review of laboratory procedures basic laboratory rules and equipment this is an fyi presentation and...
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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.
ThxThx
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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
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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
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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
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Laboratory EquipmentLaboratory Equipment
SyringesSyringes BalancesBalances FlasksFlasks PipettesPipettes Test TubesTest Tubes CentrifugesCentrifuges
Water bathWater bath Ice bathIce bath RefrigeratorRefrigerator RotatorsRotators ShakersShakers
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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
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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)
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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. .
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GraduateGraduatedd
VolumetrVolumetricic
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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
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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%
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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
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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
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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
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Types of Test TubesTypes of Test Tubes
PolypropylenePolypropylene Cloudy PlasticCloudy Plastic
PolystyrenePolystyrene Clear PlasticClear Plastic
GlassGlass ClearClear
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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
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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
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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
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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.
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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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