C HAPTER 3: T HE B ASIC S KILLS OF THE B IOTECHNOLOGY W ORKPLACE Introduction to Biotechnology, BIOL1414 Austin Community College, Biotechnology Dept

L EARNING O UTCOMES Determine the most appropriate tool for measuring specific volumes of masses Describe how to select, set, and use a variety of micropipets within their designated ranges to accurately measure small volumes Convert between units of measure using the B S rule and appropriate conversion factors Recognize the different expressions for units of concentration measurements and use their corresponding equations to calculate the amount of solute needed to make a specified solution Describe what pH is and why it is important in solution preparation Note about this PowerPoint – There are several links in this PPT that allow you to explore more into different topics. Some of these links are animations, movies, or exercises. Please note, you must be in the slide show to activate the links. You can press F5 any time to active the slide show and “Esc” to exit.

M EASURING V OLUMES IN A B IOTECHNOLOGY F ACILITY Volume is a measurement of the amount of space something occupies Volume is measured in Liters (L) Milliliters (mL) Microliters (  L) Different tools are used to measure volume Graduated cylinder Pipet Micropipet or Micropipette

C ONVERTING U NITS o Often volumes are measured in one unit of measurement and reported in another o Converting between metric units o Conversion factor o To measure volumes larger than 10 milliliters, technicians usually use a graduated cylinder

Reading a graduated cylinder. Before using a graduated cylinder, make sure you know the total volume it will hold and the value of each of the graduations. In the lab, common graduated cylinders include 10 mL, 25 mL, 100 mL, 250 mL, 500 mL, and 1 L. Pipets are available that measure volumes between 0.1 mL and 50 mL. Shown from left to right are 25-, 10-, 5-, and 1-mL pipets.

P IPETS Laboratory instrument used to transport a measured volume of liquid. Three types of pipets used in the laboratory Volumetric Mohr Serological Mechanical – Micropipets or Micropipettes

P IPETS Serological and Mohr pipets have different markings to assist in identifying them as they are used differently. Serological pipets are TD = to deliver. To accurately dispense the measured volume the last bit must be blown out. Mohr pipets are TC = to contain. These pipets are designed to dispense the correctly measured volume, so there will be a minute amount of liquid left in the tip.

M ARKINGS TD pipets will have an etched or colored ring at the top of the pipet. TC pipets will have no rings although there may be a colored bar to indicate the volume.

V OLUMETRIC Designed to transfer a fixed amount of liquid when filled to the mark, e.g. 10 mL and only 10 mL. There is generally only one "fill-line" on a volumetric pipet. For example a 5 mL volumetric pipette has one marking on it. This marking measures exactly 5 mL. of liquid, no more, no less.

V OLUMETRIC Designed to deliver a single volume precisely, the volume will be indicated near the top of the pipet, At the top of the pipet is an etched ring. Fluid must be drawn up the pipet to above the ring indicating the volume and then released slowly until the bottom of the meniscus is exactly at the ring. To transfer this volume to a second container, touch the pipet tip to the inside of the new container and allow the liquid to drain out.

S EROLOGICAL – T O D ELIVER

S EROLOGICAL - TD A pipet marked TD (the more common type of pipet) has been calibrated " to deliver " a specified volume of liquid. These pipets have no base mark, the graduations continue onto the tip and are graduated to deliver which means that ALL the measured liquid in the pipet must be delivered – blow out.

M OHR – TC – TO CONTAIN A pipet with this marking has been calibrated to contain a specified volume of liquid. These pipets have a single painted or frosted ring at the top and are allowed to simply drain with the tip placed against the side of the receiving vessel. To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer.

M OHR - TC To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer. Near the top of this type of pipet you will find the total volume indicated and the size of the smallest gradations

P ROPER U SE When filling a pipet, the tapered end is held beneath the surface of the liquid at all times. The liquid is drawn into the pipet by suction until the level is equal to or greater than the volume of liquid to be delivered. Since serological pipets are labeled with the zero mark at the top of the pipet you will need to subtract the amount you are going to pipet from the total volume of the pipet to determine the exact mark to fill the pipet to.

P ROPER U SE When reading the volume, ALWAYS view the pipet dead-on at eye level with the pipet held vertically, perpendicular to the ground. Pipets are designed to be used with a hand pump or bulb, of which there are many varieties. Never use your mouth with a pipet!

M ICROPIPET Mechanical micropipets can be set to draw and dispense different volumes or be preset to deliver an exact volume. Used to accurately deliver very small volumes, microliters, of liquid. Although 0.1 mL could be delivered by a serological pipet most biotechnology labs use micropipets.

