PRINCIPLES OF BIOLOGY (BIOL 51)
Biology 51
Laboratory
Manual
Fall 2010
Table of Contents
General Lab Procedures 3
Request for Review of Grading 7
Loss of Laboratory Points 9
Safety in the Biology Laboratory 10
Lab 1 – Classification & Evolution 13
Lab 2 – Population Genetics & the Hardy-Weinberg Theorem 23
Lab 3 – Microscopes & Mitosis/Meiosis 43
Lab 4 - Bacteriology I 57
Lab 5 – Bacteriology II & Scientific Investigation 69
Lab 6 – Protists, Fungi, and Bryophytes 71
Lab 7 – Vascular Plant Reproduction and Diversity 83
Lab 8 – Plant Anatomy 93
Lab 9 – Introduction to the Animals: Cnidaria, Platyhelminthes & Annelida 99
Lab 10 – Mollusca and Arthropoda 113
Lab 11 - Echinodermata and Chordata 125
PRINCIPLES OF BIOLOGY (BIOL 51)
General Lab Procedures
Fall 2010
General Procedures
Before coming to the lab, read the exercise to be done during that week's lab period. Mark in your lab manual any sections which you do not understand. If you still have questions after your lab instructor has explained the material, ASK QUESTIONS. If you are not clear on a concept, others probably are not either and will appreciate your questions.
Do not attempt to answer specific questions in the lab manual until after you have examined the material.
Get to the lab on time. Instructions are usually given during the first part of the lab and it is a waste of time for the lab instructor and for others in the lab if instructions must be repeated (see Attendance and Grading below).
You must complete the lab exercise in the time allotted. You may neither stay past the end of your scheduled lab period nor come to another lab session later in the week.
Attend the lab section in which you are registered. Failure to attend your lab section will be counted as an unexcused absence (see Attendance and Grading below). For exceptionally compelling reasons (e.g., a University-sponsored event) permission to attend another lab may be granted. This permission can only be granted by Ms. Walker or Dr. Brunell (not your lecturer or your lab instructor) and must be obtained on an EMERGENCY ADMITTANCE TO LAB form.
Lab Attendance and Grading
For attendance and grading guidelines, see “General Information” for Bio 51.
Procedures in the Laboratory
Forbidden Substances: No food, drinks, chewing gum, candy, tobacco, etc. may be consumed or used at your laboratory station.
Turn off all phones and any devices that make noise. Don’t use your cell phone during lab, either in the room or in the hall.
Contact Lenses: Since the specimens used in the laboratory are often treated with preservatives that may give off potentially harmful chemical fumes, we recommend that you do not wear contact lenses in the lab. If you must wear contact lenses, be sure to clean them thoroughly after the lab.
Safety glasses/goggles and closed-toed shoes are required for all labs marked with an * on the schedule. The safety goggles used in chemistry labs will be adequate for biology labs as well. Goggles may be acquired at hardware stores, home centers, discount stores, etc. Failure to bring eye protection and the correct shoes will result in point deductions.
General Policy: You are encouraged to talk with your instructor about any problems or questions you have concerning the lab exercises. Visit other lab benches and see how their experiments and slides compare with yours. If you have questions and the instructor is busy, see if another student can answer your question. Walk about the laboratory a lot; observe! Remember what Yogi Berra said: “You can observe a lot by just watching”. Talk a lot about the lab material. A silent laboratory is usually a poor one. And finally, many questions can be answered by simply consulting the index in your lecture book.
Help your instructor to help you: If you want help finding a structure on a microscope slide, get up out of your chair and let the instructor sit down and find the structure. Also, your instructor may want to check your work/answers in the lab manual before you leave lab. Have the lab manual out and ready for inspection at all times.
Drawings: Many exercises require you to record your observations as simple drawings. These drawings serve two purposes. One is simply that they allow your instructor to check your progress and comprehension of material extremely rapidly. Secondly, and this is much more important, they require you to make critical observations and interpretations. Each time you make a line on the paper, you have made a decision about an organism, which means you are thinking about them…and that is our goal.
Drawing requires no artistic ability, in fact artistic styling only adds features that are not really present and distorts the representations. You should aim to be as accurate as your natural endowment allows. Follow these pointers:
1. To draw objects as large as possible; make them fill the space available in the manual.
2. To start by drawing an outline diagram of the specimen that you wish to illustrate, and then show detail of only the specific parts indicated.
3. That an accurate drawing of a small portion of a specimen is far more informative than an inaccurate drawing of a complete specimen. For instance, a student who draws two cells of stem cortex accurately, gains a conception of the nature of this plant tissue, and spends only a few minutes making her drawing. On the other hand, a student who tries to draw fifty or more cells always performs a degenerate scribble which therefore does little to aid her conception of the tissue. Not only that, it occupies considerably more time.
4. That all drawings must be titled and all structures shown must be labeled, otherwise your instructor will not be able to evaluate your understanding of the exercises. Also, remember that untitled and unlabeled diagrams will be valueless when you try to study them.
5. Your drawings consist of your own observations, not copies that follow the textbook or photographic atlas. Remember, that would be plagiarism.
Digital Camera: Many students find a camera to be a useful tool in lab. Students are encouraged to use a camera to capture images of specimens, equipment, and slides (even though not perfect, holding the camera up to the microscope ocular will allow for a decent image to be captured). A word of advice on using a camera: it is not a substitute for study, observation, or careful drawing and labeling. Snapping a few photos and then heading out the door is not a effective strategy for performing well in lab.
Required laboratory supplies: The following items will be needed at various times during the semester and should be brought to the appropriate labs:
1 three-ring binder for your laboratory manual
2 #2 drawing pencils
1 eraser
lab goggles
Request for Review of Grading
BIOL 51 - Principles of Biology
Complete this form, staple it to your exam and return it and the exam to your lab instructor or to one of the professors within one week of the day that the exam was returned to the class.
Your Name ____________________ Phone __________ Date ________________
Pacific ID No. 988-____-________ Lect Section __________ Lab Section __________
Student’s Email address:________________________
I wish to have the following question(s) reviewed on the attached examination:
________________________________________________________________________
Why do you feel that the grading needs to be reviewed? Why do you feel that your answer/answers is/are correct even though it/they has/have been counted wrong?
|Action taken |Date |Initials |
Loss of Laboratory Points
BIOL 51 – Principles of Biology
(D. Walker & M. Brunell)
Student’s name ______________________________________ Lab section _________
Date ____________________________
____ (3 pts) Microscope not properly put away: reason
____ (5 pts) Table not cleaned properly
____ (5 pts) Disrespectful behavior or unprofessional conduct
____ (5-10 pts) Misuse/abuse of laboratory equipment
____ (5 pts) Arriving > 5 minutes late OR left early without completion of lab exercises
____ (2-5 pts) Using cell phone during lab or allowing cell phone to ring during lab
____ (2-5 pts) Not being prepared for lab or not being on task in the laboratory
____ (_____pts [deduction scaled to situation]) Other incidents
Lab instructor: ________________________________________
Date: ______________________________________
Copies to: Student, Mrs. Walker, Dr. Brunell (circle)
Safety in the Biology Laboratory:
by
Santos (1994), Byrne (1997), Christianson & Walker (2000)
Be safe. It is extremely important for you to establish good safety practices when working in the lab. You should always be aware of the nature of the materials that you are using in the lab. Always use care in carrying out experiments with these materials. Ultimately, it is to your benefit to always be informed about materials you are using and to establish good safety practices.
Safety is a combination of common sense and familiarity with existing hazards and potential dangers. Accidents in the lab are most often the result of careless or improper handling of materials. In addition to following basic safety procedures in the lab, you must also strive to anticipate hazardous situations and use foresight to prevent injuries to yourself and others.
Laboratory Safety Rules
These rules are designed to ensure that all work done in the lab will be safe for you and your fellow students. These rules will be rigidly enforced in the laboratories. Any student violating any of these rules will be asked to leave the lab and an unexcused absence will be recorded for that day.
1. Maintain a wholesome, safe attitude. Horseplay or other careless acts are prohibited.
2. Work in the lab only during your scheduled lab period and only when a Lab Instructor is present. Even if the lab is not locked, stay outside until your instructor arrives to let you in.
3. Smoking, drinking, eating or chewing is not permitted in the lab at any time. Chemicals may possibly enter your mouth or lungs. Additionally, toxic chemicals on your hands may also be transferred to your mouth, eyes, etc.
4. Unauthorized experiments and/or variations of the lab experiments are not allowed. All chemicals, lab supplies and/or equipment must stay in the lab.
5. Safety goggles must be worn when dealing with potentially harmful chemicals or preservatives. It is not recommended to wear contact lenses in the lab, especially when dissecting preserved materials. If you choose to wear them, you will need to follow special safety precautions. Check with your Lab Instructor. You can wear goggles over regular glasses.
Student Responsibilities in the Lab
1. Know the location and proper use of the safety equipment in the lab such as the eyewash fountain, first aid kits, fire extinguisher and emergency evacuation procedures etc. Ask your Lab Instructor about the proper use of such equipment.
2. Inform your Lab Instructor of students using improper techniques or unsafe practices.
3. Handle all chemicals with extreme care. Never taste or sniff a chemical unless specifically instructed to do so. Poisonous substances are not always labeled in the laboratory. Avoid breathing chemical vapors.
4. Avoid direct physical contact with any chemical. If any chemical comes in contact with your eyes, mouth or skin, rinse the affected area with copious amounts of tap water (5-10 min). Use the eyewash fountain to flush chemicals from your eyes and face. Do not hesitate to use the eyewash fountains. Immediate action can prevent a serious injury.
5. Handle glassware carefully and respectfully. Many lab accidents and injuries arise from the improper handling of glassware. Such injuries are not only dangerous, but they may also provide a means for toxic substances to enter the body. Immediately report any chipped or cracked glassware to your Lab Instructor.
6. Always wash your face, hands and arms before leaving the lab especially after handling chemicals and/or preservatives.
7. Immediately report all accidents/injuries, even minor ones, to your Lab Instructor.
Handling Reagents
1. Reagents are obtained from stock bottles. Leave the reagent bottles at the supply area Bring test tubes or beakers to the supply area for transferring chemicals. Always read the labels on the reagent bottles very carefully. Serious consequences can arise from mixing certain chemicals.
2. Avoid contaminating the stock solution. Contaminated stock solution is not only detrimental to your experiment, but to others as well.
a. Do not return the excess chemicals to the reagent bottle.
b. Transfer the stock solution to a clean beaker using the pipette from the stock beaker and a pipette bulb.
c. Use a clean dry spatula to transfer solids.
d. Keep the stoppers or lids/caps of reagent bottles clean and replace them after use. Do not lay the stopper of a bottle on the table.
3. Take only the minimum quantity of the reagent required from the stock bottle.
4. Always add the more concentrated solution into water or into less concentrated solution. This is especially true with concentrated acids.
Handling Chemical Spills
1. You should clean up chemical spills immediately. Ask your Lab Instructor about the proper procedures. If the spilled chemical is extremely hazardous, alert your Lab Instructor and other students immediately. Do not attempt to clean it up yourself as only qualified personnel are allowed to handle hazardous chemical spills.
2. For a simple acid or base spill, neutralize as follows:
a. Acid on clothing - rinse off with water and neutralize with sodium bicarbonate solution.
b. Base on clothing - rinse off with water and neutralize with dilute acetic acid solution.
c. Acid or base on counters or lab tables - Dilute with water and neutralize with solid sodium bicarbonate. Clean the area thoroughly.
Waste Disposal
1. Generally, discard waste or excess chemicals as follows:
a. Discard in a waste basket - paper products only - litmus paper, filter paper, paper towels etc.
b. Discard in a sink - nonhazardous, nonflammable, nontoxic, water soluble liquids. Adjust pH to range 5-10 before discarding.
c. Discard in waste jars - water insoluble liquids, solids, hazardous/toxic wastes, volatile liquids and reactive chemicals.
d. Discard in red biohazard containers - dissected preserved specimens.
e. Discard in “sharps” container - razor blades, needles, pins.
f. Discard in broken glass container – cover slips, broken glass.
Check with your Lab Instructor regarding disposal procedures for each specific experiment.
Lab 1 – Classification & Evolution
by L. Christianson & M. Brunell
Classification & Evolution (modified from an original exercise by Robert P. Gendron, Indiana University of Pennsylvania)
Humans classify almost everything, including each other. This habit can be quite useful. For example, when talking about a car someone might describe it as a 4-door sedan with a fuel injected V-8 engine. A knowledgeable listener who has not seen the car will still have a good idea of what it is like because of certain characteristics it shares with other familiar cars. Humans have been classifying plants and animals for a lot longer than they have been classifying cars, but the principle is much the same. In fact, one of the central problems in biology is the classification of organisms on the basis of shared characteristics. As an example, biologists classify all organisms with a backbone as "vertebrates." In this case the backbone is a characteristic that defines the group. If, in addition to a backbone, an organism has gills and fins it is a fish, a subcategory of the vertebrates. This fish can be further assigned to smaller and smaller categories down to the level of the species. The classification of organisms in this way aids the biologist by bringing order to what would otherwise be a bewildering diversity of species. (There are probably several million species of which about one million have been named and classified.) The field devoted to the classification of organisms is called taxonomy [Gk. taxis, arrange, put in order + nomos, law].
The modern taxonomic system was devised by Carolus Linnaeus (17071778). It is a hierarchical system since organisms are grouped into ever more inclusive categories from species up to kingdom. Figure 1 illustrates how four species are classified using this
taxonomic system. (Note that it is standard practice to underline or italicize the names of genera and species to indicate that they are written in Latin instead of English.)
|Table 1 - Linnean Hierarchy |
|KINGDOM |Animalia |Plantae |
|PHYLUM |Chordata |Arthropoda |Anthophyta |
|CLASS |Mammalia |Insecta |Liliopsida |
|ORDER |Primate |Carnivora |Hymenoptera |Liliales |
|FAMILY |Hominidae |Canidae |Apidae |Alliaceae |
|GENUS |Homo |Canis |Apis |Allium |
|SPECIES |Homo sapiens |Canis lupus |Apis mellifera |Allium cepa |
| |(human) |(wolf) |(honeybee) |(onion) |
In the 18th century most scientists believed that the Earth and all the organisms on it had been created suddenly in their present form as recently as 4004 BC. According to this view, Linnaeus' system of classification was simply a useful means of cataloging the diversity of life. Some scientists went further, suggesting that taxonomy provided insight into the Creator's mind ("Natural Theology").
This view of taxonomy changed dramatically when Charles Darwin published “On The Origin of Species” in 1859. In his book Darwin presented convincing evidence that life had evolved through the process of natural selection. The evidence gathered by Darwin, and thousands of other biologist since then, indicates that all organisms are descended from a common ancestor. In the almost unimaginable span of time since the first organisms arose (about 3.5 billion years) life has gradually diversified into the myriad forms we see today.
As a consequence of Darwin's work it is now recognized that taxonomic classifications are actually reflections of evolutionary history. For example, Linnaeus put humans and wolves in the class Mammalia within the phylum Chordata because they share certain characteristics (e.g. backbone, hair, homeothermy, etc.). We now know that this similarity is not a coincidence; both species inherited these traits from the same common ancestor. In general, the greater the resemblance between two species, the more recently they diverged from a common ancestor. Thus when we say that the human and wolf are more closely related to each other than either is to the honeybee we mean that they share a common ancestor that is not shared with the honeybee.
Another way of showing the evolutionary relationship between organisms is in the form of a phylogenetic tree (Gk. phylon, stock, tribe + genus, birth, origin):
Figure 1
The vertical axis in this figure represents time. The point at which two lines separate indicates when a particular lineage split. For example, we see that mammals diverged from reptiles about 150 million years ago. The most recent common ancestor shared by mammals and reptiles is indicated by the point labeled A. The horizontal axis represents, in a general way, the amount of divergence that has occurred between different groups; the greater the distance, the more different their appearance. Note that because they share a fairly recent ancestor, species within the same taxonomic group (e.g. the class Mammalia) tend to be closer to each other at the top of the tree than they are to members of other groups.
Several types of evidence can elucidate the evolutionary relationship between organisms, whether in the form of a taxonomic classification (Table 1) or a phylogenetic tree (Fig. 1). One approach, as already discussed, is to compare living species. The greater the differences between them, the longer ago they presumably diverged. There are, however, pitfalls with this approach. For example, some species resemble each other because they independently evolved similar structures in response to similar environments or ways of life, not because they share a recent common ancestor. This is called convergent evolution because distantly related species seem to converge in appearance (become more similar). Examples of convergent evolution include the wings of bats, birds and insects, or the streamlined shape of whales and fish. At first glance it might appear that whales are a type of fish. Upon further examination it becomes apparent that this resemblance is superficial, resulting from the fact that whales and fish have adapted to the same environment. The presence of hair, the ability to lactate and homeothermy clearly demonstrate that whales are mammals. Thus, the taxonomist must take into account a whole suite of characteristics, not just a single one.
The fossil record can also be helpful for constructing phylogenetic trees. For example, bears were once thought to be a distinct group within the order Carnivora. Recently discovered fossils, however, show that they actually diverged from the Canidae (wolves, etc.) fairly recently. The use of fossils is not without its problems, however. The most notable of these is that the fossil record is incomplete. This is more of a problem for some organisms than others. For example, organisms with shells or bony skeletons are more likely to be preserved than those without hard body parts.
The Classification and Evolution of Artificial Organisms
In this lab you will develop a taxonomic classification and phylogenetic tree for a group of imaginary organisms called Caminalcules after the taxonomist Joseph Camin (an acarologist) who devised them. At the back of this chapter are pictures of the 14 "living" and 58 "fossil" species that you will use. Take a look at the pictures and note the variety of appendages, shell shape, color pattern, etc. Each species is identified by a number rather than a name. For fossil Caminalcules there is also a number in parentheses indicating the geological age of each specimen in millions of years. Most of the fossil Caminalcules are extinct, but you will notice that a few are still living (e.g. species #24 is found among the living forms but there is also a 2 million year old fossil of #24 in our collection).
The purpose of this lab is to illustrate the principles of classification and some of the processes of evolution (e.g. convergent evolution). We do these exercises with artificial organisms so that you will approach the task with no preconceived notion as to how they should be classified. This means that you will have to deal with problems such as convergent evolution just as a taxonomist would. With real organisms you would probably already have a pretty good idea of how they should be classified and thus miss some of the benefit of the exercise.
Exercise 1: The Taxonomic Classification of Living Caminalcules
Carefully examine the fourteen living species and note the many similarities and differences between them. On a sheet of notebook paper create a hierarchical classification of these species, using the format in Table 2. Instead of using letters (A, B, ...), as in this example, use the number of each Caminacule species. Keep in mind that Table 2 is just a hypothetical example. Your classification may look quite different than this one.
The first step in this exercise is to decide which species belong in the same genus. Species within the same genus share characteristics not found in any other genera (plural of genus). The Caminalcules numbered 19 and 20 are a good example; they are clearly more similar to each other than either is to any of the other living species so we would put them together in their own genus. Use the same procedure to combine the genera into families. Again, the different genera within a family should be more similar to each other than they are to genera in other families. Families can then be combined into orders, orders into classes and so on. Depending on how you organize the species, you may only get up to the level of order or class. You do not necessarily have to get up to the level of Kingdom or Phylum.
Exercise 2. The Comparative Approach to Phylogenetic Analysis
Construct a phylogenetic tree based only on your examination of the 14 living species. This tree should reflect your taxonomic classification. For example, let us say you have put species A and G into the same genus because you think they evolved from a common ancestor (x). Their part of the tree would look like the diagram on the right.
When there are three or more species in a genus you must decide which two of the species share a common ancestor not shared by the other(s). This diagram indicates that species E and K are more closely related to each other than either is to C. We hypothesize that E and K have a common ancestor (y) that is not shared by C. Similarly, two genera that more closely resemble each other than they do other genera presumably share a common ancestor. Thus, even in the absence of a fossil record it is possible to develop a phylogenetic tree. We can even infer what a common ancestor like y might have looked like.
Exercise 3. The Phylogeny of Caminalcules
Using a large sheet of paper, construct a phylogenetic tree for the Caminalcules. Use a meter stick to draw 20 equally spaced horizontal line on the paper. Each line will be used to indicate an interval of one million years. Label each line so that the one at the bottom of the paper represents an age of 19 million years and the top line represents the present (0 years).
Cut out all the Caminalcules (including the living species). Put them in piles according to their age (the number in parentheses). Beginning with the oldest fossils, arrange the Caminalcules according to their evolutionary relationship. Figure 2 shows how to get started.
Figure 2
Hints, Suggestions and Warnings
a. Draw lines faintly in pencil to indicate the path of evolution. Only after your instructor has checked your tree should you glue the figures in place and darken the lines.
b. Branching should always be dichotomous (involve only two lines at a time):
Like this Not this
c. Some living forms are also found in the fossil record.
d. There are gaps in the fossil record for some lineages. Also, some species went extinct without leaving any descendants (remember the dinosaurs in Fig. 1).
e. The Caminalcules were numbered at random; the numbers provide no clues to evolutionary relationships.
f. There is only one correct phylogenetic tree in this exercise. This is because of the way that Joseph Camin derived his imaginary animals. He started with the most primitive form (#73) and gradually modified it using a process that mimics evolution in real organisms. After you complete your phylogeny compare it with Camin's original.
g. There is also only one correct phylogenetic tree for any set living organisms as well. However we rarely have complete data on the evolution of any set of organisms, but we construct the best tree that we can based on the data that we have. Different taxonomists may interpret the data differently and come up with different trees. Do not be too concerned if your tree differs slightly from Camin’s original tree.
Questions 1 - 6
LIVING CAMINALCULES
FOSSIL CAMINALCULES
(numbers in parentheses indicate age in millions of years)
FOSSIL CAMINALCULES (cont’d)
Lab 2 – Population Genetics & the Hardy-Weinberg Theorem
by L. Wrischnik & M. Brunell
[Modified from: Morgan, Judith Giles & M. Eloise Brown Carter. 2002.
Investigating Biology, 5th ed. Benjamin Cummings. San Francisco, CA.]
Before coming to lab you should:
• Review the material from last week’s lab.
• Read the Introduction for this week’s lab
• Review the material on the Hardy-Weinberg theorem in your text.
• Be able to state the Hardy-Weinberg theorem and list the conditions necessary to maintain equilibrium.
Before leaving the lab you should:
• Be able to explain Hardy-Weinberg equilibrium in terms of allelic and genotypic frequencies and relate these to the expression (p + q) 2 = p2 + 2pq + q2.
• Be able to use the bead model to demonstrate conditions for evolution.
• Be able to use a Chi-square test to determine whether or not experimental values differ from expected values.
• Be able to explain the significance of the Hardy-Weinberg theorem in modern evolution.
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, San Francisco, CA.
Population Genetics & the Hardy-Weinberg Theorem
Introduction
Charles Darwin’s unique contribution to biology was not that he “discovered evolution” but, rather, that he proposed a mechanism for evolutionary change—natural selection, the differential survival and reproduction of individuals in a population. In “On the Origin of Species”, published in 1859, Darwin described natural selection and provided abundant and convincing evidence showing how populations change over time. Evolution was accepted as a theory with great explanatory power supported by a large and diverse body of evidence. However, at the turn of the century geneticists and naturalists still disagreed about the role of natural selection and the importance of small variations in natural populations. How could these variations provide a selective advantage that would result in evolutionary change? It was not until evolution and genetics became reconciled with the advent of population genetics that natural selection became widely accepted.
Francisco Ayala defines evolution as “changes in the genetic constitution of populations.” A population is defined as a group of organisms of the same species that occur in the same area and interbreed or share a common gene pool, all the alleles at all gene loci of all individuals in the population. The population is considered the basic unit of evolution.
In 1908, English mathematician G. H. Hardy and German physician W. Weinberg independently developed models of population genetics that showed that the process of heredity by itself did not affect the genetic structure of a population. The Hardy-Weinberg theorem states that the frequency of alleles in the population will remain the same regardless of the starting frequencies. Furthermore, the equilibrium genotypic frequencies will be established after one generation of random mating. This principle is valid only if certain conditions or assumptions are met:
1. The population is very large.
2. Mating is random.
3. There are no net changes in the gene pool due to mutation; that is, mutation from allele “A” to allele “a” must be equal to mutation from “a” to “A”.
4. There is no movement of individuals into and out of the population, no gene flow.
5. There is no natural selection; all genotypes are equal in reproductive success.
Basically, the Hardy-Weinberg theorem provides a baseline model in which gene frequencies do not change and, thus, evolution does not occur. By testing the fundamental hypotheses of the Hardy-Weinberg principle, evolutionary biologists have investigated the roles of mutation, gene flow, population size, nonrandom mating, and natural selection in effecting evolutionary change in natural populations.
Use of the Hardy-Weinberg Theorem
The Hardy-Weinberg theorem provides a mathematical formula for calculating the frequencies of alleles and genotypes in populations. We begin with a population with two alleles at a single gene locus (a dominant allele “A”, and a recessive allele “a”) and let the frequency of the dominant allele be represented by “p”, and the frequency of the recessive allele by “q”. Therefore, since the sum of all the frequencies in any population must equal one (p + q = 1), if the frequency of one allele, p, is known for a population, the frequency of the other allele, q, can be determined by using the relationship q = 1 - p.
During sexual reproduction, the frequency of each type of gamete produced in a population is equal to the frequency of the alleles in the population. If mating between the individuals in a population is random, then the gametes combine at random and the expected frequency of homozygous AA individuals in the next generation is p2 and the expected frequency of homozygous aa individuals is q2. The heterozygote can be obtained two ways, one with the male parent providing a dominant allele and another with the female parent providing the dominant allele, so the expected frequency of Aa would be 2pq.
|Results of Random Mating |
|in a Population at Hardy-Weinberg Equilibrium |
| |freq A in eggs = p |freq a in eggs = q |
|freq A in sperm = p |freq AA = p2 |freq Aa = pq |
|freq a in sperm = q |freq aA = qp |freq aa = q2 |
These genotypic frequencies can be obtained by multiplying (p + q) by (p + q). The general equation then becomes
(p + q)(p + q) = (p + q) 2 = p2 + 2pq + q2
To summarize:
p2 = expected freq AA
2pq = expected freq Aa
q2 = expected freq aa
Follow the steps in this example.
1. Assume that we have a population of some organisms containing 500,000 diploid individuals, which gives us a total of 1,000,000 genes (organisms with 2 alleles of each gene are “diploid”). Let us assume that 600,000 of those genes are allele “A” and the other 400,000 are allele “a”. The frequency of allele “A” is therefore p = 600,000/1,000,000 = 0.6 and the frequency of allele “a” is q = 400,000/1,000,000 = 0.4.
