Biology Lab

Lab 1 ? Introduction to the Microscopy & Observation of Prokaryotic and Eukaryotic Cells Introduction Many of the cells and organisms that you testament be studying argon at the depress limits of visibility of fall micro backcloths therefore, it is extremely important that you attain critical lighting and direction. It is also important to handle the microscope ably to avoid damaging either the microscope or the preparation you atomic number 18 studying. Even students who have previously used microscopesshouldreadtheinstructionsc arfully. GuideBiolabo Using a web rowser, go to the following web site http//salinella. bio. uottawa. ca/biolabo/ (you can try it from home). Under Microscopy you will find links to pages that describe both fount of microscopes you will use this semester, as well as how to set up and use them. It is strongly recommended that you visit these pages prior to attendingyourfirstlab. ImageJ/Qcapture Although you can contact all your reflexions by watching directly through the oculars, it also can be d peerless on the computer screen utilise the digital television camera attached to each microscope.For that, you will use the Image J program together with a capture plugin called Qcapture. Visit the lab website to go over how to use Image J (link on the homepage). All observations can be made on yourcomputer screen or in the oculars. Each manner has its advantages and drawbacks you will have to choose which oneitmoreappropriate(ortheoneyouprefer) Oculars Screen ? Greaterresolution ? Wider field of studyofview ? Canshareobservationwith separates ? Morecomfortableforusers ? Takepictureswhileobserving Lab1? Microscopy TheCompoundMicroscope On the Guide Biolabo page click on the CX41 Compound Microscope link then on Parts and Function. This will bring up a labelled line diagram of your microscope. Familiarize yourself with the various components shown in this figure. Then, click on Setup and adroit field alignment in graze to fill s tunnedhowtouseandhandlethemicroscope. Now, nail down your change microscope in the cupboard below the sink of your workstation. break through it on the counter between the omputer and the endofthecounter. Besurethatwheneveryoutransportthemicroscope,it is al airs kept upright the ocular genus Lens will fall out if the scope is tilted or swung. Even though you dont need the dissecting microscope right now, take it outofthecupboardandinstallitbesidethecompoundmicroscope. Connect one firewire cable to each of the cameras installed on top of the microscopes. Thisway,everythingissetupforfurtherobservationsbothon yourcomputerscreenandthroughtheoculars. PartsofthecompoundmicroscopeThe microscope consists of a brass of lens systemes, a light source, and a geared mechanism for adjusting the distance between the lens system and reject being chanced. There are a tot of important components and it is essential that you be able to identify them and understand their function beforeyoucanp roceed. Bygoingthroughthe polarmodulesinBiolabo and exploitation the microscope you will develop a competency for bright field microscopy. Identify the following components employ Biolabo (Parts and functions figure)andyourmicroscopeREVOLVINGNOSEPIECESupportsthevariousintentions? Youwillonlyuse the4x,10xand40x preyivesintheBIO1140labs(notthe100x). STAGE Supports the model being observed. A system of knobs on the side of the form allows you to move the specimen under the objective lens on theXandYaxes. Tryandmovethestage. COARSE FOCUS KNOB Permits rapid change in distance between the specimen and the objective thereby allowing for rough way Do not usewhenfocusingwiththe40xobjectiveFINE FOCUS KNOB Permits small changes in distance between the specimen and the objective and thereby allows for final focussing of the image. 