GENERAL BIOLOGY II LABORATORY MANUAL
GENERAL BIOLOGY IILABORATORY MANUALThird EditionBREVARD COMMUNITY COLLEGEPALM BAY CAMPUS1
TABLE OF CONTENTSLab 1: Orientation and Safety .3Lab 2: Microscopy .7Lab 3: Natural Selection and Classification .17Lab 4: Bacterial Identification .29Lab 5: Protists.32Lab 6: Fungi .53Lab 7: Plant Diversity Part 1.61Lab 8: Plant Diversity Part 2.57Lab 9: Animal Diversity Part 1.68Lab 10: Animal Diversity Part 2.82Lab 11: Animal Diversity Part 3.87Lab 12: Turkey Creek Field Trip.90Lab 13: Samsons Island Field Trip.93Lab 14: Geocaching with GPS.102Lab 15: Field Ecology.109Lab Practical 1 Review .111Lab Practical 2 Review .1122
LAB 1: GENERAL LABORATORY PRACTICES AND LAB SAFETY PROCEDURESINTRODUCTIONThis laboratory manual has been developed to accompany the Biology II course. Thecoursework, lecture and lab, are designed to provide the student with a wide range ofinformation about living organisms and systems.The experiments contained in this lab manual accompany the lecture information in such a wayso as to illustrate and demonstrate. The lab portion of the course is a hands-on experience thatwill enhance the course material.Working in a laboratory environment requires that the student observe certain housekeepingprocedures and safety precautions. These procedure and precautions are outlined below. Thestudent needs to review and follow the procedure so as to ensure the safety of himself/herself,the instructor, fellow students, the equipment and the facilities.Safety in the lab demands that lab directions be followed carefully. Please read these 'GeneralLaboratory Practices' carefully and be sure you understand each one. If you aren't sure, ASK!!!GENERAL LABORATROY PRACTICES1.No food or drinks in the Lab.2.No smoking.3.Wear all safety equipment as required by the lab procedure or your instructor. Learn thelocation of all lab safety equipment. Wear safety glasses or goggles at all times. DO NOTWEAR CONTACT LENS AT ANY TIME.4.Do not mix any chemicals except as instructed. Do not do unauthorized experiments.5.Use fume hoods when required.7.Tie long hair back to keep it out of flames or harmful liquids.8.Wear shoes that cover all of your feet. No open toed shoes or sandals.9.Do not taste any chemical.10.Do not smell chemicals directly. Use your hand to waft the odor to your nose.11.Do not pipet solutions by mouth. Use a rubber suction bulb or special pipet filler.12.Do not work in the laboratory in the absence of your instructor or his authorizedrepresentative.13.Handle glass tubing and thermometers with care.3
14.Wash your hands before leaving the laboratory.15.NO chemicals are to be flushed down a drain unless specifically instructed to do so by thelab procedure.16.Wastes are to be poured into the appropriately labeled waste container (e.g., solventwaste, halogenated solvent waste, etc.).17.DO NOT mix wastes from different categories.18.Clean up broken glass immediately. DISPOSE OF IN SPECIFIED "BROKEN GLASS"CONTAINER ONLY.19.Clean up solid and liquid spills immediately, but only after checking with your laboratoryinstructor about possible safety hazards.20.Take containers to the stock of chemicals. Do not bring stock chemicals to yourlaboratory table.21.Read the label on chemical bottles carefully. Insure that you have the correct chemical.22.Do not insert a pipet or medicine dropper into a stock bottle. Avoid contamination bypouring a small quantity into a flask or beaker before taking a sample.23.Use special care when handling stoppers or tops of bottles so as not to pick upcontamination.24.Take no more of a chemical than an experiment requires.25.Never return as unused chemical to a stock bottle. Dispose of it as waste.26.Set up your glassware and apparatus away from the front edge of your laboratory bench.27.Follow any other housekeeping, safety, or disposal rules given by your instructor.Federal and State regulations require compliance with strict guidelines for handling,storage, and disposal of hazardous chemicals wastes. Because of the wide variety ofchemicals used in a teaching laboratory, it is imperative that all students follow proper disposalprocedures so as to not pollute our environment and groundwater supply.Willful violations of these rules can result in a zero being given for that lab. Repeatedwillful violations can result in an "F" being given for the course.Every effort has been made to minimize the hazardous chemicals used in the Lab. Allchemicals provided can be used safely by following correct procedures. However, any chemicalcan be dangerous if used improperly. Material Safety Data Sheets (MSDS) will be provided forany chemical on request.4