2. Then freq A + freq a = p + q = 0.6 + 0.4 = 1. The sum of all the frequencies will always equal one.
3. Once allelic frequencies are known for a population, the genotypic makeup of the next generation can be predicted from the terms of the general equation. In this case,
(p + q) 2 = p2 + 2pq + q2
(0.6 + 0.4)2 = (0.6)(0.6) + 2(0.6)(0.4) + (0.4)(0.4) = 0.36 + 0.48 + 0.16
The genotypic frequencies in this population are specifically
freq AA = p2 = 0.36
freq Aa = 2pq = 0.48
freq aa = q2 = 0.16
Note that the formula produces the same values seen in the Table below.
|Results of Random Mating in a Population at Hardy-Weinberg Equilibrium |
| |freq A in eggs |freq a in eggs |
| |p = 0.6 |q = 0.4 |
|freq A in sperm |freq AA |freq Aa |
|p = 0.6 |p2 = 0.36 |pq = 0.24 |
|freq a in sperm |freq aA |freq aa |
|q = 0.4 |qp = 0.24 |q2 = 0.16 |
4. Starting with these genotypic frequencies (AA = p2 = 0.36, Aa = 2pq = 0.48, aa = q2 = 0.16), we can recalculate the allelic frequencies of this generation. Since the allele frequency of “A” = p, we can calculate the value of p from the genotype frequency by taking the square root of 0.36, which is 0.6. The allele frequency of “a” can be calculated in the same way. Notice that for a population at Hardy-Weinberg equilibrium, the allelic frequencies remain p = 0.6 and q = 0.4.
Let’s look at a second example. If we know that a population is at Hardy-Weinberg equilibrium and we know that, for example, 4% (0.04) of the population is albino (a recessive trait, so we’ll designate these individuals as “aa”), then the frequency of the albino allele “a” (q) could be calculated as the square root of 0.04.
1. Frequency of “aa” albino individuals = q2 = 0.04 (genotypic and phenotypic frequency); therefore, q = √0.04 = 0.2 = freq “a”.
2. Since p + q = 1, the frequency of the dominant allele “A” (designated p) is (1 - q), or 0.8. So 4% of the population is albino, and 20% of the alleles in the gene pool are for albinism and the other 80% are for normal pigmentation. (Note that you could not determine the allele frequency of “A” by taking the square root of the frequency of all normally pigmented individuals! This is because you cannot distinguish the “AA” homozygote from the “Aa” heterozygote for this trait.)
3. Now that we know the allele frequencies, the genotypic frequencies of the next generation now can be predicted from the general Hardy-Weinberg theorem. First determine the results of random mating by completing the table below.
| |
|Results of Random Mating in a Population at Hardy-Weinberg |
|Equilibrium |
| |freq A in eggs |freq a in eggs |
| |p = ___ |q = ___ |
|freq A in sperm |freq AA |freq Aa |
|p = ___ |p2 = ____ |pq = ____ |
|freq a in sperm |freq aA |freq aa |
|q = ___ |qp = ____ |q2 = ____ |
What will be the equilibrium genotypic frequencies from generation to generation, provided that the frequencies of the alleles p and q remain in genetic equilibrium?
freq AA = ____
freq Aa = ____
freq aa = ____
The genetic equilibrium will continue indefinitely so long as the conditions of the Hardy-Weinberg theorem are met.
Although natural populations may seldom meet all the conditions, Hardy-Weinberg equilibrium serves as a valuable model from which we can predict genetic changes in populations as a result of natural selection or other factors. This allows us to understand quantitatively and in genetic language how evolution operates at the population level.
Testing the Conditions of Hardy-Weinberg Equilibrium
Working in pairs, you will test Hardy-Weinberg equilibrium by simulating a population using colored beads. The bag of beads represents the gene pool for the population. Each bead should be regarded as a single gamete, the two colors representing different alleles of a single gene. Each bag should contain a total of 100 beads of the two colors in the proportions specified by the instructor. Record in the space provided below the initial frequencies for your gene pool.
|Bead Color |Allele |Allelic Frequency |
|Red |A | |
|yellow |a | |
How many diploid individuals are represented in this population? _________________________
What would be the color of the beads for a homozygous dominant individual? ______________
What would be the color of the beads for a homozygous recessive individual? ______________
What would be the color of the beads for a heterozygous individual? ______________________
Assuming that the population is at Hardy-Weinberg equilibrium, what genotypic frequencies would one expect to see maintained in the population?
freq AA = _____, freq Aa = _____, freq aa = _____.
Experimental Procedure
Before generating the data for your experiment, determine the expected frequencies of genotypes and alleles for the population. To do this, use the original allelic frequencies for the population provided by the instructor (recall that freq A = p and freq a = q). Calculate the expected genotypic frequencies using the Hardy-Weinberg equation: p2 + 2pq + q2. The number of individuals expected for each genotype can be calculated by multiplying 50 (total population size) by the expected frequencies. Record these results in Table 2.1 below.
| |
|Table 2.1. Expected Genotypic and Allelic Frequencies |
|for the Next Generation Produced by the Model |
|Parent Population |Next Generation Expected |
|Allelic Frequency |Genotypic Count Expected |Allelic Frequency Expected |
|freq A |Freq a |# AA |# Aa |# aa |freq A |freq a |
| | | | | | | |
| | |Genotypic Frequency Expected | | |
| | |freq AA |freq Aa |freq aa | | |
| | | | | | | |
1. Now, generate your data. Without looking, randomly remove two beads from the bag. These two beads represent one diploid individual in the next generation. Record the diploid genotype (AA, Aa, or aa) of the individual formed from these two gametes.
2. Sample with replacement. Sampling with replacement means that after removing one pair of beads (representing the genotype of one individual) from the bag, you replace that pair in the bag before removing the next pair. Return the beads to the bag and shake the bag to reinstate the gene pool. By replacing the beads each time, the size of the gene pool remains constant and the probability of selecting any allele should remain equal to its frequency. For example, if you remove one “AA” homozygote from the bag, you will take out two red beads. If you do not replace the beads, you will be removing an individual from your artificial population, altering your population size from 50 to 49, and you will also change the allelic frequencies.
3. Repeat steps 1 and 2 (select two beads, record the genotype of the new individual, and return the beads to the bag) until you have recorded the genotypes for 50 individuals who will form the next generation of the population.
Results
|Individual |
|# |
|Parent Population |Next Generation Observed |
|Allelic Frequency |Genotypic Count Observed |Allelic Frequency Observed |
|freq A |freq a |# AA |# Aa |# aa |freq A |freq a |
| | | | | | | |
| | |Genotypic Frequency Observed | | |
| | |freq AA |freq Aa |freq aa | | |
| | | | | | | |
Chi-Square tests
How close did your observed values match your calculated, expected values? In science, we can’t just say “they were pretty close” or “they didn’t really match”, and leave it at that. Instead, we use statistics to help us analyze our results and generate a meaningful number that helps us decide how well our observed data matches our prediction. Here we will use the Chi-square test (or X2 test), which is a statistical procedure that can be used to compare observed data with expected values. Due to the effects of random variation, experimental data (observed data) rarely correspond exactly with theoretical values (expected data). Imagine flipping a quarter 100 times – you should get heads 50% of the time and tails 50% of the time, but how likely are you to get exactly 50 heads and 50 tails when you do this? A statistical test like the Chi-square test is useful in determining whether observed experimental values differ significantly from theoretical expected values.
Before doing an experiment, we can make predictions about the possible outcomes. These predictions are called hypotheses, and there are two types: the null hypothesis, symbolized HO, states that there is no difference between observed and expected values, and the alternate hypothesis, symbolized HA, which states that there is a difference. For coin flipping, state the null and alternate hypotheses below:
HO:_____________________________________________________________________
HA:_____________________________________________________________________
The Chi-square test will allow us to either accept or reject these hypotheses. We calculate a Chi-square value using the following formula:
[pic]
where oi is an observed value and ei is the associated expected value. The table below shows the calculation of the Chi-square statistic for a sample problem involving a population of 50 individuals with allele frequencies of A = 0.6 and a = 0.4.
|Hypothetical Chi-Square Test of Results from the Model |
| |# AA |# Aa |# aa |
|Observed value (oi) |22 |22 |6 |
|[Note: these are actual counts, not frequencies or | | | |
|percentages) | | | |
|Expected value (ei) based on the given allele |(0.6)2(50) = 18 |(2)(0.6)(0.4)(50) |(0.4)2(50) = 8 |
|frequencies | |= 24 | |
|Difference = (oi - ei) = di |22 - 18 = 4 |22 - 24 = -2 |6 - 8 = -2 |
|di2 |(4)2 = 16 |(-2)2 = 4 |(-2)2 = 4 |
|di2/ei |16/18 = 0.89 |4/24 = 0.16 |4/8 = 0.50 |
| | | | |
|[pic] = test statistic |Χ2 = 0.89 + 0.16 + 0.50 = 1.55 |
| |This is the number we calculate based on our experimental data. |
|Degrees of freedom1 |2 |
|Critical value at p < 0.05 obtained from the table on |Χ2α=0.05, df=2 = 5.99 |
|the next page. (See below for an explanation of this |We get this number from the table shown below, and it is determined by our |
|number) |degrees of freedom (df) and how sure we want to be about our data |
|Decision |Since our test statistic is smaller than the critical value, (1.55 < 5.99), we |
| |conclude that the observed values do not differ significantly from the expected |
| |values. |
| |The null hypothesis is accepted; the alternate is rejected. |
|1 degrees of freedom = df = [number of categories - 1] = 3 (aa, AA, Aa individuals examined) - 1 = 2 |
We want to find out how closely our observed numbers match up to our expected numbers. In other words, we know that the two sets of numbers are not a perfect match, but are they close enough to say “Yes, they do match!” or are they differ enough for us to say that “No, they do not match”? We start our analysis by choosing a “probability”, or p-level, value from the Chi-square table. By convention, we usually start with a probability value of 0.05 (5%) or less. (This value says that there is less than a 5% chance that we will make a mistake by saying that our observed values are different from our theoretical values; in other words, we are 95% confident that if we calculate a significant difference, then one really exists). Since we have 2 degrees of freedom in our experiment, and we’ve chosen a probability value of 0.05, our “critical value” from the table is given as 5.99. If we calculate a Chi-square value lower than 5.99, we can say that our observed numbers are NOT significantly different from the expected values; however, if we calculate a Chi-square value that is higher than 5.99, we must conclude that our observed numbers are significantly different from our expected numbers (and the differences between them are due to something other than random variation). A convenient way to remember the meaning of Chi-square is that the more of a mismatch between observed and expected, the larger the Chi-square value will be.
Since our computed Chi-square value of 1.55 is less than the critical value (at p = 0.05), we can conclude that our observed values are NOT significantly different from the theoretically expected values (the mathematical way of saying “yes they do match”).
|Degrees |Chi-Square Probability (p-level) |
|of | |
|Freedom | |
| |0.95 |0.90 |
You can now take the data from Tables 2.1 and 2.2 and use the Chi-square test to compare your observed results (from Table 2.2) with those expected (Table 2.1). Table 2.3 below will assist in the calculation of the chi-square test.
| |
|Table 2.3. Chi-Square Test of Results from the Model |
| |# AA |# Aa |# aa |
|Observed value (oi) | | | |
|Expected value (ei) | | | |
|Difference = (oi - ei) = di | | | |
|di2 | | | |
|di2/ei | | | |
|[pic] = test statistic | |
|Degrees of freedom |2 |
|Critical value at p < 0.05 |5.99 |
|Decision - Were the observed frequencies significantly| |
|different from the expected frequencies? | |
| |Null hypothesis accepted or rejected? |
Is your calculated Χ2 value greater or smaller than the given Χ2 critical value (see table)? ______
Were your observed results consistent with the expected results based on your statistical analysis? If not, can you suggest an explanation? ______________________________________
______________________________________________________________________________
Compare your results with those of other students. How variable are the results for each team?
______________________________________________________________________________
Do your results match your predictions for a population at Hardy-Weinberg equilibrium? _____
What would you expect to happen to the frequencies if you continued this simulation for 25 generations? __________________________________________________________________
Considering the definition of evolution in modern biology, is this population evolving? Explain your response. ________________________________________________________________
_____________________________________________________________________________
Consider each of the conditions or assumptions required for the Hardy-Weinberg model. Does this model meet each of those conditions?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
Simulation of Genetic Drift
Genetic drift is the change in allelic frequencies in small populations as a result of chance alone. In a small population, combinations of gametes may not be random, owing to sampling error. (If you toss a coin 500 times, you expect about a 50:50 ratio of heads to tails; but if you toss the coin only 10 times, the ratio may deviate greatly in a small sample owing to chance alone.) Genetic fixation, the loss of all but one possible allele at a gene locus in a population, is a common result of genetic drift in small natural populations. The allele which is retained is said to be “fixed”. Genetic drift is a significant evolutionary force in situations known as the bottleneck effect and the founder effect.
A bottleneck occurs when a population undergoes a drastic reduction in size as a result of chance events (not differential selection), such as a volcanic eruption or hurricane. (Bad luck, not bad genes!) Passing through a bottleneck results in unpredictable proportions of genes passing to the next generation on the other side of the bottleneck. These genes would constitute the beginning of the next generation.
When a small group of individuals becomes separated from the larger parent population, the allelic frequencies in this small gene pool may be quite different from those of the original population as a result of chance alone. This occurs when a group of migrants becomes established in a new area—for instance, the colonization of an island—and is therefore referred to as the founder effect.
Each group will choose ONE of the following two experiments: bottleneck effect OR founder effect. You will compare your results with those of other groups later in lab.
Bottleneck Effect
Procedure
1. To investigate the bottleneck effect, establish a starting population containing 50 individuals (how many beads?) with a frequency of 0.5 for each allele (Generation 0).
2. Without replacement, randomly select five individuals (10% of the population) two alleles at a time. This represents a drastic reduction in population size. On a separate sheet of paper, record the genotypes and the number of A and a alleles for the new population.
3. Count the numbers of each genotype and the numbers of each allele. Using these numbers, determine the genotypic frequencies for AA, Aa, and aa and the new allelic frequencies for A (p) and a (q) for the surviving five individuals. These are your observed frequencies. Enter these frequencies in Table 2.4, Generation 1.
4. Using the new observed allelic frequencies, calculate the expected genotypic frequencies (p2, 2pq, q2). Record these frequencies in Table 2.4, Generation 1.
5. Reestablish the population to 50 individuals using the new allelic frequencies. For instance, if you drew 8 red and 2 yellow beads, the allele frequencies are p=0.8 and q=0.2, respectively. The new population in the bag should therefore have 80 red beads (0.8 x 100 beads) and 20 yellow beads (0.2 x 100 beads).
4. Repeat steps 2, 3, 4, and 5. Record your results in the appropriate generation in Table 2.4.
5. Reestablish the gene pool with new frequencies after each generation until one of the alleles becomes fixed in the population for a couple of generations.
6. Summarize your results in the “Lab 2 Questions” section at question 3.
|Table 2.4. Changes in Allelic and Genotypic Frequencies for |
|Simulations of the Bottleneck Effect |
|First, record frequencies based on the observed numbers in your experiment. Then, using the |
|observed allelic frequencies, calculate the expected genotypic frequencies. |
|Generation |Genotypic Frequency Observed |Allelic Frequency Observed |Genotypic Frequency Expected |
| |
|Calculate expected frequencies based on the actual observed numbers in your experiment. |
|Generation |Allelic frequency |Genotypic Frequency |
| |p |q |p2 |2pq |q2 |
|0 |0.5 |0.5 |.25 |.50 |.25 |
|1 | | | | | |
|2 | | | | | |
|3 | | | | | |
|4 | | | | | |
|5 | | | | | |
|6 | | | | | |
|7 | | | | | |
|8 | | | | | |
|9 | | | | | |
|10 | | | | | |
|11 | | | | | |
|12 | | | | | |
|13 | | | | | |
|14 | | | | | |
|15 | | | | | |
Results of Experiment
How many generations did you simulate? ___________________________________________
Using the graph paper at the end of the Lab Topic, sketch a graph of the change in p and q over time. You should have two lines, one for each allele. Plot allelic frequencies on the vertical axis and time on the horizontal axis. Be sure to label the axes on your graph.
Did one allele go to fixation in that time period? If so, which allele? Remember, genetic fixation occurs when the gene pool is composed of only one allele, i.e., the frequency of one of the alleles goes to 1.0. The other alleles have been eliminated (or lost), i.e., their frequencies have gone to 0.0.
Did the other allele ever appear to be going to fixation?
_____________________________________________________________________________
Did any of the expected genotypic frequencies go to fixation? If none did, why not?
_____________________________________________________________________________
*On completion of the above simulation, choose one of the two remaining scenarios to investigate. All scenarios should be completed by at least one team in the laboratory.
Complete the Lab 2 Worksheet.
| |
|Mitotic Stage |Predicted |Tally Marks |Count |Percent |Time |
| |% | | |% |(minutes) |
|Prophase | | | | | |
|Metaphase | | | | | |
|Anaphase | | | | | |
|Telophase | | | | | |
Total number of cells in mitotic sample: ________
Do all stages of mitosis take the same amount of time to complete? Of course you could just say yes or no, but remember that we need to use a more formal, scientifically acceptable way of presenting this data. After figuring your observed stage times, you can perform the Chi-Square (X2) Test to address this question. In this test, you will calculate expected values from the hypothesis (prediction) that: the 4 mitosis stages will take an equal amount of time to complete, that is, the counts of cells at each stage should be identical. Use the Chi-Square (X2) Test to see if statistically significant differences exist in the stage timings, that is, to formally state whether the numbers of cells at each stage are equal or not.
To perform the test, you will first need to calculate what the expected stage counts would be if your hypothesis is true, i.e. that all stages take the same amount of time. Add up the total number of cells that you counted at all stages and divide that sum by 4. The result will be the expected number of cells at each stage. The minute values in the table above are termed the observed stage times. You will now compare the observed and expected values. Based on your knowledge of the Chi-Square (X2) Test from the Hardy-Weinberg lab, you should be able to fill in the following Table 3.4:
|Table 3.4 – X2 test |
|Stage |Prophase |Metaphase |Anaphase |Telophase |
|Observed number of cells | | | | |
|at each stage (O) | | | | |
|Expected number of cells | | | | |
|at each stage (E) | | | | |
|(O-E)2/E | | | | |
|Total X2 (sum of | |
|individual X2 values) | |
|Degrees of freedom | |
|Critical value at p< 0.05| |
Once you have calculated the total X2 value, compare it to the “critical value” for this test (taken from the Chi-Square table in Lab 2). If your total X2 value is less than or equal to this number, then you can accept your null hypothesis (prediction) as true, meaning that all stages have equal lengths (we could also say that the alternative hypothesis that the stages do not have equal lengths would be rejected). If your total X2 value exceeds the critical value, then you may reject your null hypothesis and accept the alternative hypothesis, meaning that significant differences exist in the timing of the stages. How sure are we about all of this being correct? The way the test is set up (with our critical value chosen by convention as p < 0.05), we will make an incorrect conclusion about which hypothesis to accept about 1 in 20 times, which is an acceptable error rate.
Questions 4, 5
Animal Cell Mitosis – Whitefish Blastula prepared slides
Examine the DEMO slide of the whitefish blastula prepared slide. Compare and contrast the animal mitosis with the plant mitosis you studied above.
Meiosis
Each species contains cells with a specific and characteristic number of chromosomes. A certain fungus might have a single set of 5 chromosomes, and we would say this fungus is haploid (Gr. haplos, single), with n = 5 chromosomes. Most animals have 2 sets of chromosomes per cell, and we say that these organisms are diploid (Gr. diplos, twofold) and these organisms have 2 of each type of chromosome). For example, humans have 46 chromosomes, 2 sets of 23 (designated 2n = 46), while the fruit fly (Drosophila) has 8 total chromosomes, 2 sets of 4 (2n = 8). Many plant species can have even more than 2 sets of chromosomes (for example, bananas are triploid, with 3 sets of chromosomes, while domestic wheat is hexaploid, with 6 sets of chromosomes per nucleus). The cells of all of these organisms can undergo mitosis: no matter what the chromosome number, each can be duplicated for mitotic cell division. However, most organisms actually have chromosomes that come in even-numbered sets. Why? So they can have sex and create more genetic variability!
Let’s focus on a diploid organism like ourselves. Within almost all of our cells we have 2 sets of 23 chromosomes, so each of our chromosomes comes in a pair. The members of a pair of chromosomes are called homologues, and each homologous pair consists of 2 chromosomes which are structurally the same, i.e. they contain the same genes in the same order along the chromosome (which means they control the same physical traits), they are the same length, they have their centromeres in the same place, etc. If at a certain position on one homologue there is a gene affecting eye color, then on the other homologue, in that same place, there is also a gene affecting eye color. Are the 2 homologous chromosomes exactly equivalent? NO! In humans, the gene for earlobe attachment is involved in the production of enzymes associated with either free or attached ear lobes. Let us use “F” for a version of this gene that makes free ear lobes and “f” for the version of this gene that causes attached ear lobes. Since each person contains 1 pair of each chromosome, that person can have up to 2 versions of this earlobe gene. Alternate versions of the same gene are called alleles of that gene, and in our example a person may have 2 “F” alleles, 2 “f” alleles, or 1 “F” and 1 “f” allele. If a person gets either 1 or 2 copies of the “F” allele, they make an enzyme that cuts their earlobe away from their head (more or less)! (Because of this we say that the “F” allele is dominant to the “f” allele, and we’ll talk about this more in Biol 61). Because the 2 alleles may be different for any of the genes along the homologous chromosomes, the 2 homologues can differ in many ways.
The majority of living organisms reproduce sexually; that is, 1 cell from 1 organism (for example, a sperm produced in a testis from a male) combines with 1 cell from a different organism of the same species (for example, an egg produced in an ovary from a female) to produce a single-celled zygote that will grow into the adult organism. With this method of sexual reproduction it would not be possible to maintain a constant chromosome number unless at some time there is a reduction in the number of chromosomes. Think about it – if 2 fruit flies mated, and if their gametes (eggs and sperm) each contained 8 chromosomes, then fertilization would produce a zygote containing 16 chromosomes, not 8 like the adults! Meiosis is the process of 2 back-to-back divisions to produce cells having half the number of chromosomes as the adult organism. Meiosis occurs during the formation of gametes in animals or during formation of spores in plants. Keep in mind that all of the cells of an organism will have this same complement of chromosomes with the exception of certain cells (ova, sperm, etc.) involved in sexual reproduction. For our fruit fly example, we see that the flies are diploid with a total of 2n = 8 chromosomes. Every cell in their bodies will be diploid, with 2n = 8 chromosomes, EXCEPT their sperm or eggs, which were generated by meiotic cell division. The sperm and eggs will have half the number of chromosomes, but not just a random collection of any 4 chromosomes. Meiosis will generate fly eggs and sperm that each contain 1 complete set of 4 chromosomes (n = 4); in other words, these fly gametes are haploid, containing 1 of each pair of homologues.
There are 2 back-to-back cell divisions that occur during meiosis: meiosis I & meiosis II. Meiosis I is quite different from mitosis, while meiosis II is very similar to a mitotic division. Meiosis I consists of prophase I, metaphase I, anaphase I, and telophase I. As with mitosis, the chromosomes are replicated well before the start of meiosis. Prophase I is a long stage during which the chromosomes become shorter and thicker and visible, the nucleus breaks down, and the spindle forms. An unusual aspect of this meiotic prophase is synapsis, the "pairing-up" of homologous chromosomes. During prophase I, the homologous chromosomes are brought together and lined up side-by-side (“pairing”). Just as in mitosis, each single chromosome is composed of 2 sister chromatids; so during pairing there will be 4 strands (or “chromatids”) lying side-by-side forming 4-stranded figures called tetrads. Portions of genetic material may also shift from one homologous chromosome to the other through a process called crossing over at this time.
Metaphase I of meiosis is strikingly different from mitosis. In mitosis, when the chromosomes line up in the middle of the spindle, each chromosome lines up by itself, with the spindle attached to both sister chromatids. In meiosis I, BOTH homologous chromosomes line up together, and the spindle is attached to just 1 sister chromatid from each homologous chromosome (the centromere of each chromosome continues to hold the 2 sister chromatids together, and will continue to do so through meiosis I). During anaphase I, each homologous chromosome of a pair will move to opposite poles of the spindle, and each chromosome still consists of 2 sister chromatids. Telophase I and cytokinesis then follow. The 2 daughter cells formed from meiosis I now have only 1 chromosome (composed of 2 sister chromatids) from each pair. This condition is called haploid. In humans, which have 46 chromosomes (23 pairs), and the cells at the end of meiosis I have only 23 chromosomes, each composed of 2 sister chromatids held together by a centromere.
There is no interphase between meiotic divisions, so no cell growth or chromosome doubling occurs in the 2 daughter cells. These haploid cells enter prophase II of meiosis II, and this division is like mitosis. At metaphase II the individual chromosomes line up by themselves, and the 2 sister chromatids are pulled to opposite poles of the spindle during anaphase II. Telophase II follows and the nuclei reform. The products of this meiosis II are 4 haploid cells, each containing a single set of chromosomes (composed of a single “sister chromatid”).
In order to picture the meiotic process and show the reduction in chromosome number, we will set up models using colored pop beads. Assume you are looking at meiosis in a diploid cell with 2n = 4 chromosomes (a cell that has 2 homologous pairs of chromosomes). Each pair should consist of 2 different colors of pop beads (why?) of the same length. Remember that the replication of chromosomes has taken place before meiosis I starts and therefore each chromosome has 2 chromatids held together by a centromere when prophase I begins.
Make your second pair of chromosomes distinctly different from your first pair so you can distinguish them from each (for example, make your second pair much shorter, or place the centromere near one end or the other). Again, the 2 chromosomes should be represented by 2 different colors and each should have 2 chromatids (same color). Set your chromosomes up as they would be arranged during metaphase I.
Using your pop bead "chromosomes" show how the chromosomes separate in anaphase I of meiosis and compare it to a mitotic anaphase. Take one of the cells produced by meiosis I and demonstrate anaphase of meiosis II. Recall that meiosis II is similar to mitosis, but the cells involved are haploid.
Sex is important because it helps produce variation among individuals in a population. Meiosis contributes to the generation of variation because the process helps form new combinations of chromosomes (and alleles) in the next generation. Consider a diploid animal with 2n=4 chromosomes, like our above example. Each of its cells contains 2 homologous pairs of chromosomes. One chromosome of each homologous pair originally came from the mother of this organism (and we’ll call this the maternal (m) chromosome) while the second member of this pair originated from the father of this organism (and we’ll call this the paternal (p) chromosome). Therefore we can represent these 2 pairs of chromosomes as follows: 1m and 1p for 1 pair, and 2m and 2p for the second pair. The maternal and paternal homologues are very likely to contain alleles that are different from each other. During anaphase I, the maternal and paternal chromosomes can separate in 2 different ways depending on how the chromosomes lined up during metaphase I:
|Ways of assorting chromosome |Ways of assorting chromosome 2 |Resulting combinations of|
|1 |relative to chromosome 1 |chromosomes |
|1m |2m |1m 2m |
| |2p |1m 2p |
|1p |2m |1p 2m |
| |2p |1p 2p |
The separation of the 1m/1p pair of homologues is independent from that of the 2m/2p pair. No matter how many pairs of homologous chromosomes an organism has, the separation of each pair of homologues is independent from that of every other pair! Thus, with 2 pairs of chromosomes there are 4 different combinations of chromosomes possible. With 3 pairs of homologues there are 8 different ways the chromosomes can segregate during anaphase I:
|Ways of assorting |Ways of assorting chromosome|Ways of assorting chromosome 3 |Resulting combinations|
|chromosome 1 |2 relative to chromosome 1 |relative to chromosomes 1 & 2 |of chromosomes |
|1m |2m |3m |1m 2m 3m |
| | |3p |1m 2m 3p |
| |2p |3m |1m 2p 3m |
| | |3p |1m 2p 3p |
|1p |2m |3m |1p 2m 3m |
| | |3p |1p 2m 3p |
| |2p |3m |1p 2p 3m |
| | |3p |1p 2p 3p |
Imagine the number of possible combination that one could see in an organism such as humans with 23 different pairs of homologous chromosomes!
