Review of Laboratory Procedures

1. 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

2. Basic Laboratory Rules • 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

3. Lab Rules Continued • 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

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

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

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

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

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

9. Graduated • Volumetric

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

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

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

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

14. How To Use A Pipette • 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

15. Types of Test Tubes • Polypropylene • Cloudy Plastic • Polystyrene • Clear Plastic • Glass • Clear

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

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

18. Guidelines for Using Centrifuges • Close and lock centrifuge before starting • Always balance tubes • 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 use brake unless extremely necessary • 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

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

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

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

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

23. Dilution Factor • This is the extent to which you have diluted your selected amount of stock solution and is given by the formulas: • V2/V1 or C1/C2 • 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.

24. Why Dilute? • 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

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

26. Dilution Formula • 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

27. Example Dilutions • 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 8 mg/ml 2 mg/ml = 4 units or a 1:4 ratio 1 part original 3 parts solvent

28. 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. • Example • 1000 mg/ml diluted to 1:10 and then diluted 1:100 • 1000mg/ml x 1/10 x 1/100 = 1 mg/ml

29. Conclusion • 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