Review of Laboratory Review of Laboratory ProceduresProcedures

Basic Laboratory Rules And Basic Laboratory Rules And EquipmentEquipment

This is an FYI presentation and This is an FYI presentation and will not be covered in class. will not be covered in class.

However, if you have any However, if you have any questions please let me know. questions please let me know.

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Basic Laboratory RulesBasic Laboratory Rules

No smoking, eating, or drinkingNo smoking, eating, or drinking No applying cosmeticsNo applying cosmetics No pipetting by mouthNo pipetting by mouth All work should be done on absorbent All work should be done on absorbent

paperpaper Do not let glassware accumulateDo not let glassware accumulate All radiation labels should be defaced All radiation labels should be defaced

prior to discarding in regular trashprior to discarding in regular trash Reagents should be stored in original Reagents should be stored in original

containercontainer

Lab Rules ContinuedLab Rules Continued

Wash hands before leaving work areaWash hands before leaving work area Do not lay pencils, pens, or papers on Do not lay pencils, pens, or papers on

work areawork area No loafing in work areaNo loafing in work area Do not give patients their resultsDo not give patients their results Do not talk about patients outside of work Do not talk about patients outside of work

areaarea Know decontamination rulesKnow decontamination rules

Lab Rules ContinuedLab Rules Continued Log arrival and exit of all kitsLog arrival and exit of all kits

On ArrivalOn Arrival Log date receivedLog date received Company it came fromCompany it came from Name of kitName of kit Lot numberLot number Expiration DateExpiration Date Quantity of vials/activityQuantity of vials/activity Whether damaged or not On ArrivalWhether damaged or not On Arrival

On ExitOn Exit If given to patient make list of who, what, and whenIf given to patient make list of who, what, and when Dispose of containers in proper receptaclesDispose of containers in proper receptacles

Laboratory EquipmentLaboratory Equipment

SyringesSyringes BalancesBalances FlasksFlasks PipettesPipettes Test TubesTest Tubes CentrifugesCentrifuges

Water bathWater bath Ice bathIce bath RefrigeratorRefrigerator RotatorsRotators ShakersShakers

BalancesBalances

Top LoadingTop Loading Like bathroom scalesLike bathroom scales

Double PanDouble Pan Uses counter weightsUses counter weights

Analytical BalanceAnalytical Balance Most accurateMost accurate Based on principle by comparing the Based on principle by comparing the

mass of an unknown with that of mass of an unknown with that of calibrated weightscalibrated weights

FlasksFlasks

Erlenmeyer FlasksErlenmeyer Flasks BeakersBeakers VolumetricVolumetric

Calibrated to contain preparing solutions Calibrated to contain preparing solutions of exact concentrations (used for blood of exact concentrations (used for blood volumes)volumes)

Types of PipettesTypes of Pipettes There are two types of pipettesThere are two types of pipettes

GraduatedGraduated A calibration mark decides how much liquid can be A calibration mark decides how much liquid can be

dispensed--come in whole number denominationsdispensed--come in whole number denominations VolumetricVolumetric

Most accurate of glasswareMost accurate of glassware Always calibrated to measure and/or deliver exact Always calibrated to measure and/or deliver exact

volumetric quantities of liquid substancesvolumetric quantities of liquid substances Limited to measurement of a fixed, single volume.Limited to measurement of a fixed, single volume.

Generally, these pipettes deliver their inscribed volume Generally, these pipettes deliver their inscribed volume by complete drainage of the pipette from an etched by complete drainage of the pipette from an etched mark. They are normally used for the accurate transfer mark. They are normally used for the accurate transfer of 1.0, 2.0, 5.0, 10.0, and 25.0 ml of liquidof 1.0, 2.0, 5.0, 10.0, and 25.0 ml of liquid. .