The significance of this random segregation is that it results in different combinations (or assortments) of chromosomes, producing new combinations of alleles and traits in the eggs and sperm, which ultimately produces more genetic diversity in the next generation. The term for this phenomenon is independent assortment, reminding us that the maternal and paternal chromosomes of each homologous pair move independently during meiosis I. Most of the variation that one sees in a population is due to independent assortment of the existing genetic material.
Model meiosis completely for 3 pairs of chromosomes showing the different possible combinations of maternal and paternal chromosomes that can form. Be sure to make your homologous pairs distinguishable from each other.
Complete the remaining questions and table on your worksheet.
Lab 4 - Bacteriology I
By K. Land
(Modified from: Morgan, Judith Giles & M. Eloise Brown Carter. 2002.
Investigating Biology, 4th ed. Benjamin Cummings, San Francisco, CA.)
Before arriving to the laboratory you should:
• Review the material from last lab.
• Review the material on bacteria in your text.
• Compare and contrast prokaryotic and eukaryotic cell architecture.
• Print out the following website on bacterial colony morphologies and take it to the laboratory:
Before leaving the lab you should be able to:
• Describe bacterial structures: cell and colony morphologies.
• Describe the principle of Gram staining and discuss its implication to cell wall biochemistry.
• Perform aseptic techniques producing bacterial streaks, smears, and lawns of pure and mixed cultures.
• Describe the ecology and control of bacteria, applying these concepts to everyday life situations.
• Understand the scientific process and how investigations are conducted.
• Design an investigation using bacteria or other microbes to answer basic questions.
References:
• Campbell, Neil A. and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, San Francisco, CA.
Introduction and background
Taxonomy is an important branch of biology that deals with naming and classifying organisms into distinct groups or categories. Much of the work of early taxonomists included recording characteristics of organisms and grouping them based on appearance, habitat, or perhaps medicinal value. As scientists began to understand the processes of genetics and evolution by natural selection, they realized the value of classifying organisms based on phylogeny or evolutionary history. Information about phylogeny was obtained from studies of embryonic development or homologous features—common features resulting from common genes. In recent years, scientists have begun using biochemical evidence—studies of nucleic acids and proteins—to investigate relationships among organisms, leading to revisions in the taxonomic scheme.
At present, classification of the diversity of life is in a state of flux. Historically, the broadest taxonomic category has been the kingdom, and since 1969, most systematists have followed the five-kingdom scheme proposed by Robert Whittaker. Recently, some researchers have proposed that living organisms be classified into categories with a higher taxonomic level than kingdom, called domains: the Bacteria, the Archaea, and the Eukarya (with multiple kingdoms, including the Plantae, Fungi, and Animalia).
Although many argue that biological categories are subjective in nature, and the criteria for designating the kingdoms of life have been modified by scientists historically, it is nonetheless true that scientists have set definite criteria or guidelines that form the basis of taxonomic classification, which assists us in understanding the evolutionary and ecological relationships among living organisms. Characteristics of bacteria are catalogued in the multi-volume set of books called Bergey’s Manual of Systematic Bacteriology. Before the era of the internet and computer algorithms to quickly identify bacteria, bacteriologists would use this manual to identify bacteria. The manual is organized very much like a dichotomous key, a systematic method for classifying organisms. You will gain practice in using a dichotomous key in later lab exercises.
Please read the relevant sections in your textbook on prokaryotic cells. In short, prokaryotes are small, single-celled organisms which lack a nucleus. Prokaryotes, from the Greek word for “prenucleus,” have existed on Earth longer and are more widely distributed than any other group of organisms. They are found in almost every imaginable habitat: air, soil, and water, in extreme temperatures and harsh chemical environments. They can be photosynthetic, using light as an energy source, or chemosynthetic, using inorganic chemicals as the source of energy. Most prokaryotes are heterotrophic, absorbing nutrients from the surrounding environment. Prokaryotes are instrumental in nutrient cycling. The metabolic diversity of these fascinating organisms has led to many applied uses and manipulations of bacteria. One example is bioremediation, where bacteria capable of metabolically inactivating toxic compounds are used in cleaning up environmental contamination.
Technical precautions when working with bacteria
When you are working with bacteria, it is very important to practice certain aseptic techniques to make sure that the cultures being studied are not contaminated by organisms from the environment and that organisms are not released into the environment.
Please review the following procedure before arriving to the laboratory. You should perform this protocol each time you are working with bacteria in this laboratory.
• Wipe the lab bench with disinfectant before and after the lab activities.
• Wash your hands before and after performing an experiment.
• Using the alcohol lamp, flame sterilize all nonflammable instruments used to manipulate bacteria or fungi before and after use.
• Place swabs and toothpicks in the disposal container immediately after use. Never place one of these items on the lab bench after use!
• Wear gloves and a lab coat, a lab apron, or a clean old shirt over your clothes to lessen chances of staining or contamination accidents.
The bacteria used in these exercises are non-pathogenic (i.e. they are not disease-causing); nevertheless, please use appropriate aseptic technique and work with care! If a spill occurs, notify your instructor. Since gloves and soiled towels are not pathological and not a biohazard, dispose of them in the trash and not in the expensive autoclavable plastic bag.
Background on classifying prokaryotes
Because of the small size and similarity of cell structure among different bacteria, techniques used to identify bacterial species are different from those used to identify macroscopic organisms. Staining reactions and properties of growth, nutrition, and physiology are usually used to make final identification of species. The structure and arrangement of cells and the morphology of colonies contribute preliminary information that can help us determine the appropriate test necessary to make the final identification. In the laboratory, you will use the tools at hand, microscopes and unaided visual observations, to learn some characteristics of bacterial cells and colonies.
One important property of bacteria used in their classification is the nature of their cell walls. Most prokaryotes have a cell wall, a complex layer outside the cell membrane. The most common component found in the cell wall of the members of domain Bacteria is peptidoglycan, a complex protein-carbohydrate polymer which helps to form part of the cell wall structure of bacteria. There are no membrane-bound organelles in prokaryotes and the genetic material is not bound by a nuclear envelope. Prokaryotes do not have linear chromosomes as studied in the mitosis lab; their genetic material is a single circular molecule of DNA. They divide by a process called binary fission, in which the cell duplicates its components and divides into two daughter cells. These cells usually become independent, but they may remain attached in linear chains or grape-like clusters. In favorable environments, individual bacterial cells rapidly proliferate, forming colonies consisting of millions of cells. Bacteria of the genus Bacillus and Clostridium can form spores, which are dormant cells that can withstand extreme heat, desiccation (drying-out), or other adverse environmental conditions. When the conditions are favorable, these cells convert back to their active stages. These dormant stages have even been found in mummies that are thousands of years old! The ability to form spores or not is useful in classifying bacteria.
In this lab, you will study prokaryotic organisms commonly called bacteria. In the five-kingdom scheme, bacteria were placed in the kingdom Monera. In the three-domain, multi-kingdom system, the common bacteria are classified in the domain Bacteria. Prokaryotes that tend to live in extreme environments (such as high or low temperatures, high salt concentrations, etc) are classified in the domain Archaea.
Colony morphologies
Differences in colony morphology and the shape of individual cells are important distinguishing characteristics of prokaryotic species. A bacterial colony grows from a single bacterium and is composed of millions of cells. Each colony has a characteristic size, shape, consistency, texture and color (colony morphology), all of which may be useful in preliminary species identification. Bacteriologists use specific terms to describe colony characteristics. Use the figure from the web site given at the beginning of the exercise to become familiar with this terminology and describe the bacterial species provided. Occasionally, one or more fungal colonies will contaminate the bacterial plates. Fungi may be distinguished from bacteria by the fuzzy appearance of the fungal colony. The body of a fungus is a mass of filaments called hyphae in a network called a mycelium. Learn to distinguish fungi from bacteria.
Go to the following website and print out the bacterial colony morphology description sheet. Use this sheet to describe your colonies.
In the following exercise, working independently, you will observe and describe the morphology of colonies and individual cells of several bacterial species. You and your lab partner will compare results of all lab studies.
Procedure:
1) Wipe the work area with disinfectant and wash your hands.
2) Set up your stereoscopic microscope.
3) Obtain one of the bacterial plates provided. Leaving the plate closed (unless otherwise instructed), place it on the stage of the microscope.
4) Examine a typical individual, separate colony. Measure the size and note the color of the colony and record this information in Table 4.1 in your lab question worksheet. Using the diagrams provided, select appropriate terms that describe the colony from your printed internet site.
5) Repeat steps 2 to 4 with two additional species. Your lab partner should examine three different species different from those that you observe.
Morphology of Individual Cells
Microscopic examination of bacterial cells reveals that most bacteria can be classified according to three basic shapes: bacilli (rods), cocci (spheres), and spirilla (spirals, or corkscrews). In many species, cells tend to adhere to each other and form aggregates, but each cell still maintains its independence. Examine the DEMO ( prepared slides) of bacteria that illustrate the three basic cell shapes and sketch the three types in Fig 4.1 in the lab worksheet.
Examining bacteria involved in periodontal disease and plaque
Protein and carbohydrate materials from food particles accumulate at the gum line in your mouth and create an ideal environment for bacteria to grow. This mixture of materials and bacteria is called plaque. To investigate the forms of bacteria found on and around your teeth, prepare a stained slide of plaque.
Procedure:
1) Set out a clean slide.
2) Place a drop of water on the slide. Make the drop of water very small.
3) Using a fresh toothpick, scrape your teeth near the gum line and mix the scraping in the drop of water.
4) Spread this plaque-water mixture into a thin film and allow it to air-dry.
5) When the smear is dry, hold the slide with a clothespin and pass it quickly over the flame of an alcohol lamp several times at a 45° angle. This should warm the slide but not cook the bacteria. Briefly touch the warm slide to the back of your hand. If it is too hot to touch, you are allowing it to get too warm.
6) Place the slide on the support of a staining tray and apply 3 or 4 drops of crystal violet stain to the smear. Crystal violet will permanently stain your clothes, and it may last several days on your hands as well. Work carefully!
7) Leave the stain on the smear for 1 minute.
8) Wash the stain off with a gentle stream of water from a wash bottle so that the stain goes into the staining pan.
9) Blot the stained slide gently with a paper towel. Do not rub hard or you will remove the bacteria.
10) Examine the plaque bacteria and determine bacterial forms. Use the 40X magnification on your compound microscope.
Gram staining and its relationship to cell wall biochemistry
Gram stain relies on the use of three stains: crystal violet (purple), Gram’s iodine, and safranin (pink/red). Gram-positive bacteria (with the thicker peptidoglycan layer), retain the crystal violet/iodine complex and appear blue/purple. Gram-negative bacteria (with a thin peptidoglycan layer and a second, outer, lipid-filled layer) lose the blue/purple complex but retain the safranin and appear pink/red. Your laboratory instructor will set up a demo of Gram+ & Gram- with 100x objective so that you and the other students can see it better.
In this lab study, you will prepare and stain slides of two different bacterial species. One member of the lab team should stain Micrococcus and E. coli. The other member should stain Serratia and Bacillus.
Procedure:
1) Prepare smears as directed for the plaque slide, substituting the bacterial species for the plaque. DO NOT add a drop of water, just use the bacterial broth. Label the slide with your initials and the name of the bacterial species being investigated.
2) Support the slide on the staining tray and cover the smear with 3 or 4 drops of crystal violet. Wait 1 minute.
3) Rinse the stain gently into the staining pan with water from the squirt bottle.
4) Cover the smear with Gram iodine for 1 minute, which sets the stain.
5) Rinse it again with water.
6) Destain (remove the stain) by dropping the 95% alcohol/acetone mixture down the slanted slide 1 drop at a time. At first a lot of violet color will rinse away. Continue adding drops until only a faint violet color is seen in the alcohol rinse. Do not overdo this step. You should be able to see some color in the smear on the slide. If not, you have destained too much. The alcohol/acetone removes the crystal violet stain from the gram-negative bacteria. The gram-positive bacteria will not be destained.
7) Using the water wash bottle, rinse immediately to prevent further destaining.
8) Cover the smear with safranin for 30 to 60 seconds. This will stain the destained gram-negative bacteria a pink/red color.
9) Briefly rinse the smear with water as above. Blot it lightly with a paper towel and let it dry at room temperature.
10) Examine each slide using the highest magnification on your microscope.
|Table 4.2. Differences between Gram-positive and Gram-negative Bacteria |
|Gram-Negative Bacteria |Gram-Positive Bacteria |
|more complex cell wall |simple cell wall |
|thin peptidoglycan cell wall layer |thick peptidoglycan cell wall layer |
|outer lipopolysaccharide wall layer |no outer lipopolysaccharide wall layer |
|retain safranin |retain crystal violet/iodine |
|appear pink/red |appear blue/purple |
INVESTIGATING SPECIFIC ENVIRONMENTS
Introduction
Your laboratory instructor will assign team numbers to sets of four students. Each team of students will sample bacteria and fungi from one of five environments: food supply, soil, air, stream water, and human hands. Read the instructions carefully for all investigations. Think about the following questions and, before you begin your investigation, hypothesize about the relative growth of bacteria and fungi in the different environments. (1) Where in the environment would bacteria be more common, and where would fungi be more common? or (2) Would any of these environments be free of bacteria or fungi?
Food Supply
1) Holding the lid in place, invert an agar plate and label the bottom “chicken”.
2) Open the dish containing the piece of chicken, and swab the surface using a sterile cotton swab. Avoid touching the chicken with your hands. Use the swab. Always wash your hands thoroughly after touching raw chicken, owing to the potential presence of Salmonella, a bacterium that causes diarrhea.
3) Isolate bacteria by the streak plate method (Fig 2).
4) Carefully lift the lid of the agar plate to no more than 45° and lightly streak the swab back and forth across the top quarter of the agar. Close the lid and discard the swab in the receptacle provided. Minimize exposing the agar plate to the air.
5) Flame the bacterial inoculating loop using the alcohol lamp. Allow the loop to cool and, starting at one end of the swab streak, lightly streak the microorganisms in the pattern shown in Figure 2. Do not gouge the medium.
6) Reflame the loop and continue to streak as shown in Figure 2. By the end of the last streak, the bacteria should be separated and reduced in density so that only isolated bacteria remain. These should grow into isolated, characteristic colonies.
7) Write the initials of your team members, the lab section, and the date on the bottom of the petri dish.
8) Seal the dish with Parafilm and place it in the area indicated by the instructor.
9) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
|Fig 2. Isolating bacterial colonies using the streak technique |
|[pic]. |
|(modified from Morgan and Carter, 2002) |
|(a) Streak the swab over the top quarter of the agar plate, region 1. |
|(b) Using the newly flamed and cooled loop, pick up organisms from region 1 and streak them into region 2. |
|(c) Reflame the loop and let it cool, and then pick up organisms from region 2 and streak them into region 3. |
Soil
1) Holding the lid in place, invert an agar plate and label the bottom “soil.”
2) Using a cotton swab, pick up a small amount of soil from the sample.
3) Prepare a streak culture by following step 3 in the procedure for “Food Supply”.
4) Write the initials of your team members, the lab section, and the date on the BOTTOM of the petri dish.
5) Seal the dish with Parafilm and place it in the area indicated by the instructor.
6) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
Air
1) Holding the lid in place, invert an agar plate and label the bottom “air.”
2) Collect a sample of bacteria by leaving the agar plate exposed (lid removed) to the air in some interesting area of the room for 10 to 15 minutes. Possible areas might be near a heat duct or an animal storage bin.
3) Write the initials of your team members, the lab section, and the date on the BOTTOM of each petri dish.
4) Seal the dish with Parafilm and place it in the area indicated by the instructor.
5) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
Stream Water
1) Holding the lid in place, invert an agar plate and label the bottom “stream water.”
2) Using a sterile cotton swab, take a sample from the stream water.
3) Prepare a streak culture by following step 3 in the procedure for “Food Supply”.
4) Write the initials of your team members, the lab section, and the date on the BOTTOM of the petri dish.
5) Seal the dish with Parafilm and place it in the area indicated by the instructor.
6) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
Hand Washing
1) Draw a line across the center of the bottom of an agar plate. Write “unwashed” on the dish bottom on one side of the line and “washed” on the other side of the line.
2) Select one person who has not recently washed his or her hands to be the test subject. The subject should open the petri dish and lightly press three fingers on the agar surface in the half of the dish marked “unwashed.” Do not break the agar. Close the petri dish.
3) The same subject should wash his or her hands for 1 minute and repeat the procedure, touching the agar with the same three fingers on the side of the dish marked “washed.”
4) Write the initials of your team members, the lab section, and the date on the BOTTOM of the petri dish.
5) Seal the dish with Parafilm and place it in the area indicated by the instructor.
6) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
INVESTIGATING THE ENVIRONMENT OF YOUR CHOICE
Introduction
In the previous experiment, you tested specific environments for the presence of bacteria and fungi. In this lab study, you will study an environment of your choice. If extra agar plates are available, you may choose to investigate bacteria in an environment before and after some treatment, such as bacteria on the water fountain before and after cleaning. DO NOT SWAB BODY REGIONS because pathogenic bacteria may be present.
Procedure:
1) Decide what environment you will investigate. It might be some environment in the lab room or somewhere in the biology building. Carry the sterile cotton swab and agar plate to the environment, and use the swab to collect the sample. If you are collecting from a dry surface, you should first dip the cotton swab in the sterile water and then swab the surface. If you apply any treatment to the surface, describe the treatment in the margin of your lab manual. Do not do throat or ear swabs! Pathogenic bacteria may be present.
2) Open the agar plate and lightly streak the swab back and forth across the agar. Discard the swab in the receptacle provided.
3) Label the bottom of the agar plate to indicate the environment tested. Record the environment tested in the Results section.
4) Write the initials of your team members, the lab section, and the date on the BOTTOM of the petri dish.
5) Seal the dish with Parafilm and place it in the area indicated by the instructor.
6) Incubate the culture until next lab period and then observe results from the entire class during the next laboratory period.
CONTROLLING THE GROWTH OF BACTERIA
Prokaryotes are found almost everywhere on Earth, and most species are directly or indirectly beneficial to other organisms. They are necessary to maintain optimum environments in animal and plant bodies and in environmental systems. However, even beneficial species, if they are reproducing at an uncontrolled rate, are potentially harmful or destructive to their environment. In addition, several species of bacteria and fungi are known to be pathogenic, that is, to cause disease in animals and plants (though, so far, no member of the archaea has been found to be a human pathogen). Their growth must be controlled. Agents have been developed that control bacterial and fungal growth. In this exercise, you will investigate the efficacy (effectiveness) of three of these growth-controlling agents: antibiotics, antiseptics, and disinfectants.
Using Antibiotics to Control Bacterial Growth
An antibiotic is a chemical produced by a bacterium or fungus that has the potential to control the growth of another bacterium or fungus. Many antibiotics are selective, however, having their inhibiting effect on only certain species of bacteria or fungi. In this lab study, you will apply an assortment of antibiotics to a lawn culture of a bacterial species. Working in pairs, you will determine which antibiotics are able to control the growth of the bacteria. Each pair of students in a group of eight should culture a different bacterium. All four species should be cultured.
A lawn of bacteria is like a lawn of grass—a uniform, even layer of organisms covering an entire surface. Prepare the lawn of bacteria carefully. The success of this experiment will largely depend on the quality of your lawn.
Procedure:
1) Label the bottom of an agar plate with your initials, the lab section, the date, and a word to indicate the experiment (such as “antibiotic”).
2) Begin preparation of a bacterial lawn by inserting a sterile swab into the bacterial culture in liquid nutrient broth.
3) Allow the swab to drip for a moment before taking it out of the culture tube, but do not squeeze out the tip. The swab should be soaked but not dripping.
4) Carefully lift the lid-of the agar plate to about 45° and swab the entire surface of the agar, taking care to swab the bacteria to the edges of the dish.
5) Rotate the plate 45° and swab the agar again at right angles to the first swab. Close the lid.
6) Carry the agar plate swabbed with bacteria to the demonstration table.
7) Remove the plate lid, place the antibiotic disk dispenser over the plate, and push down on the handle to dispense the disks. (Each disk has been saturated with a particular antibiotic. The symbol on the disk indicates the antibiotic name. Your instructor will provide a key to the symbols.)
8) Replace the lid, seal the plate with Parafilm, and place the plate in the area indicated by the instructor. They will be incubated at 37° C for 24 to 48 hours and then refrigerated.
9) Next lab, examine cultures to determine bacterial sensitivity to antibiotics. Measure the diameter of the zone of inhibition (area around disk where bacteria growth has been inhibited) for each antibiotic.
10) Record the measurement for your bacterial species and each antibiotic in Table 4.5. If the antibiotic had no effect on bacterial growth, record the size of the zone as 0.
Using Antiseptics and Disinfectants to Control Bacterial Growth
Agents other than antibiotics are often used to control bacterial growth. Those used to control bacteria on living tissues such as skin are called antiseptics. Those used on inanimate objects are called disinfectants. Antiseptics and disinfectants do not kill all bacteria, as would occur in sterilization, but they reduce the number of bacteria on surfaces.
Procedure:
1) Holding the lid in place, invert a sterile agar plate and label the bottom with your initials, the lab section, and the date. Draw four circles on the bottom. Number the circles.
2) Using the same bacterial culture as you used in the antibiotic investigation, prepare a lawn culture.
3) Carry the closed agar plate swabbed with bacteria to the demonstration table.
4) Open the agar plate and, using forceps soaking in alcohol, pick up a disk soaked in one of the antiseptics or disinfectants, shake off the excess liquid, and place the disk on the agar above one of the circles. Repeat this procedure with two more antiseptics and/or disinfectants. Place a disk soaked in sterile water above the fourth circle to serve as a control.
5) Record the name of the agent placed above each numbered circle in Table 4.6, in the Results section. (Example: 1 = Lysol, 2 = Listerine, and so on.) Seal the plate with Parafilm.
6) Place the agar plate in the area indicated by the instructor. Incubate the agar plates at 37°C for 24 to 48 hours and then refrigerate them.
7) Next lab, examine the cultures to determine the bacterial sensitivity to disinfectants and antiseptics. Measure the diameter of the zone of inhibition for each agent.
8) Record the measurement for your bacterial species and each inhibiting agent in Table 4.6. If the agent had no effect on bacterial growth, record the size of the zone as 0.
9) Using your results and the results from other teams, complete Table 4.6. Record sizes of the zone of inhibition for all species of bacteria and all antiseptics and disinfectants.
Scientists measure the effectiveness of antiseptics and disinfectants in controlling bacterial growth by a standard called the phenol coefficient (PC). PC compares a germicidal agent (antiseptic or disinfectant) with phenol, a disinfectant used since the 1860s. A PC of “1” means that the germicide is as effective as phenol in controlling the growth of germs. A substance with a PC greater than “1” is more effective than phenol, and a substance with a PC less than “1” is less effective than phenol. Salmonellosis (caused by ingesting Salmonella sp.) is one of the most serious food-borne diseases of our time. Salmonella bacteria may be found in any food substance, but are particularly common on poultry and eggs.
|Table 4.7. Phenol Coefficients of Some Common Antiseptics and Disinfectants Used to |
|Control Staphylococcus and Salmonella Growth* |
|Germicide |Staphylococcus |Salmonella |
|Phenol |1.0 |1.0 |
|Iodine |6.3 |5.8 |
|Lysol |5.0 |3.2 |
|Clorox |133.0 |100.0 |
|Ethyl alcohol |6.3 |6.3 |
|Hydrogen peroxide |— |0.01 |
|Formalin |0.3 |0.7 |
|*Modified from Table 22.1 in Alcamo, E. I. Fundamentals of Microbiology, 5th ed., 1997.|
Introduction to Scientific Investigation
Your instructor will introduce the Scientific Method and will discuss how a scientific investigation is performed. You will be provided with a choice of investigations that can be performed with bacteria and agar plates. You will grow these experiments and observe the results in the next lab. For the next lab, you will also write up your results in a scientific format.
Lab 5 – Bacteriology II & Scientific Investigation
During the last lab period, growth of bacteria was initiated and in this lab you will observe the results. Your instructor will provide details of how to write up the results of your scientific investigation. You will also discuss a paper that was assigned last lab.
Lab 6 – Protists, Fungi, and Bryophytes
by M. Brunell
Before coming to the lab you should:
• Review the material from last lab.
• Be able to define all bold-faced terms in the lab exercise.
After completing the lab exercise you should be able to:
• Recognize and be able to distinguish between the groups you have studied.
• Understand the forms of nutrition in fungi.
• Recognize the groups studied and know their structures, and if discussed, their functions.
• Understand the basic life cycles of the fungi and plants.
• Understand the differences between bryophytes and vascular plants
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, Menlo Park, CA.
• Van de Graaff, Kent M. and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ. Co., Englewood, CO.
Domain Eukarya: PROTISTS
The Protista are an artificial group composed of a diverse set of over 16 clades that are only distantly related to each other. The taxonomy of protists is in a state of flux and the relationships among the member clades, and between protists and other organisms, is under active study. Recently, scientists have proposed that the various groups of protists should be divided into several new candidate kingdoms, but because the research is still in progress, we will refer to informal “Protista” as the group containing all the protists.
When a eukaryotic organism cannot be classified as either animal, fungus, or plant, it is placed into the Protista. Many are unicellular, but there are numerous colonial, filamentous, and multicellular forms. Most have typical cells with a single nucleus, but some have multinucleate bodies. They range in size from microscopic organisms to brown algae that may be 100 feet long. Protists move chiefly by means of flagella, cilia or pseudopodia. Protists have a variety of feeding modes. There exist heterotrophic (animal-like) protists that ingest food materials, generally called protozoa, and plant-like photosynthetic autotrophs, generally termed algae. But there are many protist species that fit into more than one feeding category. For example, some “mixotrophs” like dinoflagellates and euglenoids can carry out photosynthesis AND absorb nutrients like an animal. Other protists are autotrophic and largely self-sufficient but have very specific requirements for certain nutrients – these are termed “auxotrophs”. Protists typically reproduce asexually, reserving sexual reproduction for times of stress.
