A. The Metric System (this section can be completed before or during lab)

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1 Lab I: INTRODUCTION TO MEASURING TECHNIQUES The basis for obtaining scientific knowledge is systematic observation and experimentation. A key component of most scientific investigations is measurement. By using a common system of units of measurement and by making precise and accurate measurements, scientists ensure that procedures and results can be reported and repeated anywhere in the world. The two systems of measurement used by scientists are the English system and the Metric system. However, as you are probably aware, most scientific data is reported in metric units. It is essential that students be familiar and competent in identifying the units of the metric system, making conversions among metric units, and making conversions between English and Metric units. Throughout your undergraduate career you will be asked numerous times to measure mass and volume. Thus, familiarity with devices routinely used to measure mass and volume are essential for the successful completion of a number of laboratory exercises. A measurement of mass is obtained using a balance or scale, and the method is straightforward. However, various devices are available for the measurement of volume. These include various kinds of pipets, graduated cylinders, and volumetric flasks. It is important to know which device is most appropriate for a given situation. Your efficiency, accuracy and precision in making such measurements will improve as you gain experience. To prepare you for future Biology courses, you will complete a set of simple introductory exercises that will familiarize you with the metric system, conversions between English and metric systems, measurement of mass and volume, and application of precision and accuracy to measurements. A. The Metric System (this section can be completed before or during lab) In the metric system, the basic units are the meter (m) for length, the liter, (l or L) for volume, and the gram (g) for mass. Prefixes used with the basic metric system units indicate either a fraction of a unit (e.g is represented by the prefix centi-) or a multiple of a unit (e.g is represented by the prefix kilo-). As you probably know, metric prefixes always express a power of 10 by which the basic unit (either meter, liter or gram) has been multiplied. For example, the prefix nano- means "billionth" or Thus, a nanometer is 10-9 meters or meter. Table 1 provides commonly used prefixes.

2 Table 1 Prefixes Commonly Used With Basic Metric Units Prefix Meaning Size Relative to the Base Unit Length Volume Mass Nano- Billionth 10-9 Nanometer(nm) Nanoliter(nl) Nanogram(ng) Micro- Millionth 10-6 Micrometer(µm) Microliter(µl) Microgram(µg) Milli- Thousandth 10-3 mm ml mg Centi- Hundredth 10-2 cm Kilo- Thousand 10 3 km kg Mega- Million 10 6 Giga- billion 10 9 The real challenge for most students begins when they are asked to compare metric units with English units. You would have no problem envisioning an 8 ounce cup of water, but what is the metric equivalent? On the same hand, can you envision the amount of water equivalent to 500 ml? Being fluent with the metric system and conversion between metric and English units is essential to a biologist. 1. Convert the following measurements from one unit of metric measurement to another as indicated (refer to Table 1): 1 cm = m 1 mm = nm 1 cm = mm 50 µm = cm 20 µl = ml 15 l = ml 10 g = µg 20 mg = µg 2. If you were dieting, would you rather lose 10 pounds or 5 kilograms? (show work and provide reasoning)

3 3. When measuring mass, length, or volume of objects, scientists may elect to write the number in scientific notation. A number is expressed in scientific notation when it is written as a product of a decimal number between 1 and 9 and the number of 10 raised to the proper power. For example: 1000 = 1 x ,000,000,000 = 5.68 x = 4.4 x 10-7 When a number is larger than 10, the decimal point must be moved left. When the decimal point is moved left, the power of 10 is positive and equal to the number of decimal places moved. When the number is smaller than 1, the decimal point must be moved right, and the power of 10 is negative and equal to the number of decimal places moved. Convert the numbers in the following table to scientific notation, and then convert to the metric units indicated in the second column. Scientific Notation Metric Unit conversion g l 2,323,000 m 10 µg g l m µg mg ml µm kg B. Measuring Length and Mass Students often ask how many decimal places a given measurement can include. Numbers that are experimentally measured are not exact because of small errors or uncertainties associated with the laboratory measurement. Scientists indicate these uncertainties by using an appropriate number of significant digits. A significant digit is one whose value is reasonably reliable. This is accomplished by recording all digits that are known with certainty, and then adding one additional digit that has uncertainty (e.g. is estimated by the observer). In other words, report digits that can be clearly determined with the measuring device. Usually this will give a value that includes digits whose certainty is known (e.g. is marked or indicated on the device) and one estimated digit.

