Scrm Tissue Culture Facility Handbook

SCRM TISSUE CULTURE FACILITY HANDBOOK

The tissue culture service aims to provide and maintain a high quality tissue culture facility for all SCRM users. We can supply training, help and advice as required.

This handbook provides basic guidelines for operation of the facility. Please see Theresa O’Connor if you have any further questions.

Academic supervisor: Ian Chambers

Facility Manager: Theresa O’Connor

(ext.: 519523, email: )

Technicians: Helen Henderson and Marilyn Thomson

(ext.: 519584/519585)

(Email: / )

General Rules

1. All new users must have individual instruction and training from an authorised person within their group and complete a short induction course given by Theresa O’Connor.

1. No outdoor coats must be worn or brought into tissue culture.

1. No white lab coats should be worn in tissue culture.

1. Users must arrange to get 2 blue tissue culture lab coats from stores before commencing work. You are encouraged to keep lab coats clean. Robbie (stores) can arrange laundry collection on a Monday every week.

1. The SCRM tissue culture facility is communal. The clean side is for culturing established cell lines only and the ‘primary’ side is for culturing primary cells. Cultures may be moved between rooms on the same side, and can move from the ‘clean’ to the ‘primary’ side. However, cultures should NEVER be moved from ‘primary’ to the ‘clean’ side.

1. Users are entitled to book into any ‘human’ or ‘mouse’ hood using the booking sheets provided on each hood. Users must ensure that only the necessary time is booked in a hood and that they work within that period.

1. Hoods and rooms should be left clean and tidy and any spillages cleaned up, including under the base plates.

1. All users are on a rota to clean the hoods on a weekly/monthly basis, instructions are posted in every room, please sign the sheet when you have finished.

1. Pipette jars must not be overfilled to prevent unnecessary breakage of glass pipettes. Broken glass poses a risk to tissue culture staff when they clean the pipettes.

1. Orange bags must not be overfilled, please change the bag at the end of your TC session.

1. Ensure all media, solutions and culturing vessels are clearly labelled.

Never use other people’s media or solutions.

1. Do not use autoclave tape on glass bottles, use the coloured tape provided. All tape must be removed before putting bottles into wash up.

1. New cell lines must NOT be introduced to the facility without the manager’s knowledge. All new cell lines must be quarantined in the ‘Quarantine room’ (1.53) on the primary side until tested for mycoplasma by TC staff.

Any cell lines undergoing treatment for mycoplasma must do so in a designated incubator in the ‘Quarantine room’.

1. Any chemicals being ordered for use in tissue culture should have COSHH assessment completed.

1. Any hazardous chemicals should be notified to the manager before commencing work to prevent chemicals being handled inadvertently by tissue culture staff.

1. LARGE SCALE EXPERIMENTS. Please give tissue culture staff one week’s notice of any large scale experiments so they can ensure adequate quantities of stock solutions and plastics.

1. If in doubt ask!

PLASTICS/CONSUMMABLES

Tissue culture supplies a number of different plastics for use within the facility. Currently the main supplier is Corning other suppliers used are Falcon, NUNC and Sterilin.

Plastics are stored in the Plastic consumables cupboards on both the ‘Clean’ and the ‘Primary’ corridor’s. Plastics taken from these cupboards must be signed for in the red folder provided. Groups are charged for these on a monthly basis.

The plastics listed below are charged for:

Flasks: 25cm2,75cm2, 75cm2 vented and 150cm2.

Plates:4 well, 6 well, 12 well, 24 well, 48 well, 96 well, 96 ‘U’ well and 96 ‘V’ well.

Dishes: 30mm, 60mm, 90mm, 140mm, 60mm organ culture, 35mm treated, 100mm treated and 150mm treated.

Pipettes: 2ml aspiration, 5ml, 10ml, 25ml and 50ml.

Tubes: 1ml cryotubes, 1.8ml cryotubes, 15ml centrifuge tubes, 50ml centrifuge tubes.

Well inserts

Electroporation cuvette

Cell scrapers

Cell strainers

The items listed below are provided free of charge:

Tubes: 20ml and 150ml universals.

Syringe filters (0.2uM)

Pipette tips: 200ul, 200ul square cut and 1000ul.

Gloves: XS, S, M, L and XL.

Syringes: 1ml, 2ml, 5ml, 10ml, 20ml and 50ml.

Needles: 18G, 19G, 21G, 23G, 25G and 26G.

Glass Pasteur pipettes

White roll

70% IMS

Detergents: 1% Mucosol, 1% Chemgene and Presept tablets.

SOLUTIONS AND CHEMICALS

The following solutions are available in the prep room (1.49).

