Lab SkillsLab Skills

Unit B SummaryUnit B Summary

Safety RulesSafety RulesNo eating or drinking in the laboratory. No gum chewing. No makeup No eating or drinking in the laboratory. No gum chewing. No makeup application.application.Wear safety apparel, such as safety glasses, gloves, lab coats, and other Wear safety apparel, such as safety glasses, gloves, lab coats, and other protective clothing as necessary. Tie hair back if using Bunsen burners.protective clothing as necessary. Tie hair back if using Bunsen burners.Know the location of fire exits, fire extinguishers, and safety showers.Know the location of fire exits, fire extinguishers, and safety showers.Wash hands regularly, especially after working with microorganisms or Wash hands regularly, especially after working with microorganisms or chemicals.chemicals.Be aware of potential dangers. Before using products or equipment, carefully Be aware of potential dangers. Before using products or equipment, carefully read labels, experimental protocols, and equipment instructions and read read labels, experimental protocols, and equipment instructions and read literature. Know the location of and how to read Material Safety Data Sheets literature. Know the location of and how to read Material Safety Data Sheets (MSDS).(MSDS).Contaminated samples (chemical, biological, and glass) must be disposed of Contaminated samples (chemical, biological, and glass) must be disposed of in appropriate containers. Do not pick up broken glass with your hands. in appropriate containers. Do not pick up broken glass with your hands. Learn the specific methods from your lab supervisor.Learn the specific methods from your lab supervisor.Label all samples and reagents clearly with the name of the item, the name Label all samples and reagents clearly with the name of the item, the name of the person who prepared the sample, and the date of preparation.of the person who prepared the sample, and the date of preparation.Know emergency phone numbers and the best way to contact facility safety Know emergency phone numbers and the best way to contact facility safety officer.officer.Report spills and accidents to your lab supervisor or safety officer Report spills and accidents to your lab supervisor or safety officer immediately.immediately.

MSDS sheetsMSDS sheets– Chemical Name– Stability– Reactivity– Physical Data– Toxicity– Health Effects and First Aid– Storage and disposal

What does each area mean?– Red: Flammability– Yellow: Reactivity or instability– White: Special hazard– Blue: Health and Hazard

Occupational Safety & HealthAdministration ensures worker Safety and protection

Environmental Protection Agency is responsible for protecting the environment

Department of Transportation need to know what they are transporting

Personal Protection Equipment– Lab coat– Safety Glasses / Goggles– Gloves– Face shieldClosed toe shoesNo contactsNo loose or hanging clothes such as tiesMinimal jewelryLab coat, safety glasses and gloves most commonGlasses required for all liquid experiments

Prokaryotic cells (bacteria) – no nucleus or membrane-Prokaryotic cells (bacteria) – no nucleus or membrane-bound organellesbound organelles

Eukaryotic cells (all others) – may have the following Eukaryotic cells (all others) – may have the following

organelles:organelles:

Cell StructuresCell Structures

– Nucleus – controls activities in the cellNucleus – controls activities in the cell– Cytoplasm – solution outside nucleus but inside cellCytoplasm – solution outside nucleus but inside cell– Ribosomes – makes proteinsRibosomes – makes proteins– Er (endoplasmic reticulum) – passageway for protein transportEr (endoplasmic reticulum) – passageway for protein transport– Golgi – packages the proteinsGolgi – packages the proteins– Mitochondria – converts food to energy for cellMitochondria – converts food to energy for cell– Chloroplast – coverts sunlight to food in plant cellsChloroplast – coverts sunlight to food in plant cells– Vacuole – storage of water, enzymes, wasteVacuole – storage of water, enzymes, waste– Lysosomes – digests foreign material or bad cell partsLysosomes – digests foreign material or bad cell parts– Cell (plasma) membrane – controls what comes in / out of cellCell (plasma) membrane – controls what comes in / out of cell– Cell wall – external support for plant cellsCell wall – external support for plant cells

