BACTERIA ERADICATION

LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.3/2.2

TITLE: BACTERIA ERADICATION

INTRODUCTION

The protocol describes the procedure for eradication of bacteria from cell cultures with antibiotics.

PROCEDURE

One ampoule is thawed and cells are recovered according to laboratory procedure AHC/1998/3/2.5/2.

Cells are cultured in the presence of the chosen antibiotics. Antibiotics applied are:

a) Penicillin (100IU/ml) and Streptomycin (0.1mg/ml) or

b) Gentamicin (0.05mg/ml)

The culture is maintained in the presence of antibiotics for 14 days passaging as required. Each passage should be performed at the highest dilution of cells at which growth occurs.

After treatment culture cells without antibiotics for at least 2 passages or 7 days.

One flask is submitted for microbial detection assays for microbial and fungi contamination (AHC/1998/3/3.3/2.1, AHC/1998/3/3.3/2.2).

If no bacterial or fungal contamination is detected a master cell bank is produced according to protocol AHC/1998/3/2.2/2. Reserve 5ml supernatant for antibody testing (AHC/1998/3/3.5/2).

If cells are still contaminated the procedure has to be repeated with another antibiotic, i.e. if eradication has been unsuccessful with penicillin/streptomycin the procedure has to be repeated using Gentamicin.

If attempts to eradicate fail, a new deposit may be obtained from the depositor or the cell line has to be taken out of the collection.

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