Pipetting 101 Developed by BSU CityLab

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1 Discver the Micrbes Within: The Wlbachia Prject Pipetting 101 Develped by BSU CityLab Clr Cmparisns Pipetting Exercise #1 STUDENT OBJECTIVES Students will be able t: Chse the crrect size micrpipette fr the vlume f liquid t be mved Demnstrate hw t crrectly set the vlume n different sizes f micrpipettes Demnstrate the sequence f steps fr using a micrpipette t draw up liquid frm ne cntainer and transfer it t anther Demnstrate the prper use (and care fr) these finely-tuned instruments BACKGROUND INFORMATION Micrpipettes are used t accurately measure micrliter vlumes f liquid. The prefix micr means millinth, and ne micrliter is ne millinth f a Liter. In decimal ntatin, ne micrliter is L. In scientific ntatin, ne micrliter is 1 x A micrliter is abbreviated with the symbl!l, which cmes frm the Greek letter! (prnunced mew ). When thinking abut a micrliter, it may be helpful t first cnsider a milliliter (ml). A typical transfer pipette, when filled t the base f the bulb as in the diagram, will hld abut 1.0 ml f liquid. One thusand micrliters will ccupy the same ne milliliter space! Small plastic tubes with attached caps, called micrcentrifuge tubes (r micrfuge tubes) are used in this activity, and they hld nly abut 1.5 ml (r 1,500 micrliters). In fact, the largest micrpipette hlds 1,000!L f liquid a single milliliter! When yu use a measuring instrument, yu are cmparing an unknwn quantity f smething with a knwn quantity, called a standard. In the case f micrpipettes, the standard is the micrliter. If yu draw up 350!L f liquid in a micrpipette, the instrument will cntain a precise amunt indicated with a number (350) and a unit (!L). Measurements with numbers are described as quantitative measurements. A transfer pipette, filled t the base f the bulb (as indicated by the arrw), hlds abut ne milliliter f liquid (1.0 ml).. Pipetting 101: Clr Cmparisns

2 Sme standards are less specific and cannt be assigned a number value. Fr example, if yu have fur identical cntainers f water, and each cntains a different number f drps f blue fd clring, it might nt be bvius which blue is darkest in clr, which is the lightest, and where the ther tw fit in between them. Measurements like these are mre subjective (in the eye f the bserver) and are called qualitative measurements. Yu have been intrduced t the basic peratin f the adjustable micrpipettes, and nw it is time t becme cmfrtable using them. Refer ften t the diagrams and details in the handuts prvided by the teacher. In this activity, yu will use micrpipettes t measured fd-clred water. The first step is t make three new slutins accrding t the cncentratins given in Table #1 f the prtcl. The lab prtcl is simply the step-by-step instructins yu will fllw (the written prtcl has small bxes t be checked ff as yu cmplete each step). The three new slutins are the clr standards yu will use fr cmparisn in the secnd step. Fllwing the cncentratins prvided in Table #2, yu will pipette different amunts f liquids frm the riginal cntainers t make five mre clr cmbinatins and then cmpare them t the three riginal standards. Clr Standards As yu wrk, keep in mind any surces f errr yu encunter alng the way. When carrying ut experiments, scientists always strive t perfrm each step in exactly the same way every time they d that step. Any change in the prtcl, r a change in technique, is cnsidered a variable smething that culd change the utcme f the experiment. Whenever yu use a measuring instrument, sme degree f variability is assumed. Fr example, if yu are asked t measure ut a cup f water, and then a classmate is asked t use the same measuring cup t d the same task, the chances are gd that yu will NOT measure ut the EXACT same amunt f water as yur classmate! Variability describes the range f pssible values r amunts f smething, and all measuring instruments have variability (althugh the variability range in a micrpipette is quite small). Micrcentrifuge Tube Pipetting 101: Clr Cmparisns

