10/31/13 A Refresher on Biomedical Research Laboratory Skills: Safety, Measurements and Calculations Dr. Lycely Sepúlveda Universidad Central del Caribe January 13, 2012 http://nobel.scas.bcit.ca/debeck_pt/science/safety.htm 1 10/31/13 General Safety Rules http://water.me.vccs.edu/courses/env211/lesson1_print.htm No drinking, eating or applying make up No mouth pipetting Wear appropriate clothing. Use the right protective equipment. https://www.msu.edu/~nixonjos/teaching/bio/safety/safety03.html 2 10/31/13 A word on gloves… they are a MAJOR source of laboratory contamination! •
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Choose the gloves that will provide a reasonable amount of protection. Replace gloves often. Never touch your face while having gloves on. Remove gloves before touching surfaces that will be touched without gloves by yourself or by others. http://web.princeton.edu/sites/ehs/labsafetymanual/sec6c.htm http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html Avoid using contact lenses. http://auburn.edu/research/dynamic-­‐dosing-­‐with-­‐contact-­‐lenses/ Choose the right protective equipment. http://www.company7.com/uvp/safety.html 3 10/31/13 Handle glass and sharps carefully. http://www.healthsafe.uab.edu/pages/biosafety/bbp/pages/page12.htm http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html Use secondary containers for transport. en.wikipedia.org Use the “hoods” when necessary. Biosafety cabinet http://nanostructure.usc.edu/lab/bio2/bio9.jpg Chemical hood http://www.allometrics.com 4 10/31/13 Do not use the fume hood as a storage area. http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html Lower the hood’s door to the correct level. http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html 5 10/31/13 Learn how to operate all equipment before using it for the first time. http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html •  Be aware of the hazard and how to avoid them. •  Inspect electric wires periodically. •  Unplug the equipment prior to servicing and before moving to a different location. Laboratory animals and radioactive materials must NOT be handled unless you have received the proper training. http://www.ecouterre.com 6 10/31/13 Learn the warning symbols for specific chemical hazards. http://chemistry.about.com/od/healthsafety/ig/Laboratory-­‐Safety-­‐Signs/index.05.htm Label all solutions with permanent ink: •  identity and concentration of each reagent •  your name •  date Inform the laboratory supervisor as soon as an accident happens. http://www.udel.edu/ehs/chemspillkit/chemspillkit.html •  Clean up spills immediately. http://www.enware.com.au/Fast-­‐Act-­‐-­‐-­‐Lab-­‐Spill-­‐Response/default.aspx 7 10/31/13 Know the location of the first aid kit and the laboratory emergency equipment. http://water.me.vccs.edu/courses/env211/lesson1_print.htm •  Keep the equipment clean, fully operational and readily accessible. http://www.utexas.edu/safety/ehs/lab/manual/4_guidelines.html Be prepare for emergencies: •  Do you know what to do in case of a fire or an earthquake? •  What is the closest evacuation route? •  Is there a meeting point outside the building? •  Are the emergency numbers readily accessible? http://www.dtc.dla.mil/smt/Industrial/exits/egresslargepic.htm http://blog.firenzelodging.it/emergency-­‐numbers/ 8 10/31/13 Avoid working in the laboratory alone. Plan your experiments carefully: •  Work in a logical, clean and clutter-­‐free manner. •  Always clean up after your work is done. http://brentdanley.com Safety Signs and Equipment http://www.ergo-­‐safe.com/services.htm http://www.chem.sc.edu/faculty/handy/Labpics.htm 9 10/31/13 Safety Signs – Meanings of Colors and Shapes Regulatory – an order is in place Hazard alert – nature, consequences and how to avoid danger Information Regulatory signs •  Prohibition – an action is forbidden •  Mandatory – an action is required