VirusesViruses

NonlivingNonlivingComposed of Nucleic acid and proteinComposed of Nucleic acid and proteinGrouped according to: Presence of Capsid and envelope – shape Grouped according to: Presence of Capsid and envelope – shape AND RNA or DNA, single or double stranded – structureAND RNA or DNA, single or double stranded – structureCan replicate only by invading host cell and using its enzyme and Can replicate only by invading host cell and using its enzyme and organelles.organelles.BacteriophageBacteriophage – viruses that infect bacteria; Used to study viruses – viruses that infect bacteria; Used to study virusesLytic CycleLytic Cycle– Viral genome is released into the host cellViral genome is released into the host cell– Replication follows immediatelyReplication follows immediately– Cellular components used to make new virusesCellular components used to make new viruses– Viral enzyme kills cell.Viral enzyme kills cell.

Lysogenic CycleLysogenic Cycle– Nucleic acid of virus becomes part of the host cell’s chromosomeNucleic acid of virus becomes part of the host cell’s chromosome– Nucleic acid remains in the cell in this form for many generationsNucleic acid remains in the cell in this form for many generations

BiomoleculesBiomolecules

Water - polar, good solvent, sticks together, Water - polar, good solvent, sticks together, resistant to heat change, inorganicresistant to heat change, inorganicOrganic – made of carbonOrganic – made of carbonCarbohydrate – used for energy, sugars, starch, Carbohydrate – used for energy, sugars, starch, CC66HH1212OO66, building block is monosaccharide, building block is monosaccharideLipid – used as barrier in membrane, fats, oils, Lipid – used as barrier in membrane, fats, oils, long carbon chain, building block is fatty acidlong carbon chain, building block is fatty acidProtein – used for enzymes and structure, Protein – used for enzymes and structure, building block is amino acidsbuilding block is amino acidsNucleic acids – DNA and RNA, building block is Nucleic acids – DNA and RNA, building block is nucleotidesnucleotides

MetrologyMetrology

Units define measurements & give the numbers valueUnits define measurements & give the numbers value

Accuracy is how close an individual value is to the true or Accuracy is how close an individual value is to the true or accepted valueaccepted value

% error% error = True value – measured value X 100% = True value – measured value X 100%

True valueTrue value

Precision is the consistency of a series of measurementsPrecision is the consistency of a series of measurements

Take an average of the deviation, so it is the Take an average of the deviation, so it is the average average deviationdeviation from the mean from the mean

Standards are Standards are Measurements made in accordance with Measurements made in accordance with an external authorityan external authority

MetrologyMetrology

Verification - Verification - Check of the performance of an instrument or Check of the performance of an instrument or method without adjusting it.method without adjusting it.CalibrationCalibration - Bringing a measuring system into accordance - Bringing a measuring system into accordance with external authority, using standardswith external authority, using standardsToleranceTolerance - Amount of error that is allowed in the - Amount of error that is allowed in the calibration of a particular item.calibration of a particular item. Traceability - Traceability - The chain of calibrations, genealogy, that The chain of calibrations, genealogy, that establishes the value of a standard or measurementestablishes the value of a standard or measurementErrorError is responsible for the difference between a measured is responsible for the difference between a measured value and the “true” valuevalue and the “true” valueThree types of error:Three types of error:– Gross (blunders)Gross (blunders)– RandomRandom– SystematicSystematic

VolumeVolume

Graduated cylinders – over 10 mlGraduated cylinders – over 10 ml10 ml serological pipets10 ml serological pipets5 ml serological pipets5 ml serological pipets2 ml serological pipets2 ml serological pipets1 ml serological pipets1 ml serological pipetsP1000 micropipetsP1000 micropipetsP100 micropipetsP100 micropipetsP10 micropipetsP10 micropipetsMultichannel pipetsMultichannel pipets

PipetsPipets

Use the right instrument to make the Use the right instrument to make the correct measurementscorrect measurements

Verify and calibrate micropipets with water Verify and calibrate micropipets with water which has a density of 1 g for every mlwhich has a density of 1 g for every ml

Maintenance micropipets by cleaning and Maintenance micropipets by cleaning and storing properly and recording.storing properly and recording.