3 Discver the Micrbes Within: The Wlbachia Prject Pipetting 101 Clr Cmparisns Prtcl Name: Date: MATERIALS 200!L Micrpipette 1000!L Micrpipette Bx f Micrpipette tips fr 200!L Micrpipette Bx f Micrpipette tips fr 1000!L Micrpipette Used tip dispsal cntainer Waste liquid dispsal cntainer Micrcentrifuge tube rack cntaining: Eight, empty 1.5 ml micrcentrifuge tubes with attached caps Test Tube r Centrifuge rack with: One capped test tube r cnical centrifuge tube cntaining 15 ml blue dye One capped test tube r cnical centrifuge tube cntaining 15 ml red dye One capped test tube r cnical centrifuge tube cntaining 15 ml yellw dye Permanent marker Student Handuts, ptinal Safety gggles fr each student Glves fr each student PROCEDURE Step #1 Take three f the micrcentrifuge tubes (with attached caps), and label them #1, #2, and #3 with the permanent marker. Set them aside in the micrcentrifuge tube rack. D the same with yur ther five micrcentrifuge tubes, but label them A, B, C, D and E. Read the next fur items in the prtcl befre ding anything else! Create yur reference tubes, r standards, by cmbining the liquids accrding t the amunts given in Table #1 belw. Fr example, t make standard #1, yu will mve 100!L f blue dye int micrcentrifuge tube #1. Then change the micrpipette tip and mve 100!L f yellw dye int micrcentrifuge tube #1. Snap the cap shut and lightly tap tube #1 n the table t mix the tw clrs. Returnthemicrtubet therack. Write yur results in the third clumn f Table #1. Pipetting 101: Clr Cmparisns

4 Make sure that all members f yur grup take turns cmpleting the exercises. Things t remember each time yu use the micrpipette: First, determine which style and size micrpipette t use! Secnd, check r change the vlume setting! Third, use a new tip fr each new clr! Table #1, Clr Cmparisns, Making Standards STANDARD COMBINE # #1 100!L blue dye 100!L yellw dye #2 100!L red dye 100!L yellw dye #3 100!L red dye 100!L blue dye RESULTINGCOLOR Step #2 Read these instructins befre beginning: Cmbine liquids accrding t the amunts given in Table #2 t make five (A thrugh E) new clr cmbinatins. Use the same prtcls yu fllwed t create standards #1 thrugh #3 in Step #1. Make sure that all members f the grup take turns cmpleting the exercises. Write yur bservatins in the tw clumns n the right side f the table. The last clumn asks yu t cmpare the clr f each new liquid t the crrespnding standard frm Step #1. Fr example, if yu create a green liquid in Tube A, cmpare its intensity with the green in Tube #1, and make ntes abut whether it is lighter, darker, r the same shade as the standard. Pipetting 101: Clr Cmparisns

5 Discver the Micrbes Within: The Wlbachia Prject Table #2, Clr Cmparisns, New Cmbinatins TUBE COMBINE RESULTING COLOR COMPARE TO STANDARD A 50!L blue dye 50!L yellw dye #1, (Fr example: lighter than, darker than, same as ) B 37!L red dye 37!L yellw dye #2, C 658!L red dye 658!L blue dye #3, D 100!L yellw dye 25 ul red dye (4 times) #2, E 25!L red dye 25!L yellw dye (4 times) (4 times) #2, Ntes: Pipetting 101: Clr Cmparisns

6 OBSERVATIONS 1. In Table #1, what was the ttal vlume f each standard yu made? 2. What were the vlume prprtins f the tw clrs (what was their rati) in each standard? 3. In Table #2, what vlume prprtins f each clr were used t create each new slutin (A-E)? 4. Can yu suggest three r mre variables (surces f errr) that may accunt fr yur bservatins in the last clumn? REVIEW QUESTIONS 1. What fractin f a Liter is a micrliter? 2. If the windw n the 20!L micrpipette shws the numbers t the right, what vlume f sample will be delivered? 3. Why did yu change micrpipette tips each time yu changed sample clr? Pipetting 101: Clr Cmparisns

