IST 342 Bioscience Research Methods

1. IST 342 Bioscience Research Methods Introduction and Safety

2. Contacts • murraye@marshall.edu • 1-304-696-3515 (office) • 1-304-696-3736 (lab) • 1-304-617-6198 (cell phone)

3. Purpose of this course • This course is designed to help you become competent to work in the research lab at a job or internship. • You will learn the fundamentals of how to work with the scientific instrumentation in our lab and some of the standard applications.

4. IST342: Course Materials

5. How we will use the text • Source material for online assessment quizzes • Source material and reference for open book tests. • You can take the online assessment quizzes open book. However, you should take them by yourself and not ask for help from classmates.

6. I. Laboratory Safety and Dishwashing • Review basic of general laboratory safety rules and risks

7. Safety: Risk Reduction in the Lab • Reduce the presence of hazards. • Eliminate the hazardous material when possible. • Substitute a less hazardous equivalent. • Reduce the risk of inevitable hazards with good laboratory design. • Establish good laboratory practices for handling hazards. • Use personal protective equipment (PPE).

8. Safety: Risk Reduction in the Lab • Remove the worker from the hazard (automation, shielding). • Contain the hazard (fume hood). • Dilute or reduce the volume of the hazard (chemicals). • Store material appropriately (Flammable cabinet).

9. Safety: Risk Reduction in the Lab • Establish good laboratory practices for handling hazards. • Provide employee training. • Practice good housekeeping. • Label (with initials and contents) and date everything not in original containers.

10. Clothes and shoes • General Dress: long pants or skirts, avoid dangling jewelry,flammable clothes, long loose hair, neck ties. • Lab coats: barrier to harmful agents and prevent contamination of street clothes, fire resistant. Rubber apron for corrosives. • Close toed shoes, don’t wear sandals. • Use personal protective equipment whenever necessary.

11. Gloves Correctly used, gloves protect you from: • Corrosive or toxic chemicals • Biological contaminants • Sharps or animal bites • Extreme Temperatures

12. Gloves Improperly used, gloves can cause you problems. • Allergies from latex gloves • Holes in gloves and you can have biological or chemical contaminants • Wrong glove for the chemical you are using. • Change gloves regularly. • Double-glove is necessary in some situations.

13. Gloves What gloves do we use? • Thin walled gloves for dexterity: latex or nitrile (hypoallergenic) • Heavy black rubber gloves for handling corrosives • Insulated gloves for hot or cold material. • Yellow/blue Playtex gloves for dishwashing

14. Use Eye Protection! • Danger of explosion of flying particles • Glassware under vacuum • Corrosive liquids: acids, bases • Liquid nitrogen • Blood or fluids with infectious particles • UV light and other radiation • Compressed gases • Liquid may splash your eyes

15. Use Eye Protection! • Goggles • Safety glasses with side protection • Face Shields • Contact lenses are NOT eye protection. • Know where eyewash station is and use it to rinse eyes.

16. Hearing Protection • Long-term exposure to high levels of noise can cause hearing loss. • Disposable or personal earplugs should be used with some very noisy equipment like sonicators. Follow manufacturer’s recommendations.

17. Masks and Respirators • Laboratories generally have basic respiratory protection equipment • Masks, which filter dirt or large particles from the air and provide splash protection. • Air purification or filtration respirators. • Self-contained breathing systems.

18. Clothes for working in the research lab • What should I wear? • Close toed shoes! • Slacks, jeans, skirts or shorts • Shirts or T-shirts • People are casual but not too bare! You can spill chemicals on yourself, and you may be working with animals. • It looks odd to wear shorts under a lab coat but it is done. • Safety glasses or glasses are good.

19. Go to the following webpage • http://www.practicingsafescience.org • Sign in as a colleague and visitor. • Say you study genetics • Say yes to all the questions. • Do the webpage and take the test. Print the screen for the final grade on the test OR save it as a screenshot and e-mail it to me.

20. How to do this • Press Print Screen. • Press windows symbol button (2nd from left on bottom of keyboard). • This saves the screen into your buffer- you can paste it into another program like word or into PAINT.

21. IST 342 Bioscience Research Methods Documentation in the Lab

22. What Is Documentation? • Documentation is a system of records, essential to any quality system. • If a scientist can’t show evidence of their results, those results are not credible. • Biotech and pharmaceutical companies really care about documentation. • So do research labs.

23. Functions of Documentation • Record what an individual has done and observed. • Establish Ownership for patent purposes. • Tell workers how to perform particular tasks. • Establish the specifications by which to evaluate a process or product.

24. Functions of Documentation • Demonstrate that a procedure was performed correctly. • Record operating parameters of a lab instrument. • Demonstrate by an evidence trail that a product meets its requirements.

25. Functions of Documentation • Ensure traceability: track every component of the product and its origins. • Establish a contract between a company and its customers (certificate of analysis, label contents). • Establish a contract between a company and the regulatory agency (FDA).

26. Common Lab Documents • Laboratory Notebooks. • Standard Operating Procedures (SOPs). • Forms. • Protocols. • Reports. • Equipment/Instrument Logbooks. • Recordings from Instruments.

27. Common Lab Documents • Electronic documents. • Numbering systems (bar codes, lot numbers). • Chain of custody forms. • Training reports. • Labels.

28. Laboratory Notebook • A chronological record of an individual’s work- the primary document in a research laboratory.

29. Laboratory Notebook: Why Worry? • Intellectual Property for patents. • Recording what you did so you or others can repeat the experiments. Good records make it easier to publish your results. • Troubleshooting- careful notes help you find mistakes, bad materials etc.

