SECTION 1 Lab Safety

In an emergency

Call Campus Heath Service 548-4848
Room NN256A

go directly to the
University Hospital Emergency Room

call 9-911

http://depts.washington.edu/hhpccweb/CampusHealth.html

http://www.ehs.washington.edu/forms/IncidentFillin.pdf

Tissue Culture Safety

1. Every lab member should attend the yearly Blood Borne Pathogen review class. http://www.ehs.washington.edu/forms/classes/bpecrform.htm

2. Everyone who works in the tissue culture room must:

Have taken or be taking the Hepatitis B vaccination (685-1071)

Sign a form with the occupational safety nurse declining the offer (548-6117).

3. No food or drink is ever allowed in the tissue culture room.

4. Gloves and lab coats should be worn when working in the tissue culture room.

5. If you are working with human tissue, you should also wear goggles.

6. Mouth pipetting is never allowed.

7. The “Biohazard Safety Manual” is located on the shelf over the bench by Peter’s office and in the K-089 tissue culture room. Familiarize yourself with its contents.

8. Contaminated sharps go into the red sharps biohazard container. This waste must be autoclaved before disposal.

9. Waste -- All tissue culture flasks must be treated with bleach for at least 10 minutes before disposal.

All waste in biohazard bags must be autoclaved before disposal.

NEEDLE STICKS --
Wash immediately with soap and lots of water.

Before 4:00 pm go to room NN 256A Campus Health Service

(548-4848).

After 4:00 pm go directly to the University Hospital Emergency Room

Fill out an Incident report http://www.ehs.washington.edu/forms/IncidentFillin.pdf

Pay attention to what you are doing when you work with needles and you won’t be stuck!!!!!

1. ALWAYS know the potential hazards of a chemical BEFORE you work with it. You can get this information from the MSDS (Material Safety Data Sheets). These are filed in alphabetical order in the MSDS book located above the bench by Peter’s office. You can obtain on-line MSDS information through the LSS system (see next page) or by calling 3-0467.

2. Check the Yellow Pages of the University Chemical Safety Manual on the shelf by Peter’s office for procedures concerning commonly used chemicals

3. Use the fume hood when working with toxic or carcinogenic chemicals.

4. Wear gloves, goggles, and a lab coat when working with chemicals.

5. Do not wear open toed shoes in the lab.

6. Mouth pipetting is never allowed.

7. Emergency eyewashbottles at every sink or use big hose on the sink.

8. Emergency shower– entry way K-081.

9. Spills – We have solutions to neutralize chemical spill on the chemical bench. One is for acids, one is for bases and one is for organic substances and toxins.

10. The “Chemical Safety Manual” is located on the shelf located above the bench by Peter’s office. Familiarize yourself with it’s contents.

11. Chemical inventory. Room and location in the front of the MSDS book list all of our chemicals and in the red binder on freezer 4. They are also listed on-line in the LSS.

12. LOG IN ALL INCOMING CHEMICALS ON THE INVENTORY SHEET ON THE
REFRIGERATOR.

Chemical control

Orders-- order the smallest reasonable amount of carcinogens or toxins. When possible, order the hazardous substance premixed so you do not have to weigh the powder.

Waste -- Hazardous waste disposal forms are in the front of the MSDS book. USE THEM.

Sink logs -- Federal and Washington State laws require a record of chemicals poured down the drain. Use the sink logs by the sinks to record your waste.

In an emergency

Call Campus Heath Service 548-4848 Room NN256A

go directly to the University Hospital Emergency Room

call 9-911

Chemical Spills

Copied from: http://www.ehs.washington.edu/Services/Spill_Response.htm

Chemical Spill Basics

Hazardous material spills that do not endanger workers in the immediate area may be cleaned up by area personnel who have been trained and are properly equipped to clean up the spilled material safely. Spill kits are available from University Stores during business hours.

Our spill kit is under the sink by the fumehood HSB K-081

The neutralizing absorbents are on the chemical bench in K-081

Hazardous material spills that cannot be safely adsorbed, neutralized, or otherwise controlled at the time of release by employees in the immediate release area are considered to be emergencies requiring outside assistance by the Seattle Fire Department (SFD), Environmental Health & Safety (EH&S), and possibly a spill cleanup contractor.

