Obtain approval for the usage of Core Facility Quarantine Room (Christina Tu htu@uci.edu)
Post signage on door of who to contact regarding samples in room (names with phone numbers & date range of assigned usage)
Clean the incubator before and after each phase of usage: For H2O2 incubators, follow the manual; for the regular UV incubators, follow the Core protocol below. Post dated notice on incubator door indicating date of most recent cleaning.
Comply with the Core Tissue Culture Room Guidelines (below) and pre-approved procedure from individual lab (example below) for usage of the room.
Use of the room is predicated on verifying/establishing mycoplasma free lines. Accordingly, a two incubator transfer system is in operation. No lines cultured in quarantine after initial receipt may be used outside this space until two mycoplasma tests have been passed. Tests must be submitted to Christina Tu for before alternative space in the stem cell center core facility will be allocated.
Incubators are not to be used to maintain mycoplasma positive cell lines, if infection is detected, lines must be disposed of and new samples thawed/obtained for testing. Notify Christina Tu immediately, and arrange for decontamination of incubators/hoods.
Provide copy of approvals indicating Core Room 1201 as one location and names of personnel
Safety Regulations
No food or drinks; wear closed toe shoes;
Entering Tissue Culture Room
Shower prior; wear clean clothes
Step firmly on the sticky pad at the entrance of the room; use shoe covers if needed
Wear lab coat designated for the tissue culture room only, remove and hang it on the hook upon leaving the room
Wear gloves; Tie long hair; Wear mask in flu and allergy season or as needed
CO2 Incubator
Product manual is available
Wear gloves to handle cultures
When the light indicating RH PAN is on, add Arrowhead distilled water to the tray
Wipe clean any spill as soon as possible and clean with Bacdown detergent
Close door gently and wait for CO2 to come back to 5% before leaving
Check CO2 source frequently; report when tanks are empty; Discard any old cultures
Bio-safety Hood
Product manual is available
Turn on hood by raising sash and turning on fan; spray entire hood space with 70% ethanol thoroughly and wait for 3 minutes warm up time; align sash if needed
Keep only necessary items (pipettes, pipette aid and racks) in the hood; put away your own items (tips) to avoid cross-contamination from sharing
Wipe clean any spill with 2% Bacdown detergent; if the spill goes under the panel, clean up by lifting up the panel and wiping it clean; recharge the pipette aid as needed
Upon finishing, rinse vacuum line with BD detergent before turning off the vacuum and wipe area clean with BD detergent including rim and sash, turn off hood by turning off fan and close sash.
Vaccum
Spray and wipe tubing with 70% ethanol and bring into the hood; always turn on vacuum before connecting to aspirating pipet to avoid back flow of air;
After usage of the hood, rinse inside of tubing with 2% Bacdown detergent (not ethanol) and hang it on the hook outside of the hood; shut off vacuum
Empty the Receiver Bottle Frequently
Empty liquid waste through a strainer in the sink; discard solid in trash; rinse clean and fill the receiver with at least 60ml Bacdown detergent; clean up sink and strainer
Water Bath
Refill with Millipore water if the water level is low.
Squirt some detergent into the water bath.
If there are any spills into the water bath, empty, clean and refill with water.
Red Container, Trash Bin, and Blue Recycle Bin
Deposit lab wares that come in contact with human cultures (pipettes, tips or plates that touch the human cells…..). Aspirate liquid before discarding.
If the red container is full, tie and place the red bag into the big red container outside of the tissue rooms (Do not over-fill the big container). Replace with new red bag.
Any other (empty containers, pipettes or tips that transfer un-used medium) can be disposed into the trash bins.
Re-sheath serological pipets back to envelope before discarding to red or trash bins
Media bottles that have not touched any cells may be discard in the recycle bin
Floor
Please wipe clean any spill or dirt and pick up tips, parafilm and any trash. Swiffer sweeper is available for wiping. Please keep the floor area as clean as possible!
Refrigerator and Freezer
Discard old media; be organized
Liquid Nitrogen Storage
Face shield, thermal gloves and closed-toe shoes are required. Do not place the lid or rack on the floor, instead, place them on the cart.
Use black ruler to confirm level of LN
Emergency Numbers
911 = UCI Police If any emergency situation arises that requires 911 assistance, please call 911, which will connect to UCI Police UCI Facilities 824-5444
Cleaning the Sanyo CO2 Incubator - SCRC Core 2011
Please also refer to the manual.
Turn off unit. Take out all shelves, supports, water tray and fan, and place them on a clean cart or counter. Be gentle with UV lamp and water sensor (p27-29 Sanyo Manual)
Detail clean all attachments with 2% Bacdown detergent and thoroughly rinse or wipe off with distilled water
Thoroughly spray the attachments with 70% alcohol; wipe off excess ethanol with clean paper towels; leave the parts for completely drying in a bio-safety hood (do not place lining sheets in the hood).
Wipe clean the inside wall and doors of the incubator with Bacdown detergent, then distilled water, then alcohol for sterilization. Wipe off excess liquid with dry paper towels; leave the door half-open for drying.
Gently clean the water sensor with Kim wipes containing alcohol.
When dry, reassemble all attachments; always insert the fan on the motor shaft surely
Fill the humidifying pan with sterile Millipore water and replace cover
Wipe clean the outside of the incubator with Bacdown detergent, then distilled water.
Example Quarantine Procedures(Anderson Lab) - Receiving New Cell Lines
Quarantine
After receiving a cell line, thaw and incubate (or store) in assigned quarantine incubator to prevent potential contamination of all other current working cell lines in the lab.
Quarantine Incubator (Assigned Incubator A)
Received cells will go into “Receiving” Incubator A, and should be tested for mycoplasma immediately, followed by karyotyping, and screening for human pathogens.
Derivation Incubator (Assigned Incubator B)
Once cells pass above tests, they can move to “Derivation” Incubator B. In Derivation incubator, they can be grown as neurospheres, or into concentrations sufficient for freezing. Then cells can move upstairs after passing a second mycoplasma test.
Note: Cell line may NOT move into any other assigned space until they have passed the human pathogen free testing, have normal karyotype, and two (2) mycoplasma tests.
Incubators are not to be used to maintain mycoplasma positive cell lines, if infection is detected, lines must be disposed of and new samples thawed/obtained for testing. Notify Christina Tu immediately, and arrange for decontamination of incubators/hoods.
During the Period of Quarantine
Mycoplasma testing should be implemented as a regularly: Upon new cell line arrival and before transferring to a new location (i.e. incubator, culture room), also once a month as routine verification of contamination free cultures.
Refer to Mycoplasma Testing for techniques/protocols Mycoplasma Kits:
MycoProbeTM
Mycoplasma Detection Kit
RnD systems
Cat No. CUL001B
Also, at this time Karyotyping can also be implemented