www.safetysigns.com 10 10/31/13 Hazard alert general hazard low temperature high temperature magnetic field biohazard laser warning optical radiation radioactivity non-­‐ionizing radiation UV light carcinogen toxic explosive flammable corrosive www.safetysigns.com explosion oxidizing material hot surface electric shock high voltage electric fire broken glass chemical burn hand crush eye & ear hazard Inhalation hazard foot crush environmental hazard compressed gas www.safetysigns.com 11 10/31/13 Information signs •  Health and safety http://chemistry.about.com/od/healthsafety/ig/Laboratory-­‐Safety-­‐Signs/ •  Fire equipment http://chemistry.about.com/od/healthsafety/ig/Laboratory-­‐Safety-­‐Signs/ 12 10/31/13 Safety equipment Fire extinguishers •  Class A -­‐ ordinary combustible materials (paper, wood, cardboard, most plastics) •  Class B -­‐ flammable or combustible liquids (gasoline, kerosene, grease, and oil) •  Class C -­‐ electrical equipment (appliances, wiring, circuit breakers, outlets) •  Class D -­‐ combustible materials often found in chemical laboratories (magnesium, titanium, potassium, sodium) http://www.chem.sc.edu/faculty/handy/Labpics.htm First aid kit, evacuation plan and general laboratory rules First aid kit, MSDs and chemical hygiene plan http://www.chem.sc.edu/faculty/handy/Labpics.htm 13 10/31/13 Spill kits for mercury, acids, bases and solvents http://www.chem.sc.edu/faculty/handy/Labpics.htm Use the proper disposal container for the proper waste. http://www.chem.sc.edu/faculty/handy/Labpics.htm http://www.ehrs.upenn.edu/programs/labsafety/chp/chemicalA.html http://www.thefind.com/office/info-­‐biohazard-­‐waste-­‐containers 14 10/31/13 Handling Laboratory Reagents http://www.haqchem.com/ USDOT definition of hazardous materials •  Any substance or material could adversely affect the safety of the public, handlers or carriers during transportation. Materials with properties conducive to explosions Substances that fulfill the definition for the gas state of matter 15 10/31/13 Flammable and combustible liquids Flammable and combustible solids Oxidizer and organic peroxides Toxic and infectious substances Radioactive materials Corrosive Miscellaneous http://ehs.gmu.edu/plans_manuals/LaboratorySafetyManual.pdf 16 10/31/13 The National Fire Protection Agency (NFPA) Safety Diamond http://www.compliancesigns.com/nfpadiamonds.shtml Hazardous Material Identification Guide http://www.quakerchem.com/safety/white_papers/wp_hazardous_matls.html 17 10/31/13 http://www.twu.edu/rm/hazard-­‐communications.asp Material Safety Data Sheet (MSDS) •  Document depicting how to handle chemicals in a safe manner •  Some of the information provided §  physical data §  toxicity §  health effects §  first aid §  reactivity §  storage §  disposal §  personal protective equipment §  spill clean up 18 10/31/13 Chemical Incompatibility Mixing chemicals from different hazardous classes? fire http://homepage.mac.com/charambura/MrC/web-­‐content/
scholars1.html explosion http://pubs.acs.org/cen/news/83/i21/8321ohio.html polymerization http://www.reptox.csst.qc.ca/documents/
simdut/guideang/htm/GuideAng09.htm toxic gas http://www.reptox.csst.qc.ca/documents/simdut/guideang/htm/GuideAng09.htm Measurements in the Biomedical Research Laboratory http://www.atomscientific.com/ http://www.microscopesblog.com/ http://gallery.hd.org/ http://www.graduated-­‐cylinders.com/ 19 10/31/13 Significant Figures •  Precision – consistency of a measured value across repeated measurements •  Accuracy – nearness of the measurement to the correct value Rules for recording significant figures: 1.  Record as many digits as are certain plus one digit that is estimated 2.  All nonzero digits are significant 3.  All zeros between nonzero digits are significant 4.  Zero digits to the right of a nonzero digit but to the left of an assumed decimal point may or may not be significant 5.  