green algae) lack distinct chloroplasts and the
photosynthetic apparatus consists of a system of cytoplasmic membranes. The pigments of
phytosynthesis are chlorophyll a, four different carotenoids, several kinds of phycocyanin (blue
pigment) and phycoerythrins (red pigment). The range of colours of these algae are due to the
different concentration of these pigments.
The eukaryotic Chlorophyta (green algae) are the only group of algae which contain the full
complement of photosynthetic pigment characteristic of the terrestrial plants. Other algae groups
features as yo
9-7
include simpler algal groups such as Golden, Brown, Red Algae and Diatoms. There is little doubt that
the green algae and land plants are related. Members of the Chlorophyta exhibit a wide variety of
shape, anatomical structures and life cycles.
i) Cyanobacteria (Blue-green Algae)
Procedure
1.
Prepare a wet mount of the cyanobacteria, Anabaena. It is a very narrow uniseriate (one cell
wide) filament. It is important to note the colour to facilitate comparisons to the other algal
groups that you will examine.
The filament is composed of round vegetative cells surrounded by a gelatinous sheath. In
cyanobacteria there are no membrane bound organelles. The photosynthetic lamellae float
free in the cytoplasm (they are not bound onto chloroplasts) and the DNA is in the form of fine
fibrils. Examine Figure 27.10 in the text.
2.
Put only one SMALL drop of culture on a dry slide and cover with a cover slip. Too much
liquid will cause the cover slip to float and the whole thing will jiggle with every vibration.
3.
Use the 4x lens on your microscope and shut the light way down (use the iris diaphragm lever).
Search for uniformly thin, dark, fairly straight, hair-like filaments. Center a specimen in the
field of view and go to 10x. Increase the light. You should be able to see the outline of the cells
and the blue-green colour.
4.
Center the specimen in the field of view and go to 40x. Focus carefully through the cells. Use
the iris diaphragm lever to vary the light. At low light you should be able to see the transparent
gelatinous sheath (it looks like an aura or halo when you adjust the focus). With brighter
light, and careful focusing, you can see granular looking inclusions inside the cells, these are
gas vacuoles. With brighter light the blue-green pigment is more obvious.
Almost all the cells you will see are the photosynthetic small, bead-like vegetative cells.
Larger heterocytes (lighter cells with thick cell walls, they are nitrogen fixing cells with very
little chlorophyll) and granular oval cells the akinetes (resting cells which contain energy
reserves for up to 30 cell divisions and functions to ensure survival through unfavourable
conditions) are rare in these cultures.
Draw a segment of an Anabaena filament at 400x on your microscope. Do not use oil
immersion. (Any movement of the cover slip, such as when the 100x lens contacts the oil
droplet, will cause the specimen to move out of the field on view).
5.
Assignment 4:
Draw a section of an Anabaena filament and label the underlined words as well as the cell wall and
cytoplasm.
9-8
ii) Chlorophyta (Green algae)
Procedure
1.
Prepare a wet mount of Zygnema, a filamentous chlorophyte. Unlike Anabaena, there are
membrane bound organelles. Again, note the colour of the cells. This is also a uniseriate filament.
2.
Increase the power to 400x and examine one cell. The cells of the filament are short and
cylindrical. At either end of the cell there is a large stellate (branching) chloroplast. The nucleus is
in the center but not easy to see. Like Anabaena, the filament is surrounded by a gelatinous sheath,
which is easiest to see in reduced light.
Assignment 5:
Draw a section of a Zygnema filament and label the underlined words as well as the cell wall and
cytoplasm.
Assignment 6:
Complete the Assignment Questions that follow.
Assignment Questions:
1. Gram-positive and Gram-negative bacteria differ in the nature of their cell walls. Explain why
Gram-positive bacteria trap crystal violet stain and appear purple and why Gram-negative bacteria do
not and thus appear red. _______________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
2. What physical features of bacteria (macroscopic and microscopic) are used to identify different
kinds of bacteria? _____________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
3. What cellular detail is apparent in the yeast? ____________________________________
___________________________________________________________________________
___________________________________________________________________________
9-9
4. What cellular detail is apparent in the mold? _____________________________________
___________________________________________________________________________
___________________________________________________________________________
5. How do the two types of fungi differ? __________________________________________
___________________________________________________________________________
___________________________________________________________________________
6. How do the two algae types differ? ____________________________________________
___________________________________________________________________________
___________________________________________________________________________
7. Divide the organisms you observed into prokaryotes and eukaryotes and explain your division.
Prokaryotes: _________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
Eukaryotes: _________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________