GraduateGraduatedd

VolumetrVolumetricic

To Deliver Versus To To Deliver Versus To ContainContain

To Deliver (TD)To Deliver (TD) Allows for some Allows for some

liquid always liquid always remainingremaining

To Contain (TC)To Contain (TC) Desired volume is Desired volume is

present, but it is present, but it is impossible to get impossible to get outout

Calibration of PipettesCalibration of Pipettes

Pipettes are calibrated at the factory Pipettes are calibrated at the factory with Mercury at 20 degrees Celsius with Mercury at 20 degrees Celsius with an error percentage of +/- 1%with an error percentage of +/- 1%

Blood versus Liquid PipettesBlood versus Liquid Pipettes

Blood PipettesBlood Pipettes Thin walledThin walled Bigger boreBigger bore Read the top of the Read the top of the

meniscusmeniscus

Liquid PipettesLiquid Pipettes Thick walledThick walled Small boreSmall bore Read the bottom of Read the bottom of

meniscusmeniscus

MeniscusMeniscusA meniscus is where surface tension attracts A meniscus is where surface tension attracts liquid, creating a bubble looking lineliquid, creating a bubble looking line

How To Use A PipetteHow To Use A Pipette

Never pipette by mouthNever pipette by mouth Use a vacuum creatorUse a vacuum creator Hold pipette close to mouth piece Hold pipette close to mouth piece

(top)(top) Dispense straight up with tip on glass Dispense straight up with tip on glass

deep enough, so as not to splashdeep enough, so as not to splash Perform quality control dailyPerform quality control daily

Types of Test TubesTypes of Test Tubes

PolypropylenePolypropylene Cloudy PlasticCloudy Plastic

PolystyrenePolystyrene Clear PlasticClear Plastic

GlassGlass ClearClear

CentrifugesCentrifuges Centrifuges are used as separatorsCentrifuges are used as separators

Non-refrigeratedNon-refrigerated RefrigeratedRefrigerated Ultra-centrifugeUltra-centrifuge

Super fast--38,000 RPMSuper fast--38,000 RPM Micro-centrifugeMicro-centrifuge

Holds only small tubesHolds only small tubes Hematocrit-centrifugeHematocrit-centrifuge

Holds only glass capillariesHolds only glass capillaries

CentrifugesCentrifuges

Centrifuges operate on centrifugal Centrifuges operate on centrifugal forceforce Heavier objects go to the bottom due to Heavier objects go to the bottom due to

being thrown to the outsidebeing thrown to the outside Most samples require 5,000 RPMMost samples require 5,000 RPM RCF is used by determining the radius and RCF is used by determining the radius and

the speed (RPM)the speed (RPM) RCF is measured in g, as in g-force, or RCF is measured in g, as in g-force, or

gravitygravity

Guidelines for Using Guidelines for Using CentrifugesCentrifuges

Close and lock centrifuge before startingClose and lock centrifuge before starting Always balance tubesAlways balance tubes

Balance with like substance if possible, resulting in Balance with like substance if possible, resulting in the same mass and center of gravity in each tubethe same mass and center of gravity in each tube

Do not open while centrifuge is goingDo not open while centrifuge is going Do not use brake unless extremely necessaryDo not use brake unless extremely necessary

Most damage of sample is caused by either an Most damage of sample is caused by either an improper balance, too high RPM, or brakingimproper balance, too high RPM, or braking

Do not place hands in centrifuge while it is Do not place hands in centrifuge while it is goinggoing

Collection TubesCollection Tubes

The type of tube that a blood sample The type of tube that a blood sample is drawn in determines whether the is drawn in determines whether the straw colored fluid of a settled straw colored fluid of a settled centrifuge sample is serum or centrifuge sample is serum or plasma.plasma.

Serum contains no clotting factor, Serum contains no clotting factor, while plasma contains an anti-while plasma contains an anti-coagulant.coagulant.

Types of Collection TubesTypes of Collection Tubes Red StopperRed Stopper

No anti-coagulantNo anti-coagulant Yellow StopperYellow Stopper

Acid Citrate DextroseAcid Citrate Dextrose Green StopperGreen Stopper

Sodium HeparinSodium Heparin Purple StopperPurple Stopper

EDTA (Ethyline diamine tetric anticoagulant)EDTA (Ethyline diamine tetric anticoagulant) Grey StopperGrey Stopper

Sodium FluorideSodium Fluoride

Problems With A Blood Problems With A Blood SampleSample

HemolizedHemolized Damaged RBC’s are mixed in the serum Damaged RBC’s are mixed in the serum

plasma resulting in a pink colorplasma resulting in a pink color LypholizedLypholized

White milky serum/plasma due to eating a White milky serum/plasma due to eating a fatty meal before drawing bloodfatty meal before drawing blood

IctericIcteric Bile in the blood due to liver disease; Bile in the blood due to liver disease;

serum/plasma turns a yellow green colorserum/plasma turns a yellow green color

Dilution TerminologyDilution Terminology DilutionDilution

Making a weaker solution from a stronger oneMaking a weaker solution from a stronger one Dilute solutionDilute solution

