Lab Skills

1. Lab Skills Unit B Summary

2. Safety Rules • No eating or drinking in the laboratory. No gum chewing. No makeup application. • Wear safety apparel, such as safety glasses, gloves, lab coats, and other protective clothing as necessary. Tie hair back if using Bunsen burners. • Know the location of fire exits, fire extinguishers, and safety showers. • Wash hands regularly, especially after working with microorganisms or chemicals. • Be aware of potential dangers. Before using products or equipment, carefully read labels, experimental protocols, and equipment instructions and read literature. Know the location of and how to read Material Safety Data Sheets (MSDS). • Contaminated samples (chemical, biological, and glass) must be disposed of in appropriate containers. Do not pick up broken glass with your hands. Learn the specific methods from your lab supervisor. • Label all samples and reagents clearly with the name of the item, the name of the person who prepared the sample, and the date of preparation. • Know emergency phone numbers and the best way to contact facility safety officer. • Report spills and accidents to your lab supervisor or safety officer immediately.

3. MSDS sheets • Chemical Name • Stability • Reactivity • Physical Data • Toxicity • Health Effects and First Aid • Storage and disposal • What does each area mean? • – Red: Flammability • – Yellow: Reactivity or instability • – White: Special hazard • – Blue: Health and Hazard

4. Occupational Safety & HealthAdministration ensures worker Safety and protection • Environmental Protection Agency is responsible for protecting the environment • Department of Transportation need to know what they are transporting

5. Personal Protection Equipment • – Lab coat • – Safety Glasses / Goggles • – Gloves • – Face shield • Closed toe shoes • No contacts • No loose or hanging clothes such as ties • Minimal jewelry • Lab coat, safety glasses and gloves most common • Glasses required for all liquid experiments

6. Prokaryotic cells (bacteria) – no nucleus or membrane-bound organelles • Eukaryotic cells (all others) – may have the following organelles:

7. Cell Structures • Nucleus – controls activities in the cell • Cytoplasm – solution outside nucleus but inside cell • Ribosomes – makes proteins • Er (endoplasmic reticulum) – passageway for protein transport • Golgi – packages the proteins • Mitochondria – converts food to energy for cell • Chloroplast – coverts sunlight to food in plant cells • Vacuole – storage of water, enzymes, waste • Lysosomes – digests foreign material or bad cell parts • Cell (plasma) membrane – controls what comes in / out of cell • Cell wall – external support for plant cells

8. Viruses • Nonliving • Composed of Nucleic acid and protein • Grouped according to: Presence of Capsid and envelope – shape AND RNA or DNA, single or double stranded – structure • Can replicate only by invading host cell and using its enzyme and organelles. • Bacteriophage – viruses that infect bacteria; Used to study viruses • Lytic Cycle • Viral genome is released into the host cell • Replication follows immediately • Cellular components used to make new viruses • Viral enzyme kills cell. • Lysogenic Cycle • Nucleic acid of virus becomes part of the host cell’s chromosome • Nucleic acid remains in the cell in this form for many generations

9. Biomolecules • Water - polar, good solvent, sticks together, resistant to heat change, inorganic • Organic – made of carbon • Carbohydrate – used for energy, sugars, starch, C6H12O6, building block is monosaccharide • Lipid – used as barrier in membrane, fats, oils, long carbon chain, building block is fatty acid • Protein – used for enzymes and structure, building block is amino acids • Nucleic acids – DNA and RNA, building block is nucleotides

10. Metrology • Units define measurements & give the numbers value • Accuracy is how close an individual value is to the true or accepted value • % error = True value – measured value X 100% True value • Precision is the consistency of a series of measurements • Take an average of the deviation, so it is the average deviation from the mean • Standards are Measurements made in accordance with an external authority

11. Metrology • Verification - Check of the performance of an instrument or method without adjusting it. • Calibration - Bringing a measuring system into accordance with external authority, using standards • Tolerance - Amount of error that is allowed in the calibration of a particular item. • Traceability - The chain of calibrations, genealogy, that establishes the value of a standard or measurement • Error is responsible for the difference between a measured value and the “true” value • Three types of error: • Gross (blunders) • Random • Systematic

12. Volume • Graduated cylinders – over 10 ml • 10 ml serological pipets • 5 ml serological pipets • 2 ml serological pipets • 1 ml serological pipets • P1000 micropipets • P100 micropipets • P10 micropipets • Multichannel pipets

13. Pipets • Use the right instrument to make the correct measurements • Verify and calibrate micropipets with water which has a density of 1 g for every ml • Maintenance micropipets by cleaning and storing properly and recording.