All zeros to the right of a decimal point and to the right of a nonzero digit before a decimal place are significant 6.  All zeros to the left of a nonzero digit and to the right of a decimal point are not significant unless there is a significant digit to their left General rules for rounding off 1. If digit dropped is less than 5 -­‐ no change to preceding digit (12.64 rounds to 12.6) 2. If digit dropped is greater than 5 -­‐ preceding digit is increased by one. ( 12.66 becomes 12.7) 3. Working with numbers that have various levels of significant figures -­‐ round up to the accuracy of the number with least number of significant figures 20 10/31/13 Practical tips for the biomedical research laboratory •  Reports all the digits from digital measurements (e.g., pH meter, balance) •  Look closely at the scale of analog instruments -­‐ report all the certain digits plus one estimated digit (e.g., 3.62 ml dispensed by a pipette with 0.1 ml markings) •  When adding or subtracting -­‐ retain the number of decimal places found in the original number with the least amount of decimal places (98.000 + 7.9878 + 56.2) = 162.2 (rounded up from 162.1886) •  In multiplication or division -­‐ keep as many significant figures as are found in the number with the least amount of significant digits (0.54678 × 0.980 × 7.899) = 4.23 (rounded up from 4.23263) •  Always round up after performing the calculations Units of Measure International System Measured Quality Unit Symbol Length meter m mass kilogram kg substance amount mole mol time second s electric current ampere A luminous intensity candela cd temperature Kelvin K 21 10/31/13 Metric units commonly used in Biology mass – gram (g) http://www.marshu.com length – meter (m) http://gottatopic.com volume – liter (L) Temperature – Celsius (C) http://www.waterboydist.com 22 10/31/13 Measuring Solids •  Mass – The amount of matter in an object expressed in grams •  Weight – Force of gravity on an object Person with a mass of 45.36 kg will have a weight of 100 lb. on Earth http://pillownaut.blogspot.com 23 10/31/13 Laboratory balances •  Range – Span from lightest to heaviest weight that can be measured •  Capacity – Heaviest sample that the balance can weigh •  Sensitivity – Smallest value of weight that causes a change in the response of the balance http://www.meterforall.com •  Readability – The value of the smallest unit of weight that can be read Sample specifications for electronic balances Sample specifications for analytical balances www.fishersci.com 24 10/31/13 http://www.dartmouth.edu/~chemlab/techniques/a_balance.html Measuring Liquids Common instruments used to measure volume in the research laboratory Device Best volume range Accuracy Usefulness for repetitive measurements Pasteur pipette 30 μl – 2 ml low/medium very good Graduated cylinder 5 – 2000 ml medium good Volumetric flask 5 – 2000 ml high good Burette 1 – 100 ml high very good Pipette 0.1 – 25 ml high very good micropipette 0.2 μl – 1 ml high very good microsyringe 0.5 -­‐ 50 μl high good Erlenmeyer flask/
beaker 25 – 5000 ml very low good 25 10/31/13 Glassware calibration •  To contain (TC) – will contain the specified amount when filled to the capacity mark •  To deliver (TD) – will deliver the specified amount when filled to the capacity line and transferred to a different container Siedman and Moore 2000 Siedman and Moore 2000 http://explore.ecb.org 26 10/31/13 Using pipettes and pipette aids Pipettes – hollow tubes used to draw liquids through one end. Generally used to measure volumes on the 0.1 – 25 ml range. •  Serological or “blow out pipette” -­‐ the last drop of liquid needs to be blown-­‐out of the tip to deliver the full volume of the pipette. http://www2.hawaii.edu Typical information provided in a serological pipette •  2 colored rings -­‐ calibrated as “blow out” pipette •  capacity – 10 ml in this example •  measurement graduations – 1/10 or 0.1 