10 Lab1? Microscopy OCULAR OR EYEPIECE A expatiateing element in the microscope, usually 10X. It is through the ocular, or eyepiece that one looks at the spe cimen. All our microscopes are parfocal, so that when an object is in focus with one objective, the focus will not be completely lost when changing to the nextobjective. OBJECTIVESThemagnifyingelementwhichis hand-to-handtothespecimen.See figure1tofindoutabouttheengravingsonthesideofeachobjective. CONDENSER System of lenses that concentrates the light furnished by the illuminator. Itdoesnotmagnifytheobject. CONDENSER HEIGHT ADJUSTMENT KNOB Allows one to focus the concentratedlightontothespecimen. APERTUREIRISDIAPHRAGM affairdtoreduceglarefromunwantedlightby adjustingtheangleoftheconeoflightthatcomesfromthecondenser ProductionofImagebyaCompoundMicroscope The most important part of a microscope is the objective.All the other partsoftheinstrumentaredesignedtohelptheobjectiveproducethebest possibleimage. Thebestimageisnotthe enlargedstitistheclearest. Thereis no pass judgment to a high overstatement. If the resolution is poor you will have no betterunderstandingofthespecimen. lightbeam ocularlens overstatement quantitativeaperture(NA) Determines the resolving poweroftheobjective* Optical tube length / max. coverslipthicknessinmm prism objectivelens specimen condenserlens Figure1Objectivesengravings lightsourceFigure2Imageproductioninacompoundmicroscope. 11 Lab1? Microscopy *Resolvingpoweristheabilitytoseedeuceobjectsthatareverycloseastwo separateobjects. Thehumaneyewillresolvingpowerisabout100m. Usingthecompoundmicroscope Always handle the microscope GENTLY It is an expensive, delicate and heavy instrument. Carry it with two hands, one hand on the arm, and the other hand under the base. If the ocular or objective is dirty, wipe it clean using ONLY Kimwipes or special lens tissue and cleaning fluid supplied.If you use anything else you may scratch the lens. cut through up any cleaning fluid immediately otherwise it will dissolve the glue which holds the lens in place. REMEMBER,yourdemonstratorisheretohelp,so beg 1. Make sure that the power cord is plugged into the back of your microscopeandintoapoweroutlet. 2. Usingtheletteremicroscope dislocateprovided,followsteps2through13 in the Setup and Bright field alignment procedure of Biolabo. Remember, observationcanbedoneonscreenorthroughtheoculars. Orientationand working(a)distance . Starting your examination with the 4X objective, position the letter e seashoreonthestage. 2. Drawwhatyouseeinthemicroscope_________________ 3. Whatwoulda mistakewiththelettertlook resemblingunderthemicroscope? _________________ 4. Usingtheknobslocatedonthesideofthestageandlookingthroughthe microscope, move the slide slowly to the right, then to the left. Record yourobservations. ___________________________________ 5. Now, move the slide slowly away from you, then towards you while observingthroughthemicroscope.Recordyourobservations ____________________________________ 6. Focusontheslideat10X. Checkthedistancebetweentheobjectivelens andyourslide(=theworkingdistance,seealsothereferenceattheendof this chapter). Now switch to the 40X objective and check the working distance. What happens to the working distance as your magnification increases? 12 Lab1? Microscopy Depthoffield( abstrusenessoffocus) Lenses have a depth of focus. It is the number of planes in which an object appears to be in focus.Extend your fist at arms length in front of you and hold your thumb up. Concentrate on your thumb and beak that the objectspastyourthumbontheothersideoftheroomarenotclearlyseen. Similarly with a microscope, when it is focussed on one surface, the surfaceslowerorhigherwillbeoutoffocus. 1. Position a prepared slide with coloured threads upon the stage. At low power,4X,focusontheareawherethethreadscross. 2. Usingthe handsomefocusadjustment,focusupand agglomerateslowly. 3. Repeat using different objectives.What can you say about the depth of field at different magnifications? Has it increased or decreased? (i. e. , can youseemorethreadsinonefocalplaneat4Xor40X? ) ________________________________________ ____________________ Magnification Themagnificationgivenbyobjectivesandocularsisengravedonthem. The total magnification for any combination of objective and ocular is the productofthemagnificationofeachlens. Objectivemagnification Ocularmagnification TotalMagnification Lightintensity Workingdistance 4x 10x 40x High 22mm 10x 10x 100xMedium 10. 