Protist groups we will study in lab include (refer to pages E-1 & E-2 in Campbell):
Supergroup Unikonta
Clade Amoebozoa
Group Gymnamoeba (Gr. gymnos, naked + Gr. amoeb, a change or alteration): the amoebas (Amoeba), aquatic heterotrophs that move using pseudopodia.
Group Myxogastrida (Gr. myxa, mucus + Gr. gaster, the belly): plasmodial slime molds; heterotrophic diploid multinucleate plasmodium, amoeboid movement by streaming.
Supergroup Archaeplastida
Clade Chlorophyta (Gr. chloros, grass green + Gr. phyton, a plant): green algae (Chlamydomonas, Volvox); photosynthetic with chlorophyll a and b present; unicellular, filamentous, or multicellular. Many species with flagella.
Supergroup Chromalveolata
Clade Alveolata
Group Ciliophora (L., cilium, small hair + Gr. phoros, carrier, to bear): ciliates (Paramecium) heterotrophic; unicellular with cells of fixed shape; two or more nuclei; many cilia present.
Clade Stramenopila
Group Bacillariophyta (L. bacillus, little stick): diatoms; photosynthetic; unicellular; many with double shell of silica.
Gymnamoeba – The Amoebas
Using a clean slide, place a small drop of water from the culture of Amoeba proteus on the slide, and cover with a cover glass. First observe under scanning power (40x) to locate a cell, and then move to the low (100X) or high power (400x) magnifications. Note the finger-like projections of the cell membrane called pseudopodia (Gr. pseudo, false + Gr. podion, a foot). They are used for locomotion as well as engulfing prey. What are the general anatomical features of an amoeba? What are their functions?
On your worksheet, draw an amoeba.
Myxogastrida – The Slime Molds
Obtain a Petri dish containing a growing specimen of Physarum and examine the organism under the dissecting microscope. The multinucleate mass is called a plasmodium. Examine the series of veins visible on the plasmodium.
On your worksheet, draw a representation of Physarum.
Chlorophyta – The Green Algae
Make a temporary mount of Chlamydomonas using Detain to slow their movement. Observe the cells under low power (100X). To observe greater detail, switch to high power (400x). Be careful not to crush the slide. What is the shape of these cells in three dimensions? Determine this by carefully focusing up and down with the fine focus knob.
The most spectacular green alga is probably Volvox, a hollow sphere made up of a single layer of 500 to 60,000 tiny, biflagellate, Chlamydomonas-like cells. Obtain a depression slide and place a drop of Volvox culture in the depression. Observe under scanning, low, and high power. If you observe carefully, you can see that each cell has 2 flagella and that these are beating, causing the colony to roll.
On your worksheet, draw a single Volvox colony.
Ciliophora – The Ciliates
Mount a drop of water from one of the living cultures of Paramecium caudatum on the front table onto a slide. Add a small drop of Detain to slow their movement, add a cover glass and observe the culture under scanning and then low power. What do you see?
On your worksheet, draw a single Paramecium cell.
Bacillariophyta – The Diatoms
Mount a drop of water from one of the living diatom cultures on the front table and observe it under high power. The diatom cell is surrounded by a glass-like cell wall called the frustule. The frustule consists of two halves that fit together like a Petri dish or a shoe box. The larger of the two halves is called the epitheca, the smaller the hypotheca. Notice the color of the cell. It should be green with either a yellow or brownish cast. The non-green colors are caused by the pigment fucoxanthin, which is found in a few algal phyla.
On your worksheet, draw a single diatom cell.
Domain Eukarya: FUNGI
The fungi include a large variety of heterotrophic organisms that obtain their food by secreting digestive enzymes onto the food & then absorbing the simple organic molecules released by the enzymatic action. Among the fungi, two types of heterotrophs are found: parasites, which obtain food from living organisms; and saprobes, which obtain their food from nonliving organic materials. The fungal body is formed by a network of tiny filaments called hyphae which may be packed together to form complex solid structures called mycelia (sing. mycelium). The cell walls of fungi are composed of chitin (as opposed to cellulose in plants).
The phyla of fungi are distinguished by their reproductive structures. We will study all but one of them (the Glomeromycota) in this lab.
Supergroup Unikonta
Kingdom Fungi
Phylum Chytridiomycota (Gr. chytridion, earthen pot + Gr. myces, fungus): chytrids; hyphae lack septa and form flagellated spores called zoospores.
Phylum Zygomycota (Gr. zygon, yoke): black bread mold (Rhizopus); fusion of hyphae leads directly to the formation of a zygote; hyphae lack septa.
Phylum Ascomycota (Gr. ascos, wine-skin or bag): yeasts (Saccharomyces, Candida), cup fungi (Peziza), truffles, morels (Morchella); hypha fusion leads to formation of a stable dikaryotic mycelium that forms zygotes in sac-like structures called asci which form inside a fruiting body called an ascoma; hyphae are septate.
Phylum Basidiomycota (L. basidium, small pedestal): puffballs, jelly fungi, shelf fungi, rusts, smuts, mushrooms (Agaricus campestris), toadstools; sexual reproduction similar to Ascomycota, but zygotes formed in club-shaped reproductive structures called basidia which form in a fruiting body called a basidioma; hyphae are septate.
Imperfect Fungi (Deuteromycetes; Gr. deuteros, the second + Gr. myces, fungus): fungi in which sexual reproduction has never been observed; probably includes members of all three phyla above; Penicillium, Aspergillus, ringworm, athlete’s foot.
Phylum Chytridiomycota – The Chytrids
Formally considered protists, the chytrids are a largely aquatic phylum of saprobic and parasitic fungi. They represent a very early lineage in the kingdom, and are the only flagellated fungi. Some chytrids are filamentous but others possess a globose body which will eventually be converted into a reproductive organ.
In today’s lab, we will study a prepared slide of Synchytrium endobioticum, a terrestrial parasite of potatoes. This organism causes the disease “Potato Wart Disease”, which is manifested as large wart-like lesions on potato tubers, rendering them unusable.
Obtain a prepared slide of Synchytrium endobioticum and view at low power (100x). On the slide is a section of an infected potato. Note that most of the cells in the center of the section possess large purple-colored starch grains. Ignore these for now. Look to the outer edges of the section and notice the large, red-colored blotches embedded in the tissue. These are the reproductive bodies of the fungus. Each chytrid body is a single cell, each residing within a single potato cell; the fungus lives within the potato cell and feeds on it, eventually killing it. The parasite will then enlarge, undergo mitosis but not cytokinesis, and produce zoospores bearing flagellae in a multi-parted organ called a sorus. Each of the red bodies on the slide is a sorus, which is internally divided into 4 to 9 segments. Faint lines distinguish these segments – look for them. Each segment will produce 200 to 300 zoospores, which swim away when the soil is wet and infect new tubers.
On your worksheet, draw the section at 100x and show both normal and infected potato cells. Label the parasites structures as sori, and also label the normal cells.
Phylum Zygomycota – The Molds
Except for unicellular yeasts, the basic unit that makes up the fungal body is the hypha (pl. hyphae), which is a filamentous cell or chain of cells. In the Zygomycota, the filament normally lacks cross walls called septa (sing. septum). The fungal body is thus, in effect, one large multinucleate cell (a coenocyte). A mass of hyphae is termed a mycelium, which can resemble a mass of fuzz or cotton. We will study the widespread common black bread mold, Rhizopus stolonifer, as an example of this phylum.
On the front table are agar plates that were sown with a suspension of spores about 15 hours before the lab period. Using the dissecting microscope, study one of these plates and observe the germinated spores.
The short thread that emerges from the spore wall elongates and branches in further growth. Horizontal, surface hyphae are called stolons (as in strawberry runners). Note the absence of septa (cross walls) in the hyphae. The medium on which the fungi are growing is agar, which has been combined with dextrose, a sugar. The agar itself is not available as food for the fungi.
Questions 1, 2, 3
Obtain an older culture and examine it under low power (dissecting microscope). Note how the stolons advance over the inside of the dish top. Where the stolon touches the lid, special anchoring structures called rhizoids develop. Directly opposite the rhizoids will be found upright stalks called sporangiophores; these bear terminal sacs (the sporangia) which vary in color according to age.
Questions 4, 5
Remove some of the sporangia from the culture and mount them in 1% detergent solution. The sporangia contain the asexual reproductive bodies, the spores. Observe young, old, and open sporangia. On your worksheet, draw a single enlarged sporangium labeling: sporangium; sporangium wall; spores; columella (the balloon-like cross wall that separates the sporangium from the rest of the hyphae).
Prepared slides of Rhizopus made from cultures that have been sown with the two mating strains are available for observation (view at 100x). Note that the sporangia, spores, and mycelia of the two strains look alike, but where the mycelia meet, a special type of spore is formed. The slides were made with hyphae from this meeting area. Look for the large dark zygosporangia resulting from the fusion of the two types of hyphae. Inside of the zygosporangium is a large cell called the zygospore, which is multinucleate. It is in this cell that fusion of nuclei and meiosis occurs, leading to sexual spore production. In this phylum the hyphae act as two sexual cells.
Question 6
On each side of a zygosporangium you will observe an enlarged structure, the suspensor, which leads back to an ordinary hypha. With this in mind, look for younger stages of reproduction.
The youngest stage will show two cigar-shaped structures, the progametangia, which are touching. A second stage will show small cells called gametangia isolated at the end of each suspensor by a septum; a third stage should show the gametangia fusing, and the final stage is a mature zygosporangium. Frequently the suspensors are unequal in size, but note that the gametangia are always alike. On your worksheet, draw and fully label stages in sexual reproduction of Rhizopus.
Phylum Ascomycota – The Sac Fungi
Peziza and its relatives are among the most conspicuous ascomycetes in wooded areas. The material supplied for study is a cup fungus, which occurs in great numbers during the spring. The inner surface of the cup, or ascoma, contains the fertile asci (sing. ascus), which occur in a layer called the hymenium, which is often brightly colored in living specimens. Each ascus contains eight (8) ascospores which result from meiosis. The first part of fertilization (plasmogamy) commonly takes place in the soil when two sexually compatible hyphae come in contact with each other and exchange nuclei. Fusion of the nuclei (karyogamy) occurs in the asci as they form. Karyogamy is followed by meiosis and mitosis, producing 8 ascospores.
Question 7
Locate the fresh or preserved Peziza on display. On your worksheet, draw the fungus indicating where the food absorbing mycelia would be attached.
Obtain a prepared slide of Peziza and observe it with your microscope at low power (100x). The spore bearing sacs or asci form a regular layer on the inner surface of the cup, the hymenium. The hymenium also contains slender sterile hairs, the paraphyses, which stick out like fingers among the asci. Switch to high power (400x) and on your worksheet draw a single ascus and beside it a single paraphysis to show their relative sizes.
Dutch elm disease and chestnut blight have been serious diseases caused by parasitic members of this group. They have essentially wiped out the American chestnut and seriously threaten the American elm. Ergot (Claviceps purpurea) is a nuisance in rye crops because of its poisonous nature, but a drug made from it has been a service to people in stopping dangerous hemorrhaging. The drug LSD has been isolated from ergot, and various ergot toxins are the cause of St. Anthony’s Fire (an infection of the skin characterized by bright red, hot, swollen, shiny rash that reminded people of fire).
Yeasts have been of economic interest for centuries in connection with baking and, of course, the brewer's art. They differ from the typical fungi in being unicellular organisms and usually reproducing only asexually.
Two kinds of ascomycetes are edible and counted among the rarest of gourmet delights: morels (Morchella) and truffles (not the chocolate kind). Observe a specimen of Morchella.
Question 8
Observe the DEMO of a longitudinal section of an ascocarp of a morel showing the position of the hymenium. Draw the section on your worksheet.
Deuteromycetes - imperfect fungi (sexual stage is unknown)
The deuteromycetes are thought to be mainly ascomycetes; however few of them are known to reproduce sexually so their placement in the correct phylum is problematic. The genera Penicillium and Aspergillus, both deuteromycetes, belong to a group commonly called the blue-green molds and are of great importance to human life. Spoilage of food such as bread and fruit, staining of lumber, deterioration of leather, and lung infections are only a few of the destructive aspects of these fungi. On the beneficial side, Penicillium roqueforti and P. camemberti give the characteristic odor, flavor and color to Roquefort and Camembert cheeses. Alcohol, citric acid and certain other organic acids are manufactured by use of some species of Aspergillus. Penicillium notatum is another important deuteromycete. This organism synthesizes the antibiotic penicillin, which is especially valuable in treating certain kinds of pneumonia, mastoiditis, gonorrhea, syphilis, and numerous throat infections. This drug, the first antibiotic ever known, was discovered by Alexander Fleming in 1928, and it was first used in 1941 during World War II.
Phylum Basidiomycota – The Club Fungi
Basidiomycetes are among the most familiar fungi. Commonly seen examples are puffballs, edible and poisonous mushrooms, smuts, bracket fungi, and rusts. Despite their tremendous diversity in form and size, all members of this phylum share the one trait: the basidium. This is usually a club-shaped cell that produces four external basidiospores, which are held on tiny stalks termed sterigmata (sing. sterigma). (In the ascomycetes, you will recall, the eight spores were borne inside a terminal, sac-like cell called an ascus.) The large fruiting body that holds all the basidia is called the basidioma.
The basidiomycetes that bear gills, best known for their use as food, are important agents in the decay of forest litter. We will use the common cultivated mushroom, Agaricus bisporus, in laboratory because it is readily obtainable. What we refer to as the “mushroom” is actually the basidioma.
The mycelium of Agaricus bisporus grows in the soil and assimilates food from the organic debris. When sufficient food has been accumulated by the mycelium and when environmental conditions are suitable, a fruiting body, the basidioma or “mushroom” is formed just below the surface of the soil. It emerges from the soil by the elongation of the stipe and expands its cap above the surface of the soil. Note a ring of tissue, the annulus, on the stipe of an older specimen.
Examine the character of the undersurface of the cap. The fertile layer or hymenium is on the surface of the gills.
Obtain a prepared slide of a cross-section of a Coprinus cap and view at 100x. On the gills, which should be easy to see, are located the basidia and basidiospores. The gills should radiate out from the central stalk like spokes on a hub. Next, switch to 400x and observe the tiny, dark basidiospores attached to the gill surface. The gills are lined with basidia, constituting the hymenial layer. Look carefully at the basidia to which the spores attach. Two pointed projections, called sterigmata, extend from the basidium and bear the basidiospores. On your worksheet, draw the 400x view of the gill, and label the gill, hymenial layer, basidium, basidiospores, and sterigmata.
Domain Eukarya: PLANTS
Plants are green alga-like organisms characterized by being autotrophic, having cellulose cell walls, and a sporic life cycle (alternation of generations). They can be divided into two major groups: bryophytes (Gr. bryon, moss + Gr. phyton, a plant) and vascular plants (L. vasculum, vessel or duct). Most plants are vascular plants, which possess an internal system of specialized cylindrical cells extending from the tips of their roots to their leaves for the movement of water, minerals and dissolved foods. One type of vascular tissue, xylem, carries water and dissolved minerals upward from the roots, while another type of tissue, phloem, conducts carbohydrates in solution away from the points where photosynthesis occurs. Because of this specialized transport and support system, the vascular plants can be larger and are the most conspicuous of the terrestrial plants. Vascular plants include ferns, gymnosperms and angiosperms. The other major plant group, the bryophytes, includes many species that entirely lack vascular tissues and move material by simple cell to cell diffusion. There are many bryophytes, however, that possess simple vascular tissues, but these tissues differ from those found in the vascular plants. The bryophytes are generally smaller than vascular plants and possess a simpler body.
The following classification includes the ten phyla of living plants:
Supergroup Archaeplastida
Kingdom Plantae
Bryophytes (non-vascular plants)
Phylum Hepatophyta (Liverworts, Marchantia)
Phylum Anthocerophyta (Hornworts)
Phylum Bryophyta (Mosses, Polytrichum, Mnium)
Vascular Plants
Seedless Vascular Plants
Phylum Lycophyta (Club Mosses, Spike Mosses, Quillworts)
Phylum Pterophyta (Whisk Ferns, Horsetails, Ferns)
Seed plants
Gymnosperms
Phylum Ginkgophyta (Ginkgo or Maidenhair Tree)
Phylum Cycadophyta (Cycads)
Phylum Gnetophyta (Mormon Tea, Welwitschia)
Phylum Coniferophyta (Conifers)
Angiosperms
Phylum Anthophyta (Flowering Plants)
In this lab, we will study two bryophyte phyla. Note that the term “bryophyte” has a different meaning than “Phylum Bryophyta”. The former term is an informal name for all of the mosses, liverworts, and hornworts. The latter term is a formal term for the mosses only.
Phylum Bryophyta – The Mosses
The members of the Phylum Bryophyta are small plants that often live in moist areas. Their gametophyte generation is the most conspicuous stage of the life cycle in mosses. (The sporophyte generation is the most conspicuous stage of the life cycle in vascular plants.) The moss gametophyte resembles a tiny fern or bottle-brush, and organs resembling stems, leaves, and roots are present. These organs are not the true stems, leaves, and roots found in vascular plants. Despite this difference, the “stems” are usually called stems, and “leaves” called leaves, however the root-like structures are termed rhizoids and have only an anchorage function, not absorptive as in true roots.
The moss sporophyte generation is very simple, consisting of a single unbranched axis bearing a single sporangium (spore container) at its tip. In all bryophyte phyla (mosses, liverworts and hornworts), the sporangium is called a capsule, which is the site of meiosis and spore production. The capsule is supported by a slender seta, which surmounts a tiny foot that is embedded into the gametophyte tissue. The gametophyte nourishes the sporophyte by passing nutrients through this foot.
Examine a specimen of Polytrichum. Male plants (gametophytes) have a series of leaves at the tip which form a cup. Sperm, formed by specialized gamete-producing structures called antheridia, are released into this cup and can be splashed out by rain or dripping water. If they land near a female archegonium, they can swim down into it and fertilize the egg it contains. The zygote formed in the archegonium will grow into the embryo, or young sporophyte, which will rise out of the archegonium. As the sporophyte rises, it often tears off the top of the archegonium. This torn piece is called the calyptra and forms a cap on top of the capsule. The female gametophyte lacks the cup shaped leaves. If available look at and distinguish between male and female plants.
Use a prepared slide of Polytrichum to study the male (antheridia-bearing) plants and a slide of Mnium to study the female (archegonia-bearing) plants. Antheridia are elongate and oval in shape and consist of a sterile layer of cells (sterile jacket cells) which surround the spermatogenous tissue, which produces the sperm cells. Archegonia are flask shaped and consist of a swollen basal portion, the venter, which contains the egg, and an elongate neck with a row of cells in the center called the neck canal cells. When the archegonium is mature and the egg ready to be fertilized, the neck canal cells disintegrate leaving a neck canal through which the sperm can swim to reach the egg. On your worksheet, draw one archegonium and one antheridium labeling: neck, neck canal cells, venter, egg, sterile jacket cells, and spermatogenous tissue.
Question 9
Examine specimens which illustrate the mature sporophytes of Polytrichum. Identify the calyptra covering the capsule, and remove it. The very tip of the capsule is covered by a small lid or operculum. Carefully remove this structure using a dissecting needle. View the exposed opening using a dissecting microscope. Note the small peristome teeth covering the opening in the capsule. These teeth collectively form the peristome. The function of the peristome is to assist spore dispersal. Note the present configuration of the peristome teeth and then allow the capsule to dry out. You should see a change in their position.
Question 10
On your worksheet, draw the moss sporophyte showing and labeling: calyptra, operculum, peristome teeth, capsule, seta.
Next, locate and view the Polytrichum mature capsule l.s. slide. This slide shows a capsule section, and several structures are visible. Inside you should see the central columella (like a pillar), and then the sporogenous tissue, which may appear as two broad longitudinal strands. This is the tissue that will undergo meiosis and produce the spores. Then there is the capsule wall (the outermost tissue layer). At the top you will see the operculum and some complex structures beneath it. Those structures are the peristome and the apex of the columella which has expanded. On your worksheet, draw a scanning power view (40x) showing the general shape and structure of the capsule.
Examine the examples of several other bryophytes available on the side bench. Know that these are mosses and be able to identify them to phylum level.
Phylum Hepatophyta – The Liverworts
Examine the living and preserved specimens of liverworts on the front table, noting that there are two growth forms of liverworts: those which may be called leafy and those which are thalloid. Leafy liverworts resemble mosses from a distance, but differ by having a flattened appearance. Thalloid liverworts have a ribbon-like body, with rhizoids on the underside that anchor the body to the soil. Be able to recognize the two growth forms and be able to identify these plants to phylum level.
Obtain a prepared slide of the thalloid liverwort Marchantia (mature sporophyte, longitudinal section), and observe at scanning power. The slide is a section through an archegonial head, which is an umbrella-shaped structure that arises from the thallus (body) of the liverwort, and consists of a cluster of archegonia which, when fertilized, produce sporophytes. Locate a well formed sporophyte and observe the shallow, wide foot, and the short thick seta, and the oblong capsule which is full of spores. Also notice the long, thin elaters found interspersed with the spores. The elaters are sensitive to moisture and will twist and bend with humidity changes and push out the spores.
Question 11
Draw the Marchantia sporophyte and label the foot, seta, capsule, elaters, and spores.
Lab 7 – Vascular Plant Reproduction and Diversity
by M. Brunell
Before coming to the lab you should:
• Review the material from last lab and be able to define all the bold-faced terms in the lab exercise.
• Know the names of the phyla in the Kingdom Plantae and the common names of the organisms in each phylum.
• Know the major differences between bryophytes and vascular plants.
• Know the difference in function between xylem and phloem.
• Know the adaptive evolutionary significance of a seed.
After completing the lab exercises you should be able to:
• Be able to recognize, name and explain the function of all the bold-faced terms in the exercise.
• Describe the reproductive cycle of a club moss, spike moss, horsetail, fern, gymnosperm, and angiosperm.
• Be able to compare and contrast the structures of monocots and eudicots.
• Be able to identify the various major types of angiosperm fruits using the provided key.
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings. San Francisco, CA.
• Van de Graaff, Kent M., and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ. Co., Englewood, CO
INTRODUCTION
In the previous lab, the pattern of diversity among the members of the Clade Plantae was discussed, and we looked at the bryophytes called mosses and liverworts. Today we shall examine some of the vascular plants. The vascular plants have an internal system of specialized cylindrical cells extending from the tips of their roots into their leaves for the movement of water, minerals and dissolved foods. One type of vascular tissue, xylem, carries water and dissolved minerals upward from the roots, whereas phloem tissue conducts carbohydrates in solution away from the points where photosynthesis occurs, usually the leaves. Because of this specialized transport and support system, the vascular plants can be larger and are the most conspicuous of the terrestrial plants.
Vascular plants include lycophytes, ferns, gymnosperms and angiosperms. They are divided into two major groups: the lycophytes & ferns (seedless vascular plants) which produce only spores; and gymnosperms and angiosperms (seed plants) that produce seeds. Note that all plants make spores, but seed plants make seeds in addition to spores.
Seedless vascular plants– Phylum Lycophyta (lycophytes)
The earliest vascular plants lacked seeds and produced haploid spores. This mode of reproduction is seen in such modern groups as club mosses, spike mosses, quillworts, whisk ferns, ferns, and horsetails. Of these groups, the Phylum Lycophyta (the lycophytes) consists of perhaps the earliest group of vascular plants. They have been distinct from the fern lineage for probably more than 350 million years. They formerly covered the terrestrial earth as a major component of the swampy coal forests of the Carboniferous period. Today, as a result of extinction, they are poorly represented in the earth’s flora. The few remaining forms are the club mosses, spike mosses, quillworts, and a few other unusual, rare plants.
One of the more abundant lycophytes is the club moss, Lycopodium. This herbaceous plant is common on the east coast of the U.S., but less common in the west. The sporophyte resembles a large moss plant, and bears an underground rhizome, small leaves that possess only a single, unbranched vein, and a terminal strobilus on the tip of the stem. The strobilus is a cone-like structure that produces the spores. The spores disperse in the air or water and will germinate and grow to produce the tiny bisexual gametophyte, which is usually underground and carrot-shaped.
Obtain and view a slide of Lycopodium strobilus l.s. This is a longitudinal section through a strobilus that will show a central axis bearing sporophylls, each of which bears a single sporangium on its upper surface. A sporophyll is a type of leaf that is reproductive, that is, will produce a sporangium or spore container. Each sporangium will contain many small spores, which are occasionally in groups of four. Note that all of the spores are the same size. Plants producing single-sized spores usually produce bisexual gametophytes. The sporangium is connected to the sporophyll by a thick stalk. Many of the sporophylls will not show a stalk - this is caused by the plane through which the section was made.
Under scanning power, make a drawing of the Lycopodium strobilus, and label the central axis, sporophyll, sporangium, and spores. Then switch to 100x and draw a single sporangium, showing the stalk, spores, and sporangium jacket (wall).
Another often encountered lycophyte is the spike moss, Selaginella. Spike mosses usually have a more feathery, wispy appearance than do club mosses. The well-known “resurrection plant” is a type of desert spike moss. The strobili on spike mosses are hard to locate because the sporophylls closely resemble normal leaves. A major difference between the club moss and spike moss is that the latter produces two types of spores, large and small, within its strobili. Plants that produce two sizes of spores usually produce unisexual gametophytes, that is, male and female plants each bearing sperm and egg, respectively. Locate the living or preserved spike moss on display and make a sketch.
Seedless vascular plants - Phylum Pterophyta
The other major seedless vascular plant lineage consists of the pteridophytes, classified into the phylum Pterophyta. This is a diverse group of plants that was formerly classified into several phyla, but has recently been combined into a single phylum because all members are thought to be closely related. Seed plants are thought to have evolved from pteridophyte ancestors. The pteridophytes include the whisk ferns, horsetails, and ferns, which are themselves an extremely diverse plant group.
Seedless vascular plants - Phylum Pterophyta (Horsetails)
A commonly encountered representative of the pteridophytes is the horsetail, Equisetum. Horsetails generally grow near water and possess either a highly-branched or unbranched body, and tiny, scale-like leaves. The sporophyte consists of stems that are green and hollow, and the nodes at which the leaves arise are jointed and can be easily popped apart by hand. Around campus, the common species is unbranched and looks like a pipe arising from the ground. The tip of the stem often bears a single strobilus that bears the spores. Horsetail gametophytes are tiny green blobs that grow on the mud surface.
Obtain and view a slide of Equisetum strobilus l.s. at scanning power. The strobilus of the horsetail is different from that of the Lycophyta in that there are no sporophylls. Instead, the sporangia are borne on the underside of T-shaped structures called sporangiophores, which are arranged spirally on the central axis. The sporangia are located on the underside of the sporangiophore, facing the strobilus axis. Usually you can see two oblong sporangia “dangling” beneath each sporangiophore. Located within each sporangium are spores (of one size). You should see along with the spores some curly filaments called elaters. There are four elaters connected to each spore.