ml •  calibration – “to deliver” water at room temperature (20 °C) http://www2.hawaii.edu 27 10/31/13 Measuring 3.2 ml in a serological pipette Siedman and Moore 2000 • Mohr pipettes – calibrated “to deliver” but the liquid in the tip is NOT part of the measurement as is not blown out Siedman and Moore 2000 28 10/31/13 Other types of pipettes •  Pasteur pipettes – made of glass, good for balancing tubes for centrifugation, removing or transferring liquids, commonly used with aspirators http://www.thesciencefair.com •  Transfer pipettes – plastic, disposable, with built-­‐in bulb. Used for balancing tubes, transferring cells or removing liquids that stick to glass http://www.benmeadows.com • Capillary pipettes – small glass tubes that load microliter volumes by capillary action http://www.labchem.com.my • Volumetric pipettes – calibrated to deliver one specified amount http://www.sks-­‐science.com 29 10/31/13 Pipette aids – instruments used to draw liquid into and expel it from the pipette • simple bulb – inexpensive, not easily controlled •  pump – one-­‐handed pipetting done by rotating the thumb wheel Siedman and Moore 2000 • 3 valve bulb – has 1 valves to displace air from the bulb, another to draw liquid into the pipette and a third valve to expel the liquid •  electronic pipettor – allows fine control and ease of use Siedman and Moore 2000 30 10/31/13 •  Bottle-­‐top dispenser – mainly used to dispense repetitive amounts of solvents http://www.directindustry.com Micropipettes •  Positive displacement – syringes and similar devices where the sample comes in contact with the plunge and the inner wall http://www.tradekool.com •  Air displacement – there is an air cushion between the pipette and the sample such that the sample only comes in contact with a disposable tip http://www.rubilabor.com 31 10/31/13 Factors affecting the performance of micropipettes • Operator’s technique – learn to use the micropipette correctly, consistently and smoothly • Physical and chemical properties of the liquid – designed for aqueous solutions. Pipetting volatile and viscous liquids can be challenging • Environment – Low humidity can cause quick evaporation of small amounts of liquids, differences in temperature can cause inaccurate readings Using a micropipette 32 10/31/13 Choosing the right micropipette for the volume to be transferred http://www.gilson.com •  Learn to set and read the volume Gilson micropipettes http://www.gilson.com 33 10/31/13 Eppendorf micropipette P200 http://lithiumr-­‐oses.blogspot.com P1000 P20 http://www.biosci.msu.edu •  Select the right tip for your pipette http://www.jtote.com http://arenterprises.tradeindia.com http://www.sigmaaldrich.com http://www.alibaba.com 34 10/31/13 http://www.gilson.com •  Plan ahead and organize your bench http://www.gilson.com 35 10/31/13 •  Use the proper technique http://www.gilson.com http://www.gilson.com 36 10/31/13 http://www.gilson.com •  Eject the tip and store the pipette in a vertical position to prevent liquids from entering the shaft. http://www.gilson.com 37 10/31/13 •  Be careful to avoid contaminating your samples or your micropipette http://www.gilson.com •  Follow the manufacturer’s recommendations for decontamination, routine maintenance and recalibration http://www.gilson.com 38 10/31/13 Micropipettes without a tip ejector button Seidman and Moore 2000 Other micropipetting devices Fixed-­‐volume micropipette Repeater micropipette Multichannel micropipette http://www.gilson.com 39 10/31/13 Measuring Temperature •  Temperature – average kinetic energy of a system •  Liquid expansion thermometer filled with Mercury or dyed alcohol •  Useful range of -­‐38 °C to 250 °C for Mercury and -­‐200 °C to 250 °C for alcohol Siedman and Moore 2000 http://www.globalspec.com 40 10/31/13 Other ways to measure temperature •  Metallic resistance thermometers – use metallic wires on which resistance increases