5mm 40x 10x 400x Low 0. 56mm knock back1 . Comparison magnification, working distance and brightness at collar different objectivemagnifications. Youalsocancalculatethemagnificationofyourpictureusingthefollowing face Magnificationfactor=measured size of itofobject=(X) real(a)sizeofobject 13 Lab1? Microscopy Specimen size and Magnification of thepicture Before you start this exercise, make sure you have carefully read the website section relevant to the software you will use to take digital pictures(ImageJ/Qcapture).The goal of this section is to teach you different techniques that will allow you to go out the size of objects youre observin g under the microscope. The general principle is fairly simple 2 objects have the same sexual intercourse size (expressed as a ratio) in the real world and under the microscope. actualsizeofobjectA=on? screensizeofobjectA? A1=A2 actualsizeofobjectBon? screensizeofobjectBB1B2 Thefollowingexercisesareapplications ofthisformula. Placeaslideunder the microscope.Choose the right objective and adjust the focus and light level. Then,chooseastructureyouwanttomeasureandtakeapicture. A? commencement exercise method Measuring an object using the field of view(FOV) The simplest way to determine the size of an object is to use the known sizeofthewholefieldofview(FOV,thewholepicturefromlefttoright). 1? On the computer screen (using a ruler and without writing anything of thescreen),measuretheobjectofwhichyouwanttodeterminethesize(= A2) 2? Then,measurethe widthofthewholepictureonthescreen(=B2). ? Refer to table 2 on page 20 to know the actual size of the field of view fortheobjectiveyoureusing(= B1) 4? Usethefollowingformula Actualsizeoftheobject(A1)=ActualsizeoftheFOV(B1)xon? screensizeoftheobject(A2) on? screensizeoftheFOV(B2) Example On a snapshot using the 4x objective, an biting louse has an on? screen lengthof10cm. Thewholepictureis20cmwide. Whatistheactualsizeoftheinsect? ______________________________ 14 Lab1? Microscopy B? Second method Measuring an object using a ordered series barfileFrom Image J (using the file / open command), open the file that contains the relevant scale bar in the (T/BIO/BIO1140) new10X. jpg for the 10x objective,andnew40X. jpg(forthe4xand40xobjectives). Then, using a ruler measure the following distances directly on the computerscreen 1? The on? screen length (or width) of the object whose size you want to determine(=A2) 2? Thewidthofthescalebaronthescreen(=B2) Younowcancalculatetheactualsizeoftheobjectusingtheformula actualsizeofobject=on? creenlengthofobjectxactualsizeofscalebar* on? screenlengthofscalebar ?A1=A2xB1 B2 *The actual siz e of the scale bar is indicated on the scale bar file (ex on the new10x. jpg file,thebarrepresents0. 2mmat10xor0. 02mmat100x)=B1 ExampleItookapictureofasmallinsectlarva,usingthe4xobjective. The larva length is 60mm on the screen. The scale bar on the new40x. jpg is 30mmandrepresents0. 2mm. Whatistheactualsizeofthelarva? _________________________Donotputthecompoundmicroscopebackinthecupboardyouwillneedit laterthisafternoon. Pointstorememberconcerningmicroscopes 1. Always work with a clean microscope. Use only the lens base provided. Dont bartocleantheslidetoo 2. Always locate the specimen under low power and work your way up to thehighpowerobjective. 3. Never use the coarse focusing knob when the high power lens is in position. Useonlythefinefocusknob. 4. Neverusethe100xin1styearlabs(wedidntteachyouhow) 5.Always readjust illumination whenever you change the objective. Too muchlightwillgiveyouablurryimagethatyoucannotfocuson. 15 Lab1? Microscopy Thestereoscopicmicroscope (dissectingm icroscope) The stereoscopic microscope, also called stereoscope or dissecting microscope, is used to view objects that are too large or too thick to observeunderthecompoundmicroscope. Stereo microscopes are always equipped with two oculars producing a stereoscopic or three? dimensional image. Unlike the compound microscope,theimageisnotinverted.Our stereo microscopes provide magnification in the range of 6. 