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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/1.2


TITLE: PREPARATION OF CELL CULTURE MEDIA AT ECACC


INTRODUCTION

Cell culture media are prepared by the Resource Centre in response to internal request forms (see AHC/1998/3/1.2 Appendix 1). It concerns also preparation of inactivated foetal bovine serum. Preparation of supplement for slow growing cell lines such as hybridomas is given as well as growth supplement for poorly growing lymphoblastoid lines at any stage of transformation. Media are tested for microbial contamination.

Media can be prepared upon specific customer requests.

This is the basic method used for media production - the supplements added at 5 may vary.

PROCEDURE

  1. Get a cell culture medium preparation form (see AHC/1998/3/1.2 Appendix 2) and the next sequential batch number to be allocated to the new batch of medium. Unless requested otherwise, make up media in batches of 10 x 500ml bottles.
  2. Collect 10 x 500 ml bottles (with the same manufacturer batch number) of the required medium (e.g. MEM, RPMI etc.). Label each bottle adequately.
  3. Add inactivated Foetal Bovine Serum (FBS), (see procedure AHC/1998/3/1.2 Appendix 4), to the required percentage (see AHC/1998/3/1.2 Appendix 3) to each bottle using a 50 ml tube to measure volume.
  4. Add 5 ml of L-Glutamine (x 100) to each 500 ml bottle, if media does not already contain glutamine.
  5. Add any other requested supplements
  6. NB. All MEM preparations must have non-essential amino acids (NEAA) added.

  7. Mix each bottle of media carefully, forming a vortex to completely mix the constituents. Stick on label stating percentage FCS and any extra additions, expiry date and QC status.
  8. To test for microbial contamination, perform the following sterility checks:

  1. For each bottle of media label 2 x Thioglycollate universals and 2 x TSB universals with batch and bottle number, e.g. 2000/1 to 2000/10.
  2. With a 10ml pipette remove approximately 6ml from each media bottle; add 1ml to each appropriate TSB universal and 2ml to each appropriate Thioglycollate universal.
  3. For each media bottle, put 1 x Thioglycollate universal and 1 x TSB universal at room temperature and 1 x Thioglycollate universal and 1 x TSB universal at 37C.
  4. Check all broths after 7 days and record information on the QC of prepared medium form (see AHC/1998/3/1.2 Appendix 2).
  5. If more than one sterility test is contaminated, discard the appropriate bottle(s) and record on reverse side of preparation sheet.
  6. If only one sterility test of a pair is contaminated, set up the contaminated control again and check after another 7 days. If any further signs of contamination is seen then discard the appropriate bottle(s).

N.B. Do not discard any sterility tests until batch of media has been used.

See also:

AHC/1998/3/1.2 Appendix 5 Sterility and fertility of testing of broths - ECACC -

AHC/1998/3/1.2 Appendix 6 Preparation of HB supplement - ECACC -

AHC/1998/3/1.2 Appendix 7 Preparation of GMF supplement - ECACC -

 


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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