Question 1
Make a drawing of the horsetail strobilus and label the following: central axis, sporangiophores, sporangia, spores, and elaters.
Seedless vascular plants - Phylum Pterophyta (Whisk Ferns)
Whisk ferns are an unusual group of pterophytes that superficially resemble the most primitive vascular plant fossils, like Cooksonia, which lacked roots and leaves and had simple, dichotomously branching aerial shoots that bore round sporangia. Because of this resemblance, whisk ferns were considered the most primitive living vascular plants for many years. Recently, however, DNA data has shown that these seemingly simple plants are actually related to ferns and are quite advanced.
Locate the living specimen of the whisk fern called Psilotum nudum. Notice the green aerial branches that regularly fork into two equal branches (dichotomous branching pattern). Also notice the tiny round spore-containing structures located along the stems. Each of these structures is a group of three sporangia fused into one unit called a synangium. Notice the lack of leaves, and if you could see under the soil, a lack of roots. (Only a few species of vascular plants lack roots.) Below ground is simply a horizontal stem (rhizome) from which the aerial stems arise.
Question 2
Make a sketch of the whisk fern and clearly show the dichotomous branching pattern and the synangia.
Seedless vascular plants - Phylum Pterophyta (Ferns)
The most abundant group of seedless vascular plants are the ferns. The conspicuous fern sporophyte is diploid and produces haploid spores. These spores disperse and grow into a tiny inconspicuous gametophyte which, in turn, produces eggs and sperm. Since the sperm must swim through a water film to the egg, reproduction is restricted to wet areas or wet seasons.
Examine the samples of fern sporophytes. Look on the undersides of the fronds for the sori (sing. sorus) which are clusters of sporangia where the spores are produced. Make a sketch of a typical fern leaf, and label the sori.
View at scanning power the prepared slide of fern prothallium with antheridia and archegonia, w.m. This is a typical fern gametophyte. The gametophyte is bisexual, tiny, and heart-shaped. In general, these gametophytes are termed prothalli (singular: prothallus or prothallium). At the top of the “heart” is a notch, and just below it is a mass of crowded gametangia (gamete-producing structures: antheridia and archegonia). Below the gametangia are many rhizoids. You are looking at the ventral surface of the prothallus (the underside, which faces the soil). Therefore, the gametangia face downward. The archegonia (producing eggs) are very tiny and dark, and are crowded just beneath the apical notch. The antheridia (producing sperms) are usually below and to the sides of the archegonia, and are very round and lighter in color. Inside each antheridium are the spermatogenous cells. An antheridium looks much like a tiny gumball machine. At low power, draw the entire prothallus showing all of the structures mentioned.
Question 3
After fertilization occurs, the zygote forms in the archegonium, which subsequently develops into the young sporophyte (embryo). The embryo will grow into the mature sporophyte and take root, leaving the gametophyte to shrivel up and perish.
Question 4
Seed plants - Introduction
The gametophytes of plants that produce seeds consist of only a few cells. The female gametophytes develop completely within the sporophyte from megaspores (larger, female spores) and are dependent upon the sporophyte for all their nutritional needs. The megaspore is produced inside of a megasporangium which is in turn wrapped in an integument. After development of the female gametophyte is complete, the integument-wrapped unit is called the ovule. The male gametophytes are pollen and develop from microspores (smaller, male spores).
A seed results when the egg in the female gametophyte is fertilized, and the resulting zygote grows into a tiny embryo. The seed is therefore an embryo (young sporophyte) which is wrapped in several tissue layers, with the outer integuments forming the protective seed coat. Seeds are much better protected against desiccation than spores and the evolution of a seed allows the plant to exist under drier conditions. In addition, the advent of pollen has eliminated the need for external water during reproduction.
Among the seed plants known as gymnosperms (Gr. gymnos, naked + sperma, seed) the ovule, which becomes the seed, rests exposed (hence, naked) upon an ovuliferous scale when it is fertilized. In angiosperms (Gr. angeion, vessel + sperma, seed) the ovule is enclosed in other sporophyte tissues at the time of fertilization, and is therefore no directly exposed to the environment.
Gymnosperm Seed Plants - Phylum Cycadophyta (Cycads)
Cycads are very ancient plants that superficially resemble palm trees or tough, leathery-leaved tree ferns. Generally, a little-branched trunk is crowned by a whorl of frond-like leaves. Cycads produce large cones that bear either ovules and seeds (female cones or megastrobili) or pollen (male cones or microstrobili). Usually each scale of the female cone will produce two large seeds after pollination and fertilization. Cycads are typically slow-growing and rare, and are found primarily in tropical regions. The common “Sago Palm” is an Asian species of cycad.
Cycads are well known in the fossil record as they formed the dominant vegetation on earth during the reign of the dinosaurs. Today, they are restricted to a few regions and many species are near extinction. Therefore, cycads are shrinking in numbers of individuals and species over time. There is only one species of cycad native to the U.S., occurring in Florida.
View the living cycad on display, and make a sketch showing the trunk, leaves, and any cones present.
Gymnosperm Seed Plants - Phylum Ginkgophyta (Ginkgo biloba)
You may have heard of the herbal supplement called Ginkgo biloba extract which allegedly improves memory function. This product is derived from seeds of the ginkgo, or maidenhair tree, which is the sole living representative of the phylum Ginkgophyta. Many other species are known only as fossils. In fact, even Ginkgo biloba is extinct in nature, existing only as a cultivated tree.
View the fresh or preserved specimens of ginkgo on display. The plant is a heavily branched tree bearing distinctive fan-shaped leaves. The male trees bear small, loose cones that produce pollen, whereas the female tree produces many stalked pairs of ovules rather than female cones. Make a sketch of the ginkgo materials on display, showing the stems and leaves, and any reproductive structures present.
Question 5
Gymnosperm Seed Plants - Phylum Coniferophyta (Conifers)
Conifers include woody plants like pines, spruces, hemlocks, junipers, redwoods, firs, and many others. Recognizing a conifer is simplified by the fact that most conifers bear needle-like leaves and woody or papery cones, although exceptions do exist. In this section we will study the pine tree Pinus.
The pine tree produces two kinds of cones (strobili) on the same tree. Each cone is composed of a central axis that bears spirally-arranged spore-bearing scales. The smaller male or pollen cones produce pollen, and the larger female or seed cones will produce eggs and develop seeds.
Examine the specimens of male cones on display. Next, view the slide Pinus male strobilus l.s. under scanning and low power. Note the sac-like microsporangia arranged on the lower surfaces of the scales. The scales, or microsporophylls, are attached spirally to the cone axis. The microsporangia will give rise to the microspores which, in turn, give rise to pollen grains by mitotic division. Draw an overview of the cones, showing the cone axis, scales (microsporophylls), microsporangia, and pollen grains. Next, switch to 400x and view individual pollen grains. Notice the “ears” on the grains. Draw an individual, well formed pollen grain.
Question 6
In Pinus the pollen grains are released from their cones toward the end of May. At the same time the young seed or female cones open and the pollen, by sifting down through the cone scales, comes to lie in the chamber at the base of the micropyle, which is a tiny hole in the ovule wall. The pollen grains germinate producing a slender pollen tube, containing the male gametes, which digests its way to the archegonium where fertilization takes place. Because growth of the pollen tube and development of the megagametophyte is quite slow, fertilization does not take place for as long as thirteen months and the seed with its embryo (young sporophyte) does not mature until late summer in the year after pollination.
Examine some of the examples of seed or female cones available.
Question 7
At low power (100x), examine a slide of an older female cone of Pinus and locate a scale (ovuliferous scale) and examine the ovule attached to its upper surface. You will see an inner layer of tissue, the megasporangium, surrounded by an outer layer, the integument, except for a small opening, the micropyle, at the end facing the cone axis. In some slides you may be able to see pollen grains inside the integument opposite the micropyle. Draw one ovuliferous scale with an ovule attached and label: cone axis, ovule, megasporangium, megaspore (if seen), micropyle, integument.
Observe the demonstration slide of a Pinus ovule with the female gametophyte developed and an archegonium present. There are usually two archegonia at the micropyle end of the gametophyte. The egg is produced by the archegonium.
At low power, examine a slide of a Pinus seed or embryo. Locate, draw and label: embryo, cotyledons (seed leaves), stem apex, root apex.
Angiosperm Seed Plants - Phylum Anthophyta (Flowering Plants)
The flowering plants are termed “angiosperms” because their ovules are enclosed in ovary wall tissues at the time of fertilization. The ovary wall eventually becomes the fruit containing the mature seeds. In terms of distribution, abundance and numbers of species, they are clearly the most successful of the modern plants. The diploid sporophyte is the large mature organism bearing flowers that one easily recognizes. The haploid gametophyte is reduced to a pollen grain that produces the sperm or an embryo sac that produces the egg within the ovule.
The flower is a modified stem bearing modified leaves. Its evolutionary significance is as an attractant for insects to facilitate pollination. Flowers and insects have coevolved for millions of years and form an important mutualistic relationship in which the insect pollinates the flowers and the insects receive pollen and nectar as food rewards.
Regardless of their size or shape, all flowers share certain major features. The conspicuous and showy parts of the flower are typically attached in circles or whorls. The outermost whorl, called the calyx, is composed of the sepals. The next whorl, called the corolla, is composed of the petals. The calyx and corolla taken together are called the perianth. The sepals are often green and leaf-like and the petals are typically colored and attract pollinators such as insects and birds. Sepals and/or petals may be absent or sepals may be colored like petals in certain species.
The third whorl is called the androecium (Gr. andros, male + oikos, house) and is composed of stamens. Each stamen consists of a pollen-bearing anther at the end of a stalk or filament.
At the center of the flower is the innermost whorl called the gynoecium (Gr. gynos, female + oikos, house) consisting of one or more carpels or pistils. (The name pistil is derived from the similarity of the structure to a pestle used to grind materials in a mortar.) The swollen base of the carpel is the ovary which contains one to many ovules. The ovary will eventually develop into the fruit, and the ovules will develop into the seeds. The tip of the carpel is the stigma, the place where the pollen grains will collect and begin their trip toward the ovule. The structure between the ovary and the stigma is the style.
The pollen grains germinate on the stigma and form a pollen tube which extends down through the style and ovary and enters the ovule through a micropyle, much in the same way as in gymnosperms. Of the two sperm nuclei that come down the pollen tube, one will unite with the egg to produce the zygote, and the second unites with another cell which will produce a nutrient-rich tissue called the endosperm. The endosperm serves as a food source for the developing embryo. Fertilization in angiosperms thus is a double fertilization, one producing a zygote and the other producing nutritive endosperm.
Flowers, fruits and endosperm are unique to angiosperms.
Systematists are actively studying the relationships among flowering plants, and from this work they have discovered several major lineages within the group. Two of the largest, most commonly encountered lineages are the monocots and eudicots. Monocots include familiar plants like grains, grasses, lilies, palms, and orchids, whereas eudicots include oaks, maples, roses, beans, etc. In the following exercise, you will compare and contrast these two groups of angiosperms.
Structure of monocot and eudicot flowers.
Carefully remove a single flower from an inflorescence of Gladiolus and snapdragon. Examine the flowers carefully and be sure you can recognize:
calyx (a collective term for sepals)
corolla (a collective term for petals)
perianth (a collective term for sepals + petals)
androecium (a collective term referring to the male parts or stamens)
gynoecium (a collective term referring to the female parts or carpels)
Questions 8 - 14
The word monocot means “one cotyledon”, whereas the “dicot” part of eudicot refers to “two cotyledons”. A cotyledon is the first leaf produced by the embryo and stores food materials in the seed and then expands as the seed leaf when the seed germinates. In addition to this characteristic, these two groups may be distinguished on the basis of the following characters:
|Monocots |Eudicots |
|One cotyledon (seed leaf) per embryo |Two cotyledons (seed leaves) per embryo |
|Flower parts in threes or multiples thereof |Flower parts in sets of four or five or multiples thereof |
|Leaves with parallel venation |Leaves with net-like venation |
|Pollen grains usually with one aperture |Pollen grains usually with three or more apertures |
|Multiple rings of vascular bundles in stem |One ring of vascular bundles or cylinder of vascular tissue in stem |
Distinguish the monocot from the eudicot. (Hint: see Van De Graaff and Crawley, 2005)
Question 15-16
Hold the Gladiolus flower in a position so that you can look down into it, and at the same time see the outside of the ovary. Label and draw the following structures: calyx, sepals, corolla, petals, perianth, androecium, (stamen, anther and filament), gynoecium (carpel, stigma, style and ovary). Note that the sepals and petals are similar, and the sepals are outermost whorl.
Cut a cross section of the ovary and use a needle to dig the ovules from one locule. Note their point of attachment or placenta (plural: placentae). Make a drawing of a cross section of an ovary and label the following structures: ovary wall, ovule, placentae.
The seed develops from the ovule and contains the embryo and endosperm. A fruit is a mature ripened ovary plus any associated tissues and thus contains the seeds. The fruit provides protection for the seed and, since fruits serve as food for many organisms, may also be involved in seed dispersal. Fruits may be either dry or fleshy. Dry fruits are surrounded by a tough, hard wall and often split at maturity to release their seeds.
Use the following key to identify the representatives of some of the common types of fruits.
Artificial Key to Fruits (Question: Is this a dichotomous key?)
(The term receptacle, seen below, refers to sterile tissue that grows upward from the base of the flower and surrounds the ovary. Often, the receptacle will become fleshy and contribute to the majority of the fruit flesh, whereas the ovary wall will contribute little to the flesh.)
1a. Fruit fleshy at maturity
2a. Fruit simple (develop from flower with single pistil)
3a. Flesh derived primarily from receptacle……………………………… pome
3b. Flesh derived primarily from ovary
4a. Fruit with single seed enclosed in hard pit……………………….. drupe
4b. Fruit with more than one seed; seed not enclosed in hard pit (berries)
5a. Fruit with thin skin……………………………………………. true berry
5b. Fruit with leathery skin containing oils, or a rind
6a. Skin leathery, easily peeled………………………………. hesperidium
6b. Skin with rind, not easily peeled…………………………. pepo
2b. Fruit compound (develop from flower with more than one ovary or from more than one flower)
7a. Fruit derived from single flower with more than one ovary…………. aggregate fruit
7b. Fruit develops from cluster of flowers……………………………….. multiple fruit
1b. Fruit dry at maturity
8a. Fruit compound…………………………………………………………… multiple fruit
8b. Fruit simple
9a. Fruit splits at maturity
10a. Fruit splits along one carpel edge only…………………………. follicle
10b. Fruit splits along two carpel edges
11a. Fruit with one chamber, seeds attached to carpel edges…….. legume
11b. Fruit with two chambers, seeds attached to angle between partition and ovary wall
12a. At least 4X as long as wide……………………………… silique
12b. Less than 4X as long as wide……………………………. silicle
10c. Fruit splits variously, but not along two carpel edges………….. capsule
9b. Fruit does not split at maturity
13a. Fruit without wings and seed and fruit walls tightly fused……… caryopsis
13b. Fruit with wings, or fruits with seeds loosely attached to fruit wall
14a. Fruit wing formed from outer tissues……………………….. samara
14b. Fruit without wing
15a. Fruit with a heavy, tough wall not easily broken…… nut
15b. Fruit with a thin wall, easily broken………………… achene
| |
|Identify the following |
|fruits: |
|A |(17) |
|B |(18) |
|C |(19) |
|D |(20) |
|E |(21) |
|Fruit |Example |
|Pome |Apple, pear, quince |
|Drupe |Peach, apricot, plum, cherry, coconut, olive, almond, walnut |
|True berry |Blueberry, cranberry, tomato, grape, eggplant, pepper, banana |
|Hesperidium |Orange, tangerine, lime, lemon, kumquat |
|Pepo |Pumpkin, squash, melon, cucumber, gourd |
|Aggregate fruit |Strawberry, raspberry, blackberry, rose hips |
|Multiple fruit |Mulberry, pineapple, Osage orange, fig, Liquidambar “gumball” |
|Follicle |Milkweed, larkspur, Magnolia |
|Legume |Pea, bean, soybean, peanut |
|Silique; silicle |Brassicaceae (Mustards) - Cabbage, broccoli, radish, shepherd’s purse |
|Capsule |Iris, lily, orchid, snapdragon, poppy |
|Caryopsis (grain) |Poaceae (Grasses) - Corn, wheat, barley, rye, oat, rice, sugarcane, bamboo |
|Nut |Chestnut, filbert (hazelnut), acorn (Note: walnut, almond, coconut, peanut are not nuts) |
|Achene |Sunflower, buttercup, buckwheat, Mountain Mahogany |
|Samara |Maple, elm, ash |
Lab 8 – Plant Anatomy
by M. Brunell
Before coming to lab you should:
• Review the material from last lab.
• Be able to define the bold-faced terms in these lab exercises.
• Know the major types of plant tissues and the general derivatives and functions of each.
• Know the differences between primary and secondary growth and apical and lateral meristem.
• Know the major functions of xylem, phloem and vascular cambium.
After completing the lab exercise you should be able to:
• Be able to recognize, name and identify all the bold-faced structures in the exercise.
• Be able to recognize, name and identify all the major structures in sections of monocot and eudicot stems and roots.
• Be able to recognize, name and identify all the major structures in sections of wood.
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings. SF, Boston, NY
• Van De Graaff, Kent M., and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ. Co., Englewood, CO
A vascular plant consists of a root system and a shoot system. The shoot system, which is usually above ground, consists of the stems, leaves, and reproductive organs. The leaves are the primary centers of photosynthesis, whereas the stem serves as a framework for positioning the leaves and as a means of conducting materials throughout the body. Cones, flowers, and fruits have evolved from leaves and are thus part of the shoot system. The root system, which is usually below ground, anchors the plant and penetrates the soil where it absorbs water and other nutrients.
All plants start out life with an herbaceous body, that is, they are soft and flexible, and many plants live out their lives this way. Other species, however, will produce stiff, hard, woody tissue later in life. The herbaceous part of the plant body is termed the primary plant body, whereas the woody part is termed the secondary plant body. The tissues making up the primary plant body are all derived from the dividing cells of the apical meristem, an embryonic region that exists at the tip of each shoot and root axis. Primary growth, resulting from the proliferation of apical meristem cells, increases the length of stems and roots. The tissues making up the secondary plant body are all derived from meristems called lateral meristems, of which the vascular cambium produces the wood tissue and the cork cambium produces the periderm (bark). Secondary growth, resulting from the proliferation of lateral meristem cells, adds width to stems and roots.
Whether tissues are primary or secondary, all tissues can be classified into three tissue systems: dermal, ground, and vascular. The dermal tissue forms the epidermis, which is the outer protective layer of the plant. Ground tissue consists mainly of parenchyma cells, functioning in storage, photosynthesis and secretion. Vascular tissue includes two types of conducting tissue: xylem, which conducts water and dissolved minerals upwards from the roots; and phloem which carries food materials in the form of carbohydrates, hormones, amino acids and other substances necessary for growth.
|Tissue System |Function |
|Dermal |Primary growth (from apical meristem): forms the epidermis, which is the outer protective layer of|
| |the plants; also forms guard cells of stomata |
| |Secondary growth (from lateral meristem): this system is generally not represented in secondary |
| |tissues |
|Ground |Primary growth (from apical meristem): consists mainly of parenchyma cells, functioning in |
| |storage, photosynthesis and secretion |
| |Secondary growth: (from lateral meristem): contributes to the formation of the periderm (bark), |
| |which protects and waterproofs the plant body |
|Vascular |Xylem tissue |Primary growth (from apical meristem): thin-walled, easily crushed tissue that conducts water and |
| | |dissolved minerals upwards from the roots |
| | |Secondary growth (from lateral meristem): heavy-walled, rigid tissue that can either conduct water|
| | |and minerals (sapwood) or be non-functional (heartwood); forms major structural support to plant |
| | |body |
| |Phloem tissue |Primary and secondary growth: thin-walled, easily crushed tissue that carries food materials in |
| | |the form of carbohydrates, hormones, amino acids and other substances necessary or growth |
Cross-section of Eudicot Stem (Helianthus, Sunflower).
At low power (100x), observe a prepared slide of Helianthus stem and distinguish the pith, the central region of thin-walled (parenchyma) cells, the vascular bundles, cortex and epidermis. Note that the ring of vascular bundles separates the interior pith from the exterior cortex. In a vascular bundle, differentiate an outer region of red-stained sclerenchyma (a type of lignin-rich ground tissue that supports the stem), an inner region of red-stained xylem, a region of green-stained, thin-walled phloem cells (sieve tubes and companion cells), and the vascular cambium (use high power for cambium) between the xylem and phloem. The vascular cambium may have the appearance of a distorted line or blurry area. The thick-walled sclerenchyma cells provide mechanical support for the stem, the phloem carries dissolved food material internally through the plant and the xylem carries water and dissolved minerals from the soil into all parts of the plant. The vascular cambium is a layer of cells between the xylem and phloem that divides to produce secondary xylem and phloem, thus it is a lateral meristem. In some species the vascular cambium may extend out and meet between the vascular bundles to form a complete ring.
Diagram a section of the sunflower stem, and label: pith, vascular bundle xylem, cambium, phloem, sclerenchyma, cortex, epidermis.
Cross-section of Monocotyledon Stem - Corn
Remembering that monocots lack a vascular cambium, examine a cross-section of corn stem at 100x and look for the distribution of vascular bundles. Notice also that the ground tissue is not separated into cortex and pith, but exists as a general region of ground tissue in which the vascular tissues are embedded.
Notice the structure of the vascular bundles. Each is surrounded by a fiber sheath, and consists of a distinct phloem region and a larger xylem region. Located to the epidermis-side of the bundle is the phloem, which appears as an oval mass of small, thin-walled cells. Examine some phloem tissue in detail. The food conduction in phloem is carried out through specialized cells called sieve-tube members. These are elongated cells with perforated sieve plates at either end and are joined end to end to form sieve tubes. Each sieve-tube member is associated with an adjacent parenchyma cell called a companion cell. The companion cell apparently carries out some of the metabolic needs for the sieve-tube member and its cytoplasm is connected to that of the sieve-tube member by numerous small holes (plasmodesmata) in their cell walls.
Further inward you will find the xylem, which possesses usually two or three extremely large cells and numerous smaller cells. The innermost xylem has been torn apart by stem elongation and changes in stem shape, resulting in the formation of an air space (protoxylem lacuna) at the innermost position in the vascular bundle. It is often said that the maize vascular bundle looks like a monkey’s face (air space the mouth, large xylem cells the eyes, and phloem the forehead).
Make a drawing of a monocot stem at 100x showing the distribution of vascular bundles and label: epidermis, ground parenchyma, vascular bundles. Increase power to 400x and draw a single vascular bundle and label: fiber sheath, phloem, xylem, protoxylem lacuna. Then, make a drawing of several phloem cells, labeling: sieve-tube member (note: cytoplasm present, therefore darker than a xylem cell), sieve plate (if seen), companion cell (some show nuclei).
Questions 1-3
Secondary Growth – cross-section of eudicot stem (Tilia - lime linden, basswood)
As we saw above, the vascular bundles of a eudicot form a ring within the stem, and the vascular cambium is a tissue layer between the phloem and the xylem. The vascular cambium lays down new secondary tissue on its inner (xylem) and outer (phloem) sides. Xylem tissue laid down by the vascular cambium is termed secondary xylem, whereas the phloem tissue is termed secondary phloem. The vascular cambium is the boundary between the wood (xylem) and the bark of the stem. In addition to the phloem, bark consists of the remaining cortex and a second cambium layer, the cork cambium. Cork cambium, like the vascular cambium, lays down cells both on the inside and the outside of itself. The outer of these layers develops into thick-walled cork cells that are rich in suberin, a waxy material that serves to waterproof the cell wall and retard water loss from the stem.
View a Tilia stem cross-section slide at 100x, noticing the lines of lighter cells radiating out from the center. These lines are termed rays and are sets of parenchyma cells produced by the vascular cambium that remain alive for several years and carry water and dissolved nutrients between the secondary xylem and the secondary phloem. Within the xylem the ray is called a xylem ray or wood ray, whereas the ray in the phloem is termed a phloem ray.
Starting at the outermost region of the stem, notice the flattened, dark cork cells. Below this layer is the cork cambium, which is a thin line just below the innermost cork cells (very hard to see). Beneath the cork cambium is the parenchyma of the cortex. Next, look into the vascular tissues. It starts with a region of interdigitated triangle-like areas. The light-colored triangles are the phloem rays, whereas the dark triangles are the secondary phloem tissue. Within the phloem tissue notice the many heavy-walled phloem fibers. Notice that the fibers form alternating layers with thin-walled phloem cells. The phloem rays largely consist of parenchyma tissue. Below the phloem region is a dark line of compact cells representing the vascular cambium. Further in from the vascular cambium you will see the secondary xylem which contains large vessel-members and smaller tracheids. This secondary xylem is the most recently made xylem. The oldest xylem lies further inside the stem. As you progress inward, you will see an abrupt change – the xylem cells suddenly get very tiny. This sudden change produced an annual ring. The line that forms between the large-celled area and the small-celled area is where the vascular cambium spent the winter, dormant. In the following spring, it produced the outermost xylem. The xylem possessing large-diameter cells is called spring wood (also called early wood), whereas the xylem possessing small-diameter cells is called summer wood (also called late wood). Together, the spring and summer wood make up one season of growth, therefore one annual ring. In the very center of the stem you will likely see the pith, composed of parenchyma cells.
Diagram a small segment of the stem. In this drawing, represent the general regions; the only cells which you should draw are the large vessel-members. Label: cork cells, cortex, phloem fibers, secondary phloem, phloem ray, vascular cambium, vessel-members, tracheids, xylem ray, spring (early) wood, summer (late) wood, annual ring, pith.
Question 4
Cross-section of a Eudicot Root (Ranunculus (buttercup)).
Examine a cross section of a eudicot root (Ranunculus) at 100x and compare and contrast its structure with that of a eudicot stem. The outermost layer is termed the epidermis while the area internal to the epidermis is the cortex. Note the cross-shaped complex of xylem cells at the center of the stem. The phloem is not a continuous ring but exists as usually four clusters of cells located between the arms of the cross. The vascular cambium is located between the xylem and phloem. A darker-staining ring of endodermis surrounds the above complex of cells. Also notice that the endodermis is the innermost layer of cells in the cortex, and forms a line between the cortex and vascular tissues. The thin layer of cells immediately internal to the endodermis is the pericycle, a cylinder of parenchyma cells that contributes to the vascular cambium in the areas immediately outside of the tips of the xylem arms. The pericycle also contributes to the bark that forms on the woody root.
Make a diagram of the cross-section of the eudicot root and label: epidermis, cortex, endodermis, pericycle, phloem, vascular cambium, xylem.
Cross-section of a Monocot Root (Smilax (Greenbriar)).