Terminology for weighingTerminology for weighing

RangeRange: : – the span from lightest to heaviest weight that the span from lightest to heaviest weight that

a balance is able to measurea balance is able to measure

CapacityCapacity: : – the heaviest sample that a balance can weighthe heaviest sample that a balance can weigh

SensitivitySensitivity::– the smallest value of weight that will cause a the smallest value of weight that will cause a

change in the response of the balance.change in the response of the balance.

Proper weighing procedureProper weighing procedure

Make sure the balance is Make sure the balance is levellevelAdjust the balance to Adjust the balance to zerozeroTareTare the weighing container or weigh the the weighing container or weigh the empty containerempty containerPlace the sample in a the weighing Place the sample in a the weighing container and container and read the weightread the weightRemoveRemove the sample the sampleCleanClean the balance and surrounding area the balance and surrounding area

Proper weighing techniquesProper weighing techniques

Always use a Always use a calibratedcalibrated weight to verify weight to verify the scale is in proper working order (daily)the scale is in proper working order (daily)

Always use a Always use a weigh boat or weigh paperweigh boat or weigh paper; ; do not place materials directly on the pando not place materials directly on the pan

Do not touchDo not touch the chemicals or material the chemicals or material being weighedbeing weighed

Do not return unusedDo not return unused chemicals to their chemicals to their storage bottle storage bottle (unless you use a sterile spatula or (unless you use a sterile spatula or spoon)spoon)

Calibration of BalancesCalibration of Balances

First step is to zero the balance. It should read First step is to zero the balance. It should read zero every time you press the zero buttonzero every time you press the zero buttonSecond calibration point is taken at the upper Second calibration point is taken at the upper end of the capacity of the balanceend of the capacity of the balance– Place a certified weight on the balance and verify it Place a certified weight on the balance and verify it

reads the correct weightreads the correct weightSome scales will prompt you to enter the weight Some scales will prompt you to enter the weight

A third reading can be made using a lighter A third reading can be made using a lighter calibrated weight and verifying it reads the calibrated weight and verifying it reads the proper weightproper weight

Equipment Log BooksEquipment Log Books

Notebooks or binders used to maintain Notebooks or binders used to maintain operating procedures, calibration records, operating procedures, calibration records, verification checksverification checks– Example: the equipment log book in the back Example: the equipment log book in the back

of the roomof the roomIncubator temp chartsIncubator temp charts

Refrigerator temp chartsRefrigerator temp charts

Pipet calibration recordsPipet calibration records

Balance calibration and verification chartsBalance calibration and verification charts

SolutionsSolutions

SolutionSolution: a homogeneous mixture in which one or more : a homogeneous mixture in which one or more substances are dissolved in another.substances are dissolved in another.

SoluteSolute: substances that are dissolved; : substances that are dissolved; units are often g, mg, or µgunits are often g, mg, or µg

SolventSolvent: substances in which solutes are dissolved (: substances in which solutes are dissolved (often often

times this is water or a buffertimes this is water or a buffer))units are often L, ml, or µlunits are often L, ml, or µl

ConcentrationConcentration: amount per volume mass/vol: amount per volume mass/volunits are g/L, g/ ml, mg/ml, molarunits are g/L, g/ ml, mg/ml, molar

Solution PrepSolution Prep

Mass/volumeMass/volume

% mass/volume% mass/volume

MolarityMolarity– molarity (M) is equal to the number of moles molarity (M) is equal to the number of moles

of solute that are dissolved per Liter of solventof solute that are dissolved per Liter of solvent

Dilution - C1xV1 = C2xV2Dilution - C1xV1 = C2xV2

AcidsAcids

Definition: Definition: electrolyte that electrolyte that donatesdonates hydrogen ions hydrogen ions

Properties: Properties: – Acids in water Acids in water conduct electricityconduct electricity– The stronger the acid the stronger the conductivityThe stronger the acid the stronger the conductivity

– Acids react w/metals to Acids react w/metals to produce Hproduce H22 gas gas

– Acids are indicators; they cause reversible color changesAcids are indicators; they cause reversible color changesPhenolphthalein and litmus are two examples of acid-base indicatorsPhenolphthalein and litmus are two examples of acid-base indicators