All solutions taken from the fridge/freezer must be signed for in the RED folder provided in the wash up room. Groups are charged for these on a monthly basis.

Stock SolutionsStoredStock SolutionsStored

GMEM (500ml)Fridge1% GelatinFridge

PBS (-Mg/-Ca) (100/500ml)ShelfTrypsin (TVP)Freezer

Glutamine/PyruvateFreezerTestersBench

Non-essential amino acidsFridgePuromycinFreezer

2-MercaptoethanolFridgeHygromycin BFreezerPen/Strep Freezer G418 Freezer

LIFFreezerFoetal calf serumFreezer

KO serum replacementFreezer

Solutions are tested in advance of being put out for users.

Others chemicals e.g. Leishman’s and Geimsa stain arealso available from the prep room (1.49).

FOETAL CALF SERUM

Batch testing of Foetal calf serum will be carried out as required (usually once per year) and the relevant batch reserved. Records are available from tissue culture.

LIF

This will be prepared as required however, if users are going to perform a scheme of work which may use a large quantity of this solution tissue culture staff should be notified in advance. This will allow new stock to be prepared.

MYCOPLASMA

Mycoplasma screening is provided by the tissue culture service. All cell lines new to the facility must be quarantined in the ‘Quarantine room’ (1.53) on the primary side until they have been tested for mycoplasma by TC staff. All cell lines in culture (without antibiotics and which are not under selection) should be tested every 3 months.Mycoplasma screening is carried out weekly on a Wednesday; please notify Helen or Marilyn if you have samples for testing by 3pm on the Tuesday.

For screening, cells should be approx. 70% confluent and 1 ml of culture supernatant should be passed to TC staff for the test.

KARYOTYPING

A chromosome counting service is provided for mouse ES cell lines. Please give at least one weeks notice and set up cells at 2x106 in a 25cm2 flask for chromosome preparation to be carried out the next day.

CLASS II CABINETS

The cabinets must be booked by the user on the day and for the time required.

The cabinets should be sprayed with alcohol and wiped clean with white roll, before and after use.

After completing work the user should ensure the cabinet has been washed with alcohol, that the pipette gun is in good working order, and that the aspirator line is clean and the reservoir not overfull.

The use of gloves for working in the cabinet is optional, but recommended. The alcohol spray can be used to wash hands and forearms before and during work. For example if, during the course of your work, you leave the hood and touch door-handles etc. you should sterilise your hands again before re-commencing work.

All items to be used in the cabinet should be first sprayed with alcohol and wiped down with white roll before placing in the cabinet.

Individuals should ensure that any spillages are cleaned up immediately.

A general clean of the Class II Safety cabinets used in Tissue Culture are carried out weekly by users on a rotation system. The cleaning rota is on the back of the door in every room.

Both glass and plastic pipettes are available. However, only plastic pipettes and plastic aspiration pipettes should be used when culturing human cells; the university health and safety department recommends that glass pipettes should not be used for culturing human blood or tissue.

Only plastic pipettes should be used when pipetting hazardous chemicals.

INCUBATORS

The incubators are cleaned and maintained by TC staff on a rotational basis.

Users should inform TC staff ASAP if an incubator is dirty, there has been a media spill or if they notice an infection so the incubator can be shutdown and cleaned/sterilised.

Ensure both inner glass and outer doors are shut firmly after use.

Users should inform TC staff immediately if they notice any problems with an incubator, (low water level, low CO2 or temperature).

GAS SUPPLIES

SCRM has a central CO2 and nitrogen gas supply; the gas supply lines to the incubators are maintained by the tissue culture staff.

Users should inform TC staff immediately if they notice any issues/problems with the gas supply/supply lines.

WATERBATHS

These are cleaned by TC staff on a monthly rota. If the water levels are low users should fill them with RO water available in the wash-up room (1.50).

WASTE DISPOSAL

Users are responsible for disposing of their own waste please ensure all waste is disposed of correctly.

Unwanted cultures/culture media should be aspirated into a flask using the central vacuum system. The contents of the flask should be decontaminated using Presept tablets before disposal.

Aspirators should be flushed with 70% IMS before and after use. When the aspirator bottle is ½ full the contents should be decontaminated and disposed of. Disconnect the aspirator jar, add Presept tablets (4 tablets per 5 Litres), leave for 30-60mins then take to the TC wash room and dispose of the liquid in the designated sink. Please make sure you run a lot of water down the drain after emptying the aspirator and make sure the sink is clean when you have finished. Place half a Presept tablet into the empty flask before it is reused.

FOR DISPOSAL OF ALL OTHER WASTE PLEASE SEE (page 8 & 9):

SCRM TISSUE CULTRE WASTE DISPOSAL LOCAL RULES

If you are unsure of how to dispose of a reagent or chemical the Little France health and safety advisor, Lindsay Murray, should be consulted before commencing work.