Examine a cross-section of a monocot root (Smilax) at 100x and compare and contrast its structure with that of a monocot stem and a eudicot root. Notice the epidermis bearing a few root hairs and the thick cortex filled with starch grains (small bodies within cortex cells). Further inside will be a large circular region composed of both small and large cells. The outermost cell layer to this region is the endodermis (again, its the innermost layer of the cortex). Below the endodermis are several layers of pericycle tissue. Inside of the pericycle is a mixture of phloem and xylem, and pith in the very center of the root. Look carefully at the vascular tissues. The phloem exists as a ring of small cell clusters just beneath the pericycle. Interdigitated with the phloem clusters are the arms of the xylem tissue. Notice that the number of xylem arms is greater than in the eudicot root.
Question 5.
Each xylem arm consists of smaller cells to the outside and larger cells to the inside. To the inside of the largest xylem cells is the pith, composed of parenchyma tissue. The vascular cambium is usually absent in monocots.
Diagram a cross-section of a monocot root and label: epidermis, root hairs, cortex, endodermis, pericycle, xylem, phloem, pith.
Onion Root Tip – longitudinal-section.
Examine a longitudinal section of an onion root tip (Allium) at 100x. Locate the region showing the various stages of mitosis. This zone of cell division is an apical meristem. Beyond this region toward the end of the root is a region of cells with nuclei all in interphase (not dividing). This is the root cap. Follow back from the meristematic region and note the gradual transition in cell size into the zone of elongation where the individual cells are increased in length. Another region of the root tip, termed the zone of maturation, may or may not be present on your slide. In this zone, the cells take on specialized functions and develop into their mature form. Slender epidermal cell extensions called root hairs are indicative of this zone.
Diagram an onion root tip and label the following: root cap, zone of cell division, zone of elongation, (zone of maturation/root hairs if present).
Question 6
Observe the demonstrations of radish seedlings and note the prominent root hairs.
Questions 7-8
Cross-section of the eudicot leaf (Nerium oleander (Oleander))
The leaf is an organ that attaches to the stem at the node. Between nodes is a region termed the internode. When internodes are short the leaves are densely set on the stem, whereas when the internodes are long the leaves are well spaced. Evolutionarily, the leaf is thought to represent a branch system, where several stems have oriented themselves in a plane and have become joined by webbing tissues, in a way resembling a hand with webbed fingers. The veins in the leaf represent the stems, and the tissue between the veins represents the webbing.
To gain a basic understanding of the structures within a leaf we will study a cross-section of the oleander, a plant adapted for dry climatic conditions (a xerophyte). View a slide of oleander leaf x.s. at 100x, orienting yourself to the upper and lower surfaces of the leaf. The upper epidermis has a fairly flat, thick cuticle layer and a few hairs called trichomes, whereas the lower epidermis has abundant trichomes and invaginations called stomatal crypts.
Question 9
Notice that the upper epidermis actually consists of several layers of epidermal cells – typical for a xerophyte. Below the upper epidermis you will see two very well organized layers of palisade parenchyma (palisade = “a fence of stakes”) which contains abundant chloroplasts. The palisade parenchyma comprises the palisade mesophyll layer of the leaf. You may see some big crystals here and there near the palisade parenchyma layers. Below the palisade layer is the spongy parenchyma, making up the spongy mesophyll layer of the leaf. Within this layer you will vascular bundles (veins) running here and there. Going further down toward the lower epidermis, you will see the stomatal crypts which are full of large trichomes. These stomatal crypts are pockets of lower epidermis that form cavities in which the stomata are located. Look at the lining of the crypts to find the dark-colored guard cells of the stomata. Much of the lower epidermis also has several layers of cells, and finally a cuticle covering.
At 100x, draw the entire cross-section, avoiding the midrib of the leaf, labeling all of the following structures: upper epidermis, cuticle, lower epidermis, trichomes, stomatal crypts, palisade parenchyma, palisade mesophyll, spongy parenchyma, spongy mesophyll, vascular bundles, and guard cells.
Lab 9 – Introduction to the Animals: Cnidaria, Platyhelminthes & Annelida
By A. Moore and M. Brunell
Before coming to lab you should:
• Review the material from last lab.
• Be able to define all the bold-faced terms in this lab exercise.
• List the characteristics of the phyla which serve to distinguish them from other phyla.
After completing the lab exercise you should be able to:
• Be able to tell whether an organism is an animal or not, just by looking at its body plan.
• Recognize, name, and state the major distinguishing characters of the various classes of cnidarians, platyhelminths and annelids.
• Recognize, name, and describe the function of all the anatomical structures exhibited in the lab exercise.
• Describe the general life cycles of the members of each phylum.
• Describe the major structural differences between the phyla and the evolutionary and adaptive significance of these differences.
Relevant sections in text:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, San Francisco, CA.
• Van de Graaf, Kent M., and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ. Co., Englewood, CO.
Domain Eukarya: ANIMALS
The myriad of different ways that animals are put together is mind boggling. Yet, just like humans, each animal has to keep the cells in its body alive and produce viable offspring in order to be represented in the living world today. The next series of labs will explore animal diversity. It will also introduce you to the system of organization that biologists use to keep track of all the similarities and differences among all these animals.
At the end of this series of labs you should be able to recognize most members of the major phyla, classes, etc. and classify them. You should know the general appearance and structure of organisms in the major phyla and classes so that you can compare and contrast their structures and distinguishing characteristics. For example, when you hear the name "sea cucumbers" you should be able to name the phylum and class to which sea cucumbers belong and to remember the major characteristics of that phylum and class and how sea cucumbers differ from other animals. You should also know what other phyla and classes are closely related to sea cucumbers and why they are regarded as being closely related, i.e., what characteristics they have in common. In addition, you should know what animals are not closely related to sea cucumbers and why they are regarded as not being closely related, i.e. how they differ from sea cucumbers.
Another aspect of our study of animal diversity will involve an examination of the major life processes of animals. For example, you will learn about the ways in which animals obtain nutrients and distribute them throughout the body. What types of animals have special organs to facilitate these processes? Under what conditions are special organs necessary? Under what conditions are no special organs necessary? If an animal does not have an organ, for instance, a heart, that is quite necessary for a human, how does that animal survive without it? How does the general principle of surface/volume ratios relate to these processes?
PHYLUM CNIDARIA
The phylum Cnidaria (Gr. knide, nettle) is a large and diverse group of more than 10,000 species, most of which are marine. The members of this phylum are characterized by the presence of radial symmetry and specialized stinging organelles called nematocysts. Cnidarians may exhibit either of two different body forms, polyp or medusa. A polyp is a tubular form, closed at the aboral end (usually attached to the substrate) and has a mouth surrounded by tentacles at the oral end (usually directed upwards). Polyps are generally sessile or very slow moving. Medusae have umbrella- or bell-shaped bodies. The mouth of the medusa (oral side) is located at the end of a central projection called the manubrium on the lower surface. Tentacles are arranged around the margin of the medusal umbrella. The general structure of a cnidarian is the same regardless of whether the oral surface is directed upwards or downwards. Cnidarians are diploblastic; the cells of the body are organized into two major tissue layers derived from embryonic ectoderm and endoderm. Cnidaria display a tissue grade of organization. That is to say, unlike the sponges, their specialized cells act collectively to perform specific tasks. For instance, the gastrodermis digests and absorbs food. There are true muscle cells arranged in bands and innervated by neurons. But there are no complete muscle organs with tendon or nerves. Being a radially symmetric animal, the cnidarian has no single side that requires more sensory structures or nervous tissue. Therefore it does not have any central nervous system. Nevertheless, cnidarian behavior is quite complex, as it must be since nearly all cnidarians are predators.
The phylum Cnidaria is composed of four classes. A major difference between the classes is which of the two body forms is present or dominant. There are also other morphological distinctions, which you will discover as the lab progresses. We will not study the Cubozoans in lab.
Supergroup Unikonta
Kingdom Animalia (Eumetazoa)
Phylum Cnidaria
Class Hydrozoa: (Gr. hydros, water + zoon, animal) hydrozoans (Hydra, Gonionemus, Obelia, Physalia, etc.); solitary or colonial animals in which the polyp stage is dominant and the medusa stage is absent or reduced; medusae (when present) with a velum (iris-like diaphragm that is used to create a powerful water jet for movement); freshwater or marine.
Class Scyphozoa: (Gr. skyphos, cup + zoon, animal) larger jellyfish (Aurelia, etc.); solitary animals in which the medusa stage is dominant and the polyp stage is reduced or absent; medusae without a velum; marine.
Class Anthozoa: (Gr. anthos, flower + zoon, animal) anemones (Metridium, etc.) and corals; solitary or colonial animals completely lacking a medusa stage; marine.
Class Hydrozoa
Using a glass dropping pipette, obtain a specimen of the common freshwater hydrozoan, Hydra, from your lab instructor and place it in depression slide containing fresh water.
Question 1
Using a dissecting microscope, observe the cluster of tentacles about the mouth at the oral end. Tap the dish or the slide and observe the amount of rate of contraction exhibited by the animal.
Question 2
Add several cladocerans (phylum Arthropoda, subphylum Crustacea, Daphnia) to the slide or dish and see if your Hydra will eat.
Question 3
Now cover your living Hydra carefully with a cover slip. Examine your Hydra under the compound microscope with reduced light and locate the wart-like cnidocytes on the tentacles. The cells contain the nematocysts, the stinging cells that are peculiar to the members of the Phylum Cnidaria. When an object contacts the trigger-like cnidocil located at the edge of the cnidocyte, the membrane permeability of the system is altered in such a way that the coiled thread-like structure within the nematocyst everts (turns inside out and extends) and the thread shoots out from the nematocyst.
On your worksheet, make a drawing of your living Hydra and indicate the relative positions of the following structures: tentacles, mouth, body stalk, oral and aboral sides.
The nematocyst can also be fired in response to chemical stimuli. While watching the Hydra through the microscope, have your lab partner add a small drop of dilute acetic acid at the side of the cover slip and draw it beneath the cover slip with a small piece of paper towel. This reaction occurs extremely quickly, so you must be watching the hydra at the instant that the acetic acid makes contact. Observe the reaction of the nematocysts as the pH changes.
Question 4
If available, view the video and observe the movement and behavior of Hydra.
Hydra can reproduce either by budding or by sexual reproduction. Examine a slide containing budding Hydra.
Question 5
Hydra has very unusual sexual structures for a cnidarian. In sexual reproduction, the male hydra produces sperm in structures known as spermaries, which are small conical outgrowths from the side of the body. The sperm are released from the male's body and travel through the water to fertilize the ova which are located in the female hydra's ovaries. The ovaries resemble spermaries, however they are more rounded in shape. The embryo develops within the female’s ovary and after 10-70 days a young, miniature hydra is released. Examine prepared slides of sexually reproducing Hydra.
On your worksheet, draw sexually reproducing Hydra and label the following structures: bud, gastrovascular cavity, ovary, and spermary.
Examine a prepared cross-section of Hydra. Note that the body wall surrounding the gastrovascular cavity is composed of only two layers--an outer epidermis (derived from ectoderm) and an inner gastrodermis (derived from endoderm) lining the gastrovascular cavity. Between the two layers is a region filled with gelatinous mesoglea. The mesoglea is an acellular and fiber-less layer that serves to support the body, functioning somewhat like an elastic skeleton.
On your worksheet, draw the cross-section of Hydra and label the following structures: gastrovascular cavity, mesoglea, ectoderm, and endoderm.
Although Hydra does not have a medusa stage, most hydrozoans do. We will examine a marine hydromedusa of the genus Gonionemus. Place a specimen in a shallow dish and examine it under a dissecting microscope. Note the thin, muscular membrane called the velum extending inward from the margin of the umbrella-like bell. The velum is found only in members of the Class Hydrozoa and will serve to distinguish hydrozoan medusae from the jelly fish in the Class Scyphozoa.
The medusa usually swims with its convex side (aboral side) upwards and with the tentacles hanging downward. Hanging down from the inside of the bell is the manubrium which contains the mouth. A gullet leads from the mouth to the gastrovascular cavity at the base of the manubrium. Canals lead from the large cavity at the base of the manubrium to the base of the bell and to smaller canals leading into the tentacles. The entire system from the gullet to the canals in the tentacles makes up the gastrovascular cavity.
Question 6
The sexes are separate in Gonionemus, as in most cnidarians, and the gonads are located along the radial canals. Eggs and sperm are simply released into the sea and fertilization occurs externally.
On your worksheet, draw a Gonionemus medusa and indicate the relative positions of the following structures: bell, velum, tentacles, manubrium, gullet, mouth, radial canal, gonad, oral surface, aboral surface.
Unlike Hydra and Gonionemus which are solitary, most hydrozoans are colonial. A common colonial hydrozoan found on both the Atlantic and Pacific coasts of North America is Obelia.
Examine a small piece of an Obelia colony under a dissecting microscope. The plant-like colony consists of numerous branches, each of which terminates in one of two kinds of polyps. A feeding polyp, called a hydranth, will have tentacles and somewhat resemble Hydra. A reproductive polyp, called a gonangium, is somewhat club-shaped and lacks tentacles. The entire colony, including all the branches, is covered by a transparent, non-cellular sheath called the perisarc. The portion of the perisarc that surrounds the hydranth is the hydrotheca, while that which surround the gonangium is called the gonotheca. The hydrocaulus, the stem that bears the polyps, is a hollow tube composed of a cellular body wall which surrounds the gastrovascular cavity. As is typical of colonial cnidarians, the single gastrovascular cavity extends throughout the entire colony.
Study a prepared slide of a whole mount of an Obelia colony under the dissecting microscope. On your worksheet, draw the Obelia colony and indicate the relationship of the following parts: hydrocaulus, perisarc, gastrovascular cavity, hydranth, hydrotheca, gonangium, gonotheca. Identify a single polyp and its oral and aboral ends.
Reproduction in Obelia involves the budding off of medusae from the gonangia and the release of these medusae into the water. The medusa then reproduces sexually and the zygote develops into a swimming ciliated larva called a planula. The planula fastens itself to a surface, then develops first into a polyp and eventually into a complete colony. Examine a prepared slide of the free-swimming medusa of Obelia. On your worksheet, draw the medusa and label the following structures: tentacles, manubrium.
Question 7
Examine a demonstration specimen of the floating colonial hydrozoan Physalia, sometimes called the Portuguese man-o-war. This "animal" is actually a colony of polyps like Obelia. In Physalia, some of the polyps have taken on different body shapes to better specialize in one function for the colony. In addition to feeding polyps and reproductive polyps, this colony also has defensive polyps and one giant, gas-filled polyp that keeps the entire colony afloat on the high seas. Note the long tentacles (the defensive polyps) covered with nematocysts that serve to catch prey as well as deter predators. These tentacles can catch and kill fish as big as Physalia's float. A full grown Physalia float can be two to four feet long.
Class Scyphozoa
The Class Scyphozoa includes the so-called "true jellyfish", in which the polypoid (= shaped like a polyp) stage is minute or lacking. Scyphozoan medusae can be more than 2 m in diameter, although most are considerably smaller. A few scyphozoans, such as Cyanea, grow tentacles that are as long as blue whales! How do they do this when they are only two cell layers thick? Let's just say it involves lots and lots of mesoglea.
Study a specimen of Aurelia, one of the common coastal jellyfish. Compare the structure of this scyphomedusa with that of the hydromedusa examined earlier. Remember, look for what is similar, because they are both cnidarians, and what is different, because they are in different classes. On your worksheet, draw the Aurelia medusa and label the following structures: gastrovascular cavity, velum, mouth, manubrium, and gonads.
Question 8
Study the demonstration of reproduction in Aurelia. The medusa reproduces sexually giving rise to a planula larva which forms a small polyp. The polyp, in turn, reproduces asexually and forms immature medusae termed ephyrae, which later develop into mature medusae.
Class Anthozoa
Members of the Class Anthozoa, the corals and sea anemones, have only the polyp stage and have a flower-like appearance. They can be distinguished from polyps in other cnidarian classes because their gastrovascular cavity is partitioned by mesenteries or septa (sing. septum) that are inward extensions of the body wall.
Examine the structure of dissected specimens of the sea anemone Metridium. Compare and contrast the structure of Metridium with that of the hydrozoan polyps (e.g., Hydra) studied previously. Again, look for similarities, because they are in the same phylum, and differences, because they are in different classes. Use the similarities you have found to fill in the table at the beginning of your worksheet. Remember that you should observe the character in all three classes in order for it to be a shared character for the whole phylum.
Question 9
In groups of four, observe a living sea anemone. As you observe, remember the tissue grade organization that this animal is made of. It has several bands of longitudinal muscles and several bands of circumferential muscles along with a non-centralized nerve net. Try to figure out which of these muscles and neurons have to work in order to achieve the motions that you see. First of all, get oriented. Identify the mouth, oral disk, pedal disk, body column and tentacles.
Question 10
Take a small piece of krill and touch it to one of your anemone’s tentacles. What happens?
Question 11
There are several types of corals in the Class Anthozoa, all of which produce an internal or an external skeleton of calcium carbonate. The stony corals produce an external skeleton and occur in large colonies, eventually giving rise to coral reefs. At night, the tentacles of the polyp extend out through openings in the external skeleton and capture suspended food materials from the water. Examine the demonstrations of coral skeletons. Choose one skeleton and identify where the polyp went when the coral was alive.
Question 12
INTRODUCTION TO WORMS
The animals that you will see in the next exercise are worms (Greek root helminth, Latin root vermis). Any animal that is longer than it is wide and has no obvious appendages can be called a worm, so the word "worm" doesn't narrow the description by very much. The worm shape has its advantages, as appendages tend to stick out from the body, and get stuck when the animal is squeezing through narrow burrows and passageways. A straight, long, bendable body is an excellent shape for digging through sediment or compost or even living flesh. Many (but not all) of the worms you will see today live in just these habitats. Today you will study 2 of the 17 worm phyla that exist.
PHYLUM PLATYHELMINTHES
The members of this phylum can be recognized by their dorsoventrally flattened body and are thus given the common name of flatworms. They are all triploblastic, that is, a third major tissue layer, mesoderm, develops between the embryological ectoderm and endoderm. Flatworms have a recognizable head and tail and are bilaterally symmetrical. Like the sponges and members of the phylum Cnidaria, flatworms lack a body cavity between the gut and the outer body wall and are therefore termed acoelomates. They have well-developed nervous, excretory, muscular, and digestive systems. We say that they have the organ-system level of complexity. As in the cnidarians, the digestive tract is called incomplete in that it has only one entrance, a mouth/anus, instead of having a mouth at one end of the tract and an anus at the other. Some of the parasitic flatworms are particularly specialized for living within the gut of their host and absorb their nutrients when they have been already digested by their host; these flatworms completely lack a digestive tract.
The phylum Platyhelminthes (Gr. platys, flat + helminthes, worm) is commonly divided into four classes. You will explore three of these classes in lab today:
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Lophotrochozoa)
Phylum Platyhelminthes
Class Turbellaria: (L. turba, crowd, stir [currents produced by cilia]) free-living (= not parasitic) flatworms (Planaria); mouth usually ventral; body undivided; development usually direct (= no larval stage present).
Class Trematoda: (Gr. trematos, hole) flukes (Clonorchis), etc.; parasitic; mouth usually anterior; body undivided; one or more suckers for attachment within their host.
Class Cestoda: (Gr. kestos, girdle) tapeworms (Taenia); parasitic; digestive tract absent in adult; body usually divided into segments (called proglottids), each of which contain a complete reproductive system; usually with anterior scolex for attachment.
Class Turbellaria
The free-living flatworms are best represented by the small freshwater animals called planarians. Place a living planarian in a dish of fresh water and examine it under the dissecting microscope, taking care to keep the animal moist.
Planaria move by a ciliary-mucus mechanism. Their ventral side is covered in cilia. They also have small glands that secrete a mucus lubricant to help the worm glide smoothly along the substratum.
Questions 13 & 14
Now, let's take a closer look at the planarian body plan. Examine prepared cross-section slides of stained planarians. The first thing you need to do is to get oriented. Try to relate the section you see in the slide to the planarian you were just watching in your dish. Where is dorsal? Where is ventral? As you look at this section, try to figure out how far back or forward in the worm it was taken - that is, how far along the anterio-posterior axis are you looking at?
Now that you know which way is up, look for the following items in your section: The epidermis (derived from ectoderm) covering the outside of the body is composed of cuboidal cells and interspersed unicellular glands secreting mucus or adhesives.
Next, locate the two layers of muscle (derived from mesoderm) tissue immediately beneath the epidermis.
Questions 15 & 16
The large columnar cells (which may not be distinguishable) that line the gut are collectively called the gastrodermis and are derived from endoderm. The tissue between the gastrodermis and the muscle layers is filled with parenchyma (cells derived from mesoderm). (The open spaces seen in the parenchyma are artifacts that develop when the tissue rips while it is being sectioned. These spaces should not be confused with a coelom.)
On your worksheet, draw a cross-section of a planarian showing the relative position of the following structures or areas: dorsal and ventral sides, epidermis, gastrodermis, muscle layers, parenchyma, gut.
Class Trematoda 650-504-7482
All the members of this class are parasites and most inhabit the body of a vertebrate during some stage of their life cycle. Flukes have at least one sucker, usually surrounding their mouth, which keeps them from losing their position within their host. They also have a tough outer epidermis that keeps them from being digested while in the gut of their host. Many species of flukes have larval stages that live in one or more species of intermediate host and an adult stage that lives in a different species (termed the definitive host). (How would one decide which one of these stages is the adult stage?)
Examine a prepared slide of the liver fluke Clonorchis sinensis. (Since these worms can infect humans, there will be no living specimens in the lab.) Locate the oral sucker surrounding the mouth at the anterior end of the body. Locate the ventral sucker posterior to the mouth. A muscular pharynx and a short esophagus lead to a two-branched intestine.
The conspicuous dark-staining structure spreading through most of the center of the animal's body just posterior to the ventral sucker is the uterus. The single ovary is near the midline of the body and just posterior to the uterus. Ova pass from the ovary, through the uterus and exit through a small genital pore just anterior to the ventral sucker. The testes spread throughout most of the posterior one-fourth of the body posterior to the ovary. Ducts lead from the testes to the genital pore. Even though they are hermaphrodites, most trematodes are cross-fertilizing so that the sperm from one animal fertilizes the ova of another.
On your worksheet, draw a fluke in ventral view showing the relative positions of the following structures: anterior and posterior ends, oral sucker, mouth, ventral sucker, testes, ovary, genital pore, and uterus.
Examine the demonstration materials of flukes paying particular attention to the variation and complexity seen in the life cycles of the various animals.
Compare the two different classes of Platyhelminthes, so that, if you are shown a new and different flat worm, you'll be able to tell whether it is a turbellarian or a trematode.
Class Cestoda
The members of the class Cestoda are called tapeworms and are all internal parasites. The adult lives in the digestive tract of some vertebrate (the definitive host) and the larva lives in the tissues of some other animal (the intermediate host). The definitive host feeds on the intermediate host, and thereby ingests the larvae of the tapeworm. The pig tapeworm (Taenia solium), for example, has a pig as its intermediate host and a human as its definitive host.
Examine the demonstration specimens of tapeworms. Note the knob-like scolex at the anterior end of the body which bears suckers and hooks.
Question 17
The body of the tapeworm is made up of a series of segments called proglottids, which form by transverse budding in the neck region. The young proglottids contain a full compliment of organs of excretion (flame cells), nervous system (longitudinal nerve cords), and reproduction. Because each proglottid is self-contained, a tape worm is very similar to a colony of individual flatworms that live attached in a line.
As the proglottids age, everything except the reproductive system degenerates and they become little more than sacs of sex organs. The male reproductive organs mature first, then the female organs. Since new proglottids are produced at the neck region, the farther away you move from the neck, the older is the proglottid. So, hermaphroditic proglottids are found most posteriorly, males in the middle and proglottids with all their vegetative (non-reproductive) organs are nearer to the neck. The ova in hermaphroditic proglottids are fertilized by sperm from some other tapeworm or from a proglottid located farther anteriorly in the same worm. Thus self-fertilization and cross-fertilization are both possible. The proglottids eventually break off and are released from the host's digestive tract with the feces. At this point the proglottid breaks up and the eggs are released. Some of the fertilized eggs are eaten by the intermediate host and the hatched larva burrows through the intestinal wall and travels by way of the lymphatic and blood vessels to skeletal muscle. The larva then forms a cyst in the skeletal muscle and remains there until it is eaten by the definitive host.
Examine some stained and mounted proglottids under magnification. The scattered, dot-like structures are the testes whose sperm is carried by a sperm duct to the genital pore near the center of the proglottid. The ovary is a branched lobate mass near the center of the proglottid. The eggs are fertilized by sperm which reaches them by way of the vagina which leads from the genital pore.
On your worksheet, draw the general structure of a cestode, showing the relationships of the following parts: anterior and posterior ends, scolex, suckers, hooks, testes, sperm duct, ovary, vagina, and genital pore.
Question 18
What are the similarities and what are the differences between cestodes and turbellarians? Again, look for similarities, because they are in the same phylum, and differences, because they are in different classes. Use the similarities you have found to fill in the table at the beginning of the worksheet. Remember that you should observe the character in all three classes in order for it to be a shared character for the whole phylum.
Question 19
PHYLUM ANNELIDA
The members of the phylum Annelida (L. annelus, little ring) are commonly referred to as segmented worms. This reflects the fact that the animals' bodies are divided both internally and externally into similar segments or metameres that are arranged in linear order. The margins of the segments are often marked externally by small grooves (annuli). This segmentation or metamerism is also seen in the repetitive arrangement of organs and organ systems and in the internal partitioning off of the segments by septa (internal walls). The annelids are bilaterally symmetrical, triploblastic protostomes with complete digestive tracts and closed circulatory systems. With the exception of the members of the class Hirudinea, most annelids bear small chitinous bristles called setae that function in locomotion or in holding the animal in place.
The phylum Annelida is commonly divided into three classes:
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Lophotrochozoa)
Phylum Annelida
Class Polychaeta: (Gr. poly, many + chaeta, hair) polychaetes, tubeworms, etc.; head distinct and bearing eyes and tentacles; most segments with parapodia bearing setae; clitellum absent.
Class Oligochaeta: (Gr. oligos, few + chaete, hair) earthworms, night crawlers (Lumbricus), etc.; head inconspicuous; setae present; parapodia absent; clitellum present.
Class Hirudinea: (L. hirudo, leech) leeches; head inconspicuous; parapodia and setae absent; clitellum present; anterior and posterior suckers usually present.
Class Oligochaeta
Get a live nightcrawler (Lumbricus terrestris) from the front of the class. Be sure to keep it moist and cool. (The heat from your hand can put it into heat shock, so don't hold it in the palm of your hand for too long. Also, squirt it with fresh water from time to time to keep it from drying out.) Let it crawl around on the table or in the dish provided.
Question 20
Examine a preserved specimen of Lumbricus terrestris, the night crawler, taking care not to let it dry out. About one-third of the way back from the anterior end is a thick cylindrical collar called the clitellum. The structure secretes the cocoon that will protect the eggs until hatching.