– Acids react w/hydroxide compounds to form water and salt; Acids react w/hydroxide compounds to form water and salt; this type of reaction is called “neutralization”this type of reaction is called “neutralization”

– Strong acids completely dissociate in water to Strong acids completely dissociate in water to release release hydrogen ions = Hhydrogen ions = H++

i.e. hydrochloric acid (HCl): HCL in water i.e. hydrochloric acid (HCl): HCL in water H H++ + Cl + Cl--

– Tastes SourTastes Sour

BasesBases

Definition: Definition: electrolyte that yields hydroxide ions or electrolyte that yields hydroxide ions or accepts hydrogen ionsaccepts hydrogen ionsProperties:Properties:– Bases in water Bases in water conduct electricityconduct electricity– The stronger the base the stronger the conductivityThe stronger the base the stronger the conductivity– Bases react with acids in neutralization reactions to form Bases react with acids in neutralization reactions to form

water and a saltwater and a salt– Bases cause reversible color changes in acid-base Bases cause reversible color changes in acid-base

indicators (color is pH dependent)indicators (color is pH dependent)– Bases in water solution are Bases in water solution are slippery to the touchslippery to the touch– Caution: even dilute bases can be caustic!Caution: even dilute bases can be caustic!– Strong bases completely dissociate in water to Strong bases completely dissociate in water to release release

hydroxide ions = OHhydroxide ions = OH- -

NaOH in water NaOH in water Na Na+ + + OH+ OH--

TheThe OHOH-- ions react with H ions react with H ++ to form water, thereby to form water, thereby the the concentration of hydrogen ionsconcentration of hydrogen ions

– Tastes bitterTastes bitter

pH ispH is

A way to express hydrogen ion concentration in a A way to express hydrogen ion concentration in a solutionsolutionMeasurement of the acidity/alkalinity of an aqueous Measurement of the acidity/alkalinity of an aqueous solutionsolutionpH is the –log of the HpH is the –log of the H+ + concentrationconcentrationpH is measured on a scalepH is measured on a scale– Ranges from 0 to 14Ranges from 0 to 14

Pure waterPure water– HH++ concentration is 1x10 concentration is 1x10-7-7 mole/L mole/L– The log of 1x10The log of 1x10-7-7 = -7 = -7– The – log of –7 = 7The – log of –7 = 7– The pH of pure water = 7The pH of pure water = 7

Buffer Buffer

Substance(s) that when in aqueous Substance(s) that when in aqueous solution resists a change in Hsolution resists a change in H++ concentrationconcentration even if acids or bases are even if acids or bases are addedaddedSome buffers change pH as their Some buffers change pH as their temperature and/or concentration changestemperature and/or concentration changesTris buffer is widely used in molecular Tris buffer is widely used in molecular biology; it is very sensitive to temperature biology; it is very sensitive to temperature and the pH will vary greatly at various and the pH will vary greatly at various temperatures.temperatures.

Measuring pHMeasuring pH

IndicatorsIndicators– Phenophthalein, phenol red, bromothymol Phenophthalein, phenol red, bromothymol

blue, universal indicator to name a fewblue, universal indicator to name a few

pH PaperpH Paper

pH MeterspH Meters

pH Meter pH Meter Meter / electrode system for measuring pH in laboratoryMeter / electrode system for measuring pH in laboratoryProvides greater accuracy, sensitivity than chemical Provides greater accuracy, sensitivity than chemical indicatorsindicatorsCan measure pH of a solution to the nearest 0.1 unitCan measure pH of a solution to the nearest 0.1 unitCan be used with variety of aqueous solutionsCan be used with variety of aqueous solutionsConsists of:Consists of:– Voltmeter – measures voltageVoltmeter – measures voltage– Two electrodes connected to one another (sensor Two electrodes connected to one another (sensor

probe)probe)When immersed in the sample they develop an When immersed in the sample they develop an electrical voltage that is measured by the voltmeterelectrical voltage that is measured by the voltmeter

Calibration recommended with each use, when battery Calibration recommended with each use, when battery replaced and when fluid in sensor is changedreplaced and when fluid in sensor is changed

MicroscopeMicroscope

Important Lab instrumentImportant Lab instrument

Why use a microscope?Why use a microscope?