4 Measuring Length Consider a metric ruler that is marked in centimeters (note: there should be one at your lab bench). Notice that the numbers on the ruler indicate the measurement of a centimeter. Also notice that each centimeter is further divided into 10 divisions (each is a mm). This indicates that this instrument measures with certainty to nearest whole mm. It allows the user to include in a measurement one estimated (or uncertain) digit that being 0.1 mm. Say you use this ruler to measure the length of an object, and you find that the length is between 3.3 and 3.4 cm. Every time you measure this object it falls between 3.3 and 3.4 cm. How should you report the length? As 3.3 or 3.4 cm (whichever the measurement is closest to). Or as 3.33 cm (e.g. an eyeball measurement you make that falls between 3.3 and 3.4)? Measuring Mass Scales used in laboratories to measure mass have a digital display. In addition, the scale will have a limited capacity (e.g. the maximum mass that can be weighed) and accuracy. Often, this information is provided somewhere on the instrument. The digital display represents the significant digits that can be recorded. For example, a given scale indicates that it s capacity is 1000g ± 0.01 g. The latter number represents the accuracy limit of this scale. This would indicate that the user can record the mass of an object taken with this balance to the nearest 0.01 g. 1. Examine the balance in the laboratory. What is the measuring capacity of the balance? 2. What is the accuracy limit of the balance (this information may be located on the sides or bottom of the balance)? If it is not given, note the smallest decimal place provided in the display)? 3. If you were to weigh an object on this balance, to what decimal place would you record mass? 4. To protect the balance pan when making a measurement of mass, a piece of weighing paper, a plastic weigh boat, or another container(s) (e.g. a beaker, flask or a test tube setting in a beaker) is used. This means the mass of the container must be subtracted from the total mass of the container and sample. This can be done by recording the mass of the container and then subtraction this value from the total mass or by taring the balance. Taring means that the balance is set to zero with a tare knob or button after the container is placed on the balance pan. The mass recorded upon addition of sample then reflects only the mass of the sample.

5 In the space below, describe how you would determine the mass of a spoon of water (use only the materials available at a lab bench). 5. Carry out your procedure. What is the mass (in g) of water in one spoon? 6. The following scenario to help you understand the importance of taring the balance at the appropriate time. You will only answer the question; you will not carry out the actual measurement. You place a weigh boat on the pan of the balance and notice its mass is 0.78 g. You add a spatula of sugar to the weigh boat and record a mass of 9.21 g. You just remember that you forgot to tare the balance before weighing the sucrose. You tare the balance and add another spatula of sugar. The mass is now 7.64 g. You add another spatula of sugar and now read a mass of g. Is it possible to determine the mass of the sugar at this point? If you answered yes, provide the mass. C. Measuring Volume There are several devices available for measuring volume. Selection, for the most part, is determined by the sample volume needed. In general, a measuring device should not be used to measure volumes that are less than 50% of its maximum measuring capacity. For example, a 100 ml graduated cylinder should not be used to measure volumes less than 50 ml. Pipets are usually used to measure liquid volumes of 10 ml or less, although larger pipets are available. Pipets come in two types: to deliver (usually labeled TD) and to contain (usually labeled TC). A label near the upper end of the pipet shaft indicates the type of pipet. Most pipets that you will work with (probably all) will be the TD type. There are two kinds of TD pipets - delivery and blow out. Examples are shown in the following figure:

