Use
and Etiquette for the Tissue Culture Room (Rosen 344)
REAGENTS:
Media:
Unsupplemented media is in the cold room (338) in
cardboard boxes on the TC shelves. We keep the media in
cardboard boxes, as it is light sensitive. Please
remember to turn off the light when you leave the cold
room. We maintain stocks of RPMI, IMDM and DMEM. If we
are running low please let the TC person know by writing
it on the white board and more will be ordered. Other
types of media are considered specialty media and you
are responsible for your own stocks. DO NOT HOARD MEDIA.
It is not polite to take more bottles than you are going
to use. Plus it is expensive and wasteful.
Supplements:
Add penicillin/streptomycin and l-glutamine to your
media at a 1:100 dilution. Convenient 5 ml aliquots are
kept in fridge/freezer K in the TC room (344).
Heat-inactivated fetal bovine serum (FBS) is kept in 50
ml aliquots in the same freezer. Generally FBS is used
at 5-10%. COS-7, HuT78 and CEMx174 all do very well at
5% FBS. So, for the sake of money, please use serum at
5% unless it is absolutely necessary to do otherwise. If
stocks are low, please let the TC person know and more
will be aliquotted.
Sterile
PBS: Stocks of sterile PBS are available in the
cold room on the TC shelves. We keep both 1X and 10X PBS
stocks. Please take a bottle and label it as yours. Do
not share bottles or assume that other people will use
yours. PBS can be stored at room temperature.
LSM,
Histopaque and Ficoll-Paque: LSM, Histopaque and
Ficoll-Paque are stored in the cold room on the TC
shelves. When opening a new bottle, label with date and
store in the TC refrigerator. Note that Ficoll-Paque and
LSM are supposed to be used at room temperature.
Other:
Several aliquotted reagents are stored in freezer K in
344. 1X trypsin is available in 12 ml aliquots. Be aware
that there are also aliquots of 10X trypsin. DO NOT take
thawed trypsin from other people. Thaw your own aliquot.
PHA is available in 1 ml aliquots at 0.2 mg/ml.
Recombinant IL-2 is available in 0.5 ml aliquots at
10,000 U/ml. Polybrene is available in 0.5 ml aliquots
at 100 mg/ml. Puromycin is available in 0.5 ml aliquots
at 200 mg/ml. If stocks are low, please let
the TC person know and more will be aliquotted.
INCUBATORS
Use:
Currently we have three incubators. Incubator #2 is for
common use. Incubators #1 and #3 are Mycoplasma free and
are used for maintenance of the cell lines. No primary
cultures are to be kept in the mycoplasma free
incubators. If you need to use that incubator, ask the
TC person first. Space is at a premium, so please be
considerate with your cultures. If you will not be
routinely maintaining your cultures, THROW THEM AWAY.
Leaving flasks indefinitely in the incubator is an
unnecessary hazard that threatens the cultures of the
people who are actively using the incubator. If you
detect contamination, eliminate it IMMEDIATELY.
Maintenance:
If you notice small spills from your flasks or plates,
wipe it up immediately with 70% ethanol sprayed onto a
paper towel. Do not spray ethanol directly into the
incubator, as the fumes will kill the cells. Also wipe
down your flask if you will be putting it back into the
incubator. Please only use 70% ethanol or dilute Roccal
to clean the incubator. Bleach and Wescodyne are
damaging to the incubator.
There
is a tray in the bottom of the incubator that holds
water to humidify the incubator. If it runs out of water
add 4 liters of 90% deionized water and 10% tap water
plus 2 ml Roccal. The TC person will clean the tray
every Friday.
If
the add water alarm starts beeping, this does not refer
to the humidifier tray at the bottom of the incubator.
This refers to the water jacket. Please contact the TC
person or someone else who knows how to add water to the
jacket. Alternatively, read the manual and do it
yourself. Funnel, tubing and Allen wrench are stored in
the cabinet by the sink. Remove the plug above the door
with the Allen wrench. Screw the tubing into place. Fill
jacket with deionized water. Be aware that you must
bleed the system of air in order to get the water to
enter the jacket.
Cleaning:
Remove all cells (transfer to one of the other
incubators) and turn off CO2. Take out
shelves and sides. Scrub down incubator and shelves with
dilute Roccal. Wash down everything with 70% ethanol.
Rub down walls and shelves with a small amount of
straight Roccal. Let the fumes die down. Autoclave
shelves, sides and water reservoir and put everything
back into the incubator. Fill the water reservoir with 4
liters of water (90% deionized, 10% tap) and 2 ml Roccal.