When in doubt about whether you need help or not, it is best to call for help. EH&S staff cannot clean up spills but can offer advice on how to handle spills. Call 206.543.0467.

When you need emergency help, do the following:

Alert others and evacuate all affected areas.
Pull the fire alarm if needed or report the incident to UW police:

UW Campus or UWMC dial 9-911

The UW Police will notify the Seattle Fire Department (SFD) who will respond, stabilize, and contain the spill. Environmental Health & Safety (206.543.0467) will advise SFD as needed. The incident may require use of a spill clean up contractor at the department's expense. All waste must be contained and labeled as instructed by EH&S.

Major Chemical Spills

Pull the Fire Alarm or call 9-911

A major chemical spill is:

One that has caused injury to personnel or is likely to cause injury, or
Uncontained and spreading out of the immediate area endangering other labs, or
Has the potential to cause a fire.

Pull the fire alarm if someone has been injured. This is the fastest way to get help and alert others nearby of the emergency. The Seattle Fire Department is the primary responder for major chemical spills.

Minor Chemical Spills

Call 206.543-0467

Laboratory employees are responsible for minor spills of the chemicals they commonly use. Cleanup of minor spills is part of managing your laboratory chemicals properly. EH&S can provide training and consultation but does not maintain a Hazardous Materials Response team.

If you can answer YES to the following 4 questions, it is safe for you to clean up the spill:

1. Do you know what chemical was spilled?

2. Do you know the hazards of the spilled chemical?

3. Do you have a chemical spill kit?

Can you protect yourself from these hazards?

If you answered NO to any of the above questions or need assistance with the spill cleanup, evacuate the area and call EH&S at 206.543.0467 for assistance. EH&S will help you find the answers to these questions or bring in an outside Hazardous Materials Contractor to do the cleanup for you.

Chemical Waste

Copied from: http://www.ehs.washington.edu/waste/wastechemical.htm

Routine Chemical Collection

We have a routine collection set up for 70% ethanol-30% water: Routine 2000

We have a routine collection set up for Xylene 99% ethanol 1% Routine 2022

Wastes that are generated on a regular basis may be set up as a routine collection. To qualify for a routine request pickup, the composition of the waste generated must be identical each time. This is an extremely efficient process for collection. Your routine waste is assigned a number that is kept in our database; when you call in or e-mail the routine number to be picked up, we automatically know the waste's composition, regulatory codes, disposal options, and where you are located.

To set up a routine collection you will need to fill out a Setup Routine Chemical Request Form.
If you already have a routine set up and would like to request a pickup, please fill out a Routine Chemical Collection Request Form or call us at 685-2848 with the routine number and quantity to be picked up.

Do I have a Hazardous Waste?

In general, chemicals that are caustic, corrosive, flammable, toxic, or explosive are considered hazardous. Be aware that not everything that is non-hazardous is non-regulated. Refer to the Chemical Waste Management Guide or call 685-2848 if uncertain.

Certain acids and bases may be neutralized and disposed of to the sewer by the person who generated the material. Specific instructions and neutralizing agents are available through EH&S at 685-3759.

Preparing Chemical Waste for Pickup and Disposal

Chemicals must be in an appropriate container compatible with the waste, with a securely fastened screw top lid. As a rule, containers that were designed for solid reagents should not be used for liquids.
All containers must be labeled with a UW Hazardous Waste Label (Acrobat Reader)), unless the container has the original manufacturer's label on it already. The label must state all components of the waste and their percentages (percentages must add up to 100%).
Complete a Chemical Collection Request Form and mail to Box 354400 or FAX to 685-2915. When filling out the form, do not use chemical abbreviations or empirical formulas. Be sure to list all components in the mixture using percentage by volume (percentages must equal 100%). You may list up to four containers of the same mixture per line on the form. Standard turn around time is 2-4 weeks. Be aware that large requests, volume, or number of containers may take longer to be collected.
If you have any questions, please contact the technologist collecting in your area at one of the following numbers.

Zone 3	HSB	685-2849
Zone 4	South Campus, HMC	616-3200

What if it's an unknown?

Unknowns present a serious problem for the University. Without an accurate chemical name, chemicals can neither be handled nor disposed of in a safe manner.

