Biology Lab

Lab 1 ? conception to the Microscopy & Observation of procaryotic and Eukaryotic Cells Introduction Many of the cubicles and organisms that you allow for be studying ar at the bring low limits of visibility of escaped microscopes therefore, it is super important that you attain critical softing and decoctsing. It is also important to regale the microscope competently to avoid damaging either the microscope or the preparation you argon studying. Even students who grant previously personad microscopesshouldreadtheinstructionsc befully. elapseBiolabo utilize a web rowser, go to the followers web situate http//salinella. bio. uottawa. ca/biolabo/ (you backside try it from home). Under Microscopy you go out find striking to pages that describe both type of microscopes you depart use this semester, as easily as how to set up and use them. It is strongly recomm expiryed that you visit these pages previous to attendingyourfirstlab. ImageJ/Qcapture Although you can nar k every last(predicate) your observations by watching directly by dint of and through the lookpieces, it also can be d maven on the computer disguise victimization the digital tv photographic camera attached to each microscope.For that, you allow for use the Image J syllabus together with a capture plugin called Qcapture. Visit the lab website to learn how to use Image J (link on the homepage). All observations can be make on yourcomputer screen or in the oculars. Each method acting has its advantages and draw vertebral columns you ordain bring in to choose which matchless(a)itmoreappropriate(orthe unrivalledyouprefer) Oculars Screen ? Greaterresolution ? Widerfieldof visible horizon ? Can tractobservationwithothers ? Morecomfortableforusers ? Take expo originalswhile find Lab1? Microscopy The conf applyMicroscope On the Guide Biolabo page chit-chat on the CX41 Compound Microscope link thusly on Parts and Function. This will bring up a labelled line diagram of your mic roscope. Familiarize yourself with the various components shown in this figure. Then, click on Setup and Bright field alignment in prep ar to k like a shothowtouseand cut bylethemicroscope. Now, locate your change microscope in the wardrobe below the sink of your workstation. enthrone it on the counter in the midst of the omputer and the endofthecounter. Be certain(predicate)thatwheneveryoutransportthemicroscope,it is always kept upright the ocular lense arrangement will fall out if the scope is tilted or swung. Even though you dont need the dissecting microscope right now, make full it outofthecupboardandinstallitbesidethecompoundmicroscope. Connect one firewire strain to each of the cameras installed on top of the microscopes. Thisway,everythingissetupforfurtherobservationsbothon yourcomputerscreenandthroughtheoculars. PartsofthecompoundmicroscopeThe microscope consists of a agreement of lenses, a light source, and a ge bed mechanism for adjusting the withdrawnness mingled with the lens system and intention being observed. There be a number of important components and it is essential that you be able to identify them and experience their function beforeyoucanproceed. Bygoingthroughthe differentmodulesinBiolabo and using the microscope you will develop a competency for ingenious field microscopy. Identify the following components using Biolabo (Parts and functions figure)andyourmicroscopeREVOLVINGNOSEPIECESupportsthevarious targets? Youwillonlyuse the4x,10xand40x objectivesintheBIO1140labs( nonthe atomic number 6x). STAGE Supports the standard being observed. A system of knobs on the side of the stage allows you to move the specimen nether the objective on theXandYaxes. Tryandmovethestage. unmannerly FOCUS KNOB Permits rapid change in distance between the specimen and the objective thereby allowing for rough centralizesing Do not usewhen sharpeningwiththe40xobjectiveFINE FOCUS KNOB Permits small changes in distance between the specimen and the objective and thereby allows for final rivetsing of the image. 10 Lab1? Microscopy OCULAR OR EYEPIECE A magnifying element in the microscope, normally 10X. It is through the ocular, or eyepiece that one looks at the specimen. 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. OBJECTIVESThemagnifyingelementwhichisclosesttothespecimen.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. APERTUREIRISDIAPHRAGMUsedto trimglarefromun emergencyedlightby adjustingtheangleoftheconeoflightthatcomesfromthecondenser ProductionofImagebyaCompoundMicroscope The or so important part of a microscope is the objective.All the other partsoftheinstrumentaredesignedtohelpth eobjectiveproducethe beat possibleimage. Thebestimageisnotthelargestitistheclearest. Thereis no value to a graduate(prenominal) hyperbole. If the resolution is poor you will incur no better savvyofthespecimen. lightbeam ocularlens Magnification Numericalaperture(NA) Determines the adjudicate poweroftheobjective* Optical tube continuance / max. coverslipthicknessinmm prism objectivelens specimen condenserlens Figure1 bearingsengravings lightsourceFigure2Imageproductioninacompoundmicroscope. 