as the temperature increases. Allow accurate control of temperature precision machines. http://www.labrepco.com •  Thermocouples – use 2 different metals joined together. The voltage at the junction is converted to a temperature measurement. http://www.omega.nl/ Measuring pH •  pH – degree of acidity or alkalinity of a solution §  “p” – mathematical symbol for negative logarithm §  “H” – chemical symbol of Hydrogen §  pH = -­‐log [H+] Pure water at 25 °C H2O H+ + OH-­‐ both ions at [10-­‐7 mole/L] log of 1 × 10-­‐7 = -­‐7 -­‐log = -­‐(-­‐7) = 7 pH of water = 7 = neutral 41 10/31/13 •  Acids – release H+ when added to water Hydrochloric acid in water HCl H+ + Cl-­‐ (complete dissociation) strong acid Acetic acid in water CH3COOH H+ + CH3COO-­‐ (incomplete dissociation) (mostly in this form) weak acid •  Bases – release OH-­‐ when added to water OH-­‐ + H+ H2O (from base) (from water) (forms more water) Adding more OH-­‐ = removing H+ = increase in pH in water Sodium hydroxide NaOH Na+ + OH-­‐ (complete dissociation) strong base Ammonia in water NH3 + H2O NH4+ + OH-­‐ (incomplete dissociation) (mostly in this form) weak acid 42 10/31/13 •  The pH scale is logarithmic: 1 unit increase = 10× change in concentration http://www.isa.org Measuring pH in Molecular Biology •  pH indicators – weak organic acids or bases in which the associated and dissociated forms have different colors. Useful to monitor pH changes in the 1 – 2 unit range. §  estimating pH of solutions §  determining a change of pH §  approximate the pH of intracellular compartments http://www2.chemistry.msu.edu 43 10/31/13 •  pH electrode/meter – Provide better accuracy, sensitivity and flexibility Reference electrode (not sensitive to Δ [H+], constant voltage) H+ sensitive electrode High input meter http://www.microscopesblog.com •  Basic principle behind the operation of a pH meter § The electric potential (voltage) of the glass electrode varies depending on the samples’ [H+] § The electrical potential of the reference electrode is constant § The 2 electrodes are connected through a voltage meter § The meter measures the voltage difference between the electrodes and generates an electrical signal § The signal is amplified and converted to a display of pH http://biology.hunter.cuny.edu 44 10/31/13 •  Calibrating a pH meter – tells the instrument how to translate the voltage difference between the electrodes into pH units § Should be done every day for pH meters that are used constantly or every time the pH meter is used for those meters used sporadically § 2 buffers of known pH are used to calibrate a pH meter because there is a linear relationship between the voltage measured by the system and the pH of the sample http://biotech.matcmadison.edu Sample 2 point calibration of a pH meter http://biotech.matcmadison.edu •  Slope – response of electrodes to pH •  Ideal slope ~ 60 mV/pH unit at 25 °C •  Often expressed as a percent of theoretical value to express condition of electrode (e.g., 97% slope = 58.2 mV/pH unit) 45 10/31/13 General steps for measuring pH using a pH meter •  Remove the electrode from the storage buffer, rinse with distilled water and wipe gently with lint-­‐free tissue to remove excess water •  Standardize with pH 7 buffer and another buffer (depending on pH range of solution to be measured) •  Adjust the pH with NaOH or HCl, if necessary •  Rinse the electrode, wipe with tissue and return to storage buffer •  Bring the solution to its final volume http://protocols.davidson.edu/ Effects of Temperature on pH •  Electrode’s response is temperature dependent – report temperature to the meter or use an electrode with an automatic temperature compensator http://biotech.matcmadison.edu •  pH of solution can change with temperature 46 10/31/13 http://www.flickr.com Making Chemical Solutions http://www.biochemista.com Solution – homogeneous mixture where particles of one or more