Unknowns should be processed for collection and disposal as soon as possible following discovery. Do not store these wastes in satellite areas. Any information, such as history and physical properties that can be provided to the hazardous waste staff will aid in the investigation and identification of unknowns.
Disposal companies will not accept unknown chemical waste without an analysis. To have an unknown scheduled for testing first complete a Chemical Collection Request Form and mail it to Box 354400 or FAX it to 685-2915. Please include a budget name and number on the form. Currently, the cost of analysis is approximately $83 for unknowns of less than 4L (1 gallon).
The problems presented by unknowns can be reduced by periodic examination and inventory of stock chemicals, promptly labeling new containers, and disposing of all unused and waste chemicals from a satellite area prior to a faculty or staff member's departure.

Instructions for LSS

1. To get into the system click on "Uwick” in the program files, then “SSH secure”.

2. Type curie.u.washington.edu for host name OR click on curie.u.washington.edu if it is an option.

3. Username= gollahon

Password= rablab1

5. When the tutorial screen appears, bypass by pressing return

6. At the next menu, chose Chemical Inventory Menu by choosing or typing “INV”

7. At the next menu, chose view, update, or insert

V= view

U= update (for changing, deleting, and looking at files)

I= insert (for inserting new chemicals)

9. Next screen will be blank version of screen that you will always be working with. To get to the files you want to look at or update, must enter building and room codes.

Bldg ID: HSK -- press return

Room ID: K 081 – press Shift return

10. The screen should fill with product information at this point if you are in the update mode.

11. While in update screen:

(.)= delete

(O)= save

(1)= see options

(4)= add a comment onto a product line

(7)= see the online MSDS

F3= gets you out of any screen to the previous screen

F2= help

for other commands, see the official LSS manual

TO ADD A CHEMICAL TO AN EXISTING FILE:

1. Log on, go to the INV menu and chose “I” = insert

2. Enter Bldg ID: HSK-- press return

Room ID: K 081-- press enter

3. Tab to the product name field

4. Type name of chemical and press enter on numeric keypad

5. Find the chemical from the list the will appear by using the down arrow key

6. Press enter to select the product and have it placed in the inventory

7. At the point, return acts like TAB to move between columns so that you can add info like “amount”, “unit,” “surplus,” etc.

8. In “unit” column, press enter to see available codes, use down arrow to select correct unit (e.g. ML) and press enter

*If Chemical is not in LSS, but you want to add it: at step #4 above:

1. Move cursor to blank row using down arrow

2. Type name of product, press return

3. Type “y” to confirm that you want to add, and press return

Radiation Safety

All laboratory members who use radioactive materials must take the Radiation Safety Course offered by Environmental Health and Safety

http://www.ehs.washington.edu/training/radclass.htm

http://www.ehs.washington.edu/forms/RSClass_reg_form.htm

Our laboratory is authorized to use ³H and ³²P. ³H has low energy (0.006 MeV) beta emissions and requires no shielding. The waste is regulated and must be disposed of in LSA boxes. ³²P has beta emission (0.7 MeV) and should be shielded with plastic.

The waste for ³²P is in SEPARATE boxes from the ³H.

All ³²P waste is in K-081 (main lab).

All ³H waste is in K-089 (tissue culture room).

Make sure you know where to put the waste for your isotope.

All liquid ³²P sewer waste MUST be recorded on the clipboard that is on the shelf above the PCR machines. At the present time, there is ³H in K-089 and ³²P in K-081.

The Radiation Safety Manual is on the shelf above the bench by Peter’s office.

NO FOOD OR DRINK IS ALLOWED IN ROOM K-089 or K-081. BOTH ROOMS MUST BE LOCKED WHEN NO ONE IS PRESENT. We can lose our license over these two things!!!

Performing Calibrations
http://www.ehs.washington.edu/RadSaf/Rad_Calibrations.htm

The UW Radiation Safety Office (RSO) operates an instrument calibration facility (call (206)543-6328 for more information). Costs of meter calibration at the UW facility are comparable to other calibration facilities.

It is not required that the RSO calibrate your instruments. Calibrations may be performed by any qualified agency or by the instrument owner, provided that it can be demonstrated that the calibration is performed correctly.

To insure compliance with state regulations:

All survey instruments must be calibrated at least annually.
A record of the results and the method of calibration must be kept on file in the lab.
The records must be made available to the RSO or the Washington State Department of Health.
Even if the RSO does not calibrate your instruments, it is important that you provide them with copies of your calibration records to ensure compliance with the UW's Radioactive Materials License.