7X ? 45X using a zoom? type lens system. By rotating a dial located on the right side of the stereo microscope head, the viewer obtains a continuous change of magnification. Our stereo microscopes can be used with reflected or transmitted light. Reflected light is directed unto opaque specimens from above and is reflected to the viewer. Transmitted light is used with translucent specimens and passes through the specimen from beneath the stage and intotheviewerseyes.Useofthestereoscopicmicroscope 1. On the Biolabo home page left click on Stereoscope (Dissecting microscope)andtheno nStereoscopesetup. 2. ClickonStep1andreaditcarefully. nurseastereomicroscopefromthe samecupboardasyourcompoundmicroscopeifyouhaventyet. 3. Clickonandreadsteps2through7. 4. Placeacoinonthestage. 5. Using the focussing knob on either side of the arm, lower or raise the objective until the coin is in focus. Examine it in both reflected and transmittedlight.Which is best for an opaque specimen? Try the various magnifications by traveling the zoom knob. The reflected light source is sympathetic to a spotlight anditsorientationcanbeadjustedmanually. Tryrotatingthelightupwards anddownwards. 6. Examineothermaterialssuchasbrine shrimplarvae(Artemia)inawatch glass using both reflected and transmitted light. Add 1? 2 drops of proto? slow solution to slow down the larvae. Estimate the actual size of one larva__________ 16 Lab1? Microscopy ProkaryoticandEukaryoticcellsIt has long been recognized that living organisms are composed of basic structural and running(a) units called cells. Cells c an be divided into two general types prokaryotic and eukaryotic, based on the presence of a nucleusandothermembraneboundorganellesinthelatter. Prokaryotic cells belong to 2 big groups archaea and eubacteria. They are usually smaller than eukaryotic cells (typically 1? 5m). These unicellular organismsmaybesmall,buttheyarethemostabundantorganismsonthe planet, representing about half the biomass (Biology, Brooker et al. 010, McGraw? Hill&Ryerson). They are devoid of membrane bound organelle such as the nucleus, mitochondria or chloroplasts. Their inheritable material is usually composed of one circular chromosome plus other extra chromosomalelementscalledplasmids. Eukaryotic cells are usually much larger. They possess a membrane bound nucleus, their organelles are more complex and numerous, and their genome is larger than prokaryotes. Eukaryotic organisms can be uni? or multicellular. You will have a chance to observe many eukaryotic cells duringthissemesterAmoeba,Lilly,Whitefish.In t odays exercise you will take a first look at the similarities and differences between prokaryotic and eukaryotic cells as well as the diversitywithinthesegroups. You should familiarize yourselves with a whole array of cellular structures and organelles you will probably encounter during the course of this exercise. Before your scheduled lab session, hold open down the definition and functionforeachofthefollowingtermsplasma(cell)membrane,cellwall, protoplast,cytoplasm,vacuoles,nucleus,nucleolusandchloroplasts.EukaryoticCellsElodea(plant) 1? Get a young young Elodea leaf from the jar. Mount it in a drop of water on a clean microscope slide with the convex side of the leaf uppermost. shroudthepreparationwithacoverslip. 2? Observe the preparation at 4X, then at 10X. If you see brownish oval structures on the leaf surface, ignore then. These are probably epiphytic diatoms. Concentrateyourattentiononthecellsnearthecentralribatthe baseoftheleafandonthemarginalcellsatthe go onoftheleaf. Canyoudistinguishseverallayersmakinguptheleaf? ____ ? What is the average length ______ and width ______ of the cells in micrometres? 