LABORATORY SAFETY PROCEDURESSafety is one of the concerns in any lab. Safety for humans is always a primary concern.The location of safety equipment should be common knowledge to anyone working in the lab.In order to become familiar with the safety equipment in the lab, make a map on the reverseside of this sheet showing the location of the following safety items. Indicate the fire escaperoute with an arrow.1.Emergency shower and eye wash station2.Fire extinguisher(s)3.Fire blanket4.Exits from the room5.Fire escape route6.Fire alarm boxes7.Container for broken glass8.Eletrical power cut off switch(es)9.First aid box10.Biohazardous Waste ContainerAfter you have read the 'General Laboratory Practices' and completed the mapassignment, please complete this page and turn it into your lab instructor.I have read and understand the general lab practices and procedures and am familiar with thelocation and operation of safety items in the lab.Printed NameLab InstructorSignatureClass Section5
NAMEDATE:SECTIONLAB 1: MICROSCOPE TUTORIAL QUIZ.DIRECTIONS: Before using the microscopes in the lab, the Microscope Tutorial Program(installed on the desktop in the computer lab under Biology Tutorials) and the following quizmust be completed. While running the tutorial, match the following. Words may be used morethan once or not at all.A.B.C.D.E.F.G.H.I.J.Limit of ResolutionNumerical ApertureLight MicroscopyScanning Electron MicroscopyTotal MagnificationWorking DistanceDepth of FieldField of ViewField SizeElectron GunK.L.M.N.O.P.Q.R.S.T.Condenser LensElectromagnetic LensesIon PumpSpecimen StageEDS DetectorSputter CoaterSecondary Electron DetectorPhotomicrographMicrometerObjective Lens1.Relies on the bending, or refraction of light rays.2.The visual circle of light.3.The amount of enlargement of the specimen.4.One millionth of a meter.5.Deposits a thin layer of metal over the specimen.6.Produces the primary electron beam.7.Influences intensity or brightness of light beam.8.Use magnetic fields to control primary electron beams.9.Detects x-rays emitted from an SEM specimen.10.The space between the objective lens and the specimen.11.The smallest detail which we may clearly see.12.A measure of the quality of the lens.13.A platform upon which the specimen rests.14.A picture of a magnified specimen or feature.15.The thickness of specimen that is in focus.6
LAB 2: MICROSCOPYMicroscopy Materials List Light or compound microscopePrepared slides:Letter “e”Colored threadsClean microscope slidesCover slips KnifeToothpicksIodine solutionOnionCutting BoardINTRODUCTIONThe microscope is a precise piece of equipment that should be handled with special care. Amicroscope may be seriously damaged if dropped or bumped against a hard object. Thestudent should report immediately to the instructor any defects that might occur to his or hermicroscope.The microscope should always be carried with both hands, one under the base and the other onthe arm.THE LIGHT MICROSCOPEThe following parts are the parts of the microscope that the student should be familiar with.Refer to Figure 1 to aid in locating these parts on your microscope.1. The OCULAR (eyepiece) contains the upper most lenses of the microscope. Its functionis to magnify. The part may be loose, but it should never be removed from themicroscope as such practice will allow dust to get inside the instrument. As you lookthrough the ocular, you may notice a solid line; this is a POINTER. Never attempt toclean the inner part of the ocular for you will remove the pointer.2. The BODY TUBE connects the ocular to the nosepiece. This is a tube through whichlight rays pass between the upper and lower lenses.3. The NOSEPIECE is a rotating disc on which the objectives are mounted. When movingthe nosepiece, the fingers should be placed on the disc and not the objectives.4. There may be three or four OBJECTIVES of different lengths and magnifying powersattached to the nosepiece of your microscope. These objectives, together with theocular, magnify the size of the objects that you are observing. Remember, the shorterthe objective the lower the power of magnification.7
Figure 15. The ARM supports the above parts. This is one of two structures that should be heldwhen carrying the microscope.6. The STAGE is the platform with a mechanical stage for holding the slides in place. Notethe circular opening in the center of the stage which allows light to pass through. Theobject which is to be viewed should be centered over this opening.7. The ILLUMNATOR is a small lamp located directly beneath the stage. Electrical outletsare located on the tables.8