substances (solute) are distributed uniformly through another substance (solvent) Which water to use? •  Tap water – washing glassware and laboratory equipment •  Laboratory grade water – pretreated by reverse osmosis or distillation, good for making buffers •  Reagent grade water – further purified by distillation or deionization, good for buffers, culture media and biochemical reagents •  Ultrapure reagent grade water – treated with ultrafiltration, usually endotoxin, nuclease and protease free, used for experiments with stringent requirements 47 10/31/13 Solutions commonly used in biomedical research •  Molar solutions 1 mole of substance per 1 L of total solution §  mole – 6.02 × 1023 (Avogadro’s number) atoms for elements – atomic weight in grams for compounds – formula weight in grams Seidman and Moore 2000 Seidman and Moore 2000 48 10/31/13 Seidman and Moore 2000 •  weight/volume (w/v) – simplest way to express concentration, used for small amounts of chemicals or biological reagents weight of solute volume solvent How much proteinase K is needed to make 5 ml of a 2 mg/ml solution? ? = 2 mg proteinase K 5 ml 1 ml “ cross multiply” (5 ml) (2 mg) = 10 mg 1 ml 49 10/31/13 •  percents – amount of solute per 100 units of total solution amount of solute 100 ml solution §  weight per volume (w/v) percent solution Seidman and Moore 2000 §  volume per volume (v/v) percent solution – used when the solute is liquid or while diluting a concentrated stock solution 70 % ethanol – 70 ml of absolute ethanol per 100 ml of solution §  weight percent (w/w) – weight of solute in the numerator and the weight of the total solution in the denominator. Used when dealing with viscous solvents whose volumes are difficult to measure. 50 10/31/13 •  “Parts” – mix together a given amount of each component using the stated proportion 25:24:1 phenol:chloroform:isoamyl alcohol – used to remove proteins from nucleic acid preparations § Used used in environmental applications to express the concentration of pollutants in large amounts of solvents parts per million -­‐ parts solute / 1,ooo,000 parts solution parts per billion – parts solute / 1,000,000,000 parts solution ppm = 1 μg ppb = 1 ng ml ml Other ways of expressing concentration that are not common in Biology •  Molality (m) -­‐ 1 mole of compound dissolved in 1 kg of water •  Normality (N) – number of “equivalent weights” of solute / L solution acid – 1 equivalent weight = grams of acid that react to yield 1 mole of H+ base – 1 equivalent weight = grams of base that react to yield 1 mole of OH-­‐ Seidman and Moore 2000 51 10/31/13 Seidman and Moore 2000 Making solutions from hydrated compounds •  The molecular weight of the compound listed in the bottle includes the mass of water. •  The attached water molecules contribute water to the solution – important when determining the volume of water to used to dissolve the compound. •  The amount of water must be taken in consideration when the recipe calls for the anhydrous form of the compound. 52 10/31/13 •  Mind the extra water that will be added to the solution Na3PO4 Ÿ 12 H2O sodium phosphate tribasic dodecahydrate (FW 380.12) How much water is contributed by the compound when you make a 0.5 M solution? -­‐  To make 1 L, you need 190.06 g of Na3PO4 Ÿ 12 H2O (0.5 × 380.12) -­‐  1 mole water = 18.02 ml -­‐  0.5 mole Na3PO4 Ÿ 12 H2O = 6 mole water (12/2) -­‐  6 mole water = 108.12 ml water (6 × 18.02) -­‐ Take in consideration the 108 ml of water added by the compound when setting the initial volume of water to be used to make the solution Water contributed by a hydrated compound M ×
V
×