M ICROPIPET Mechanical pipets are operated by depressing the plunger. On the downward stroke of the plunger there are TWO stops: the first offers firm resistance, and the second is a hard stop. To take up a volume in the micropipet, place a tip on the end of the pipet. Depress the plunger to the first stop and insert into the sample to be transferred. Draw the liquid into the pipet by SLOWLY releasing the plunger. To dispense the liquid from the pipet, place the tip of the pipet into the opening of the well and slowly depress the plunger all the way to the second stop. When the liquid has been dispensed withdraw the pipet tip from the well BEFORE releasing the plunger.

Micropipet. Learning to use each part of a micropipet correctly is essential. o On the micropipette shown, the plunger has two “stops.” o Pressing to the first stop evacuates air to the volume in the display. o Pressing to the second stop evacuates that volume plus another 50% or so. o To ensure accurate measurement, feel the difference between the first and second stop before using the pipette. o Inaccurate measurement could waste costly reagents and cause invalid experiment results. Watch this Video! Watch this Video! Click here! Click here! Using a Micropipet Using a Micropipet

M ULTICHANNEL P IPET A multichannel pipet allows several samples to be measured at the same time, a feature that saves time during an experiment with multiple replications and repetitive pipetting.

E LECTRONIC M ICROPIPET

A UTOMATED M ICROPIPET P IPETTING M ACHINE

M AKING S OLUTIONS o Solution preparation is one of the most essential skills of a biotechnician. o Solutions are mixtures in which one or more substances are dissolved in another substance. o Solid solutes are measured on balances or scales. o Concentration is measured in several ways: o Mass/volume o Volume/volume o % mass/volume o Molarity o Normality

T HE A NALYTICAL B ALANCE Most analytical balances measure down to milligrams, even though they usually report in grams.

T OP L OADING E LECTRONIC B ALANCE

G LASSWARE Glassware can be divided into 2 groups Non-volumetric glassware Beaker Flask Volumetric Glassware Volumetric Flask Graduated Cylinder

B EAKER Used for transferring liquid to another container or to transfer a small amount of reagent for use in procedures. Volume is not accurate, just an estimate. NEVER PLACE A REAGENT IN ANOTHER CONTAINER WITHOUT LABELING THE CONTAINER FIRST.

E RLENMEYER F LASK Features a conical base, a cylindrical neck and a flat bottom. They are marked on the side ( graduated ) to indicate the approximate volume of their contents. Volume is NOT accurate

G RADUATED C YLINDER For rapid measurement of liquid volume. They are generally more accurate and precise for this purpose than flasks. This is a semi-accurate liquid measuring vessels. NOTE: Pipet is more accurate for volumes less than 50mL - WHY?

R EADING THE V OLUME 10 mL has approx 6.62 mL 100 mL 52.7 mL 25 mL has 11.5 mL

V OLUMETRIC F LASK A volumetric flask is used to measure very precisely one specific volume of liquid This flask is used to prepare a solution of known concentration. To make up a solution, first dissolve the solid material completely, in less fluid than required to fill the flask to the mark. After the solid is completely dissolved, very carefully fill the flask to the mL mark. The top is then sealed and the flask is inverted several times to mix.

Y OUR T URN ! P RACTICE Q UESTIONS 1. What instrument would you use to measure and dispense the following volumes most accurately? 23.5  L6.5 mL125 mL 7  L2.87 mL555  L 2. Convert the following units to the requested unit: 1.7 L = _____ mL235.1  L = _____ mL 2.37 mL = _____  L

P REPARING S OLUTIONS

S OLUTIONS OF G IVEN M ASS /V OLUME C ONCENTRATIONS Mass/Volume Solution. Solvent is added until a volume of 10 mL is reached. A protein solution that has a concentration of 1 g/mL is considered fairly concentrated.