6 Figure 1 Types of Pipets TD delivery pipet TD blow-out pipet Notice that on the TD delivery pipet the scale stops before it narrows. The scale on the TD blow out pipet runs the length of the pipet (to the tip). When using the blow out variety, all of the liquid must be removed from the pipet. When using the delivery variety it is important to deliver liquid only down to the lowest line of the scale (near the tip). When using a TC pipet, the liquid is allowed to flow (due to the force of gravity) out of the pipet until the flow stops. Also note that the pipet scale is further divided into fractions of a ml. The extent of graduation is given at the top of the pipet. A typical label at the top of a pipet (as shown in the preceding figure) is 10 ml in 1/10 TD. TD tells the user that the pipet is a to deliver pipet. The maximum volume the pipet can measure is 10 ml. Further graduations are in 1/10 of a ml, or.1 ml. To determine if the pipet is delivery or blow out, you must look at the format of the scale. You should familiarize yourself with any pipet before using it to eliminate errors in measurement. 1. Several pipets have been placed at your lab bench: 1, 5, and 10 ml pipets, and a Pasteur pipet (a glass and/or plastic one). Describe the 10 ml pipet you have been given (as TD delivery or TD blow-out; refer to Figure 1) and the basis for this description: 2. A pipet aid is a mechanical device used to draw liquids into a pipet. There are two types: the bulb type and the plunger type. Both are shown in the following figure. In the pipet bulb, A must be squeezed to remove air from the bulb; S is pinched to suck up liquid (when pipet tip is below the surface of a liquid sample); E is pinched to dispense the liquid. Plunger type pipet aids come in two colors: blue and green. The blue pipet aid is designed to be used with 1 ml pipets only. The green pipet aid is designed for use with 5, 10 and 25 ml pipets. Which type of pipet aid is at your lab bench?

7 Figure 2 Types of Pipet Aids (a) bulb type and (b) plunger type You will always use a pipet aid to dispense volumes between 1 and 10 ml. Note: NEVER PIPET BY MOUTH. ALWAYS USE A PIPET AID. Practice pipetting using the available 1, 5 and 10 ml pipets. Whenever possible, pipets should not be used to measure volumes less than 50% of their total measuring capacity (e.g. a 10 ml pipet should not be selected to measure a 4.3 ml volume if a 5 ml pipet is available). Place a beaker or weigh boat on the balance pan and tare the balance. To a container add the following one at a time, recording the total weight after each addition. Recorded Weight (based on: density water = 1 g/ml) Record Weight based on the density of water = 1g/ml To a container add the following one at a time, recording the total weight after each addition Volume to add Expected Total Observed Total 0.6 ml water 0.6g 4.3 ml water 4.9g 5.6 ml water 0.3 ml water Always add water and never pour any out. 3. Offer explanations for differences between observed and expected weights.

8 Researchers in biotechnology laboratories need to measure micro volumes accurately and repeatedly. To do this, a micropipette is used. Micropipettes are devices that are used to reproducibly measure and aseptically deliver small volumes. Common ranges are 1 10 l; l; and l. Several brands of micropipettes are on the market. While each has some unique design features, all have certain common features. Each is designed to work with a disposable tip into which the liquid to be measured is drawn. Each has a mechanism for dialing in the desired volume. Each has a multiple stop plunger: the first stop is calibrated to pick up the volume that has been dialed in; the second stop is designed to deliver the entire volume of air behind the liquid to ensure complete delivery; the third stop is used to eject the tip (some micropipettes have a separate mechanism for tip ejection instead of a third plunger stop) Tips are usually color-coded for the size of the pipette and come packaged in boxes for easy insertion onto the micropipette and sterilization in an autoclave (an autoclave is an instrument that uses steam and pressure to sterilize solids and liquids). 4. Using the micropipette at your bench, insert a tip onto the micropipette. Slowly depress the plunger with your thumb until you feel the first stop point. Apply more pressure to depress the plunger and feel the second stop. If your micropipette does not have a separate mechanism for tip removal, apply more pressure and note the ejection of the tip. Ask your lab instructor to demonstrate how to use a micropipette or to confirm that you are using it correctly. 5. Place a clean and dry weigh boat on the balance pan. Next, label a microcentrifuge tube with the letter M. Take a second microcentrifuge tube and label it with the letter G. Take your 100 l volume micropipette and set the volume for 100 l. Place an appropriate tip on the micropipette. a) Place the microcentrifuge tube labeled M in the weigh boat on the balance and record the mass of the empty tube. Pipette up 100 l of water and dispense it into the microcentrifuge tube labeled M and place on the balance pan (don t forget to tare the balance with the weigh boat before adding the microcentrifuge tube). Record the weight minus the weight of the tube in the table below. Since the density of water is 1.00 g/ml at room temperature, 100 l should weigh 0.1 g, or 100 mg. b) Next add 500 l of water to the microcentrifuge tube using your large-volume micropipette ( l capacity). Record the weight minus the weight of the tube in the table below. c) Finally, add 1000 l to the microcentrifuge tube so that your final volume is approximately 1600 l. Do not pour out the water in the microcentrifuge tube but rather you should continue to add to the volume so that the final volume after the three additions is 1600 l = 1.6 ml. Record the weight minus the weight of the tube in the table below.