#H2O × 18.02 = ml H2O contributed by compound M = molarity (mole/L) V = final volume (L) # H2O = # water molecules in hydrated compound 18.02 = ml per mole of water •  Using the hydrated compound to make solutions calling for the anhydrous form Make 2.75 liters of 3.2M copper sulfate using CuSO4·∙5H2O (FW 249.7) -­‐  Subtract the water from the formula weight 5 H2O × 18.02 = 90.1 249.7 – 90.1 = 159.6 = FW anhydrous compound -­‐ Determine the % anhydrous form present in the hydrated compound 159.6 × 100 = 63.9% 249.7 -­‐  Determine the amount of anhydrous compound needed to make the solution 2.75 L × 3.2 mole/L × 159.6 g/mole = 1404.48 g -­‐  Determine the amount of hydrated compound needed to make the solution 1404.48 g = 2197.9 g 0.639 53 10/31/13 Using stock solutions Stock solutions -­‐ concentrated solutions that need to be diluted before they are used •  “X” – indicate the concentration factor 10× PBS (phosphate buffer saline solution concentrated 10 times) 20× SSC (standard saline citrate solution concentrated 20 times) C1V1 = C2V2 C1 = Concentrationstock V1 = Vstock C2 = Concentrationfinal V2 = Vfinal Dilution series – a group of solutions that have the same components at different concentrations. •  Commonly used in the laboratory to reduce the concentration of standards, reagents, bacterial or cell sample to achieve a concentration suitable for the test at hand •  Extremely important to keep the dilution factors in mind when making the calculations of the concentrations present in each sample 54 10/31/13 Independent serial dilution Seidman and Moore 2000 Dependent serial dilution Barker 2005 Making buffer solutions Buffers – solutions that keep a biological system at the proper pH •  pKa – pH at which the buffer is more stable and experiences little change of pH upon the addition of acids or bases Seidman and Moore 2000 55 10/31/13 Common Buffers Used in Molecular Biology http://www.teknova.com Strategies for making buffers 1.  The buffer is tritrated with a strong acid or base 2.  The buffer is prepared from 2 reagents, 1 more acidic and the other more basic 3.  The buffer is prepared as 2 stock solutions, 1 more acidic and the other more basic 56 10/31/13 Tris buffers (Tris(hydroxymethyl)aminomethane) – pKa of 8.06 but the pH is above 10 when dissolved in water •  Strategy 1 – Dissolve Tris base in water and bring the pH down with HCl •  Strategy 2 – Combine Tris base and its conjugated form called Tris-­‐HCl Seidman and Moore 2000 Phosphate buffers •  Strategy 3 – make individual solutions of sodium phosphate monobasic (Na2HPO4) and sodium phosphate dibasic (NaH2PO4) and mix then together to get the right pH http://www.molecularinfo.com 57 10/31/13 Sterilizing solutions Sterile – absence of living organism •  Autoclave – steam under pressure •  Filter sterilization http://www.enzymax.net http://www.directindustry.com http://www.bio-­‐equip.cn Keeping Sterility •  Use aseptic techniques •  Work in the appropriate biosafety cabinet §  Class I – protects the operator from aerosols but does not protect the sample because it draws air directly from the room http://en.biobase.cn 58 10/31/13 § Class II – Most common in the laboratory, protects both the operator and the product. Draws HEPA-­‐filtered air across the work surface and re-­‐filters the air before venting http://www.cardinalhealth.com § Class III – totally enclosed, ventilated cabinet of gas tight construction. Operations are done through attached rubber gloves http://www.scientificdealers.com Operating Centrifuges Barker 2005 59 10/31/13 Balancing the load is crucial when using a centrifuge. A difference of 0.5 g at 5000,000 × g = 250 Kg difference! http://www.stanford.edu Seidman and Moore 2000 Unbalanced centrifuge runs can ruin the rotor and the centrifuge. http://www.chem.purdue.edu/ 60 10/31/13 References Lisa Seidman and Cynthia Moore. 2000. Basic Laboratory Methods for Biotechnology: Texbook and Laboratory Reference. Prentice Hall. Kathy Barker. 2005. At the Bench: A Laboratory Navigator Updated Edition. Dany Spencer Adams. 2003. Lab Math: A Handbook of Measurements, Calculations, and Other Quantitative Skills for Use at the Bench. Cold Spring Harbor Laboratory Press. 61