Passage matters – The importance of minimizing the number of times an organism is passaged

By Carol Horton

Hand holding petri dishWhen we talk about the concept of stock maintenance, one of the most important aspects of preserving cultures is to minimize the number of times an organism is passaged in vitro. There are a lot of factors in the culture environment that can affect the sterility or performance of an organism. For example, contaminated culture media and reagents, poor aseptic technique, or mold spores present in a non-sterile environment can introduce contaminating species into a culture. Alternatively, changes in aeration or media constituents can drive the selection of clonal populations with undesired consequences. For example, some antibiotic-resistant strains carry their resistance genes on a plasmid, which might be lost when the culture is grown on solid media in the absence of an appropriate selection reagent or when using scaled-up fermentation methods.

To minimize the risk of contamination as well as genotypic or phenotypic changes within the organism, it’s essential to minimize the passage of cultures in vitrobut how many passages are acceptable? The number of times an organism can be passaged could be empirically worked out by performing both genotypic and phenotypic studies on material that has been sub-cultured numerous times; however, this process is both time-consuming and costly since it requires optimal maintenance of a growing culture and repeated testing over a very long period of time.

Since many of our strains are used as quality controls in the routine analysis of sterile and non-sterile products, ATCC generally recommends following guidance set forth by regulators within their respective industries. For example, USP chapter 51 states that microorganisms used in antimicrobial effectiveness testing be no more than 5 passages removed from the original ATCC culture1. However, even for basic research, this is still a good rule of thumb to follow when expanding both master cell and working cell banks for use in prolonged studies—using material that is consistent in terms of both passage number and maintenance is essential for repeatability of experimental data. 

  1. United States Pharmacopeial Convention, Inc. U.S. Pharmacopeia-National Formulary [USP 40-NF 35]. Chapter <51> Antimicrobial Effectiveness Testing. Rockville, MD, 2017.