General laboratory
and office waste / Uncontaminated paper and hand towels,
cardboard and packaging materials
Non chemical
Non Biological
No glass or sharps / Clear plastic bag in black plastic bin.
Cardboard to be flattened
and placed next to bin for recycling
No barcode / Collected by cleaning staff.
Plastic media bottles / Non-infectious plastic pipettes / Green recycling bin / Rinse out all plastic media bottles and place into the green plastic recycling bin in TC corridors.
GM waste
(Refer to your GMO risk assessment.
Only if your GMO risk assessment
specifies should you use GM disposal route) / GM waste from tissue culture rooms. GM contaminated tissue culture plates, pipettes etc. must either be autoclaved or
chemically disinfected by soaking in 1%w/v Virkon, or
1,000 ppm Precept (four 0.5g tablets into one litre of water)
for at least two hours before discharging any excess liquid via drains. / A clear plastic bag in a metal bucket for autoclaving followed by
and orange bag
or
Following chemical decontamination an orange bag.
Needs a barcode. / If waste is to be autoclaved:
Tie bag at the end of TC session, and label it ‘GM waste’ take the bag IN THE BUCKET to the autoclave room.
Or following chemical decontamination:
Place into an orange bag, tie orange bag at the end of TC session and place in the white plastic bin in the TC corridor.
Taken to clinical waste room by TC staff.
Clinical/biological waste
(For heat treatment) / All non-infectious disposable gloves, contaminated paper towels, eppendorf tubes, pipette tips, plasticware,
syringe bodies (NO NEEDLES!)
No glass or sharps / Orange plastic bag in grey bin (mouse TC)
or
Orange bag in a metal bucket (Human TC).
Needs a barcode / Tie at the end of TC session and place in the white plastic bin in TC corridor.
Taken to the clinical waste room by TC staff.
Plastic pipettes / All non-infectious plastic pipettes
No glass or sharps / Cardboard pipette bins
Needs a barcode / Seal when full, place cardboard bin into an orange bag. This will come ¾ up the side of the box tape on the orange bag. Place next to the white bin in the TC corridor.
Taken to the clinical waste room by TC staff.
Clinical/biological waste
(For incineration) / All non-infectious tissue, organs, blood etc.
No glass or sharps / Yellow plastic bag in a metal bucket
Needs barcode / Tie at the end of TC session. Place bucket containing bag into the white bin in the corridor and inform TC staff it is there.
Taken to designated freezer in clinical waste room by TC staff.
Sharps / Sharps including glass Pasteur pipettes, needles, scalpel blades, and broken glass.
No syringe bodies. / Yellow rigid bin with orange lid.
Needs barcode / Seal when 70% Full. Place inside large yellow sharps bin in the TC corridor.
Taken to clinical waste room by TC staff.
Cytotoxic waste
e.g. phenol, RNA isolation reagent,
Trizol. / Phenol (phenol, RNA Isolation reagent, Trizol) contaminated tips and plastics. / Yellow rigid bin with purple bin.
Needs barcode / Seal when 70% full and apply barcode. Write ‘Phenol’ on the bin. Take to yellow wheelie bins in clinical waste room.
Inform Mark Marsden.

OUT OF HOURS WORK

Any users who wish to work any time after 18:00 or before 07:00 (Monday to Friday) and any time at all over a weekend or University holiday should have completed the ‘Late & Lone Working’ Safety Training session and should have read and signed their groups Late and Lone working risk assessment.

When working out of hours users should book into the hoods using the booking sheets provided and ensure class II cabinets, water baths and microscopes are SWITCHED OFF when they finish work.

Orange bags should be changed at the end of your TC session. Orange bags should be tied and placed into the white bins in the corridor.

Do not overfill pipette jars, make use of spare jars available in each room and move full jars to the wash up room.

Rooms should be left clean and tidy!

EQUIPMENT ALARMS OR EQUIPMENT FAILURE

During working hours alarms should be immediately notified to a member of TC staff.

Out of hours (after 17.00 or at weekends), security should be notified (call 24 29289/90) and requested to call Theresa or Mark Marsden.

NO ATTEMPT SHOULD BE MADE BY USERS TO SHUT OFF ALARMS OR REPAIR EQUIPMENT.

MSDS and COSHH

Files of materials safety data sheets and COSHH forms are kept in the prep room (1.49). All users should be aware of the risks of solutions and chemicals that they use and how to dispose of them safely. All appropriate COSHH forms should be signed before work commences and new protocols introduced into tissue culture should have a COSHH form completed to cover that work.

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