Fire Safety

Fire Safety. You must leave the area when there is a fire alarm. Make sure your lab area is as safe as possible before leaving. Do not use elevators.

The fire extinguishers are located outside the lab door (room K-081) and in K-079 (flow cytometry room). You should know how to use them. There is a class for this if you would like to take it. http://www.ehs.washington.edu/forms/classes/fetform.htm

In case of a big fire, activate the fire alarm in the hall. Then leave the building.

Earthquake Safety

Class offered through EEHS http://www.ehs.washington.edu/forms/classes/edform.htm

When the Earth Shakes

If you're at WORK OR SCHOOL, DROP under a desk or sturdy table. Stay away from windows, bookcases, file cabinets, heavy mirrors, hanging plants, and other objects that could fall. Stay under COVER until the shaking stops. HOLD onto the desk or table. If it moves, move with it. Here are some additional tips for specific locations.
If you are in a HIGH-RISE BUILDING and not near a desk or table, move against an interior wall, and protect your head with your arms. Face away from windows. Do not use elevators. Do not be surprised if alarm or sprinkler systems come on.

After the Earthquake
At the University you should have a predetermined place for you and your co-workers to meet -- kiosk outside K-wing.

Evacuate slowly and carefully. Look before exiting to make sure there is no overhead danger.
As you evacuate take note of utilities. Look for wires arcing, water running, and the smell of natural gas.

· Report to the kiosk outside K-wing, and take note of who is missing and any injuries that may exist.

Call or send a runner to the Police on Boat Street to inform them of damage, missing persons, injuries, and utility damage.
At your meeting spot, assist others, and check on loved ones. Let someone know if you leave.

Hazard Hunt
Conduct a hazard hunt at work.. Most injuries occur from interior flying or falling items. Check at least the following items:

Top heavy free standing furniture, Heavy or breakable objects
Electronic equipment and appliances. Unsecured cupboard doors
Hazardous chemicals, Utilities (gas, water, electrical)

Needle Sticks

The UW Campus Health Service (CHS) is the program for occupational health needs of all UW employees, students, faculty, volunteers and other designated UW affiliates.

The CHS clinic is located at the UW Medical Center on the 2^nd floor (NN256A) next door to the Emergency Medicine Service or at Hall Health Primary Care Center.

Call (206) 548-4848

http://depts.washington.edu/hhpccweb/CampusHealth.html

http://www.ehs.washington.edu/Services/accinc.htm

In case of a needle stick.

1. Report the incident to Katy, Martin, or Peter.

2. Seek medical care in the CHS clinic or Emergency Room without delay.

3. Laboratory tests and medication for HIV post-exposure prophylaxis should be started
within 1 to 2 hours after exposure.

4. Fill out an incident report form http://www.ehs.washington.edu/forms/IncidentFillin.pdf

Problems with Cooling, Heating, the Building or Scientic Instruments

Chilled Water Failure in Flow Cytometry Room

Call Robert Davis at 5-9438

General Heating, Cooling, Fumehood, or Building Problems:

M-F call 3-3010 or put your request on line at http://www.washington.edu/admin/facserv/workrequest.html. You will need a budget number for any repair that is not heating or cooling. The air-conditioner in the flow room requires a budget # because it is not a standard part of the building.

Evenings and weekends call the University Police at 3-3010 wait for the end of the message and then dial 0. They will contact the physical plant manager who is on-call.

Autoclave Failure

Call Steris at 1-800-333-8828. You will need to give them the institution, the room number and the serial number of the autoclave. Account # 46860

Gravity Sterilizer                             Room K059        serial # 011589306
Vacumatic Sterilizer                        Room K059        serial # 011589305
Gravity Sterilizer (dirty autoclave) Room K092        serial # 012788315

Scientific Equipment Repair

   Pipetmen go to G-156 HSB along with a budget #
   Small equipment can be taken to T-287 along with a budget #
   Large equipment (eg. incubators) call 543-5580 again you will need a budget #

Laminar Flow Hood in TC

Kurt Geissel Email: kutis@u.washington.edu
Phone: direct (206) 685-9343 (with Voice Mail) department (206) 543-9510
Fax: (206) 616-3360

Telephone Problems

Call 3-0133

Hospital Laundry

Call Michelle at 206-521-1740 (for pick up call 3-6729)
Pick ups are on Fridays only

SECTION 2 Record Keeping, Animal Use
AND Computer Use

Lab Notebook

All experiments must be recorded in a laboratory notebook. Katy can supply you with a notebook.