14. Terminology for weighing • Range: • the span from lightest to heaviest weight that a balance is able to measure • Capacity: • the heaviest sample that a balance can weigh • Sensitivity: • the smallest value of weight that will cause a change in the response of the balance.

15. Proper weighing procedure • Make sure the balance is level • Adjust the balance to zero • Tare the weighing container or weigh the empty container • Place the sample in a the weighing container and read the weight • Remove the sample • Clean the balance and surrounding area

16. Proper weighing techniques • Always use a calibrated weight to verify the scale is in proper working order (daily) • Always use a weigh boat or weigh paper; do not place materials directly on the pan • Do not touch the chemicals or material being weighed • Do not return unused chemicals to their storage bottle (unless you use a sterile spatula or spoon)

17. Calibration of Balances • First step is to zero the balance. It should read zero every time you press the zero button • Second calibration point is taken at the upper end of the capacity of the balance • Place a certified weight on the balance and verify it reads the correct weight • Some scales will prompt you to enter the weight • A third reading can be made using a lighter calibrated weight and verifying it reads the proper weight

18. Equipment Log Books • Notebooks or binders used to maintain operating procedures, calibration records, verification checks • Example: the equipment log book in the back of the room • Incubator temp charts • Refrigerator temp charts • Pipet calibration records • Balance calibration and verification charts

19. Solutions • Solution: a homogeneous mixture in which one or more substances are dissolved in another. • Solute: substances that are dissolved; units are often g, mg, or µg • Solvent: substances in which solutes are dissolved (often times this is water or a buffer) units are often L, ml, or µl • Concentration: amount per volume mass/vol units are g/L, g/ ml, mg/ml, molar

20. Solution Prep • Mass/volume • % mass/volume • Molarity • molarity (M) is equal to the number of moles of solute that are dissolved per Liter of solvent • Dilution - C1xV1 = C2xV2

21. Acids • Definition: electrolyte that donates hydrogen ions • Properties: • Acids in water conduct electricity • The stronger the acid the stronger the conductivity • Acids react w/metals to produce H2 gas • Acids are indicators; they cause reversible color changes • Phenolphthalein and litmus are two examples of acid-base indicators • Acids react w/hydroxide compounds to form water and salt; this type of reaction is called “neutralization” • Strong acids completely dissociate in water to release hydrogen ions = H+ • i.e. hydrochloric acid (HCl): HCL in water H+ + Cl- • Tastes Sour

22. Bases • Definition: electrolyte that yields hydroxide ions or accepts hydrogen ions • Properties: • Bases in water conduct electricity • The stronger the base the stronger the conductivity • Bases react with acids in neutralization reactions to form water and a salt • Bases cause reversible color changes in acid-base indicators (color is pH dependent) • Bases in water solution are slippery to the touch • Caution: even dilute bases can be caustic! • Strong bases completely dissociate in water to release hydroxide ions = OH- • NaOH in water  Na+ + OH- • TheOH- ions react with H + to form water, thereby  the concentration of hydrogen ions • Tastes bitter

23. pH is • A way to express hydrogen ion concentration in a solution • Measurement of the acidity/alkalinity of an aqueous solution • pH is the –log of the H+ concentration • pH is measured on a scale • Ranges from 0 to 14 • Pure water • H+ concentration is 1x10-7 mole/L • The log of 1x10-7 = -7 • The – log of –7 = 7 • The pH of pure water = 7

24. Buffer  • Substance(s) that when in aqueous solution resists a change in H+ concentration even if acids or bases are added • Some buffers change pH as their temperature and/or concentration changes • Tris buffer is widely used in molecular biology; it is very sensitive to temperature and the pH will vary greatly at various temperatures.