17 Lab1? Microscopy 3? Focussingat40Xlocatethecellwall,thevacuole,thecytoplasmandthe numerousgreenchloroplasts. ? What important biological process takes place in the chloroplasts? _____________________________________ ? Whatpigmentisresponsiblefortheirgreencolouration? ________________________________________________ ? Whatistheshapeofchloroplasts? ____________________________________________ ? atomic number 18thechloroplastsmoving? Whatsortofmovement? _________________________________________________ ? Thephenomenonyouareobservingiscalledcytoplasmicstreaming or cyclosis. What do you think the function of such a process could be? ___________________________________________________ 4? You have probably realised that the plasma membrane cannot be seen in plant cells. It is too thin to be resolved with the compound microscope.In order to see the true l imiting boundary of the cytoplasm it is necessary to treat the cells in such a manner that the plasma membrane becomes withdrawn away from the rigid cell wall. This can be done by placing the cell in a strong salt solution. This will cause water to turn out out of the cell by osmosis, thereby decreasing the cell volume. The unaffected cell wall remainsinitsoriginalstate. Whatcanthenbeseenisaspacebetweenthe cellwallandthelimitingboundaryoftheprotoplast(thecellminusthecell wall)whichtherebybecomesvisible. Remove your Elodea slide from the microscope stage. Delicately remove the coverslip, add one drop of 5% NaCl solution then put backthecoversliponyourpreparation ? Refocus at 40x (dont forget you must first focus at 4X, then 10X andfinallyat40x). ? Are the cells plasmolyzed? (If not wait a while longer). How do they looklikenow? ______________________ ? Hasthecellwallbeenaffected? _________________ ? What becomes of the large central vacuole during plasmolysis? ______________________ _______________________________ TakeapictureofaplasmolyzedElodeacell. Howdoesitcompareto thepreviouspicture? 18 Lab1? Microscopy ProkaryoticCellsLyngbya(eubacteriacyanobacteria) 1. Take a close look at the sample in the jar. Which colour would best describeitsappearance? ___________________ 2. Preparea cockeyedmountoffreshLyngbyabythefollowingprocedure ? With forceps or an eye dropper, put a very small amount of green matteronacleanslide ? Addadropofwaterfromthejar. ? Carefully place a coverslip over it. Make sure it lies flat on the preparation.Dont worry if there are just a few air bubbles. With practice, your skills will improve. However, if too many air bubbles are present, your preparation risks to dry out very quickly during viewing,compromisingyourobservations. 3. Startingwiththe4Xobjective,focusonyourpreparation. ? Canyouseenumerousgreenfilaments? _______ ? Arethefilamentsmoving? __________ 4. Switchtothe10Xthenthe40Xobjectiveandfocususingthefinefocus knobonly ? Doyouseethei ndividualcellsmakingupeachfilament? ________ ? Estimatethewidthofonefilamentinmicrometres_______ Whatsthefilamentwidthin millimetres(mm)? ________ ? REMEMBER You are working with living cells. Work quickly and keep your specimen wet at all times. Dead, dry or damaged biologicalpreparationsareuseless. Returningthemicroscopesafteruse After completing all observations, turn and click the low power objective (4X)onthecompoundmicroscopeintoposition. Removetheslidefromthestageandreturnittoitscorrectbox. Wipethestageswithacleanpapertowel. Carefullydisconnectthecamerafromthefirewirecable.Make sure you turned off the light on each microscope, then unplug the powercordandmakealoosecoilofitaroundtheeyepieces. Returnthemicroscopeinthecupboard. 19 Lab1? Microscopy TAs will check that you properly returned the microscopes in the cupboard withthecordproperlyattachedandnoslidepresentonthe stage. You will losemarksforthislab(andotherlabs)ifyoudontdoso. Evaluation A short essay on microscope compone nts, specimen observations and measurementofobjectswilltakeplaceatthebeginningofLab2.Beontime,thequizwillstartat230. References 1? Metricsystem(seealsoappendixIVattheendoflabmanual) 1centimetrecm=10? 2metres(m) 1millimetremm=10? 3metres 1micrometre? m=10? 6metres 1nanometrenm=10? 9metres 2? Sizeofcamerafieldofviews(fov) Table2FieldsofViewOlympusCX41CompoundMicroscope Objective 4X 10X 40X 100X Camerafieldofview (widthinmm) 1. 75 0. 70 0. 175 0. 070 Table3FieldsofViewOlympusSZ61TRDissectingMicroscope ZoomSetting 0. 67X 0. 8X 1X