8. The DIAPHRAGM may be an iris or rotating disc, depending on the kind of microscope.It is located below the stage. The diameter of the diaphragm may be controlled by eithera lever on the iris or by rotation of the disc. Various objects can be seen better undercertain light conditions. When using the highest magnification, more light is needed thanwhen using lower magnifications.9. The CONDENSER is a group of lenses beneath the stage. The condenser causes lightrays from the illuminator to converge on the surface of the microscope slide. For mostmicroscopic work, it is best to keep the condenser at its highest level. Only rarely is itdesirable to lower it slightly. When the condenser is used at a lowered position, theresolving power is greatly reduced. There is a small milled wheel just under the stagethat is used to control the position of the condenser.10. The BASE is the heavy, horseshoe-shaped structure upon which the microscope rests.This is the other part of the microscope that is held when the microscope is beingcarried.11. The COARSE ADJUSTMENT is the large milled wheel on the microscope, which is usedin focusing the lenses.12. The FINE ADJUSTMENT is the smaller milled wheel on the microscope. The wheelmay be separate from the coarse adjustment wheel on some microscopes, where onothers it is the smaller, outermost portion of a dual wheel assembly.Resolution and MagnificationThe resolution limit or resolving power of a microscope lens is a function of the wavelength oflight, the design of the condenser, and the use of immersion oil with the 100X objective. Theshortest wavelengths of visible light provide the maximum resolution. It is for this reason that allmicroscopes use blue filters over the light source.The best compound microscope lenses have a resolving power of approximately 0.2 microns.This means that two small objects that are 0.2 microns apart will be seen as separate entitiesunder the oil immersion lens. If they are closer than this, a single object is seen due to thefusion of the images.Magnification of an object seen through the microscope is a function of the power of the ocularand objective. If the 10X objective is used with a 10X ocular, the magnification is 100X. Whenthe oil immersion lens is used, the magnification is 10X x 100X, of 1000X.9
THE SCANNING ELECTRON MICROSCOPEScanning electron microscopy provides a three-dimensional view of surface features. With ascanning electron microscope (SEM), a narrow electron beam is played back and forth across aspecimen's surface, which is either conductive itself or has been coated with a thin metal layer.Electron energy triggers the emission of secondary electrons in the metal. Equipment similar toa television camera detects the emission patterns, and an image is formed.In contrast to the light microscope, the scanning electron microscope has a depth of field at allmagnifications up to 500 times greater than the light microscope.SEM Parts and Functions1.Electron Gun Assembly: The electron gun assembly produces the primary electronbeam. A voltage difference is established between the negatively charged tungstenfilament (cathode) and a grounded anode plate. The filament is heated in order togenerate a flow of electrons. Then the negatively charged electrons accelerate towardthe anode. (This is referred to as "accelerating voltage".)2.Apertures: Depending on the design of the SEM, one or more aperatures may be usedto reduce and exclude extraneous electrons in the lenses. The accelerated electronsfirst pass through a hole in the anode which acts as a crude aperture. This creates asmaller, more cohesive electron beam. Decreasing the aperture size will result in anincrease in depth of field, however, a loss of relative brightness.3.Electromagnetic Lenses: The electromagnetic lenses focus the primary beam on thespecimen and consist of two genral types:a.The Condenser Lens: This is the first lens that influences the electron beam. Asthe condenser lens current is changed, the position of the focal point in thecolumn (focal length) is altered. This, in part, influences intensity of the electronbeam when it strikes a given specimen. As a result, it directly affects thebrightness of the image signal from that specimen.b.The Final Lens: A final lens is used to bring the beam into focus at the specimenby demagnifying (converging) it to a focal point at the specimen surface. Thisdetermines the diameter or spot size of the electron beam at the specimen.Specimen resolution is determined by this beam diameter.10
