Week 3 Flashcards
It is important to understand how each chemical will react in
-Storage (a safe and ventilated environmental ideal)
-Waste
-Mix (can one chemical safely react with another, what safe concentration can they do this)
Storage must be_________
Ventilated and fireproof
What are desiccants?
-Can be with or without vaccuum
-Some chemicals are affected by water vapour
-Blue or purple pellets at the base of the container to indicate water with a colour change
-Used for heavy metals like magnesium, sulfur, potassium, and lithium to prevent oxidation
Why are brown or amber glass bottles used?
-For light sensitive chemicals, to keep reaction away from light or covered with foil ex. Bradford reagent, phenol
What should be done with oxygen sensitive reagents?
-Must be preserved by removing oxygen from container and replacing with nitrogen gas, typically done in fume hood
-This must be done for purified lipid samples
What are important considerations for fume hoods?
-Keeping sash at the indicator level for optimal flow (calibrated to specific height)
-Work in the middle of the hood (6” in)
-Do not block the rear intake
-Keep at least 50% clear
-Not a storage area for chemicals
What are some considerations for acids and bases?
-Adding them to water
-Kept in a fire safety cabinet unless small volumes are decanted (10 mL)
-Waste containers will be specific to the type of acid and type of base, never mix them
-Follow waste handling protocols
What are the hazards of acrylamide?
-Used in protein electrophoresis gel but is a Neurotoxin (wear mask and gloves)
What are the hazards of ethidium bromide?
-Used to label nucleic acids, but is a mutagen and you should wear gloves
What are the hazards of phenol?
-Used for DNA extraction but is corrosive so you should wear chemical resistant gloves in fume hood
What are the hazards of phenylmethyl-sulfonate fluoride (PMSF)?
-Used for protein isolation to prevent proteinase activity, but extremely toxic if absorbed through skin– wear gloves
What are the hazards of Sodium dodecyl sulfate (SDS)?
-A detergent used in protein electrophoresis, but an extreme irritant if inhaled, wear a mask and gloves
What are the hazards of Methanol?
-Poisonous if ingested, irritant to skin
-Used in several biotech protocols ex. transfer buffer for immunoblotting
-Use in fume hood to decant, wear gloves
What are the hazards of 2-mercaptoethanol?
- Toxin causes pain on skin, irritant to lungs
-Used to denature protein samples before electrophoresis
-Used in fume hood, wear gloves
What are the requirements for chemical waste?
-Clear and concise records of how much and what kind of waste is in each container
-Dates and signage
What are the different kinds of waste
-Chemical waste
-Hazardous waste
-Radioactive waste
-Biohazard
-Trash/recycling
What is specific activity in regards to radioactivity?
-How many molecules decay each second (HOT probe)
What is half life?
-Time needed for initial number of radioactive atoms to reduce by half
-Shorter half-life indicates greater specific activity
What are some types of emissions?
-Beta particles (use a geiger counter to detect)
-Gamma x-ray particles (require background thyroid scan, gamma monitor)
What are isotopes?
-Elements that share the same amount of protons but different number of neutrons
What are radioisotopes?
-Radioactive form of the element (ex. carbon has 6 protons but the radioisotope has 8 neutrons 14C)
Sale, transportation, use and disposal of radioactive compounds is regulated by
The Federal government
How are radioactive compounds stored?
-Mostly cold storage ranging from 4 to -80*C
-There will be a designated space in fridge or a separate fridge in radioactive room for stock and samples
What is iodination?
An aerosol forming compound that requires a fume hood, lead vest and shield
Disposal of it will be costly
What are the hierarchy of controls?
-Elimination, substitution, engineering controls, administrative controls, PPE
Shielding can be
Acrylic and/or lead (for gamma rays)
PPE for reducing external exposure
Have a separate lab coat for radioactive work
Wear your dosimeter
Controlling the duration of exposure
Have the label added the latest possible in the procedure
Do a “dry” run without label to make sure everything is easily accessible
Controlling the distance from the source
Keep as far away as possible from the work area
Proper monitoring includes
Thyroid scans (for iodine), submit dosimeters for evaluation
What are dosimeters?
A badge that tells how much radioisotope you’ve been exposed to
What is scintillation counter?
Scintillation counter is robotic and measures nuclear probes, helps amplify signal, used for point inspections swab places where you touch something to make sure not “HOT”
Liquid samples in scintillants create an emission of light in pulses
Used for beta emitters
Gamma emitters require a crystal and no scintillants
Used also for point check in spot inspections
What is the PPE?