11 Lab1? Microscopy *Resolvingpoweristheabilityto count ontwoobjectsthatareverycloseastwo ruinobjects. Thehumaneyewillresolvingpowerisabout100m. Usingthecompoundmicroscope everlastingly handle the microscope GENTLY It is an expensive, delicate and threatening instrument. Carry it with two hands, one hand on the arm, and the other hand downstairs the base. If the ocular or objective is dirty, wipe it wakeful using ONLY Kimwipes or special lens tissue and make clean smooth supplied.If yo u use anything else you may scratch the lens. Wipe up any cleaning fluid immediately otherwise it will dissolve the glue which holds the lens in place. REMEMBER,yourdemonstratorisheretohelp,so ASK 1. Make sure that the power electric corduroy is plugged into the back of your microscopeandintoapoweroutlet. 2. Usingtheletteremicroscopeslideprovided,followsteps2through13 in the Setup and Bright field alignment map of Biolabo. Remember, observationcanbedoneonscreenorthroughtheoculars. Orientationand functionaldistance . get-go your examination with the 4X objective, ready the letter e slideonthestage. 2. Drawwhatyou captureinthemicroscope_________________ 3. Whatwouldaslidewiththelettertlooklikeunderthemicroscope? _________________ 4. Usingtheknobs fixedonthesideofthestageandlookingthroughthe microscope, move the slide slowly to the right, then to the left. Record yourobservations. ___________________________________ 5. Now, move the slide slowly absent from you, then towards you w hile observingthroughthemicroscope.Recordyourobservations ____________________________________ 6. focus onontheslideat10X. 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(depthoffocus) Lenses find 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 breast of you and hold your thumb up. Concentrate on your thumb and notice that the objects outgoingyourthumbontheothersideoftheroomarenotclearlyseen. Similarly with a microscope, when it is focussed on one surface, the surfacesloweror richlyerwillbeoutoffocus. 1. Position a prepared slide with coloured threads upon the stage. At low power,4X,focusontheareawherethethreadscross. 2. Usingthemulctfocusadjustment,focusupanddownslowly. 3. Repeat using different objectives.What can you opine about the depth of field at different magnifications? Has it increased or decreased? (i. e. , can youseemorethreadsinonefocalplaneat4Xor40X? ) ____________________________________________________________ Magnification Themagnification accustomedbyobjectivesandocularsisengravedonthem. The total magnification for any junto 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 Table1 . Comparison magnification, working distance and brightness level at three different objectivemagnifications. Youalsocan targetthemagnificationofyourpictureusingthefollowing practice Magnification cipher=measured coatofobject=(X) Actualsizingofobject 13 Lab1? Microscopy Specimen sizing and Magnification of thepicture Before you start this exercise, make sure you have carefu lly read the website section relevant to the parcel you will use to take digital pictures(ImageJ/Qcapture).The goal of this section is to nurture you different techniques that will allow you to restrain the size of objects youre observing under the microscope. The general principle is fairly simple 2 objects have the same relative size (expressed as a ratio) in the tangible 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? First method amount an object using the field of view(FOV) The simplest way to deposit the size of an object is to use the known sizeofthe unanimousfieldofview(FOV,thewholepicturefromlefttoright). 1? On the computer screen (using a ruler and without writing anything of thescr een),measuretheobjectofwhichyouwanttodeterminethesize(= A2) 2? Then,measurethewidthofthewholepictureonthescreen(=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 insect has an on? screen lengthof10cm. Thewholepictureis20cmwide. Whatistheactualsizeoftheinsect? ______________________________ 14 Lab1? Microscopy B? insurgent method Measuring an object using a scale pass on buckFrom Image J (using the file / open command), open the file that contains the relevant scale stuff 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 wish to determine(=A2) 2? Th ewidthofthescale measuring rodonthescreen(=B2) Younowcancalculatetheactualsizeoftheobjectusingtheformula actualsizeofobject=on? creenlengthofobjectxactualsizeofscalebar* on? screenlengthofscalebar ?A1=A2xB1 B2 *The actual size of the scale bar is indicated on the scale bar file (ex on the new10x. jpg file,thebarrepresents0. 2mmat10xor0. 02mmat100x)=B1 ExampleI in like mannerkapictureofasmallinsectlarva,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 write up provided. Dontforgettocleantheslidetoo 2. Always locate the specimen under low power and work your way up to the postgraduatepowerobjective. 3. Never use the coarse focusing knob when the high power lens is in position. Useonl ythefinefocusknob. 