IST 342 Bioscience
Research Methods
Introduction and Safety
Contacts
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murraye@marshall.edu
1-304-696-3515 (office)
1-304-696-3736 (lab)
1-304-617-6198 (cell phone)
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.
IST342: Course Materials
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.
I. Laboratory Safety and
Dishwashing
• Review basic of general laboratory
safety rules and risks
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).
Safety: Risk Reduction in
the Lab
1. Remove the worker from the
hazard (automation, shielding).
2. Contain the hazard (fume hood).
3. Dilute or reduce the volume of the
hazard (chemicals).
4. Store material appropriately
(Flammable cabinet).
Safety: Risk Reduction in
the Lab
1. Establish good laboratory
practices for handling
hazards.
2. Provide employee training.
3. Practice good housekeeping.
4. Label (with initials and
contents) and date everything
not in original containers.
Clothes and shoes
1. General Dress: long pants or skirts, avoid
dangling jewelry,flammable clothes, long
loose hair, neck ties.
2. Lab coats: barrier to harmful agents and
prevent contamination of street clothes, fire
resistant. Rubber apron for corrosives.
3. Close toed shoes, don’t wear sandals.
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Use personal protective equipment
whenever necessary.
Gloves
Correctly used, gloves protect you
from:
1. Corrosive or toxic chemicals
2. Biological contaminants
3. Sharps or animal bites
4. Extreme Temperatures
Gloves
Improperly used, gloves can cause you
problems.
1. Allergies from latex gloves
2. Holes in gloves and you can have
biological or chemical contaminants
3. Wrong glove for the chemical you are
using.
4. Change gloves regularly.
5. Double-glove is necessary in some
situations.
Gloves
What gloves do we use?
1. Thin walled gloves for dexterity: latex or
nitrile (hypoallergenic)
2. Heavy black rubber gloves for handling
corrosives
3. Insulated gloves for hot or cold material.
4. Yellow/blue Playtex gloves for
dishwashing
Use Eye Protection!
1.
2.
3.
4.
5.
6.
7.
8.
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
Use Eye Protection!
1.
2.
3.
4.
5.
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.
Hearing Protection
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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.
Masks and Respirators
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Laboratories generally have basic
respiratory protection equipment
1. Masks, which filter dirt or large particles
from the air and provide splash
protection.
2. Air purification or filtration respirators.
3. Self-contained breathing systems.
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.
Go to the following
webpage
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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.
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.
IST 342 Bioscience
Research Methods
Documentation in the Lab
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.
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.
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.
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).
Common Lab Documents
• Laboratory Notebooks.
• Standard Operating Procedures
(SOPs).
• Forms.
• Protocols.
• Reports.
• Equipment/Instrument Logbooks.
• Recordings from Instruments.
Common Lab Documents
• Electronic documents.
• Numbering systems (bar codes, lot
numbers).
• Chain of custody forms.
• Training reports.
• Labels.
Laboratory Notebook
• A chronological record of an
individual’s work- the primary
document in a research laboratory.
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.
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.
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.
Laboratory Notebook
Rules
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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.
Laboratory Notebook
Rules
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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.
Laboratory Notebook
Rules
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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.”
Laboratory Notebook
Rules
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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.
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.
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.
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:
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
IST 342 Bioscience
Research Methods
Washing Dishes
Washing Dishes
• Key part of any lab.
• Cleaning of glassware/plastic ware
can affect experiments.
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
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.
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.
Dishwashing: How to tell if
glassware is clean
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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.
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.
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.
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
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
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
Dishwashing:
Washing Dishes by Hand
• Wear gloves.
• Wash containers in hot tap water with
laboratory grade, non-phosphate detergent.
• Rinse at least three times with LOTS of tap
water to remove detergent.
• Rinse three times with distilled water to remove
traces of minerals from tap water..
• Oven dry or hang on rack to air dry (no towels).
• Cool in contaminant-free environment.
• Cover to avoid exposure to dust.
Dishwashing:
Washing Dishes by Machine
• Do all the Pre-dishwashing steps.
• Dishwashers clean with jets of water
and HOT water (150-180o C).
• Loading dishwasher carefully is
essential:
– Don’t put in fragile glass pieces.
– Wash test tubes and small items in wire
baskets.
– Don’t interfere with water jet arms.
Dishwashing:
Washing Dishes by Machine
• Our lab dishwasher is in 212
• We do the cycle without soap.
• Make sure the arm can move freely
around the glassware.
• Before running the dishes, you must
rinse them 5 times (minimum) in water
from that tap.
• This removes water softeners, chlorine
and other additives to our drinking
water.
Dishwashing: Our Dishroom
• Dirty to the right of the sink, clean to
the left.