At the extreme anterior end of the body one finds a small, fleshy projection called the prostomium which overhangs the mouth. (The prostomium is not regarded as a separate segment.) At the very posterior end of the body is a vertical slit termed the anus. Note that there is no tail since the anus is terminal.
Several small openings should be visible on your specimen with a dissecting microscope. A pair of rather large and conspicuous male genital pores are located laterally on the ventral side of segment number XV, while a pair of smaller female genital pores are located on the ventral surface of segment XIV. The small paired openings of the seminal receptacles are located between segments IX and X and between X and XI. (The location of the above openings varies with respect to segment numbers from one species to another and the description applies only to L. terrestris.) Even if you can’t find the pores on your worm, you should know where to expect them. If you are having trouble finding the pores, try the living worm...they should be easier to see. Oligochaetes are hermaphroditic, but not self-fertilizing.
Question 21
Cut across your specimen about three or four cm posterior to the clitellum. Save all pieces of your worm until the end of the exercise. Keep the worm moist with wet toweling.
From the posterior part of your worm cut a piece containing two or three segments and examine both the anterior and the posterior ends of the segments. Remove a second set of segments and section it dorsoventrally along the midline of the body (sagittal section). The body of the worm is essentially a tube-within-a-tube, the gut within the body wall. The space between the tubes (the coelom) contains the various organs and is divided into compartments by the septa. Locate the fold of tissue called the typhlosole which extends down into the gut from its dorsal side.
Annelids have a lot of internal body parts that are easy to destroy by cutting too deeply. You are about to make a longitudinal incision in the anterior half of your Lumbricus. Examine an anatomical model or a demonstration dissection of the anterior portion of a worm before you start your own dissection. Get an idea of where the body parts are so that you can avoid them as you cut. Use the instructions below to open up the front half of your worm:
1) Along one side of a dissecting tray, place your specimen dorsal side up. At the posterior end, put two pins through only the ventral body wall. You may want to pin the anterior end with one pin. Try to avoid the mouth and other external features.
2) Identify where the dorsal blood vessel is so that you can avoid it. You can see it from the open cross-section on the posterior end. You can follow it to the anterior part of the worm by finding the dark line visible through the dorsal body wall.
3) Fill the dissecting tray with just enough water that the worm is completely submerged. Now take a blunt probe and gently insert it along one side of the dorsal blood vessel at the posterior end of your half worm. Hold the blunt probe so that it pulls upward on the body wall just a little. Make sure you haven't trapped any internal organs between the blunt probe and the body wall.
4) Being careful to avoid damaging the dorsal blood vessel, cut the section of body wall that's held up by the blunt probe.
5) Use a dissecting needle to release the thin, transparent septa from their attachments to the digestive tract. Be careful to avoid all internal organs as you do this.
6) Go back and forth between the blunt probe and the dissecting needle to open the worm as you move anteriorly.
7) As the slit widens, pin the sides to the dissecting tray so that the internal organs become visible.
8) Gently wash away any debris around the organs with water and keep the specimen flooded with water while you are working on it.
Trace the digestive tract or gut, taking care not to damage the other organs that are in close contact with it. Begin with the mouth lying just beneath the prostomium and follow the tract posteriorly to the expanded pharynx. The pharynx, in turn, leads to a narrow esophagus (which is partially hidden by the whitish seminal vesicles). The esophagus extends through segments VI through XIV. It leads to the large, thin-walled crop, which connects to the thick-walled gizzard located approximately in segments XVII and XVIII. The remainder of the gut is the intestine, which leads eventually to the anus.
Question 22
A number of other organs can be seen as one looks at the various parts of the gut. The seminal vesicles were already seen at the margin of the esophagus. They connect with the male genital pores in segment XV, but you will not be able to see this. Just anterior to the seminal vesicles are the seminal receptacles, which are smaller and rounder than the vesicles. There should be two seminal receptacles on each side of the gut. The "brain" of the earthworm is represented by a pair of whitish cerebral ganglia just dorsal to the anterior end of the pharynx. In order to see this you need a really good dissection. Circumpharyngeal connectives lead ventrally and meet at the subpharyngeal ganglion just beneath the pharynx. Again, these structures are difficult to see. Ask the instructor for help if necessary. The ventral nerve cord extends posteriorly from this point. (The ventral nerve cord may be easier to see later on in your dissection so do not be too concerned with searching for it just yet.) The ventral nerve cord looks single, but is actually two paired nerve cords fused together.
The dorsal blood vessel, located at the mid-dorsal line above the gut, connects anteriorly with five pairs of pumping vessels (sometimes called hearts or aortic arches) extending on either side of the esophagus and partially hidden by the septa. These pumping vessels connect beneath the esophagus to form a ventral blood vessel. Look carefully for a pair of small, white, coiled tubes lying ventrolaterally in each segment. These tubes are the nephridia which filter waste from the coelomic fluid and transfer it to the nephridiopores leading to the body surface. The nephridia and nephridiopores are very difficult to see.
On your worksheet, draw a dorsal view of a dissected earthworm showing the relative position of each of the following structures: anterior and posterior ends, mouth, pharynx, esophagus, crop, gizzard, intestine, anus, genital pores, dorsal blood vessel, pumping vessels, ventral blood vessel.
Examine a prepared slide of the cross-section of an earthworm posterior to the clitellum and note the number of layers in the body wall. As usual with cross-sections, get oriented first. Figure out which is dorsal and which is ventral. (How can you tell with an earthworm?) The outermost layer, the cuticle, is quite thin and composed of thin, grayish, acellular material secreted by the epidermis. The epidermis is composed of a complex of columnar cells (plus some others) and lies deep to the cuticle. Beneath the epidermis lie two layers of muscle, a thin layer of circular muscles that appear rather fibrous and a thicker layer of longitudinal muscles that look somewhat feathery.
Questions 23 - 25
Locate the dorsal and ventral blood vessels and the ventral nerve cord on your slide. Depending upon the quality of the slide, you may also be able to see nephridia in the coelomic cavity lateral to the gut. The dorsal fold that you see extending into the cavity of the intestine is the typhlosole.
Question 26
On your worksheet, draw a cross-section of an earthworm posterior to the clitellum and indicate the relative position of the following structures: cuticle, epidermis, longitudinal and circular muscle layers; intestine, typhlosole, dorsal and ventral blood vessels, ventral nerve cord
Class Polychaeta
The polychaetes have the greatest number of species of all the annelid classes. Most of them are marine and live in burrows, under rocks, in sand or mud, etc. The "feather-duster" types of tube worms are also in this class. Examine the representative specimens of polychaetes living in our aquaria. Since many polychaetes will hide when they are disturbed, it may be easier to examine the body parts of preserved specimens. Note the development of the head and the fact that the setae are always located on lateral, paddle-like structures called parapodia.
Class Hirudinea
Leeches are rather advanced annelids. Most live in freshwater, while a few are marine and some tropical forms are terrestrial. They are predators feeding upon the blood of various other animals or scavengers feeding upon detritus. Examine the demonstration specimens of leeches on display. Note the anterior and posterior suckers, used for attachment to prey or to the substrate. The mouth is located in the anterior sucker and may or may not contain teeth.
What is similar and what is different between earthworms, polychaetes and leeches? Again, look for similarities, because they are in the same phylum, and differences, because they are in different classes. Use the similarities you have found to fill in the table at the beginning of the worksheet. Remember that you should observe the character in all three classes in order for it to be a shared character for the whole phylum.
Question 27
Lab 10 – Mollusca and Arthropoda
by
A. Moore and M. Brunell
Before coming to lab you should:
• Review the material from last week's lab.
• Be able to define all the bold-faced terms in the lab exercise.
• List the characteristics of the phyla that serve to distinguish them from other phyla.
• Explain how the members of the phyla relate to each other and to other phyla studied previously.
• Complete the tables in the exercises that describe the life processes exhibited by the members of the phyla Mollusca and Arthropoda.
After completing the lab exercise you should be able to:
• Recognize, name, and be able to list the major distinguishing characters of the members of the various classes of mollusks and arthropods.
• Recognize, name, and describe the function of the major anatomical structures exhibited in the lab exercise.
• Describe the general life cycles of the members of each phylum.
• Describe the major structural differences between the phyla and the evolutionary and adaptive significance of these differences.
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, San Francisco, CA.
• Van de Graaf, Kent M., and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ Co., Englewood, CO
PHYLUM MOLLUSCA
The phylum Mollusca (L. molluscus, soft) is an extremely diverse group of more than 50,000 named living species in addition to some 35,000 extinct forms. Molluscs range in size from submicroscopic to 250 kg and include some of the most sluggish animals and some of the most active and swiftest.
The members of the phylum Mollusca are bilaterally symmetrical, triploblastic, protostomes with complete digestive tracts and open circulatory systems (except for the cephalopods). They are characterized by the presence of a foot, a mantle, and a radula. The foot is a specialization of the ventral body wall and generally functions in locomotion, although it is variously modified in different groups. The mantle is derived from the dorsal body wall and encloses a mantle cavity and also secretes the shell. The radula is a specialized feeding organ located in the mouth of some (but not all) molluscs. You should be able to find each of these three molluscan organs in every mollusc you see today.
The phylum Mollusca is composed of many classes, but we will only examine four of them in this course:
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Lophotrochozoa)
Phylum Mollusca
Class Polyplacophora: (Gr. poly, many + placo, plate + phora, bearer) chitons; shell composed of eight dorsal plates; body flattened dorsoventrally, head reduced.
Class Gastropoda: (Gr. gastro, stomach + podos, foot) snails, slugs, limpets, nudibranchs, etc.; body asymmetrical; viscera usually in a coiled shell (which may be reduced or absent).
Class Bivalvia: (Gr. bi, two + valva, doors) clams, oysters, mussels, scallops, etc.; shell of two lateral halves that are hinged dorsally; radula absent.
Class Cephalopoda: (Gr. kephalo, head + podos, foot) squids, octopuses, nautili, etc.; head well-developed with large, conspicuous eyes; foot modified as tentacles.
Class Polyplacophora
Polyplacophorans have the common name chitons. They are the only molluscan class that you will see today whose body has not been twisted or bent throughout its evolutionary history. For that reason, it is a good mollusc on which to get oriented and to identify the typical molluscan body parts. The chitons are all dorsoventrally flattened and have a dorsal surface that bears eight articulating plates. A broad, flat, muscular foot covers most of the ventral surface. They are sedentary animals and feed upon algae which they scrape off the rocks with their radulae.
Examine the demonstration specimens of chitons. Choose one and identify its anterior and posterior ends. Find the following external organs: head, mouth, foot, mantle, mantle cavity, anus, gills and shell. How many plates of the shell can you see in the specimen you chose? Remember this basic body plan as you tour the bodies of other molluscan classes.
On your worksheet, draw the ventral side of a Polyplacophoran. Identify anterior and posterior ends as well as left and right. Label the following structures: head, mouth, foot, mantle, mantle cavity, anus, gills and shell (in some species, the shell may not be visible from the ventral side).
Class Bivalvia
In a bivalve, figuring out which way is up is one of the most difficult tasks because all bivalves have lost their heads. However, it is absolutely imperative that you figure out the three body axes BEFORE you start cutting your clam, or else all will be lost (literally). Examine a preserved bivalve, noting that the valves (the two halves of the shell) are located laterally and that the hinge is dorsal. Locate the dark, elastic hinge ligament on the dorsal margin of the valves that join them together. Toward the anterior end of each valve is a protuberance called the umbo. The umbo generally points in the anterior direction. The concentric lines that radiate from the umbo are called lines of growth. Determine the anterio-posterior axis, the dorsoventral axis and the left-right axis. Confirm these directions with your instructor before you go on.
Question 1
Now you are ready to get to the meat of the matter: Carefully, insert a scalpel beneath the right valve and separate it from the sheet-like mantle in its inner surface. Also cut the adductor muscles attached to the right valve, but do not remove the valve YET. Press the ventral margins of the valve together and release them.
Question 2
Remove the right valve completely and carefully examine the inside of the valve. Find the marks on the valve that show the positions of attachment of the adductor muscles.
Question 3
Carefully examine the left valve containing the soft body of the bivalve. Locate the margin of the mantle, which was originally attached to the ventral edge of the right valve. Follow this posteriorly until you reach two siphons. Water enters the mantle cavity through the incurrent siphon (ventral), flows over the gills and then exits via the excurrent siphon (dorsal). In some species the siphons will form long tubes that extend well beyond the margin of the valves.
The large anterior and posterior adductor muscles should be quite conspicuous in this view. The foot of the bivalve functions in locomotion. The muscular foot is extended into the substrate and then the animal is pulled up to the foot.
Question 4
The visceral mass is located between the two adductor muscles and dorsal to the foot (that is to say, it lies in the middle of the animal but wedged back near the hinge of the shell). It contains the organs of the digestive and reproductive systems (collectively called the viscera). Most of these organs are difficult to locate and can best be seen on the models provided. Look at the prepared models to identify the various viscera. The heart is located in a pericardial cavity dorsal to the visceral mass and just ventral to the hinge. The circulatory fluid is carried away from the heart by arteries, but seeps back to the heart through the interstitial spaces. Therefore, we state that molluscs have an open circulatory system and we call the circulating fluid hemolymph instead of blood. The mouth is located between the labial palps just dorsal to the foot at the anterior end of the body. Follow the large gills from their free end near the siphons to their points of attachment near the labial palps. You should be able to locate the mouth of your own specimen with a blunt probe. (Do not use a pin probe, or else you may make your own opening into the digestive tract.). The gut winds through the dorsal portion of the visceral mass and passes from anterior to posterior through the pericardial cavity and through the heart to terminate at the anus in the excurrent siphon.
The sexes are separate in most (but not all) bivalves. The gonads are located in the visceral mass and the eggs and sperm are simply discharged into the mantle cavity and dispersed through the excurrent siphon. Fertilization usually occurs externally, but most freshwater bivalves have internal fertilization in which the eggs are retained in the gills and are fertilized by sperm brought in through the incurrent siphon. Embryos develop into a bivalved larval stage called a glochidium, which is discharged by way of the excurrent siphon. The glochidia then attach to the gills or skin of a fish and live as parasites for several weeks before dropping off and becoming independent. Look at the prepared slides and demonstrations of glochidia.
Question 5
On your worksheet, diagram the lateral view of a bivalve with the left valve removed and indicate the relative positions of the following structures: anterior and posterior adductor muscles, hinge ligament, hinge teeth, incurrent and excurrent siphons, gills, visceral mass, labial palps, mouth, anus.
Class Gastropoda
With more than 40,000 extant and 15,000 extinct species, the gastropods are the largest and most diverse of the molluscan classes. They are all basically bilaterally symmetrical, but due to the torsion that occurs in the body during the early larval stage, the visceral mass has become asymmetric. There is a twisting of the body of 180° degrees with respect to the head and foot which puts the anus more anterior and the gills more posterior. (The torsion of the body is not the same as the coiling of the shell.) The head is anterior to the foot but is withdrawn into the shell before the foot. This means that the foot will close the opening to the shell when the body is withdrawn into it. The head always has tentacles and a radula. The shell, when present, is always composed of one piece and is often, but not always, coiled.
Get a live snail (Helix) from the front of the class. If it goes into its shell, have patience. It will come out again. IMPORTANT: If your snail starts to bubble, something is gravely wrong: Squirt it with fresh water immediately and call the instructor over.
At a snail’s pace, let your snail walk on the piece of glass. Look underneath, as it walks. Measure how fast it can go on a horizontal surface and on a vertical surface.
Question 6
Examine the demonstrations of the various types of gastropods and observe and locate the major external characters of the phylum Mollusca and the class Gastropoda exhibited by each type.
Class Cephalopoda
The cephalopods are generally regarded as the group that has changed the most from the ancestral molluscan condition, i.e., they are the most derived of all the molluscs. Squids, octopuses, and nautili are all active marine predators. They have a very well-developed nervous system and are the only molluscs to have a closed circulatory system. As the name of the class implies, the modified foot had been concentrated in the head region. The edges of the foot have been drawn out to form the sucker-bearing arms and tentacles. A portion of the foot forms the funnel through which water is forcibly expelled from the mantle cavity.
Question 7
Examine the demonstration specimens of cephalopods and observe and locate the major external characters of the phylum Mollusca and the class Cephalopoda exhibited by each type.
PHYLUM ARTHROPODA
Almost 1,000,000 species have been described within the set of animals called arthropods (Gr. arthros, joint + podos, foot), over two-thirds of the known animals on the face of the Earth. The great adaptive diversity of these organisms has enabled them to invade virtually every possible habitat and they are sometimes regarded as the most successful of all terrestrial animals.
Arthropods are bilaterally symmetrical, triploblastic, protostomes. The digestive system is complete and the circulatory system is open. They are characterized by the presence of a rigid, chitinous exoskeleton and internal and external segmentation with each segment bearing a pair of jointed appendages. The form and function of these appendages vary from one type of arthropod to another.
Owing to the great diversity and number of species in this phylum, the classification is rather complex. The living (extant) arthropods are divided into several subphyla and many classes, only a few of which are listed below. The subphyla are often listed as distinct phyla in other references.
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Ecdysozoa)
Phylum Arthropoda
Subphylum Cheliceriformes (Gr. chele, claw + keros, horn): body divided into cephalothorax and abdomen; antennae absent; anterior-most appendages (chelicerae) modified as pincers or fangs.
Horseshoe crabs; cephalothorax convex and horseshoe-shaped; long spine at posterior end of abdomen; compound lateral eyes.
Arachnids (Gr. arachne, spider): scorpions, spiders, ticks, mites; cephalothorax with six pairs of appendages (chelicerae, pedipalps, four pairs of walking legs).
Subphylum Myriapoda (Gr. myrio, numberless + poda, foot ):
Class Diplopoda (Gr. diploos, double + podos, foot): millipedes; body subcylindrical; usually two pairs of legs per body segment.
Class Chilopoda (Gr. cheilos, lip + podos, foot): centipedes; body flattened dorsoventrally; one pair of legs per body segment.
Subphylum Hexapoda (Gr. hexa, six + poda, foot):
Insects, Springtails, etc. (L. insecare, to cut into [segments]): insects; body with distinct head, thorax, and abdomen, usually with marked constriction between thorax and abdomen.
Subphylum Crustacea (L. crusta, hard shell): two pair antennae; appendages typically biramous; three or more pairs of appendages modified as mouth parts, including hard mandibles; walking legs on thorax, but abdominal appendages also present (as opposed to insects).
Decapods (Gr. deca, ten + podos, foot): crayfish, crabs, lobsters, etc.; exoskeleton of chitin hardened by calcium carbonate; dorsal cephalothorax covered with carapace.
Subphylum Crustacea, Decapods
Obtain a fresh/frozen specimen of the freshwater shrimp Macrobrachium. Examine its external parts. (N.B., This animal is very similar to the crayfish. You will find that the pictures of crayfish in your atlas will help you get oriented to your shrimp.) The body is divided into an anterior cephalothorax (fused head and thorax) and a posterior abdomen. The portion of the chitinous exoskeleton that covers the cephalothorax laterally and dorsally is called the carapace. The anterior pointed extension of the carapace between the eyes it termed the rostrum. The medial, pointy plate at the end of the abdomen is the telson. Why is the telson not a tail? The telson is flanked by a pair of uropods, the most posterior pair of appendages in these animals.
Examine the structure of the various types of paired appendages found on the shrimp. There are 19 pairs of appendages and it is easiest to find them all if you start at the posterior end with the uropods. As you find each pair, remove the right appendage and leave the left on the animal for orientation later. Tape the right appendages to a piece of paper and write the name of the appendage next to it. You will be able to compare all the appendages when you have collected one of each. Before you pull off that first uropod, look at it carefully. Each uropod consists of two flat paddle shaped projections, which are both attached to a cylindrical stalk that is connected to the body at the base of the telson. This is a typical biramous (“two branched”) appendage that all crustaceans have. In any biramous appendage there are three parts: the distal medial branch or endopodite, the distal lateral branch or exopodite and the proximal stalk or protopodite.
On your worksheet, draw the biramous uropod of your shrimp. Identify exopodite, endopodite and protopodite.
It is hypothesized that all of the paired appendages of the crustaceans have evolved through modification of primitive biramous appendages present on each body segment. (Structures that are derived from similar embryological materials in different body segments of the same animal are said to be serially homologous.) See if you can find the endopodite, exopodite and protopodite in the shrimp’s other 18 pairs of appendages.
Besides the uropods, there are 5 other pairs of appendages attached to the shrimp’s abdomen. These are called swimmerets or pleopods and are numbered 1 through 5 from anterior to posterior. Find all three parts of each of these appendages, detach the right swimmerets and label them on your piece of paper.
The thorax has 8 pairs of appendages. All 8 pairs have gills attached to their protopodites. At this time, you may want to cut the right side of the carapace off, so that you can remove the right appendage in its entirety, gill and all. The most posterior of the thoracic appendages are the 5 pairs of walking legs or periopods. In decapods, the walking legs are no longer biramous. The exopodite was lost at some time in their evolutionary past. Like the pleopods, the periopods are numbered 1 through 5, starting at the anterior end. Unlike the pleopods, however, not all periopods are alike. You will, no doubt, have already noticed the very striking 2nd periopod. Note that periopod 2 contains only the protopodite and endopodite of the leg. The claw arises because the last segment of the leg is attached at the base of the second to last segment. So technically, there’s no branching on this leg. This kind of condition is called chelate, not biramous.
On your worksheet, draw the chelate periopod 2 of your shrimp.
Carefully detach each of the right periopods, starting with the 5th, and label them on your paper. Try to keep the gills with the legs as much as possible.
The anterior-most thoracic appendages are called maxillipeds (jaw-legs) because their endopodites help to chew food and their exopodites help to place the food in the mouth. It is easy to miss one branch or the other as you remove these legs, and there are more mouth parts further anterior (that is to say, closer to the mouth). There are 3 pairs of maxillipeds and each looks more like a jaw and less like a foot as you go from posterior to anterior. Carefully identify the protopodite of the 3rd (posterior-most) maxilliped and grab the protopodite firmly with your forceps and twist slowly. The whole maxilliped should come out as one unit. Once the 3rd maxilliped is out you should be able to find the protopodite of the 2nd maxilliped and repeat the procedure for it and for the 1st maxilliped. Compare the different shapes and attach the maxillipeds to your paper.
The head of your shrimp has 5 pairs of biramous appendages. There are two pairs of maxilla, one pair of mandibles and two pairs of antennae. The eyes are not considered appendages even though they stick out of the body. The eyes’ embryological development is quite different from that of the legs. The three pairs of mouth parts attached to the head are difficult to distinguish, so you may want to compare what you pull off to what your neighbor does. (Or check with your instructor to make sure you’ve labeled the right appendages.) The two maxillae are the posterior-most head appendages. Find each protopodite, and twist off the maxillae. The endopodites should look increasingly like jaws and the exopodites should look increasingly wispy, as they serve the same purpose as our tongues. After you have detached the two right maxillae, you should be able to see the very hard mandible. The mandible may be difficult to remove as it is attached to a massive and strong muscle, which you will see when you look at the shrimp’s internal anatomy. Again, take the right mandible’s protopodite in your forceps and twist firmly. Be careful not to destroy the rest of the shrimp as you do this. Look at the medial surface of the mandible.
Question 8
There are only two pairs of appendages left, and these are the antennae. The antennae are biramous and the exopodite serves a different function than the endopodite. Find the protopodite of each of the right antennae and add them to your piece of paper.
Now that you have all 19 right appendages to look at, compare and contrast the shapes. Think about the functions of each appendage. Before you leave lab today, be sure you can tell the difference between each of the following leg categories just by looking at them: uropod, pleopod, periopod, mouthpart (includes maxilliped, maxilla and mandible together) and antenna.
Now you are ready to have a look at the inside of your shrimp. To do this, you will have to remove most of the carapace. Gently insert your blunt probe underneath the carapace on the right side, making sure that there is no soft tissue between the blunt probe and the carapace. Now insert your scissors BETWEEN the carapace and the blunt probe so that you can cut the carapace while using your blunt probe as a shield to the soft tissue below. Begin to cut the carapace from the base of the right 1st periopod. Cut along a line just posterior to the eyes, then go posteriorly as you reach the thick exoskeleton of the rostrum. After you have crossed the rostrum turn anteriorly and cut as close to the left eye as possible—all the while protecting the soft tissue with your blunt probe. Cut from the left eye to the base of the left 1st periopod. Now, very gently, lift the posterior part of the carapace from the side that you just cut. Use your blunt probe to gently detach any tissue that sticks to it. It is very easy to rip the heart out at this stage, because it is the dorsal-most organ.
Once you have the carapace off, you should be able to see the heart at the posterior end of the thorax along the medial line. The best way to see the heart morphology is to float it under water. Fill your dissecting pan with enough water to submerge the entire shrimp. The heart will then gently fill and lift a bit. Look for three pairs of small holes in the heart called ostia which open into the cavity of the heart. The hemolymph is pumped anteriorly and posteriorly from the heart through major arteries. In very clean dissections, you may be able to see several arteries leaving the heart. If you can see them in your specimen, let your instructor know so that the rest of the class can see them. Remember that we use the term "blood" to refer to the circulating fluid in a closed circulatory system and "hemolymph" to refer to the fluid in an open circulatory system. In a closed circulatory system the fluid between the cells of the body (interstitial fluid) is kept separate from the fluid of the circulatory system.
Questions 9 & 10
Anterior to the heart, you should see two massive sets of muscles. O ne set, running anterioposteriorly are the abdominal retractor muscles. What would happen to the shrimp’s body if these contracted? The second set of muscles is the mandibular muscles. These massive muscles attach to dorsal side of the carapace and travel anterioventrally and attach to a long protrusion at the end of the mandible. This kind of protrusion is called an apodeme, and is found in arthropods whenever more surface area is needed for muscle attachment. If you could pull this apodeme in the same direction that the mandibular muscle would, do you suppose it would open or close the mandible?
Carefully dissect away one of the abdominal retractor muscles and one of the mandibular muscles to expose the stomach. Note that the stomach is lined with exoskeleton as a series of hard bumps – these are collectively termed the gastric mill. The stomach is directly dorsal to the mouth. If you insert your blunt probe into the mouth of the animal, you will find that it goes through the esophagus to the stomach. Throughout the thoracic cavity is a digestive gland which serves to absorb most of the food material. This gland tends to liquefy and spread as a goopy material over the entire animal. If this goop gets excessive, rinse it away with water.
Waste is passed through the intestine to the anus, which is located on the telson. The intestine is difficult to find. Theoretically, you could follow the tiny, thin intestine through the massive muscles of the abdomen if you carefully cut away the dorsal shell on each abdominal segment. Give it a try and see if you can find the intestine. (When cooking shrimp, the intestine is called a “vein”, so it is best to be sure that your shrimp has been “de-veined” before you eat them.).
Now go back to the anterior end. The major excretory glands of decapods are the green glands located just inside the base of each antenna. They open to the outside through a duct at the antenna base. Thus, the animal gets rid of its metabolic waste anteriorly and its digestive waste posteriorly.
