Review of Laboratory Procedures Basic Laboratory Rules And Equipment This is an FYI presentation and will not be covered in class. However, if you have any questions please let me know. Thx

Basic Laboratory Rules No smoking, eating, or drinking No smoking, eating, or drinking No applying cosmetics No applying cosmetics No pipetting by mouth No pipetting by mouth All work should be done on absorbent paper All work should be done on absorbent paper Do not let glassware accumulate Do not let glassware accumulate All radiation labels should be defaced prior to discarding in regular trash All radiation labels should be defaced prior to discarding in regular trash Reagents should be stored in original container Reagents should be stored in original container

Lab Rules Continued Wash hands before leaving work area Wash hands before leaving work area Do not lay pencils, pens, or papers on work area Do not lay pencils, pens, or papers on work area No loafing in work area No loafing in work area Do not give patients their results Do not give patients their results Do not talk about patients outside of work area Do not talk about patients outside of work area Know decontamination rules Know decontamination rules

Lab Rules Continued Log arrival and exit of all kits Log arrival and exit of all kits On Arrival On Arrival Log date received Log date received Company it came from Company it came from Name of kit Name of kit Lot number Lot number Expiration Date Expiration Date Quantity of vials/activity Quantity of vials/activity Whether damaged or not On Arrival Whether damaged or not On Arrival On Exit On Exit If given to patient make list of who, what, and when If given to patient make list of who, what, and when Dispose of containers in proper receptacles Dispose of containers in proper receptacles

Laboratory Equipment Syringes Syringes Balances Balances Flasks Flasks Pipettes Pipettes Test Tubes Test Tubes Centrifuges Centrifuges Water bath Water bath Ice bath Ice bath Refrigerator Refrigerator Rotators Rotators Shakers Shakers

Balances Top Loading Top Loading Like bathroom scales Like bathroom scales Double Pan Double Pan Uses counter weights Uses counter weights Analytical Balance Analytical Balance Most accurate Most accurate Based on principle by comparing the mass of an unknown with that of calibrated weights Based on principle by comparing the mass of an unknown with that of calibrated weights

Flasks Erlenmeyer Flasks Erlenmeyer Flasks Beakers Beakers Volumetric Volumetric Calibrated to contain preparing solutions of exact concentrations (used for blood volumes) Calibrated to contain preparing solutions of exact concentrations (used for blood volumes)

Types of Pipettes There are two types of pipettes There are two types of pipettes Graduated Graduated A calibration mark decides how much liquid can be dispensed--come in whole number denominations A calibration mark decides how much liquid can be dispensed--come in whole number denominations Volumetric Volumetric Most accurate of glassware Most accurate of glassware Always calibrated to measure and/or deliver exact volumetric quantities of liquid substances Always calibrated to measure and/or deliver exact volumetric 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 by complete drainage of the pipette from an etched mark. They are normally used for the accurate transfer of 1.0, 2.0, 5.0, 10.0, and 25.0 ml of liquid. Generally, these pipettes deliver their inscribed volume by complete drainage of the pipette from an etched mark. They are normally used for the accurate transfer of 1.0, 2.0, 5.0, 10.0, and 25.0 ml of liquid.

Graduated Graduated Volumetric Volumetric

To Deliver Versus To Contain To Deliver (TD) To Deliver (TD) Allows for some liquid always remaining Allows for some liquid always remaining To Contain (TC) To Contain (TC) Desired volume is present, but it is impossible to get out

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

Blood versus Liquid Pipettes Blood Pipettes Blood Pipettes Thin walled Thin walled Bigger bore Bigger bore Read the top of the meniscus Read the top of the meniscus Liquid Pipettes Liquid Pipettes Thick walled Small bore Read the bottom of meniscus

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

How To Use A Pipette Never pipette by mouth Never pipette by mouth Use a vacuum creator Use a vacuum creator Hold pipette close to mouth piece (top) Hold pipette close to mouth piece (top) Dispense straight up with tip on glass deep enough, so as not to splash Dispense straight up with tip on glass deep enough, so as not to splash Perform quality control daily Perform quality control daily

Types of Test Tubes Polypropylene Polypropylene Cloudy Plastic Cloudy Plastic Polystyrene Polystyrene Clear Plastic Clear Plastic Glass Glass Clear Clear

Centrifuges Centrifuges are used as separators Centrifuges are used as separators Non-refrigerated Non-refrigerated Refrigerated Refrigerated Ultra-centrifuge Ultra-centrifuge Super fast--38,000 RPM Super fast--38,000 RPM Micro-centrifuge Micro-centrifuge Holds only small tubes Holds only small tubes Hematocrit-centrifuge Hematocrit-centrifuge Holds only glass capillaries Holds only glass capillaries

Centrifuges Centrifuges operate on centrifugal force Centrifuges operate on centrifugal force Heavier objects go to the bottom due to being thrown to the outside Heavier objects go to the bottom due to being thrown to the outside Most samples require 5,000 RPM Most samples require 5,000 RPM RCF is used by determining the radius and the speed (RPM) RCF is used by determining the radius and the speed (RPM) RCF is measured in g, as in g-force, or gravity RCF is measured in g, as in g-force, or gravity

Guidelines for Using Centrifuges Close and lock centrifuge before starting Close and lock centrifuge before starting Always balance tubes Always balance tubes Balance with like substance if possible, resulting in the same mass and center of gravity in each tube Balance with like substance if possible, resulting in the same mass and center of gravity in each tube Do not open while centrifuge is going Do not open while centrifuge is going Do not use brake unless extremely necessary Do not use brake unless extremely necessary Most damage of sample is caused by either an improper balance, too high RPM, or braking Most damage of sample is caused by either an improper balance, too high RPM, or braking Do not place hands in centrifuge while it is going Do not place hands in centrifuge while it is going

Collection Tubes The type of tube that a blood sample is drawn in determines whether the straw colored fluid of a settled centrifuge sample is serum or plasma. The type of tube that a blood sample is drawn in determines whether the straw colored fluid of a settled centrifuge sample is serum or plasma. Serum contains no clotting factor, while plasma contains an anti-coagulant. Serum contains no clotting factor, while plasma contains an anti-coagulant.