9 d) Next, place the microcentrifuge tube labeled G in the weigh boat on the balance and record the mass of the empty tube. Using a 1.0 ml pipette, pipette up 0.1 ml of water and dispense it into the microcentrifuge tube labeled G and place on the balance pan (don t forget to tare the balance with the weigh boat before adding the microcentrifuge tube). Record the weight minus the weight of the tube in the table below. Since the density of water is 1.00 g/ml at room temperature, 0.1 ml should weigh 0.1 g, or 100 mg. e) Next add 0.5 ml of water to the microcentrifuge tube using the same 1.0 ml glass pipette. Record the weight minus the weight of the tube in the table below. f) Finally, add 1.0 ml to the microcentrifuge tube so that your final volume is approximately 1.6 ml. Do not pour out the water in the microcentrifuge tube but rather you should continue to add to the volume so that the final volume after the three additions is 1600 l = 1.6 ml. Record the weight minus the weight of the tube in the table below. Table 1: A comparison of the accuracy of the micropipette to the glass pipette. Your results should also indicate if you have used the two types of pipettes correctly. Volume of H 2 O Pipetted Volume Total Predicted Weight Observed/Measured Weight (in mg) First micro-centrifuge tube and micropipettes. 100 µl 100 µl 100 mg µl 600 µl ( µl) 600 mg µl Second micro-centrifuge tube and glass pipettes. 0.1 ml 0.5 ml 1.0 ml

10 6. There is one more device for measuring volume that you will likely encounter as a student of biology the volumetric flask. A volumetric flask does not have graduations. It is calibrated at the factory to measure a single volume 25, 50, 100, 250, 500 or 1000 ml. A volumetric flask can be more accurate than a graduated cylinder. This is due to the narrowness of its neck, which extends the last few milliliters of solution over a longer distance than in the graduated cylinder, thereby allowing greater accuracy in matching meniscus position with the etched line on the flask. Measure (as accurately as possible) 100 ml of water in your graduated cylinder. Use one of the disposable transfer pipets at your bench to assist you in accurately measuring this volume. Carefully pour the water into a volumetric flask of the same measuring volume (also at your bench). Is the water exactly at the line on the neck of the volumetric flask? Note the meniscus position relative to the line on the neck: D. Comparing Measuring Devices In addition to the graduated cylinder, there are three other types of devices for measuring volume at your lab bench: a beaker, an Erlenmeyer flask, and a volumetric flask. If you have to make several measurements of exactly (100) ml of water, which of these containers (a graduated cylinder, a beaker, an Erlenmeyer flask, or a volumetric flask) would be most appropriate or would they all be equally appropriate? Note: you must use all four devices in your experiment! 1. Formulate a hypothesis to explore this question: 2. What is your prediction for this hypothesis? (Remember that a prediction is a statement that states an expected result when a specific action is taken; e.g. if I do (action), then I should observe (result).

11 3. Describe a procedure to test this prediction (provide enough procedural detail so that another student could carry out the instructions exactly as you will). Note: do not forget to include at least three replicate measurements! 4. Provide your data/results in a table (don t forget to include an informative title and labels for each row or column).