Turn the CO2 back on and put the cells back
when temperature and CO2 levels are back to
normal.
HOODS
Use:
There are three important things to remember when using
a tissue culture hood:
1.
NEVER EVER TURN OFF the blower for the hood. This
maintains the sterile field. If the blower has been
turned off, wash down hood thoroughly with 70% ethanol.
Turn the hood blower on. Wait a minimum of 30 minutes.
Wash the hood down again with 70% ethanol. You may now
use the hood.
2.
Don’t block the airflow. This is what keeps the
workspace sterile. Even though it is convenient, you
should never have supplies lying in the airflow tray at
the front of the hood.
3.
Keep as few things as possible in the hood while
you are working. This is part of the airflow issue.
Objects along the back of the hood block the back air
vent. The most sterile spot in the hood is right in the
center so keep your flasks well behind the airflow in
the front.
Maintenance:
Before using the hood, spray it down with 70% ethanol.
Always keep the airflow on. Spray down the hood with
ethanol when you are finished and turn off the
fluorescent light. You can leave the receptacle on but
turn off the pipetaid in the hood. Please close the
glass shield when finished to reduce noise level in the
room.
Vacuum
waste: This vacuum trap is for use with Pasteur
pipettes. A canister of sterile pipettes is kept in each
hood. Dispose of used pipettes in the sharps container
under the hood. Make sure that the glass pipettes are
completely within the sharps container. If tips are
sticking out, the next person who tries to dispose of a
pipette may stab him/herself. If the container fills up,
remove it and replace it. After you are done with the
vacuum, run some bleach through the hose. If the media
in the flask does not turn clear, indicating that
everything has been oxidized, run some more bleach
through the hose. Follow with a few squirts of ethanol
into the hose to facilitate drying and increasing
longevity of the hose. If the flask is almost full,
empty it into the sink. EVEN IF YOU DID NOT PUT MOST OF
THE LIQUID IN THE FLASK. DO NOT EVER fill it up so that
liquid flows into the safety filter.
In-hood
waste boxes:
These boxes are provided for waste pipette tips ONLY.
Pipette tips will puncture the general biohazard waste
bag and therefore must be disposed of in puncture
resistant boxes. Gloves, tubes and paper towels are NOT
puncture hazards and therefore do not need to be
disposed of in puncture resistant boxes. Please don’t
waste box space with these items. If you fill up a box,
tape it up and replace with a new one. Line with a
medium sized autoclave bag.
REFRIGERATORS
Again,
space is at a premium. Throw away unnecessary items.
GLASSWARE
Pasteur
pipettes: Use the sterile canister in the hood.
When it is empty place canister in the metal basket on
the counter top. New canisters with sterile pipettes are
in the drawer labeled glass pipettes. Let the TC
person know if we are getting low on sterile canisters.
Bottles:
Sterile glass bottles are available for your use. They
are kept on the shelves by the sink in the ELISA area.
When you are done with a bottle, RINSE IT with deionized
water and place it in the dish container near the sink.
PLASTICWARE
Tip
Boxes:
Sterile tip boxes for p20, p200 and p1000 are kept
inside the hood. Please use and keep the boxes sterile
inside the hood. When you empty a box, dispose of it
properly.
Stocks:
Most plasticware stocks are kept in the labeled cabinets
and drawers in the tissue culture room. Some stocks are
kept in the hallway outside of room 344.
Disposal:
Flasks and plates are disposed of in the large biohazard
bag. REMOVE EXCESS LIQUID before throwing away. Pipettes
are disposed of in the pipette keeper boxes. NEVER throw
pipettes into the large biohazard bag. They are a
puncture hazard. When the pipette boxes are full, tape
shut and place on the floor next to the door. DO NOT
throw full boxes into the large biohazard bags.
CENTRIFUGE
Use:
Always make sure that the buckets are properly balanced.
If you are centrifuging something that is an aerosol
hazard (blood, FIV, FeLV) use the aerosol caps on the
buckets. Turn off when done.
MICROSCOPE
Use:
Turning the microscope on and off repeatedly during a
short period of time is hard on the bulb. Therefore, do
not turn the scope off until you are completely done. To
increase bulb life, keep the light turned down to its
lowest intensity unless you are actively using it. There
are new bulbs in the drawer labeled microscope stuff.
They are taped to a sheet of paper where we are keeping
a log of bulb use – please note the date when you
replace a bulb. Always clean hemacytometer and
coverslip carefully with 70% ethanol after use. Dirty
hemacytometers greatly impair other people’s
experiments.
Questions
or concerns? Contact:
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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