Types of Collection Tubes Red Stopper Red Stopper No anti-coagulant No anti-coagulant Yellow Stopper Yellow Stopper Acid Citrate Dextrose Acid Citrate Dextrose Green Stopper Green Stopper Sodium Heparin Sodium Heparin Purple Stopper Purple Stopper EDTA (Ethyline diamine tetric anticoagulant) EDTA (Ethyline diamine tetric anticoagulant) Grey Stopper Grey Stopper Sodium Fluoride Sodium Fluoride

Problems With A Blood Sample Hemolized Hemolized Damaged RBC’s are mixed in the serum plasma resulting in a pink color Damaged RBC’s are mixed in the serum plasma resulting in a pink color Lypholized Lypholized White milky serum/plasma due to eating a fatty meal before drawing blood White milky serum/plasma due to eating a fatty meal before drawing blood Icteric Icteric Bile in the blood due to liver disease; serum/plasma turns a yellow green color Bile in the blood due to liver disease; serum/plasma turns a yellow green color

Dilution Terminology Dilution Dilution Making a weaker solution from a stronger one Making a weaker solution from a stronger one Dilute solution Dilute solution A solution that contains a relatively small amount of solute A solution that contains a relatively small amount of solute Solvent Solvent A substance in which another substance, called solute is dissolved A substance in which another substance, called solute is dissolved Solute Solute Solution component present in smaller amount than solvent Solution component present in smaller amount than solvent

Dilution Factor This is the extent to which you have diluted your selected amount of stock solution and is given by the formulas: This is the extent to which you have diluted your selected amount of stock solution and is given by the formulas: V 2 /V 1 or C 1 /C 2 V 2 /V 1 or C 1 /C 2 In other words, we can either see how many times the volume has increased after the dilution which is 100mL/50mL = dilution factor of 2 in the previous problem. You could also say the concentration halved, so 0.100uCi/0.050uCi = dilution factor of 2. In other words, we can either see how many times the volume has increased after the dilution which is 100mL/50mL = dilution factor of 2 in the previous problem. You could also say the concentration halved, so 0.100uCi/0.050uCi = dilution factor of 2. Either way results in the same answer, but keep in mind a dilution factor always needs to be greater than 1. If your final answer is less than 1, you divided the wrong way which implies you concentrated instead of diluted. This is a way to check to make sure you did the math correctly. Either way results in the same answer, but keep in mind a dilution factor always needs to be greater than 1. If your final answer is less than 1, you divided the wrong way which implies you concentrated instead of diluted. This is a way to check to make sure you did the math correctly.

Why Dilute? If the concentration of the solution that needs to be analyzed is too great to be accurately determined If the concentration of the solution that needs to be analyzed is too great to be accurately determined Dilutions are usually referred to as a ratio of 1 to 10--one unit of original solution diluted to a final volume of 10 units Dilutions are usually referred to as a ratio of 1 to 10--one unit of original solution diluted to a final volume of 10 units 1 original + 9 dilutant = 10 units 1 original + 9 dilutant = 10 units

Dilution Principle Dilution principle can be expressed mathematically: Dilution principle can be expressed mathematically: Q = V x C Q = V x C Where: Where: Q = Quantity of Tracer Q = Quantity of Tracer V = Diluting Volume V = Diluting Volume C = Tracer concentration in fluid C = Tracer concentration in fluid

Dilution Formula Original Concentration x Dilution ratio = Final concentration Original Concentration x Dilution ratio = Final concentration Original Concentration of solution Original Concentration of solution Desired Concentration of solution = Dilution Ratio* Desired Concentration of solution = Dilution Ratio* * Dilution ratio is also known as dilution factor

Example Dilutions Final concentration of an original solution of 500 mg/ml diluted to 1:25 solution Final concentration of an original solution of 500 mg/ml diluted to 1:25 solution 500 mg/ml x 1/25 = 20 mg/ml Have a 8mg/ml solution and want a 2 mg/ml solution Have a 8mg/ml solution and want a 2 mg/ml solution 8 mg/ml 2 mg/ml = 4 units or a 1:4 ratio 1 part original 3 parts solvent

Series Dilution If more than one dilution is done within a given solution, the concentration of the solution is achieved by multiplying the original concentration by the series of dilutions. If more than one dilution is done within a given solution, the concentration of the solution is achieved by multiplying the original concentration by the series of dilutions. Example Example 1000 mg/ml diluted to 1:10 and then diluted 1: mg/ml diluted to 1:10 and then diluted 1: mg/ml x 1/10 x 1/100 = 1 mg/ml 1000mg/ml x 1/10 x 1/100 = 1 mg/ml

Conclusion There is a variety of lab equipment that must have quality control performed on it daily. There is a variety of lab equipment that must have quality control performed on it daily. Safety is a concern when dealing with laboratory equipment, as well as body fluids. Safety is a concern when dealing with laboratory equipment, as well as body fluids. Dilutions may be necessary if the concentration is too high to be measured by available methods. Dilutions may be necessary if the concentration is too high to be measured by available methods. Return to the Table of Content Table of ContentTable of Content