Question 11
Remove the organs remaining in the cephalothorax, and look at the floor of the chamber for the ventral nerve cord, which lies in a compartment largely separated from the other organs of the cephalothorax. Carefully dissect through the layer of chitin in the ventral floor of the cephalothorax and locate the ventral nerve cord. Trace it anteriorly and locate the two circumesophageal connectives around the esophagus which lead to the supraesophageal ganglion ("brain") in the head. The ventral nerve cord looks single, but is actually two paired nerve cords that lie very close together.
On your worksheet, draw a lateral view of the shrimp and show the relative shapes and positions of each of the following structures: mouth, esophagus, stomach, gastric mill, intestine, anus, heart, digestive gland, green gland, ventral nerve cord, circumesophageal connectives, and supraesophageal ganglion.
Examine the demonstration specimens of other crustaceans and compare and contrast their external characteristics with those of a shrimp.
Subphylum Cheliceriformes, Horseshoe crabs
The members of this class are very closely related to the fossil eurypterids, three-meter long aquatic predators in the Paleozoic seas. Examine the demonstration specimen of the extant horseshoe crab, Limulus, found off parts of the Atlantic coast of North America. The animal gets its name from the dark-brown horseshoe-shaped carapace. Posterior to the carapace is the abdominal shield with its posteriorly-projecting telson. Look at the ventral surface and note the seven pairs of jointed appendages. Compare the anterior-most pair of appendages in Limulus to the anterior most pair of appendages in Macrobrachium.
Questions 12 & 13
Arachnids – spiders scorpions, mites and ticks
Examine the demonstration specimens of spiders and scorpions. Note that the body segments are fused into two principle regions, the cephalothorax, bearing the head and four pairs of walking legs, and the abdomen. Compare and contrast the arachnids and the insects with respect to external morphology.
Subphylum Hexapoda, Insects
The insects are easily the most abundant of all land animals. Insects differ from other arthropods in having three major body segments (head, thorax, and abdomen) only three pairs of legs and usually two pairs of thoracic wings. They are the only invertebrates that can fly and that greatly increases their ability to find food and otherwise exploit their habitat. Their chitinous exoskeleton protects them from mechanical injury and from desiccation, and their tracheal tubes permit efficient O2-CO2 exchange. Since the tracheal tubes extend deep within the body to the tissues where the O2-CO2 exchange occurs, the hemolymph does not transport a significant amount of O2 or CO2. Remember that in many animals (like humans), gases are carried in the circulatory fluid, not through tiny tubes directly to the cells.
Examine the demonstration specimens of insects and note the great variety of sizes, shapes and adaptations.
Subphylum Myriapoda, Class Chilopoda - centipedes
The centipedes are active and agile nocturnal predators that prey mainly upon other arthropods and annelids. The body is always dorsoventrally flattened and with many body segments. The appendages of the first body segment are modified to form poison claws.
Class Diplopoda - millipedes
Millipedes live in moist dark places under stones or beneath or within rotting logs. They are primarily scavengers and feed upon dead plant material, but they will also eat animal matter. The body of a millipede tends to be cylindrical.
Question 14
Clean up. Preserved specimens are to be placed in a red hazardous waste bag. Freshly-killed specimens are wrapped in plastic or saran wrap and given to the instructor for freezing. Scrub instruments with soap and water, rinse and dry them. Clean your desk top with spray cleaner.
Lab 11 - Echinodermata and Chordata
by
L. Christianson
Before coming to lab you should:
• Review the material from last lab.
• Be able to define all the bold-faced terms in the lab exercise.
• List the characteristics of these phyla which serve to distinguish them from other phyla.
• Explain how the members of these phyla relate to other phyla studied previously.
• Complete the tables in the exercises that describe the life processes exhibited by the members of the phyla Echinodermata and Chordata.
After completing the lab exercise you should be able to:
• Recognize, name, and be able to list the major distinguishing characters of the members of the various major classes of echinoderms and subphyla and classes of chordates.
• Recognize, name, and describe the function of the major anatomical structures exhibited in the lab exercise.
• Describe the general life cycle of members of the phyla Echinodermata and Chordata.
• Describe the major structural differences between these phyla and others studied and the evolutionary and adaptive significance of these differences.
References:
• Campbell, Neil A., and Jane B. Reece. 2008. Biology, 8th ed. Benjamin Cummings, San Francisco, CA.
• Van de Graaff, Kent M., and John L. Crawley. 2005. A photographic atlas for the biology laboratory, 5th ed. Morton Publ. Co., Englewood, CO.
Phylum Echinodermata
The members of the phylum Echinodermata (Gr. echinos, hedgehog + derma, skin) are all marine organisms and include such forms as sea stars, brittle stars, sea urchins, sea cucumbers, and sea lilies. They are triploblastic deuterostomes with complete digestive systems and radial symmetry. Their radial symmetry is secondarily developed in that their larvae (as well as their ancestors) are bilaterally symmetrical. The larvae of echinoderms resemble the larvae of chordates and this resemblance is one of the reasons for suggesting that chordates and echinoderms share a common ancestry. Echinoderms are sharply distinguished from the members of other phyla and can be recognized by the presence of a water vascular system, a spiny endoskeleton of calcareous ossicles, pedicellariae, and dermal branchiae. They are the only radially symmetrical group to have such complicated organ systems.
Questions 1 & 2
The living (extant) members of the phylum Echinodermata are commonly divided into six classes. You will explore four of them in lab:
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Deuterostomia)
Phylum Echinodermata
Class Asteroidea: (Gr. aster, star + eidos, form) sea stars ("starfish"); body star-shaped with five to twenty-five arms.
Class Ophiuroidea: (Gr. ophis, serpent + oura, tail + eidos, form) brittle stars; distinct central disk with flexible jointed arms.
Class Echinoidea: (Gr. echinos, hedgehog + eidos, form) sea urchins and sand dollars; body more or less globular or disk-shaped and lacking arms; compact skeleton or test with closely fitted plates and bearing moveable spines.
Class Holothuroidea: (Gr. holothurion, type of zoophyte + eidos, form) sea cucumbers; elongate, cylindrical body with no arms or spines; ossicles microscopic and buried in the muscular body wall.
Class Asteroidea
Examine a preserved specimen of a sea star. The body is composed of a central disk from which radiate five arms. The oral surface of each arm contains an ambulacral groove which leads inward toward the central mouth. The spines that are seen on the aboral surface are part of a set of small calcareous ossicles that form the endoskeleton of the sea star.
Question 3
There is a thin layer of epidermis over the ossicles and the ossicles themselves are partially buried in tissue. Thus, what appears at first glance as an exoskeleton is really an endoskeleton. The small button-like structure on the aboral surface is the madreporite and it is actually a porous plate connecting to the water vascular system. Along the edges of the ambulacral grooves one can see the ambulacral spines that extend part way across the grooves. In the ambulacral grooves are the tube feet. They are soft and slender and have expanded tips and function in locomotion.
Question 4
Remove a 2 cm2 section of the aboral surface of one arm of the sea star and examine it under a dissecting microscope. The thin, hollow, soft projections between the spines are the dermal branchiae which function as gills. The projections that look like miniature crab claws are the pedicellariae.
Questions 5-6
The two arms that are closest to the madreporite are termed the bivium, while the remaining three arms are called the trivium. Using a stout pair of scissors, cut off the tip of each of the arms in the trivium. Then cut along the sides of these arms, taking great care not to injure the delicate organs beneath. Lift and carefully remove the aboral surface of each of the trivial arms after loosening the delicate mesenteries connected to the fragile internal organs below. Cut around the central disk (but not into the bivium) and completely remove the aboral surfaces of the trivial arms. Leave the madreporite in place and undisturbed. Cut across one of the arms of the bivium to expose a cross-section.
Note that there is a fairly extensive coelom surrounding the internal organs. Most of the coelom in each arm is taken up with a pair of greenish (often reddish in preservation), branched glands called the pyloric caeca. The enzymes produced in the pyloric caeca are carried to the stomach by pyloric ducts. The two ducts from each arm join and enter the pyloric stomach approximately at the level of the base of the arm. A short, slender intestine connects the pyloric stomach to the anus on the aboral surface. Some species of sea stars lack an anus and even when an anus is present, it is very difficult to see on an undissected animal. Gently lift the aboral surface and peak beneath it to see the fragile intestine.
The mouth opens to a short esophagus which leads to the cardiac stomach, which is just on the oral side of the pyloric stomach and connected to it. The cardiac stomach can be everted though the mouth to contact and digest prey. You will probably have to remove the pyloric stomach before you can see the cardiac stomach, but be sure to look for the intestine first.
Question 7-9
Digestion in sea stars is facilitated by a number of enzymes produced in the stomach walls and the pyloric caeca. The products of the stomach’s digestion are carried through the pyloric ducts to the pyloric caeca where digestion is completed and absorption occurs. The pyloric ducts also carry wastes out to the intestine where they are eliminated through the rectum.
Cut through the pyloric ducts and remove the pyloric caecae from one arm. Look for the gonads located at the junctions of the arms beneath the pyloric caeca. During the reproductive season the gonads will almost completely fill the coelomic cavity of the arm. The gonads open to the outside through a tiny inconspicuous gonopore at the base of the arm. Sexes are separate in the starfish, but there is almost no sexual dimorphism.
On your worksheet, diagram the relationship of the following parts in both an aboral and a lateral view (from the side): mouth, esophagus, cardiac stomach, pyloric stomach, intestine, anus, pyloric caecae, gonads.
The water vascular system of the sea star consists of a number of seawater-filled ducts whose primary functions are in transport, locomotion and feeding. To expose the water vascular system, carefully remove the digestive and reproductive systems of your sea star. Water enters the system through the porous madreporite. A calcareous tube called the stone canal carries water from the madreporite to the ring canal, which is a soft structure on the medial surface of the hard ring of ossicles encircling the mouth. Radial canals lead out from the ring canal, but are located beneath the ambulacral ridge and not visible from the aboral side. Look for the radial canal in the cross-section of the bivial arm which you prepared earlier. A lateral canal extends from the radial canal to each tube foot. Water from the lateral canal collects in the ampullae seen on the floor of the coelom. Contraction of the ampullae forces water into the tube feet and causes them to elongate. Contraction of the muscles in the tube feet causes them to retract or to bend from side to side.
On your worksheet, diagram the water vascular system in aboral view showing the relationship of the following structures: madreporite, stone canal, ring canal, radial canal, lateral canal, ampullae, and tube feet.
Class Ophiuroidea
Brittle stars and sea stars are quite similar, although the arms of a brittle star are noticeably more moveable in life. Compare and contrast the external morphology of the brittle star demonstrations with the sea star. Note particularly the position of the madreporite and the pedicellariae.
Class Echinoidea
Examine the demonstrations of sea urchins and sand dollars. Their endoskeleton is a rigid shell called a test formed by the fusion of the calcareous ossicles. The tube feet are similar to those seen in sea stars and are restricted to the ambulacral areas. Examine the jaw apparatus of a sea urchin. It is a complex of five protrusible teeth and is called Aristotle's lantern.
Class Holothuroidea
Examine the demonstration specimens of sea cucumbers. The body of a holothuroid is greatly elongated in the oral-aboral axis. The calcareous ossicles are greatly reduced in most species and the body takes on a leathery appearance. The tube feet around the mouth are modified into ten to thirty tentacles. In some species the tube feet are restricted to the ambulacral areas near the mouth, in others they may be scattered over the entire surface of the body.
Phylum Chordata
The animals that are the most familiar to us probably belong to the phylum Chordata. Fishes, amphibians, reptiles, birds and mammals are all chordates and, thus, share a number of distinct characteristics. These characteristics are always found in the early embryonic stages, although they may be altered or disappear completely in the later stages of the life cycle. The four principle distinguishing characteristics of the phylum Chordata are the presence of a notochord, a dorsal, hollow nerve cord, pharyngeal slits, and a muscular postanal tail. The notochord is a longitudinal flexible rod extending along the length of the body. It serves as a simple skeleton, keeping the body from collapsing and providing a point for muscle attachment. The nerve cord is hollow and dorsal, as opposed to being solid and ventral as it is in other phyla. The pharyngeal slits open from the anterior gut to the outside of the body. Their primitive function is probably as filtering devices for feeding, but they become modified for gas exchange as gill clefts in most aquatic vertebrates. In most non-chordates, the digestive tract extends the entire length of the body so that the anus is at the extreme posterior end. In chordates, a tail extends beyond the anus.
Supergroup Unikonta
Kingdom Animalia (Eumetazoa, Bilateria, Deuterostomia)
Phylum Chordata: (L. chorda, cord)
Subphylum Cephalochordata: (Gr. cephalo, head + chord, cord) lancelets, amphioxus; notochord and nerve cord found along entire body and persisting through life.
Subphylum Urochordata: (Gr. uro, tail + chord, cord) tunicates; notochord and nerve cord only present in the free-swimming larval stage; adults are sessile (a few species are motile, e.g. salps) and encased in a thick outer covering called a tunic.
Subphylum Craniata: (Gr. cranium, skull) head present consisting of brain at anterior and of dorsal nerve cord, eyes and other sensory organs, neural crest cells, and a skull.
Class Myxini: (Gr. myxo-, mucus or slime) hagfishes; lacking true jaws, paired appendages and vertebrae.
Class Petromyzontida: (Gr. petro, stone + myzo, suck) lampreys; lacking true jaws and paired appendages. (Note: also known as Class Cephalaspidomorphi.)
Class Chondrichthyes: (Gr. chondros, cartilage + ichthyes, fish) cartilaginous fishes, sharks and rays; skeleton cartilaginous (lacking bone); five to seven separate gill openings without a bony operculum.
Class Actinopterygii: (Gr. actino, ray, beam + pterygos, wing, fin) ray-finned fishes, fins supported by long, flexible rays.
Class Actinistia (Gr. actino, ray, beam) coelacanths
Class Dipnoi (Gr. di, two + pnoi, air-breathing) lungfishes
[Actinopterygii, Actinistia and Dipnoi were traditionally lumped together into the Class Osteichthyes: (Gr. osteon, bone + ichthyes, fish) bony fishes; skeleton composed partially of bone; gill openings covered with bony operculum.]
Class Amphibia: (Gr. amphi, dual + bios, life) salamanders, frogs and caecilians; skin moist, containing mucous glands and lacking epidermal scales.
Class Reptilia: (L. reptum, to creep) lizards, snakes, crocodilians, turtles, tuataras and birds; skin dry, lacking mucous glands and covered with epidermal scales.
Class Mammalia: (L. mamma, breast) mammals; mammary glands and hair present.
We often use the name Vertebrata (L. vertere, turn, as in a joint) to refer to members of the classes from Cephalaspidomorphi through Mammalia because they all share the presence of bony or cartilaginous vertebrae surrounding the nerve cord in living forms and have a notochord in all embryos and persisting to varying degrees.
The classes from Chondrichthyes through Mammalia are often called gnathostomes (Gr. gnathos, jaws + stoma, mouth) because they all have jaws. The other classes, Myxini and Cephalaspidomorphi, are often referred to as agnaths (Gr. a, lacking + gnathos, jaws) because they lack jaws.
Subphylum Cephalochordata
Examine the demonstration specimens, slides, and models of amphioxus. Locate the four major chordate characters as they are seen in amphioxus. On your worksheet, compare and contrast the external structure of amphioxus with a fish.
Subphylum Urochordata
Examine the demonstrations specimens and models of tunicates. Examine the four major chordate characters in the larval and adult stages.
Jawless Fishes - agnathans (Classes Myxini and Petromyzontida)
Examine the demonstration specimen of lampreys and hagfishes. Note the lack of paired appendages. The gill pouches open separately to the outside. The eyes are well developed and there is only a single median nasal opening. On your worksheet, compare and contrast the mouths of lampreys and hagfishes and relate it to the modes of feeding. Note that jawless fishes lack the skeletal supports about their mouths that form the jaws of all other vertebrates.
Class Chondrichthyes
Look at the demonstration specimens of sharks. Note that the mouth is surrounded by jaws, as opposed to what we see in the jawless fishes. Look at the demonstration of the spiral valve from the intestine of a shark. This structure is analogous to the typhlosole in the gut of an earthworm.
Questions 10-11
The medial margins of the pelvic fins of male chondrichthyan fishes are modified into claspers which are elongated, grooved projections which serve to transmit sperm from the cloaca of the male into the cloaca of the female.
The Class Chondrichthyes takes its name from the fact that the skeletons of all its members are constructed solely of cartilage and lack bone. The usual condition in a vertebrate is to have the skeleton composed of a combination of both bone and cartilage.
Class Actinopterygii
This class contains a greater number of species than all the rest of the vertebrate classes combined and its members are adapted to a wide variety of habitats, feeding behaviors, locomotor techniques, etc.
Examine the demonstration specimens of bony fishes and contrast their external anatomy with those of chondrichthyan fishes. Note the gills of bony fishes are enclosed in a chamber and covered with a bony operculum, while the gills of cartilaginous fish open separately to the outside of the body and are not covered by a bony operculum.
Bony fishes have a skeleton composed of a combination of both bone and cartilage, as is typical for vertebrates.
On your worksheet, compare and contrast the gill openings of jawless, cartilaginous and bony fishes.
Class Amphibia
Examine the demonstration specimens of salamanders and frogs. Notice that the skin of an amphibian is smooth. In life it would also be moist and covered with layer of mucous. Amphibians almost always have a distinct larval stage (as do most jawless and bony fishes). Amphibian larvae, as opposed to adults, can be recognized by the presence of gills (although the gills of larval amphibians are not homologous to the gills of fishes). The adults usually lose their gills at metamorphosis.
Class Reptilia
Examine the demonstration specimens of various types of reptiles. Look for the presence of a dry (in life) scaly integument as a way of distinguishing extant reptiles from amphibians.
Question 12
Examine the demonstration specimens of birds. Almost every organ system in the body of a bird exhibits adaptations related to the ability to fly. Birds are the only animals with feathers. In addition to their importance in maintaining lift and streamlining for flight, a primary function of feathers is as insulation, retarding the flow of heat from the body and thereby facilitating the maintenance of homeothermy and endothermy.
Question 13
A homeotherm is an organism that maintains a relatively constant body temperature, while a poikilotherm's body temperature is variable. An endotherm is an organism that can maintain its internal body temperature independent of the environmental temperature. An ectotherm is an organism whose body temperature depends upon the temperature of its environment. Thus the terms "homeothermy" and "poikilothermy" refer to the relative constancy of body temperatures, while the terms "endothermy" and “ectothermy" refer to the heat source for the organism.
Some modern classification systems place birds in a taxon called Archosauria along with the dinosaurs and the crocodilians (alligators, crocodiles, etc.), indicating that all these animals are in the same evolutionary line and that birds evolved from dinosaurs.
Class Mammalia
Examine the demonstration specimens of mammals. Mammals are the only animals with hair and mammary glands. The primary function of mammalian hair is as insulation, retarding the flow of heat from the body and thereby facilitating the maintenance of homeothermy and endothermy.
Questions 14-15
Vertebrate Anatomy
Obtain a preserved specimen of a bony fish and locate and learn the major external features described below.
The cloacal aperture is the common exit of the digestive and urogenital systems and is located on the ventral surface near the base of the tail.
Question 16
The unpaired median fins include one or two dorsal fins, an anal fin posterior to the cloacal aperture, and a caudal fin on the tail. The paired pectoral fins are near the anterior end of the body, while the position of the pelvic fins varies greatly from one species to another. The posterior-most and ventral-most paired fins are the pelvic fins even though they are located near the head.
After verifying that your fish has the usual vertebrate number of eyes and mouths (two and one, respectively), examine the paired nostrils. Insert a blunt probe into the nostrils and determine where they lead.
Questions 17-18
Look for a row of light-colored dots (actually small pores) along either side of the body. These are the openings to the lateral line system which is a set of water-filled canals that detects changes in the pressure of the water around the fish.
Question 19
Using a scalpel, remove one scale from the lateral line of your fish. Keep track of which end of the scale is attached to the side of the fish. Mount the scale on a microscope slide and examine it under low power. Look for the opening of the pore of the lateral line system.
Question 20-21
The entire surface of the fish (including the scales) is covered by a thin, soft, mucous-producing epidermis which facilitates movement through the water and protects the fish from invasion by disease-causing organisms.
Question 22
Posterior to the eye is the bony operculum that forms a protective cover over the delicate gill structures. Lift the gill cover to see the structures beneath. Unlike the gills of cartilaginous fishes, the gills of bony fishes are in a common cavity on either side of the pharynx. Cut off the operculum on the left side to expose the four gill slits that lead from the pharynx to the outside. Probe through the mouth and trace the path of water across the respiratory surfaces. The soft red (or grey in preservation) filaments projecting posteriorly are the gill filaments. This is the point in the system where the O2-CO2 exchange occurs. The gill filaments are attached to the gill arch which contains a bony or cartilaginous support. The tooth-like projections on the medial (pharyngeal) margin of the gill arch are the gill rakers. They serve to protect the gill filaments from being damaged by food passing through the pharynx and also as a screen to remove large pieces of food from the water. Remove one of the gill arches and examine the relative positions of the gill filaments and the gill rakers.
Refer to the original evolutionary origin of the gills slits of chordates and explain how they still retain some of their original filtering activity, even though they have become respiratory organs in the fish.
Question 23-24
On your worksheet, diagram the pharyngeal area of the fish in lateral (side) view showing the relative positions of the following structures: gill slits, gill filaments, gill arch, gill rakers.
Carefully cut through the ventral body wall from a point just anterior to the cloaca to the area between the gills. On the left side of the body cut dorsally from either end of the previous incision and remove the entire left lateral body wall to expose the underlying organs. Take care not to disturb any of those organs as you remove the body wall.
Trace the thin tubular intestine anteriorly from the cloaca. The sac-like structure at its anterior end is the stomach. Shortly before the intestine reaches the stomach, one finds several pyloric caecae which are blind tubes that extend off the intestine. The junction of the stomach and the intestine is called the pylorus. There are usually long yellow or white masses of fat lying in the folds of the intestine. The large yellowish or reddish-brown organ (often grey in preserved specimens) that partially covers the stomach anteriorly and dorsally is the liver. Cut away the liver to expose the short esophagus that connects the stomach to the pharynx (area between the mouth cavity and the esophagus) and mouth cavity (between upper and lower tooth-bearing jaws).
Carefully cut through the esophagus and through the intestine near the cloacal aperture and remove the digestive tract from your fish. Open the entire tract and examine the contents and look for parasites.
Locate the swim bladder at the dorsal margin of the abdominal cavity. In life it is a gas-filled sac that functions as a hydrostatic organ. The fish can add or remove gases from the swim bladder and control the amount of water that its body displaces, thereby regulating its buoyancy.
Questions 25-27
The lines of dark tissue along either side of the vertebral column dorsal to the swim bladder are the kidneys. There is a tiny duct leading from the posterior end of the kidney to the base of the urinary bladder just dorsal to the intestine. (The size of the urinary bladder in your specimen will vary depending upon whether it is full or empty.) The gonads (ovaries or testes) are masses of tissue between the intestine and the swim bladder. During certain times of year, the ovary is filled with eggs and fills most of the empty space in the coelom.
Question 28
Continue your midventral incision anteriorly toward the lower jaw. This will expose the cavity in which lies the heart of your fish. The bony fish heart is small and the dissection is difficult. If you are having problems seeing the important structures, refer to the larger shark heart found in the lab room. The ventral, median, thick-walled ventricle should provide a good landmark for beginning your study. Leading anteriorly from the ventricle is the thick tube of the conus arteriosus that continues anteriorly as the ventral aorta. Trace the ventral aorta anteriorly and see where the paired aortic arches lead off to the gills. Dorsal to the ventricle one sees the thinner material that forms the wall of the atrium. Posterior to the atrium is the thin-walled sinus venosus that receives the venous blood from the body.
The dorsal aorta carries oxygenated blood posteriorly from the gills. Locate the dorsal aorta at the dorsal margin of the abdominal cavity between the kidneys and follow it anteriorly to locate the aortic arches that carry blood from the gills.
The blood flows from the venous system into the sinus venosus and then to the atrium. From the atrium it moves to the ventricle which contracts and forces the blood out through the conus arteriosus. From here the blood passes through the aortic arches and the capillaries of the gills where O2-CO2 exchange occurs. The oxygenated blood in the aortic arches then travels to the dorsal aorta and thence to arteries leading to the rest of the body. It passes through the various capillary beds of the body and then returns to the heart via the venous system.
Questions 29-31
On your worksheet, diagram a lateral view of a fish showing the relative positions of each of the following structures: cloaca, intestine, pyloric caeca, stomach, esophagus, liver, gonad, urinary bladder, swim bladder, sinus venosus, atrium, ventricle, conus arteriosus, ventral aorta, gill arch, dorsal aorta.
Cut a cross-section through the vertebral column of your fish somewhere near the middle of the body and examine the relative position of the nerve cord, notochord, and vertebrae. The notochord is the mushy material between the individual vertebrae (the notochord is best seen in sagittal section – that is, cut lengthwise into right and left halves). It forms the axial skeleton running down the length of the fish’s body during embryological development, but is gets restricted to the areas between the vertebrae after the vertebrae develop. The dorsal, hollow nerve cord runs through the neural arches on the dorsal side of the vertebrae.
On your worksheet, diagram a cross-section of a fish showing the relative positions of the vertebrae, notochord and nerve cord.
Clean up. Preserved specimens are to be placed in a red hazardous waste bag. Freshly-killed specimens are wrapped in plastic wrap and given to the instructor for freezing. Scrub instruments with soap and water, rinse and dry them. Clean your desk top.
We Hope You Enjoyed Bio 51!
-----------------------
?
58
Magnetic centromere ’!
74
?
18
73
19
?
17
Millions of Years Ago
[pic]
|Table 2 - Hypothetical Classification of |
|Some Caminalcules |
|PHYLUM CAMINALCULA |
CLASS Magnetic centromere →
74
?
18
73
19
?
17
Millions of Years Ago
[pic]
Table 2 - Hypothetical Classification of Some CaminalculesPHYLUM CAMINALCULA
|CLASS 1 |CLASS 2 |
|ORDER 1 |ORDER 2|ORDER 3|
|FAMILY 1 |FAMILY |FAMILY |FAMILY |
| |2 |3 |4 |
|GENUS |GENUS |GENUS |GENUS 4|GENUS 5|GENUS 6|
|1 |2 |3 | | | |
A |G |H |D |B |J |L |E |K |C |F |I | |
[pic]
[pic]
[pic]
[pic]
................
................
In order to avoid copyright disputes, this page is only a partial summary.
To fulfill the demand for quickly locating and searching documents.
It is intelligent file search solution for home and business.
Related download
Related searches
- what is principles of marketing
- principles of financial management pdf
- principles of management notes pdf
- weber six principles of bureaucracy
- principles of financial management
- principles of marketing notes pdf
- principles of management pdf books
- basic principles of cash management
- fundamental principles of accounting mid exam
- fundamental principles of accounting final exam
- principles of marketing pdf
- principles of marketing textbook pdf