25. Measuring pH • Indicators • Phenophthalein, phenol red, bromothymol blue, universal indicator to name a few • pH Paper • pH Meters

26. pH Meter • Meter / electrode system for measuring pH in laboratory • Provides greater accuracy, sensitivity than chemical indicators • Can measure pH of a solution to the nearest 0.1 unit • Can be used with variety of aqueous solutions • Consists of: • Voltmeter – measures voltage • Two electrodes connected to one another (sensor probe) • When immersed in the sample they develop an electrical voltage that is measured by the voltmeter • Calibration recommended with each use, when battery replaced and when fluid in sensor is changed

27. Microscope Important Lab instrument

28. Why use a microscope? • To view objects and detail too small to see with human eye. Improves resolution of object • List examples of how what you used it for: • Blood cell detail • rbc, wbc, platelets • Bacteria • Cocci, rod, bacillus • Protozoa • Trichomonas • Giardia

29. Types of Microscopes • Compound microscope • Bacteria, fungi and protozoa • Electron microscope • Required for viruses • Fluorescence • Used as a diagnostic tool for immunofluorescence tests

30. Parts of a microscope • Coarse adjustment • 1st step in focusing to change the distance between specimen and lens • Fine adjustment • To fine tune the picture • Used particularly with 100x and oil objective • Stage • Holds the slide, moved up and down with coarse and fine adjustment

31. Parts of a microscope • Objectives • Common objectives are 4x, 10x, 40x, 100x, oil • Total magnification is eye piece magnification multiplied times objective you are viewing with • 40x objective and 10x eye piece is 400x magnification • Oil objective is labeled and is always used with immersion oil • Oil increases the resolving power by focusing the light rays

32. Proper care for microscope • Always start with lowest objective to focus • Always store with lowest objective locked in place • Carry with two hands • Cover with dust cover • Be sure to clean oil off of oil objective • Use fine adjustment with 100x and oil objective

33. Spectrophotometers • When light shines on a solution, it can bounce off of the molecules (reflect), pass through the solution (transmittance), or some of the energy be absorbed by the solution. • Spectrophotometers are instruments that measure the interaction of light with materials in solution • Spectrophotometers compare the light transmitted through a sample to the light transmitted through a blank. • The blank contains everything except the analyte

34. Chromatography • Physical properties that can be used to separate molecules • Size • Shape • Density/gravity • Charge • State (solid, liquid, gas) • Phase changes (mp, bp, evap)

35. Chromatography Key Terms • Chromatography: techniques for the separation of complex mixtures that rely on the differential affinities of substances • Stationary phase: what you pack the column with or the plate/paper • Mobile phase: solvent/phase moving in the bed; fraction or sample being separated; • Effluent: the mobile phase leaving the column • Types of Chromatography • Paper • Thin Layer • Ion Exchange or Affinity • Size Exclusion • High Pressure Liquid Chromatography (HPLC)

36. Gel Electrophoresis • Definition: the process of separating molecules based on size and charge • Agarose: highly purified agar, heated and dissolved in buffer. Forms a matrix of pores for molecules to travel through. • Smaller molecules travel further • Molecules migrate towards the • positive (red) end of the chamber

37. Gel Electrophoresis • Process • Make Agarose gel • Thinner gels (0.8%) yield better results for larger DNA • Prepare samples • Restriction enzymes used to cleave at specified sites • Apply samples to gels, apply current • If samples run from positive end they will run off the gel • Stain gels to see bands • Would not be able to see bands if we did not stain

38. Gel Electrophoresis • DNA molecules have a negative charge • This allows them to migrate towards the positive end of the chamber • The samples and the electrophoresis chamber use specialized buffers. Using • TAE/TBE buffer helps stabilize the sample • and allows the reaction to occur quicker in • the chamber. • If water were in the chamber instead of TAE/TBE buffer the reaction would take much longer or migration may not occur at all • Stains: ethidium bromide will cause the bands to glow orange under UV light. Fast stain will result in blue bands

39. Uses for Gel Electrophoresis • DNA fingerprinting or profiling • Paternity testing • Crime scene sample analysis • Identification of bacteria and other pathogens • Who is credited with discovering the DNA profiling process? • Alec Jefferies in 1985

40. Cell Culture • Definition: the in vitro growth of cells isolated from multi-cellular organisms • Process: Cells will continue dividing until they fill up the container; cell to cell contact stops cell division • Uses: vaccines, research of all kinds including stem cell, recombinant DNA, production of antibodies

41. Types of Cell used • Bacterial cells • Yeast cells • Mold cells • Plant cells • Insect cells • Mammalian cells

42. Growing Bacterial Cells • Choose bacteria – E. coli most common • Make media • Petri plates use agar in media (Luria Broth, nutrient agar) • Liquid cultures use broth (LB, nutrient broth) • Sterilize media in autoclave • Pour media plates • Innoculate media • Grow cells in incubator (37oC)