Mass/Volume Concentration Equation ____ g/mL X ____ mL = ____ g of solution Concentrationvolumeto be weighed out, desireddesireddissolved in the solvent M AKING M ASS /V OLUME S OLUTIONS

____ % = ____ percent valuedecimal value of the g/mL ____ X ____ = ____ g of solute to decimaltotal volumebe measured and valuedesired (mL)added to the volume desired of solvent S OLUTIONS OF D IFFERING % M ASS /V OLUME C ONCENTRATIONS A percentage represents something that is part of 100. Mass/Volume Concentration Equation

Y OUR T URN ! P RACTICE Q UESTIONS ! 1. What mass of the protein, gelatin, is needed to make 0.5 L of a 3 g/L gelatin solution? 2. What mass of sugar is need to make 25 mL of a 25 mg/mL sugar solution? 3. What mass of salt is needed to make 150 mL of a 100  g/mL salt solution? Describe how the solution is prepared. 2. What mass of gelatin (a protein) is needed to make 0.5 L of a 3% gelatin solution? 3. What mass of sugar is needed to make 25 mL of a 2.5% sugar solution? 4. What mass of salt is needed to make 150 mL of a 10% salt solution? Describe how the solution is prepared.

Molarity Concentration Equation volumemolaritymolecularthe number of grams to be wanted (L)XdesiredXweight of the =dissolved in solvent, up to (mol/L)solute (g/mol)the total volume of solution desired S OLUTIONS OF D IFFERING M OLAR C ONCENTRATIONS

Periodic Table. The Period Table of Elements shows the elements (atoms) found in compounds (molecules). Each element is listed along with the atomic weight (mass) of each atom in the element. A NaCl molecule has a molecular weight of about 58.5 amu (atomic mass units) because the Na atom weighs about 23 amu, and the Cl atom weighs about 35.5 amu. Together, in the NaCl molecule, the atoms total approximately 58.5 amu. The mass of a hydrogen atom equals 1 amu.

This instrument is a mass spectrometer. Scientists use it to determine the molecular weight of a compound. A “mass spec” can also determine if a sample is contaminated with molecules of different molecular weights.

Y OUR T URN ! P RACTICE Q UESTIONS ! 1. What is the molecular weight of each of the following compounds? NaOHHClNaCl 2. What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution? 3. What mass of sodium hydroxide (NaOH) is needed to make 750 mL of a 125 m M NaOH solution? Describe how to prepare the solution.

D ILUTES OF C ONCENTRATED S OLUTIONS Concentrating 1 L Solution. Many chemical and biological reagents are purchased in concentrated form. Concentrated solutions can be prepared initially with a greater amount of solute to solvent, or a solution can be concentrated by removing water. A diluted solution can be prepared by adding solvent to a concentrated one.

Diluting a 100 mg/mL Stock Solution to 1 mg/mL. D ILUTION OF S TOCK S OLUTION C1 x V1 = C2 x V2 Where, C1 is concentration of stock V1 is volume of concentrated stock needed C2 is final concentration of solution desired V2 is the final volume desired

D ILUTION OF S TOCK S OLUTION How do you make 1000 mL of a 1mg/mL solution from a 100 mg/mL Stock? C1 x V1 = C2 x V2 Solve for V1 (100mg/ml)xV1 = (1mg/mL)x(1000mL) V1 = 10mL

P RACTICE ! Animation on solution preparation calculations Click here! Click here! Preparing Solutions Preparing Solutions Practice Problems Practice Problems

PHPH pH measures how many hydrogen ions are in solution pH is reported in (log) units ranging from 0-14 In pure water, most of the water exists as H 2 O, but a few ionize to H + and OH - ions. The numbers of H + and OH - ions are equal so their charges cancel out. Pure water has no charge – and is neutral = pH 7 If solutes are dissolved in water the ratio of ions change If H + ions are added the pH goes down and is considered acidic, if the OH - ions are added the pH goes up and is considered basic

PHPH In a biotech lab, proteins or DNA in cells and solutions prepared in a the lab must be made and maintained at a specific pH, otherwise a change in the molecular structure, and therefore function, can result Buffers are solutions that RESIST the change in pH They are used as DNA and protein solvents to ensure that the pH and electrical charges around the molecules stat at a constant pH Explore Calibrating a pH meter Calibrating a pH meter

R EVIEW Q UESTIONS Your Turn! Put your name at the top of a sheet of paper, answer these questions and hand in: 1. How do you prepare 40 mL of a 2 mg/mL protein solution from 10 mg/mL protein solution? 2. How do you prepare 200  L of 2X enzyme buffer from 10X enzyme buffer solution? 3. How do you prepare 500  L of 10  M NaCl solution from 5  M NaCl solution? 4. How do you prepare 3 L of 1X TAE buffer from 50X TAE buffer stock solution?

Q UESTIONS AND C OMMENTS ?

R EFERENCES 1. MLAB 1335 Immunology Serology PPT lecture, Terry Kotrla, MS, MT(ASCP)BB 2. Biotechnology: Science for the New Millennium Ellyn Daugherty.