A solution that contains a relatively small A solution that contains a relatively small amount of soluteamount of solute

SolventSolvent A substance in which another substance, A substance in which another substance,

called solute is dissolvedcalled solute is dissolved SoluteSolute

Solution component present in smaller Solution component present in smaller amount than solventamount than solvent

Dilution FactorDilution Factor This is the extent to which you have diluted This is the extent to which you have diluted

your selected amount of stock solution and is your selected amount of stock solution and is given by the formulas:given by the formulas: VV22/V/V11 or C or C11/C/C22

In other words, we can either see how many In other words, we can either see how many times the volume has increased after the times the volume has increased after the dilution which is 100mL/50mL = dilution factor dilution which is 100mL/50mL = dilution factor of 2 in the previous problem. You could also of 2 in the previous problem. You could also say the concentration halved, so say the concentration halved, so 0.100uCi/0.050uCi = dilution factor of 2. 0.100uCi/0.050uCi = dilution factor of 2.

Either way results in the same answer, but keep Either way results in the same answer, but keep in mind a dilution factor always needs to be in mind a dilution factor always needs to be greater than 1. If your final answer is less than greater than 1. If your final answer is less than 1, you divided the wrong way which implies you 1, you divided the wrong way which implies you concentrated instead of diluted. This is a way concentrated instead of diluted. This is a way to check to make sure you did the math to check to make sure you did the math correctly.correctly.

Why Dilute?Why Dilute?

If the concentration of the solution If the concentration of the solution that needs to be analyzed is too that needs to be analyzed is too great to be accurately determinedgreat to be accurately determined

Dilutions are usually referred to as Dilutions are usually referred to as a ratio of 1 to 10--one unit of a ratio of 1 to 10--one unit of original solution diluted to a final original solution diluted to a final volume of 10 unitsvolume of 10 units

1 original + 9 dilutant = 10 units1 original + 9 dilutant = 10 units

Dilution PrincipleDilution Principle

Dilution principle can be expressed Dilution principle can be expressed mathematically:mathematically:

Q = V x CQ = V x C Where:Where:

Q = Quantity of TracerQ = Quantity of Tracer V = Diluting VolumeV = Diluting Volume C = Tracer concentration in fluidC = Tracer concentration in fluid

Dilution FormulaDilution Formula

Original Concentration x Dilution ratio = Original Concentration x Dilution ratio = Final concentrationFinal concentration

Original Concentration of solutionOriginal Concentration of solution Desired Concentration of solution = Desired Concentration of solution =

Dilution Ratio*Dilution Ratio*

**Dilution ratio is also known as dilution factorDilution ratio is also known as dilution factor

Example DilutionsExample Dilutions Final concentration of an original solution Final concentration of an original solution

of 500 mg/ml diluted to 1:25 solutionof 500 mg/ml diluted to 1:25 solution500 mg/ml x 1/25 = 20 mg/ml500 mg/ml x 1/25 = 20 mg/ml

Have a 8mg/ml solution and want a 2 Have a 8mg/ml solution and want a 2 mg/ml solutionmg/ml solution8 mg/ml8 mg/ml

2 mg/ml = 4 units or a 1:4 ratio 2 mg/ml = 4 units or a 1:4 ratio

1 part original 3 parts solvent1 part original 3 parts solvent

Series DilutionSeries Dilution If more than one dilution is done within a If more than one dilution is done within a

given solution, the concentration of the given solution, the concentration of the solution is achieved by multiplying the solution is achieved by multiplying the original concentration by the series of original concentration by the series of dilutions.dilutions.

ExampleExample 1000 mg/ml diluted to 1:10 and then diluted 1000 mg/ml diluted to 1:10 and then diluted

1:1001:100 1000mg/ml x 1/10 x 1/100 = 1 mg/ml1000mg/ml x 1/10 x 1/100 = 1 mg/ml

ConclusionConclusion There is a variety of lab equipment that There is a variety of lab equipment that

must have quality control performed on it must have quality control performed on it daily.daily.

Safety is a concern when dealing with Safety is a concern when dealing with laboratory equipment, as well as body fluids.laboratory equipment, as well as body fluids.

Dilutions may be necessary if the Dilutions may be necessary if the concentration is too high to be measured by concentration is too high to be measured by available methods.available methods.

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