-Gloves, lab coat, goggles, face mask/shield, iron vest, autoclave glove, liquid nitrogen gloves
What is an iron vest used for?
-A radioactive shield worn under lab coat when working with gamma emmitters ex. iodine 131, 125
What does sterile technique require?
-PPE
-70% ethanol or other disinfectant
-Working slowly and organized to reduce air turbulence
-Autoclaved or pre-sterilized equipment
What are Biological Safety cabinets?
Also known as Laminar Flow hood
-Requires 30 minutes to warm up and current to flow
-Everything must be clean including surfaces, your lab coat and gloves, everything should be prepared so you can keep movements slow and minimal
Can you use the same biological safety cabinet for bacterial and tissue culture work?
No
What are clean rooms?
Require an employee to change clothes and enter through an air shower to prevent contamination (75% comes from people)
What is Biosafety level risk group 1?
-Low risk to lab worker and community ex. non-pathogenic E. coli
What is Biosafety level risk group 2?
-Moderate risk to worker, low risk to community ex. HIV, S. aureus
What is Biosafety level risk group 3?
-High risk to worker, low risk to community ex. West Nile virus, M. tuberculosis, SARS-COV-2
What is Biosafety level risk group 4?
High risk to worker, high risk to community ex. Ebola
What are primary cells in cell culture?
-Isolated from plant or animal tissue, typically used once
What are continuous cells in cell culture?
Transformation of primary cells, indefinite passages.
What are transformed cells in cell culture?
Tumour tissue transformed from continuous cells by chemical or tumour-inducing viral DNA, indefinite passages
What are hybridomas in cell culture?
-Fusion of antibody secreting B cells and malignant myeloma cells
What is cell culture similar to that happens in the body?
-Cell tissue culture is similar to a wound in the body, exposed to an open environment (monolayer of cells-–stressing them out because no barrier)
Suspension cells
grow floating in media, do not need to attach to culture vessel.
-typically cells from blood, spleen and bone marrow
-advantages of large yield and ease of harvesting
Adherent cells
Grow in a monolayer attached to the culture vessel (typically dermal cells such as fibroblasts and epithelial cells)
Where can cell lines be purchased?
American Type Culture Collection (ATCC)
Most industrial settings will work with a __________cell line
Standardized cell line
Eukaryotic cells are often stored in a _________ at a temperature of __________-
In media + glycerol/DMSO in a cryogenic tank (dewar) with liquid nitrogen; at -190*C
Why do cells need to be split?
To maintain a monolayer of cells
What should be noted during propagation?
Note passage numbers each split on the vial, the date, the cell type and your initial
Making stock vials should be done immediately from original vial (lowest passage number
CO2 incubator
5% CO2 regulates pH of cell media
Done in a humid atmosphere at 37*C
Minimize opening and closing, if you run out of CO2 never open the door, go immediately to find a source of CO2
Most contaminations come from_______
The water; either from water bath to heat media or at the base of the incubator (water plate)
Species commonly used in bacterial work?
E. coli and B. subtilis
Distilled water
Pre-treated through distillation or reverse osmosis
Double-distilled water
Deionized distilled water used for buffers and cell culture (once buffer has been filter-sterilized or autoclaved) (separate device in the facility)
Ultrapure dd H2O
Endotoxin free, often bought in small quantities for RNA work
What are some considerations about making buffers?
-SDS for each chemical if never used before
-Reserving stock chemicals you need
-Determine how much is required for the week
-Should the buffer be kept on the bench or kept cold
-Should buffer be made fresh every time, do components degrade?
-Label concentration, name, pH and date made
What is the purpose of aliquotting?
To prevent the contamination and freeze/thaw cycle of the stock solution
Liquid media
Specific to the strain you are growing (filter sterilized or autoclaved), all additives are sterilized separately and added just before culture is added
Solid media
Typically for bacteria growth to select for colonies
-Solidified by agar
What are some considerations for refrigerators/freeze maintenance?
-Keeping a list or map of major reagents found on each shelf on outside
-Minimize the time the doors are open
-Not all are frost free
When defrosting you should?
-Plan for storage in another location
-Keep everything cool in transport
-Inform everyone in the lab of the move/defrost maintenance
Cold storage can be done at?
4C, -20C, and -80*C