1. Use a numbered bound notebook for all entries. Number the pages and use the first few pages for an index.

2. Use pen for all entries

3. Date all entries. Write the day and full date at the top of the page. Try to keep different experiments on different pages.

4. Record ALL experiments – Successes, failures, and things you don’t understand.

5. Enter primary data immediately. Do not keep notes on scraps of paper or paper towels to enter later. Record details -- amount, concentration, how solutions were made, time and temperature of incubations, centrifuge speed, mistakes you made along the way, etc. Make sketches or diagrams if necessary.

6. Explicitly list, label, and identify your controls or standards.

7. If you run your experiment on the flow cytometer, make a note of the protocol used and the file names. It is a good idea to make a copy of the list you give to Thong with sample numbers for your notebook.

8. End each record with your conclusion and plans. Do this as soon as possible. You should have some hypothesis of what you expected from the experiment. Did this experiment confirm your hypothesis or not?

Frozen Cell Lines Log

N₂ TANK LOG INSTRUCTIONS:

The frozen cell line catalog is in the TC lab K-089 (brown binder). In an effort to minimize database disasters, you should use this catalog and not the computer to look up, record, edit or remove entries. Judy and only Judy will edit the computer logs and update this catalog as needed.

THE FIRST SECTION IS AN ALPHABETICAL LISTING BY CELL NAME.

THE SECOND SECTION IS A RACK AND BOX LISTING.

INSTRUCTIONS FOR REMOVING OR EDITING:

1. Look up your cell line in the ALPHABETICAL LISTING.

2. If you remove a line, highlight it with the attached yellow pen.

3. If you edit a line, just write the appropriate change in pen next to the entry.

INSTRUCTIONS FOR ADDING:

1. Look up the appropriate slot in the RACK AND BOX LISTING.

2. Record your new entry in pen on the appropriate line.

3. Highlight the new entry with the attached yellow pen.

ParafFin block Log

Paraffin BLOCK LOG INSTRUCTIONS:

The paraffin block catalog is in the K-081 (brown binder) under the bench in the middle bay. In an effort to minimize database disasters, you should use this catalog and not the computer to look up, record, edit or remove entries. Jeanne and only Jeanne will edit the computer logs and update this catalog as needed.

THE LISTINGS ARE BY DRAWER AND ROW

INSTRUCTIONS FOR REMOVING A BLOCK:

1. Look up your block.

2. Make a slip of paper and label it with the name of the block you will be taking.

3. Highlight the block name with the attached yellow pen. Write your initials and date by the block name

4. Remove the block and place the slip of paper in the slot so we will know where the block belongs when you return it.

INSTRUCTIONS FOR ADDING:

5. Identify the slot and drawer where the block lives. There should be a piece of paper in the slot.

6. Remove the paper and replace the block

7. In the log write returned and the date.

Computer Use

Rabinovitch Lab Computers

The computers in the Rabinovitch Lab are maintained by the Department of Pathology. For computer support or questions go to the following web site. http://www.pathology.washington.edu/tech/ or call (206) 221-5790

Access. In order to have access to a computer in the lab you must be assigned a password. Peter or Mike will send a request to info@pathology.washington.edu along with the individual’s name and email address.

Computer glitches. Send a Tech request to Computer Support http://www.pathology.washington.edu/tech/

Computer Use. The computers in the lab are research tools and as such they are to be used to write papers and reports, process data, access journal articles, correspond with colleagues concerning research information etc. They are NOT for playing games or excessive correspondence with friends.

For questions concerning UW policy see the following web site:

http://www.washington.edu/computing/rules/

Improper use of UW computers and networks can get you into trouble. It is your responsibility to know the rules. These UW guidelines and examples of the rules and laws of the state of Washington will help you to use computing and networking resources appropriately.

Revised Code of Washington (RCW) - Laws passed by the State Legislature

RCW 9a.52.110 Computer trespass in the first degree.

RCW 42.52.180 Use of public resources for political campaigns.

RCW 42.17.260 Documents and indexes to be made public.

