Review of Laboratory Procedures Basic Laboratory Rules And

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

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

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

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

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

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

Types of Pipettes n There are two types of pipettes n Graduated n n A calibration mark decides how much liquid can be dispensed--come in whole number denominations Volumetric Most accurate of glassware n Always calibrated to measure and/or deliver exact volumetric quantities of liquid substances n Limited to measurement of a fixed, single volume. n n 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.

n Graduated n Volumetric

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

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

Blood versus Liquid Pipettes n Blood Pipettes n n n Thin walled Bigger bore Read the top of the meniscus n Liquid Pipettes n n n 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 n n n Never pipette by mouth Use a vacuum creator Hold pipette close to mouth piece (top) Dispense straight up with tip on glass deep enough, so as not to splash Perform quality control daily

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

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

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

Guidelines for Using Centrifuges n n Close and lock centrifuge before starting Always balance tubes n n n Do not open while centrifuge is going Do not use brake unless extremely necessary n n Balance with like substance if possible, resulting in the same mass and center of gravity in each tube 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

Collection Tubes n n 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.

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

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

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

n Dilution Factor This is the extent to which you have diluted your selected amount of stock solution and is given by the formulas: n n n 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 100 m. L/50 m. L = dilution factor of 2 in the previous problem. You could also say the concentration halved, so 0. 100 u. Ci/0. 050 u. Ci = 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.

Why Dilute? n n n 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 1 original + 9 dilutant = 10 units

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

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

Example Dilutions n Final concentration of an original solution of 500 mg/ml diluted to 1: 25 solution 500 mg/ml x 1/25 = 20 mg/ml n Have a 8 mg/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 n n 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 1000 mg/ml diluted to 1: 10 and then diluted 1: 100 n 1000 mg/ml x 1/100 = 1 mg/ml n

Conclusion n 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. Dilutions may be necessary if the concentration is too high to be measured by available methods. Return to the Table of Content