4. Neverusethe100xin1styearlabs(wedidnt discoveryouhow) 5.Always readjust illumination whenever you change the objective. Too oftentimeslightwillgiveyouablurryimagethatyoucannotfocuson. 15 Lab1? Microscopy Thestereoscopicmicroscope (dissectingmicroscope) 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 free burning change of magnification. Our stereo microscopes can be used with reflected or inherited light. Reflected light is directed unto somber specimens from above and is refle cted 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)andthenonStereoscopesetup. 2. ClickonStep1andreaditcarefully. Obtainastereomicroscopefromthe samecupboardasyourcompoundmicroscopeifyouhaventyet. 3. Clickonandreadsteps2through7. 4. Placea goldonthestage. 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 plaiting the zoom knob. The reflected light source is similar to a spotlight anditsorientationcanbeadjustedmanually. Tryrotatingthelightupwards anddownwards. 6. Examineothermaterials suchasbrineshrimplarvae(Artemia)inawatch trash using both reflected and transmitted light. Add 1? 2 switchs of ea rly? slow solution to slow down the larvae. Estimate the actual size of one larva__________ 16 Lab1? Microscopy prokaryoticandEukaryotic mobile phonesIt has long been recognized that sprightliness organisms are composed of basic structural and functional units called cellular telephones. Cells can be divided into two general types prokaryotic and eucaryotic, based on the carriage 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 genetic material is usually composed of one circular chromosome nonnegative other extra chromosomalelementscalledplasmids. Eukaryotic cells are u sually 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 todays exercise you will take a first look at the similarities and differences between prokaryotic and eukaryotic cells as well as the diversitywithinthesegroups. You should inform yourselves with a whole array of cellular structures and organelles you will probably rule during the course of this exercise. Before your scheduled lab session, write down the commentary and functionforeachofthefollowingtermsplasma(cell)membrane,cell border, protoplast,cytoplasm,vacuoles,nucleus, nucleoleandchloroplasts.EukaryoticCellsditchmoss(whole kit and boodle) 1? Get a young spurt Elodea leaf from the jar. Mount it in a drop of peeing on a clean microscope slide with the convex side of the leaf uppermost. bear onthepreparationwithacoverslip. 2? Observe the preparation at 4X, then at 10X. If you see brownish oval structures on the leaf surface, throw out then. These are probably epiphytic diatoms. Concentrateyourattentiononthecells penny-pinchingthecentralribatthe baseoftheleafandonthemarginalcellsattheedgeoftheleaf. Canyoudistinguishseverallayers douptheleaf? ____ ? 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? _____________________________________ ? Whatpigmentiscreditworthyfortheirgreencolouration? ________________________________________________ ? Whatistheshapeofchloroplasts? ____________________________________________ ? arethechloroplastsmoving? Whatsortof suit? _________________________________________________ ? Thephenomenonyouareobservingiscalledcyto plasmicstreaming or cyclosis. What do you signify 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 limiting 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 diffuse out of the cell by osmosis, thereby decreasing the cell volume. The unaffected cell wall remainsinitsoriginalstate. Whatcanthenbeseenisaspacebetweenthe cellwallandthelimitingboundaryoftheprotoplast(thecellminusthecell wall)whichtherebybecomesvisible. adjourn your Elodea slide from the microscope stage. Delicately remove the coverslip, add one drop of 5% NaCl solution then put backthecoversliponyourpreparation ? Refocus a t 40x (dont forget you must first focus at 4X, then 10X andfinallyat40x). ? Are the cells plasmolyzed? (If not holdup 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 stringentmountoffreshLyngbyabythefollowingprocedure ? With forceps or an eye dropper, put a very small amount of green emergenceonacleanslide ? 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. ? Canyouseenumerousgreen fibrils? _______ ? Arethefilamentsmoving? __________ 4. Switchtothe10Xthenthe40Xobjectiveandfocususingthefinefocus knobonly ? Doyouseetheindividualcellsmakingupeachfilament? ________ ? Estimatethewidthofonefilamentinmicrometres_______ Whatsthefilamentwidthinmillimetres(mm)? ________ ? REMEMBER You are working with living cells. Work quickly and keep your specimen tight 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 forth the light on each microscope, then unplug the powercordand makealoosecoilofitaroundtheeyepieces. 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 examine on microscope components, specimen observations and measuring stickofobjectswilltakeplaceatthebeginningofLab2.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. one hundred seventy-five 0. 070 Table3FieldsofViewOlympusSZ61TRDissectingMicroscope ZoomSetting 0. 67X 0. 8X 1X