12 5. From your results, describe/summarize your findings. 6. Is your prediction supported? Why? 7. Do your results support your hypothesis? Explain your answer. 8. What do you conclude about the measuring device that should be used to measure liquids in future labs? E. Precision and Accuracy A professor always emphasizes that measurements should be precise and accurate. What do these two words mean? Accuracy is the degree to which an observed value corresponds to a true value. Precision is the degree to which measurements are reproducible when repeated. A measurement may be accurate but not precise, precise, but not accurate, both, or neither. Lack of precision or accuracy may be a result of the measuring device (e.g. something internal or perhaps a calibration error) or a function of the technique (e.g. the device is not appropriate for the measurement being taken). Lack of precision may also be due to experimental design. 1. How accurate was the Erlenmeyer flask compared to the graduated cylinder in Part D? 2. Were your measurements for Part D precise? Explain. 12

13 3. Referring to Part D, explain (be specific) how it might be possible for one or more of the tested measuring devices to be inaccurate but precise. 4. Referring to part D, explain (be specific) how it might be possible for one or more of the tested measuring devices to be accurate but not precise. Remember that replicate (i.e. multiple) measurements must be taken in order to comment on a measuring device s precision. 5. You measure 5 ml of water and determine its mass on a balance. You repeat this procedure four times. You try the experiment again. This time you have four new values for mass, as shown below. Choose the statement that best describes your measurements. Experiment 1: 5.7 g, 5.8 g, 5.7 g, 5.8 g Experiment 2: 4.9 g, 5.0 g, 4.8 g, 5.3 g a) The measurements in both experiments are accurate and precise. b) The measurements in both experiments are not accurate but are precise. c) The measurements in Exp. 1 are not accurate but are precise; those in Exp. 2 are accurate but not precise. d) The measurements in Exp. 1 are accurate and not precise; those in Exp. 2 are not accurate and not precise. 13

14 YOU WILL HAVE QUESTIONS FROM THIS MATERIAL ON YOUR FIRST LAB EXAM. TO PREPARE FOR THE EXAM YOU SHOULD REVIEW ALL OF THE ACTIVITIES PERFORMED IN THIS EXERCISE AND ANSWER THE FOLLOWING QUESTIONS. 1. You need 0.25 ml of a solution. What measuring device would you use to measure this amount? Explain the basis for your answer. Consider only those measuring devices that you used in this laboratory exercise. 2. You place a piece of paper on a balance and find that its mass is 0.28 g. You place some solid NaCl (sodium chloride; table salt) onto the paper and get a mass of g. You remember that you forgot to tare the balance before taking this measurement. You tare the balance, add more NaCl, and read g. You add more NaCl and read g. How much NaCl do you have on the balance in: g; mg; µg; kg (provide all answers in scientific notation) µl is equivalent to ml. 4. Which pipet aid (blue, green, or either) should be used with a 5 ml pipet? 5. A pipet is labeled as 5 ml in 1/10. What does the 5 ml indicate? What does the 1/10 indicate? Draw a representation of the graduations your expect to see on a TD (to deliver) blow-out pipet that has this label. 5 ml in 1/10 6. A pipet is labeled as 1/10 ml in 1/100. Draw a representation of the graduations your expect to see on a blow-out pipet. 7. You are using a 10 ml blow-out pipet graduated in 1/10 ml. Your pipet reads 0 at the highest marking on the stem. You have drawn 8.6 ml of liquid into the pipet and you cannot put any back 14

15 into the bottle even though you need to measure out only 6.5 ml. At what mark on the pipet is the meniscus of your liquid now located? If you release liquid from your pipet, at what line should you stop to obtain the desired 6.5 ml? Draw the pipet and, using two arrows, indicate the start and stop points. 8. You measure 5 ml of a solution and determine its mass on a balance. You repeat this procedure four times. You try the experiment again. This time you have four new values for mass, as shown below. Describe the accuracy and precision of the measurements in each experiment. Experiment 1: 5.7 g, 5.8 g, 5.7 g, 5.8 g Experiment 2: 4.9 g, 5.0 g, 4.8 g, 5.3 g 9. What device(s) is appropriate to use when measuring 2 ml? 4000 µl? (select from: 1 ml pipet, 5 ml pipet, 10 ml pipet, 10 ml graduated cylinder, 10 ml volumetric flask, transfer pipet, 10, 100 or 1000 µl micropipette, any of these) Why? 10. If you want to measure 43.2 µl with a micropipette, what should the display on the micropipette read? 11. A balance is described by the manufacturer as having an accuracy of ± 0.01 g. Explain what this means (without using the word accurate) 12. A balance is described by the manufacturer as having a precision of ± 0.01 g. Explain what this means (without using the word precise). 15