7 Discver the Micrbes Within: The Wlbachia Prject Pipetting 101 A Slutin fr the Unknwn Slutins: Starch and Idine Assay Prtcl Name: Date: MATERIALS per Grup 200!L Micrpipette Bx f Micrpipette tips fr 200!L Micrpipette Used tip dispsal cntainer Waste liquid dispsal cntainer Micrcentrifuge tube rack cntaining: Six t eight empty, unlabeled 1.5 ml micrcentrifuge tubes with attached caps One 1.5 mlcapped micrcentrifugetube, labeled starch One 1.5 ml capped micrcentrifuge tube, labeled idine One 1.5 mlcapped micrcentrifugetube, labeled W One 1.5 ml capped micrcentrifuge tube, labeled X One 1.5 ml capped micrcentrifuge tube, labeled Y One 1.5 ml capped micrcentrifuge tube, labeled Z Permanent marker StudentHanduts,ptinal Safety gggles fr each student Glves fr each student PROCEDURE Observe the micrcentrifuge tubes (in the rack) that cntain liquids. One tube cntains starch. The ther knwn tube cntains idine. The tubes are labeled, s yu knw what is in them. The ther fur tubes cntaining liquids are labeled W, X, Y and Z. They are cnsidered the unknwns, because yu d nt knw exactly what is in them. Yu d knw this, hwever: they cntain starch r idine r clred water r plain water. Using the marker, label ne empty micrcentrifuge tube as the Standard Observe the data table called A Slutin fr the Unknwn Slutins. The first rw f the table suggests that yu will use a micrpipette t cmbine 50!L f starch and 50!L f idine in the tube yu labeled as the Standard. A Slutin fr the Unknwn Slutins: Starch and Idine Assay

8 Select the apprpriate size micrpipette, set the vlume and affix a clean tip. Then g ahead and cmbine 50!L f starch and 50!L idine int the Standard tube. Observe the resulting clr and write it in the table. Read the fllwing items befre cntinuing: As yu can see, starch and idine give a distinct clr change when mixed. This clr is nw the standard, r indicatr. It tells yu that the liquid in the tube cntains bth starch and idine. Yur task is t perfrm a series f trials t figure ut what liquids are in the unknwn tubes W, X, Y and Z. They may be starch r idine r distilled water r clred water. T slve the mystery, yu will cmbine 50!L samples frm each f tw different tubes int clean, empty tubes. Be sure t label each new tube and write n the data table which tw samples yu mixed tgether. Recrd the clr f each new mixture and cmpare yur results with the standard. Test as many different cmbinatins f tw liquids as yu need t in rder t figure ut the fur unknwns. There is n set number f trials yu must run because it depends n yur prcedure. There are at least tw pssible ways t perfrm the assay, s dn t be alarmed if anther grup is ding smething different. Yu may nt need t use all the empty micrcentrifuge tubes yu have been given nr all the blank rws n the data table. Yu may even discver that yu need mre than the number f tubes and rws prvided (in this case, just ask yur teacher fr assistance). Remember, all micrpipetting will be dne in 50!L vlumes. Use yur best micrpipetting technique, and be sure t use a fresh tip each time yu pipette frm a different slutin. When yu have figured ut what slutins are in tubes W, X, Y and Z, write their names in the cnclusins sectin belw the data table. A Slutin fr the Unknwn Slutins: Starch and Idine Assays

9 Discver the Micrbes Within: The Wlbachia Prject A Slutin fr the Unknwn Slutins Data Table TUBE LABEL COMBINE RESULTINGCOLOR Standard 50!L Starch, 50!L Idine, Trial #1 Trial #2 Trial #3 Trial #4 Trial #5 Trial #6 Trial #7 Trial #8 Trial #9 Trial #10 Trial #11 Trial #12 Trial #13 Trial #14 A Slutin fr the Unknwn Slutins: Starch and Idine Assay

10 CONCLUSIONS W= X = Y = Z = THOUGHT QUESTIONS 1. Did the mixing f starch and idine cause a physical r chemical reactin? Hw d yu knw? 2. Anther wrd fr a test is an assay. The starch-idine assay is cmmnly used in chemistry and bilgy. What might be yur definitin f it? 3. Describe in detail hw yu carried ut yur assay. What strategies did yu use t determine the unknwns? Hw did the standard help yu? 4. Hw psitive d yu feel abut yur cnclusins? Describe any pssible errrs r variables in yur assay. A Slutin fr the Unknwn Slutins: Starch and Idine Assays