RCW 42.52.160 Use of persons, money, or property for private gain.

Washington Administrative Code (WAC) - Rules and regulations for all state agencies

WAC 292-110-010 Appropriate and inappropriate use of state resources. (revised 04/98)
[See UWeek article State changes guidelines on email use]

Executive Ethics Board

Frequently asked questions about email and Internet use

Washington State Attorney General's Office

Junk Email - Information from the Consumer Protection Division

Email and Computer Usage by Faculty and Staff - Notice from the Provost and Executive Vice President

When you establish a UW NetID you open a gateway to a wealth of computing resources at the UW and beyond. Remember that inappropriate use of these computing resources can result in loss of access to them.

Staff use of Uniform Access computers is subject to the approval of their departments and supervisors.

Electronic Mail (EMAIL)

UW-GS 5

(Revised) January 1998 http://www.washington.edu/admin/recmgt/uw.gs5.html

Electronic mail is a technology that allows for the written exchange of information in machine readable format. Email represents not the system, but the information communicated through the system. Email messages are public records when they are created or received in the transaction of public business. They must be retained as evidence of official policies, actions, decisions or transactions. Email messages are considered public record material with legally mandated retention requirements, and are subject to the same rules and regulations as those which govern the management of paper records. Email is managed by its content, not its format.

Purpose. Email is meant for informal correspondence and scholarly communications. It should not be used for official record-keeping purposes. (For further guidelines on the uses of email, see Knowing the Rules on the Computing and Networking page)

Electronic Management. The University of Washington does not have central processes or resources to manage email in a way that meets specific Washington State Code regulating the management of public records. Backup of folders may not exist or folders may only be kept for a very short duration, so inadvertent deletion of messages can result in loss of information.

Privacy. Confidential and sensitive information should not be sent via email. The privacy and integrity of an email message cannot be guaranteed. Also, once created, there is no guarantee that attempts to erase or delete email will be effective.

Release. Under the Public Records Act (RCW 42.17.250 et seq.), if requested by a member of the public, email must be transmitted to the UW Public Records Office for review and possible release. Tape or disk copies of deleted documents are also subject to the Public Records Act.

Litigation. Unless protected by legal privilege, email can and will be discoverable in litigation. This applies to email on disk or on a backup medium.

Legal Proceedings. Like other forms of records, and regardless of retention requirements, email pertaining to pending audits, or judicial or public disclosure proceedings must not be destroyed until the issue is resolved.

Email messages are subject to the guidelines in RCW 40.14 regulating the preservation and destruction of public records and as such are managed through records retention schedules.

Email that is considered to have no administrative, legal, fiscal, or archival requirements for its retention may be deleted as soon as it has served its reference purpose. Refer to UW-GS4 .

The following categories of messages have specific retention periods. Refer to the University General Records Retention Schedule or your Departmental Records Retention Schedule for the retention period of individual items.

These records must be printed out and saved as a paper document as it is difficult for a department or unit to retain electronic records since individuals, electronic media, and desktop hardware and software change. Backup procedures for desktop equipment are often neglected and disaster recovery routines are not practiced. The only way to assure the retention of information is to print it and file it by subject or function in the appropriate paper filing system.

Policy and Procedure Directives

Correspondence or memoranda related to official public business

Agendas and minutes of meetings

Documents related to legal or audit issues

Messages which document departmental/office actions, decisions, operations and responsibilities

Documents that initiate, authorize or complete a business transaction

Drafts of documents that are circulated for comment or approval

Final reports or recommendations

Appointment Calendars

Email distribution lists

Other messages sent or received that relate to the transaction of University business.

Animal Protocols

1. Any one who works with animals is required to attend the animal training session given by the Department of Comparative Medicine every year. See policy page: http://cer.hs.washington.edu/iacuc/policies/index.html

2. The booklet written by the Department of Comparative Medicine in on the lab safety shelf above the bench by Peter's office (Laboratory Animal Regulations).

3. You must have an approved animal protocol for any procedure you perform on animals. It is a good idea to list the number of the protocol used in your laboratory notebook when you do the procedure. Our approved protocols are in the back of the Laboratory Animal Regulations book. Protocol forms are available on-line http://cer.hs.washington.edu/iacuc/iacucforms/index.html

4. If you need help with a procedure see Katy; she has worked with animals for many years and is familiar with most animal handling techniques.

