Tips for Sterile Technique and Cell Manipulation

 

·        Media is aliquotted to 50 mL tubes which are stored in the refrigerator.  Each student should have her/his own tube of media;  label each tube that you take with your initials.  Failure to label your tubes will result in the lowering of your safety score.  Use that tube until it is empty and then take another.

·        Clean hood with ethanol before and after each sterile operation.  Spray ethanol liberally over surfaces and wipe clean with kimwipe. Store the ethanol bottle outside the hood.

·        When pipetting liquid from a container (bottles, T flasks, tubes, etc), tilt the container toward the hand with the pipet.

·        Gloves should be worn at all times.  Change gloves when you suspect contact with contaminated materials.

·        Before putting hands in the hood, spray hands with ethanol.  After spraying hands, rub them together to ensure that areas between fingers have contacted ethanol.

·        When sterilizing jars of liquid with ethanol, spray the whole bottle and up under the cap lip.  Bottles may be wiped dry with a kimwipe.

·        Try not to get liquid on “bent-up” part of T-75 flask.  Don’t turn tissue culture flask upside down or shake it around vigorously.

·        Unwrap and rewrap T flasks from their plastic-wrap sealers in the tissue culture hood.

·        To add or remove liquid from T-75 flask, unscrew and remove cap.  Hold both the T flask and its cap in one hand. Encircle cap with first finger.  Grab flask with other fingers and thumb.

·        Set equipment toward the middle to back of the hood.  Also, conduct work (aliquotting, transfer, etc) toward the middle to back of the hood.

·        If possible, line materials/containers/jars that you are working with lengthwise (x-direction) within hood (see diagram below).   Air is flowing across the surface in the y-direction;  thus, stacking items may result in a contamination between items.  Materials/containers/jars that are not being used may be stacked on the side.

 

CORRECT for manipulation of materials:

	tubes		media	T flask		XXX		XXX

                                                            front of hood                                        y                     

                                                                                                                                    x

NOT CORRECT for manipulation of materials;  OK for storage:

				“stacking”
	XXX			media				XXX
	XXX			T flask				XXX
								XXX

                                                            front of hood

 

·        Leave lids and caps ON their respective plates and/or bottles as much as possible.  If you ever need to set a lid or cap down, put it near the back of the hood.  Set a cap so that the top is down, and you can see the rings inside the cap.  Set a lid so that the top surface is down, and the walls/ledges are sticking up.

·        When opening sterile pipets, place thumb and first finger of both hands on the two flaps.  Pull out and down to expose pipet about 6 inch.  Fold flaps down to hold with one hand.  Place manual pipetter on exposed end.  Do not touch the “naked” sterile pipet.

·        Numerical markings on pipet can help guide orientation of pipet in manual pipetter. Insert pipet into manual pipetter so that you can see the necessary numbers.

·        Before beginning an operation using a pipet, loosen caps on containers (T flasks, bottles, and tube).  Then, unsheathe the pipet.  i.e., do the two handed jobs before you fill up one of your hands with the pipet.

·        Visually inspect media and T flasks for contaminants each time they are used.

·        When aspirating liquid from a T-75 flask, make sure that a sterile aspirating pipet is attached to the vacuum hose.  Remove cap from T flask.  Tilt the T-75 flask toward the aspirating pipet.  Suction liquid from bottom corner, minimizing contact of the pipet with the flask.  Do not touch pipet to mouth of T flask.  Do not touch pipet tip to bottom of flask where cells are growing.

·        The cell resuspension step during cell passaging is tricky and requires slow methodical mixing.  Initially, the pipet will be full with 8 mL of media.  Release 1 mL from pipet.  Suck back up.  Release 1-2 mL from pipet.  Suck back up.  Repeat 3-5 times until no visible cell clumps can be seen.  After that procedure, release all liquid from the pipet.  Suck up from the bottom and release liquid to the top.  (Alternatively, suck up from the top and release liquid to the bottom.)  Try to minimize mixing with air and bubble formation.  (The shear associated with bubbles destroys cells.)

·        When using well plates, leave lid on whenever possible.  Set lid down by placing top face down.  When aspirating from well plate, use glass Pasteur pipet and tilt well plate slightly toward you to pool liquid.

·        When adding cells to a 24-well plate, release liquid cell suspension in center of well.

·        When adding PBS or other wash solution to well containing attached cells, release liquid on side wall of well.  Don’t squirt liquid directly onto cells.

·        When aspirating liquid from 24-well plates, use a glass Pasteur pipet.  Do not use a disposable aspirating pipet.  Place glass Pasteur pipet as close to all of well as possible to minimize disturbance of cells attached to bottom of well.

·        Liquid biohazardous material must contact clorox before disposal down the drain.

·        Solid biohazardous material (pipets, T flasks, vials, etc) must be thrown away in red waste container.  Do NOT through biohazardous material in regular trash.