To view objects and detail too small to see with To view objects and detail too small to see with human eye. Improves human eye. Improves resolutionresolution of object of object

List examples of how what you used it for:List examples of how what you used it for:– Blood cell detailBlood cell detail

rbc, wbc, plateletsrbc, wbc, platelets

– BacteriaBacteriaCocci, rod, bacillusCocci, rod, bacillus

– ProtozoaProtozoaTrichomonasTrichomonas

GiardiaGiardia

Types of MicroscopesTypes of Microscopes

Compound microscopeCompound microscope– Bacteria, fungi and protozoaBacteria, fungi and protozoa

Electron microscopeElectron microscope– Required for virusesRequired for viruses

FluorescenceFluorescence– Used as a diagnostic tool for Used as a diagnostic tool for

immunofluorescence tests immunofluorescence tests

Parts of a microscopeParts of a microscope

Coarse adjustmentCoarse adjustment– 11stst step in focusing to change the distance step in focusing to change the distance

between specimen and lensbetween specimen and lens

Fine adjustmentFine adjustment– To fine tune the pictureTo fine tune the picture– Used particularly with 100x and oil objectiveUsed particularly with 100x and oil objective

StageStage– Holds the slide, moved up and down with Holds the slide, moved up and down with

coarse and fine adjustmentcoarse and fine adjustment

Parts of a microscopeParts of a microscope

ObjectivesObjectives– Common objectives are 4x, 10x, 40x, 100x, oilCommon objectives are 4x, 10x, 40x, 100x, oil– Total magnification is eye piece magnification Total magnification is eye piece magnification

multiplied times objective you are viewing withmultiplied times objective you are viewing with40x objective and 10x eye piece is 400x 40x objective and 10x eye piece is 400x magnificationmagnification

– Oil objective is labeled and is always used Oil objective is labeled and is always used with immersion oilwith immersion oil

Oil increases the resolving power by focusing the Oil increases the resolving power by focusing the light rayslight rays

Proper care for microscopeProper care for microscope

Always start with lowest objective to focusAlways start with lowest objective to focusAlways store with lowest objective locked Always store with lowest objective locked in placein placeCarry with two handsCarry with two handsCover with dust coverCover with dust coverBe sure to clean oil off of oil objectiveBe sure to clean oil off of oil objectiveUse fine adjustment with 100x and oil Use fine adjustment with 100x and oil objectiveobjective

SpectrophotometersSpectrophotometers

When light shines on a solution, it can bounce When light shines on a solution, it can bounce off of the molecules (reflect), pass through the off of the molecules (reflect), pass through the solution (transmittance), or some of the energy solution (transmittance), or some of the energy be absorbed by the solution.be absorbed by the solution.Spectrophotometers are instruments that Spectrophotometers are instruments that measure the interaction of light with materials in measure the interaction of light with materials in solutionsolutionSpectrophotometers compare the light Spectrophotometers compare the light transmitted through a sample to the light transmitted through a sample to the light transmitted through a transmitted through a blank.blank.The blank contains everything The blank contains everything

except the analyteexcept the analyte

ChromatographyChromatography

Physical properties that can be used to Physical properties that can be used to separate moleculesseparate molecules– SizeSize– ShapeShape– Density/gravityDensity/gravity– ChargeCharge– State (solid, liquid, gas)State (solid, liquid, gas)– Phase changes (mp, bp, evap)Phase changes (mp, bp, evap)

Chromatography Key TermsChromatography Key Terms

ChromatographyChromatography: techniques for the separation of complex : techniques for the separation of complex mixtures that rely on the differential affinities of substances mixtures that rely on the differential affinities of substances Stationary phaseStationary phase: what you pack the column with or the : what you pack the column with or the plate/paperplate/paperMobile phaseMobile phase: solvent/phase moving in the bed; fraction or : solvent/phase moving in the bed; fraction or sample being separated; sample being separated; EffluentEffluent: the mobile phase leaving the column: the mobile phase leaving the columnTypes of ChromatographyTypes of Chromatography– PaperPaper– Thin LayerThin Layer– Ion Exchange or AffinityIon Exchange or Affinity– Size ExclusionSize Exclusion– High Pressure Liquid Chromatography (HPLC)High Pressure Liquid Chromatography (HPLC)