5. If you have questions about a protocol or procedure and Katy does not know the answer, veterinarians are on call 24 hr. a day to help. Call 543-6257 for assistance.

SECTION 3 TISSUE CULTURE

Tissue Culture Media Preparation

MCDB 1531( liter)

Bottle sterile water for irrigation

Autoclaved flasks

Bell filter

MCDB 153 from Sigma (in refrigerator crisper)

Sodium Bicarbonate (7.5% solution) Invitrogen/Gibco

· Pour about half of the distilled H₂0 into large beaker.

· Add MCDB 153 powder to the beaker.

· Rinse the MCDB 153 packet with bottle sterile distilled H₂0 and add it to
the beaker.

· Fill the beaker up to about 900 ml mark

· Stir the solution

· Add 15.7 ml of NaHCO₃

· While stirring, bring the final pH to 7.2 by adding appropriate 4N HCl or
4N NaOH.

· Using 1 liter cylinder, add enough distilled H₂0 to bring it to 1 liter
solution

· Filter with bell filter into 200 ml plastic flasks.

L-15 (1 liter)

Bottle sterile water for irrigation

Two autoclaved 500 ml bottles

Bell filter

L-15 from Sigma (in refrigerator crisper)

· Pour 1 liter of the distilled H₂0 into graduated cylinder.

· Pour about half of distilled H₂0 the large beaker.

· Add L-15 powder to the beaker.

· Rinse the L-15 packet with some distilled H₂0 and add it to the beaker.

· Fill the beaker with the rest of the water from the cylinder.

· Stir the solution

· While stirring, bring the final pH to 7.2 by adding appropriate 4N HCl or
4N NaOH.

· Filter with bell filter into 500 ml bottles.

Barrett's Media Preparation

Reagent	Final Conc.	1 liter	200 ml	Stock
MCDB-153	1 X	1 package
Hydrocortisone	0.4 mg/ml	80 ml	16 ml	50 mg/ml
EGF	20 ng/ml	2 ml	400 ml	10 mg/ml
Cholera Toxin	10^-10M	84 ml	16.8 ml	100 mg/ml
Adenine	20 mg/L	200 ml	40 ml	100 mg/ml
Bovine Pituitary Extract	140 mg/ml	140 mg	25 mg	[see below]
Fetal Bovine Serum	5%	50 ml	10 ml
Penicillin-Streptomycin	100 unit/ml	10 ml	2 ml	10,000 units/ml
Amphotericin B	0.25 mg/ml	1 ml	200ml	250 mg/ml
Insulin-transferrin- selenium	5mg/ml	1ml	200ml	10 mg/ml
L-glutamine	4 mM	20 ml	4 ml

Media storage: Store in refrigerator

--------------------------------------------------------------------------------------------

Preparation of Growth Factors

1. Hydrocortisone (5 mg/ml stock) Sigma H-0396

Dissolve in water and aliquot one ml vials and freeze

2. Epidermal growth factor R&D Systems Cat.# 236-EG

3. Adenine (100 mg/ml) Sigma A-2786

1 gram of adenine + 10 ml 1N NaOH

Aliquot in 1 ml

Store in freezer

4. Cholera toxin (100 mg/ml) Sigma C-8052

0.5 mg Cholera toxin + 5 ml dH₂0

Aliquot into microcentrifuge tubes

Store in the refrigerator

5. Bovine Pituitary Extract
**Preparation depends on source of BPE**

**Test new BPE before old lot # runs out**

Invitrogen/Gibco BRL catalog # 13028-014 (25 mg in 5 mls--store frozen)

-Thaw and place into centrifuge tube. Spin down 5 minutes

Add supernat to media

- Remove 5 ml of media and resuspend BPE pellet. Spin down
another 5 minutes. Add supernat to media.

- Discard pellet

6. Fungizone (Invitrogen/Gibco)

Thaw and aliquot into 1.7ml. microfuge tubes. Refreeze at -20.

7. L-glutamine

Aliquot in 8 ml volumes.

Store in freezer. Thaw until clear, then add to media.

8. Insulin-transferrin-selenium/ITS (10 mg/ml, Invitrogen/Gibco)

Biohazardous material.

50 mg ITS + 5 ml sterile dH₂0

Aliquot 200