30. Laboratory Notebook Rules • The notebook should have permanently bound pages which are consecutively numbered and should be used by a single engineer or scientist. • Ideas, calculations and experimental results should be entered into the notebook as soon as possible, preferably the same date they occur, so that the laboratory notebook becomes a daily record of the inventor's activities. Recopying can cause errors.

31. Laboratory Notebook Rules • All entries should be made in the notebook in permanent black ink and should be as legible and complete as possible. • Write legibly. • Draw a line through all errors and date the corrections. Do not erase. Never use whiteout.

32. Laboratory Notebook Rules • Notebook entries should be made without skipping pages or leaving empty spaces at the bottom of a page. • To start an entry on a new page, draw a line through any unused portion of the previous page. • Never tear out or remove a page from the notebook.

33. Laboratory Notebook Rules • Each page should be signed with the researcher’s full name and dated. • All photos, charts or computer printouts pertinent to the project should be permanently put in the notebook with your initials and date over the tape. • No entry should be changed or added to after signature by a witness. • If the researcher has any additional information or corrections, a new entry should be made.

34. Laboratory Notebook Rules • If an additional entry is made between the initial and final pages recording an experiment, the entry should identify the page on which the previous entry for that experiment occurs. You can state: “Continued from Page 23.”

35. Laboratory Notebook Rules • Store the lab notebook in a safe location in the lab. Companies make microfilm copies. • Computer files don’t provide sufficient evidence of priority of invention, if they can be altered. They can be backed up with permanent date stamps, and then they are acceptable. • The old notebooks should stored following the company's record retention and destruction policy for such documents.

36. SOPs • Standard Operating Procedure: • A procedure that is done many times in many different protocols may have an SOP written. • For example, running the Autoclave may require an SOP. • Some labs have you fill out a form showing you performed the SOP every time you use a piece of equipment.

37. Reports • Lab reports summarize what experiment was done, who did it, why it was done and the conclusions of the study. • Lab reports are usually published in scientific journals after they are reviewed by other scientists.

38. Protocols • A protocol is detailed plan of a scientific or medical experiment, treatment, or procedure. • A step-by-step outline that tells a scientist how to perform a task or perform and experiment that is intended to answer a question. • Next is a very short protocol:

39. Protocols on Line (until November) • You can access Current Protocols from Marshall University or your home institution’s computers. • http://www.wvbrin.org • You can also access several online journals: • Nature • Cell • Journal of Immunology

40. IST 342 Bioscience Research Methods Washing Dishes

41. Washing Dishes • Key part of any lab. • Cleaning of glassware/plastic ware can affect experiments.

42. Key Aspects of Cleaning 1) precleaning handling 2) cleaning chemistry/concentration 3) time 4) temperature 5) type of agitation 6) rinsing conditions 7) drying conditions 8) postcleaning handling

43. Preliminary procedures in glassware washing • Return glassware that contains chemicals or contamination to the researcher for proper disposal. • Broken glassware. Dispose of any chipped or cracked items in a proper disposal container. • Remove labels. • Presoak or rinse glassware.

44. Dishwashing: Pre-Dishwashing Steps • Bacteria solutions or media- add 10% volume household bleach and wait 15 minutes. • Rinse or soak to remove residues. • Remove contaminants like Agarose and Acrylamide immediately- these can dry to a clear film and show up in your next experiment. • Scrub tubes with a brush.

45. Dishwashing: How to tell if glassware is clean • The glass should wet uniformly. If the meniscus pulls way from the side as the glassware drains, it isn't clean. NO dust or residues. • Look at the glassware carefully before putting something into it.

46. Dishwashing: How to tell if glassware is clean Detergent Residue Testing Using a pH Meter • pH the source water in a clean container • Rinse the glassware with 10% volume of source water. • Take pH of rinse water. Any significant increase in pH indicates alkaline detergent residue. A significant change is 0.2 or more pH units on a pH meter measuring to 0.1 pH units of sensitivity. A result of less than 0.2 pH units change indicates properly rinsed glassware.

47. Dishwashing: Types of Cleaners • Strong Alkaline Solutions (2M NaOH, KOH) • Acid Cleaners (Chromic Acid, 1M HCl) to remove protein or lipids. • Protease enzyme cleaners • Radioactivity Cleaners (chelators) • Solvents (Acetone, Alcohols) get rid of tape residue.

48. Dishwashing: Detergents as Cleaners • Detergents have a hydrophobic end and hydrophilic end. They are both water and lipid soluble. • Ionic Detergents: • Hydrophilic portion is ionized in solution. • Phosphate containing detergents (Sparkleen) can leave residues which can interfere in some tests. • Anionic Detergents: • Hydrophilic portion is NOT ionized in solution. • Alconox/Alcotabs for dishwashers

49. Dishwashing: Some Specialized Cleaners • LIQUI-NOX: phosphate-free to eliminate phosphate interference • ALCOTABS: free rinsing for difficult pipette cleaning • CITRANOX: enhanced acid for safer cleaning of trace metal residues • LUMINOX: neutral pH to eliminate alkaline waste treatment concerns • CITRAJET: low foaming enhanced acid for safer cleaning of trace metal residues

50. Assignment: Go to this web site and send off for this book and a detergent sample. I will assign 5 points if you bring in the manual or the soap to show me. Alternatively, read the pdf file of the book and answer some questions. http://www.alconox.com/static/section_customer/book.asp