4.The Vacuum System: The vacuum system allows passage of the electron beamthrough the column without interference. This is necessary because (1) a hot filamentwill oxidize and burn out in the presence of air, (2) the column must be kept clean if thebeam is to be well-focused (the vacuum prevents corrosive moisture and chargeabledust particles which can deflect the beam), and (3) air molecules will scatter electrons.5.The Specimen Stage: The specimen stage is the platform upon which the specimenrests in the column of the SEM. It is located directly below the final lens and representsa significant portion of the specimen chamber. Since, while viewing the specimen, thechamber must be under vacuum, so all manipulations of the stage are accomplished bydelicate micrometers which can move the specimen in the x, y, or z directions (figure).Further, the specimen may be tilted at least 550 or rotated 3600.6.The Signal Detection and Display Components: The signal detector forms an imageby drawing electrons toward positive charges via a collector and transferring the energyof electrons to photons. This is accomplished by using a scintillator which containsphosphors that will produce small flashes of light (photons) when struck by electrons.The photons are carried by a light pipe and converted outside the microscope column toan amplified electronic signal. The signal can then be displayed on a cathode ray tubeon the display console.PROCEDURE 1: Using the MicroscopeWhen properly used, the microscope should cause no eye strain. Try to keep both eyes openwhen working the monocular microscope and use the dominant eye to look through the ocular.If you wear glasses, it will not be necessary to use them with the microscope, since themicroscope automatically corrects for this.1. Obtain the microscope to which you were assigned from the microscope cabinet. Whencarrying the microscope, remember to place one hand under the base and the other onthe arm.2. Before the microscope is place on the desk, ample space must be provided for it. Allbooks, purses and other unneeded paraphernalia should be put away.3. Place your microscope in front of you in a comfortable working position about one inchfrom the table’s edge. The nosepiece should have the low-power (4X) objective inposition over the opening in the stage. Make sure the switch is in the off position andplug the power cord into a suitable grounded electrical outlet. Turn on the illuminator.4. Obtain a prepared microscope slide. Notice the label which describes the materialmounted on the center of the slide. Make a gross examination of the slide. (If the slideis dirty, clean it by rubbing lightly with a soft cloth or paper towel; do not use expensivelens paper for this purpose.)11
5. Secure the slide firmly in the mechanical stage. Be sure to wedge the slide BETWEENthe stage clips and NOT UNDERNEATH them. Rotate the coarse adjustment knob sothat the low-power objective is about one inch above the stage. While observing fromone side of the microscope (not through the ocular), adjust the slide so that theembedded material is in the approximate center of the opening. Make sure the side withthe cover slip is up. Move the low-power objective down as close to the cover glass aspossible without actually touching it.6. While looking through the eyepiece, move the body tube or stage by turning the coarseadjustment until you can see the mounted material clearly. If you do not see the material,re-center the slide and repeat the procedure. If you turn the coarse adjustment toorapidly you may go past the point of focus without realizing it.7. Bring the mounted material into the sharpest possible focus by turning the fineadjustment wheel about one quarter of a turn. Prepare the slide so the magnificationmay be increased by placing the material to be observed in the center of the field ofview. If the material is not in the center, it may be lost when switching to a higher powerbecause the field gets smaller.8. Adjust the interpupillary distance.a. Pull the oculars apart the maximum distance.b. While looking through the oculars slowly move the oculars together until the twoimages form into one.c. Pull the oculars apart until the images splits into two parts again. Return to thebest position. NOTE: Errors in this adjustment generally produce eye strain inone eye within 10 minutes.9. Adjust the diopter.a. With the interpupillary adjustment properly adjusted, the diopter adjustmentbecomes much easier. While the left eye is closed, carefully use the fine focus toprovide the best possible images to your right eye.b. Without touching the fine focus, open the left eye, close the right eye and rotatethe diopter adjustment slowly from one extreme to the other. Somewherebetween the extremes there will be a “best place”.c.
This laboratory manual has been developed to accompany the Biology II course. The coursework, lecture and lab, are designed to provide the student with a wide range of information about living organisms and systems. The experiments contained in this lab manual accompany the lecture information in such a way so as to illustrate and demonstrate.
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