Gel ElectrophoresisGel Electrophoresis

DefinitionDefinition: the process of separating : the process of separating molecules based on size and chargemolecules based on size and chargeAgaroseAgarose: highly purified agar, heated and : highly purified agar, heated and dissolved in buffer. Forms a matrix of dissolved in buffer. Forms a matrix of pores for molecules to travel through.pores for molecules to travel through.– Smaller molecules travel furtherSmaller molecules travel further– Molecules migrate towards the Molecules migrate towards the – positive (red) end of the chamberpositive (red) end of the chamber

Gel ElectrophoresisGel Electrophoresis

ProcessProcess– Make Agarose gelMake Agarose gel

Thinner gels (0.8%) yield better results for larger DNA Thinner gels (0.8%) yield better results for larger DNA

– Prepare samplesPrepare samplesRestriction enzymes used to cleave at specified sitesRestriction enzymes used to cleave at specified sites

– Apply samples to gels, apply currentApply samples to gels, apply currentIf samples run from positive end they will run off the gelIf samples run from positive end they will run off the gel

– Stain gels to see bandsStain gels to see bandsWould not be able to see bands if we did not stainWould not be able to see bands if we did not stain

Gel ElectrophoresisGel Electrophoresis

DNA molecules have a negative chargeDNA molecules have a negative charge– This allows them to migrate towards the positive end of the This allows them to migrate towards the positive end of the

chamberchamber

The samples and the electrophoresis chamber use The samples and the electrophoresis chamber use specialized buffers. Using specialized buffers. Using TAE/TBE buffer helps stabilize the sample TAE/TBE buffer helps stabilize the sample and allows the reaction to occur quicker in and allows the reaction to occur quicker in the chamber.the chamber.– If water were in the chamber instead of TAE/TBE buffer the If water were in the chamber instead of TAE/TBE buffer the

reaction would take much longer or migration may not occur at reaction would take much longer or migration may not occur at allall

Stains: ethidium bromide will cause the bands to glow Stains: ethidium bromide will cause the bands to glow orange under UV light. Fast stain will result in blue orange under UV light. Fast stain will result in blue bandsbands

Uses for Gel ElectrophoresisUses for Gel Electrophoresis

DNA fingerprinting or profilingDNA fingerprinting or profiling– Paternity testingPaternity testing– Crime scene sample analysisCrime scene sample analysis– Identification of bacteria and other pathogensIdentification of bacteria and other pathogens

Who is credited with discovering the DNA Who is credited with discovering the DNA profiling process?profiling process?– Alec Jefferies in 1985Alec Jefferies in 1985

Cell Culture Cell Culture

DefinitionDefinition: the : the in vitroin vitro growth of cells growth of cells isolated from multi-cellular organismsisolated from multi-cellular organisms

ProcessProcess: Cells will continue dividing until : Cells will continue dividing until they fill up the container; cell to cell they fill up the container; cell to cell contact stops cell divisioncontact stops cell division

UsesUses: vaccines, research of all kinds : vaccines, research of all kinds including stem cell, recombinant DNA, including stem cell, recombinant DNA, production of antibodiesproduction of antibodies

Types of Cell usedTypes of Cell used

Bacterial cellsBacterial cells

Yeast cellsYeast cells

Mold cellsMold cells

Plant cellsPlant cells

Insect cellsInsect cells

Mammalian cellsMammalian cells

Growing Bacterial CellsGrowing Bacterial Cells

Choose bacteria – Choose bacteria – E. coliE. coli most common most commonMake mediaMake media– Petri plates use agar in media (Luria Broth, Petri plates use agar in media (Luria Broth,

nutrient agar)nutrient agar)– Liquid cultures use broth (LB, nutrient broth)Liquid cultures use broth (LB, nutrient broth)

Sterilize media in autoclaveSterilize media in autoclavePour media platesPour media platesInnoculate mediaInnoculate mediaGrow cells in incubator (37Grow cells in incubator (37ooC)C)