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Reference Strains: How many

passages are too many?

ATCC cultures have been specified and cited in national and This updated definition is preferable. The
international standards for many years. Examples include the earlier definition left questions about
standards promulgated by the Clinical and Laboratory Standards the meaning of “an established culture.”
Institute (formerly NCCLS) for the healthcare community and by There were several questions raised on the
the United States Pharmacopeia (USP) for the pharmaceutical and PMFList as to whether the frozen or freeze-
biopharmaceutical industries. Cultures are used in performance dried vial from ATCC was an established
testing of products, as positive and negative controls, as indicator culture. To some, the phrase “established
organisms and as identification standards. culture” implied a growing culture. It is
clear, however, that these frozen or freeze-
Though the use of microbiological standards is widely accepted, dried vials of reference strains from ATCC are indeed “viable”
there is still some confusion as to specific laboratory guidelines, cultures. A passage involves growing the microorganism with fresh
especially when determining the number of subcultures allowed medium, either on solid agar or in broth. Resuscitating frozen or
beyond the reference strain. Discussions about passages freeze-dried cultures by thawing or rehydrating is not by itself
have occured on the Pharmaceutical Microbiological Mail List considered a passage. Thus subculturing the reference strain from
(PMFList).1-3 This Technical Bulletin will attempt to clear up some ATCC’s frozen or freeze-dried vial to the stock culture is the first
of the confusion about passage and microbial cuture maintenance passage. Subculturing stock cultures to working cultures is the
and provide some definitions and recommendations. second passage from the original reference strain. Any subsequent
subculture is another cumulative passage.

Strain definitions
Authenticity of cultures
The confusion starts with the different names that are ascribed
to reference strains. In various CLSI and USP publications these Another term used by commercial sources other than ATCC is
cultures are called control strains, standard cultures, reference “ATCC derived.” A culture is derived from the ATCC reference
strains, test strains, and quality control strains. These terms can strain by subculturing in other words, one or more passages.
generally be used interchangeably, though the preference seems CLSI recognizes reliable commercial sources for reference strains.
to be reference strain or reference culture. Both the CLSI and However, you must take into account that these cultures are at
USP agree that reference strains should come from a reliable least one or two passages from the ATCC reference strains.
source; both organizations cite ATCC. There is agreement that
the reference strains from ATCC are subcultured to make “stock
cultures,” which are subcultured weekly or monthly to make the Maintaining cultures
“working cultures” used daily. Working cultures are often kept as
slants, and it is these subcultures that raise the questions about The USP recommends a seed lot system to maintain reference
passages from the original reference strain. strains in laboratories. In this system the ATCC reference strain
is subcultured to several replicates at one time, all of which are
A subculture is a passage. The USP 36-NF 31 <51> Antimicrobial within one passage. These replicates of the stock culture are the
Effectiveness Testing, states: “For the purposes of the test, seed vials for the laboratory. The seed stock is subcultured—the
one passage is defined as the transfer of organisms from an second passage—to make replicates of working cultures. This is
established culture to fresh medium. All transfers are counted.”4 the same system used at ATCC. (Figure 1).

This definition was updated in the USP <1117> Microbiological But how many passages are recommended or acceptable in the
Best Laboratory Practices to read: “One passage is defined as laboratory? There is agreement that the number of passages
the transfer of organisms from a viable culture to fresh medium should be minimized to reduce the possibility of phenotypic
with growth of the microorganisms. Any form of subculturing is variations, genetic drift, and contamination as much as possible,
considered to be a transfer/passage.”5 but standards organizations differ as to how many passages are
acceptable.
 Figure 1. Seed lot system Utilizing cold storage

Storage temperature of stock and working cultures can affect

growth characteristics and viability. The CLSI recommendations
include storage at −50 to −70°C for one year or below −70°C
indefinitely,6 or −20°C or below (preferably below −70°C) for
“prolonged” storage.8 Storage of slants is recommended at 2 to
8°C for either one week8,9 or two weeks.10 The USP 36-NF 31 <51>
recommends storage in liquid nitrogen or a mechanical freezer
below −50°C.

ATCC Culture For long-term storage of frozen cultures, ATCC recommends

Passage 0 the vapor phase of liquid nitrogen or a mechanical freezer at
Working Cultures −80°C. Immersion in liquid nitrogen is not recommended. Frozen
Passage 2 cultures containing a final concentration of 50% glycerol as a
5 Replicates cryoprotectant may be kept at −20°C for short-term storage (less
than one month). Do not store frozen cultures in a freezer with a
defrost cycle; this will expose the cultures to higher temperatures.
Freeze-dried cultures should be stored at 2 to 8°C. Slants can be
kept at 2 to 8°C for up to a week.
Stock Cultures
Passage 1
5 Replicates Conclusion: ATCC recommendations

1. ATCC reference strains should be subcultured to replicate stock

Over the years the recommendations from CLSI have varied. cultures in the laboratory. Stock cultures can be subcultured
The least specific recommendations call for subculturing stock for working cultures weekly, typically kept as slants. A seed lot
cultures weekly with new working cultures subcultured monthly, system is recommended.
with no maximum number of passages noted. Another CLSI
2. A passage is defined as a subculture involving growth of
standard recommends up to three subcultures of the stock
the viable microorganism with fresh medium. Thawing or
cultures and up to three subcultures of the working cultures.6 This
rehydrating ATCC reference cultures is not a passage.
would add up to as may as seven passages from the original ATCC
reference culture (one to make the first stock culture, plus three 3. Microorganisms for standard protocols should be used within
subcultures and three additional subcultures). five passages of the ATCC reference culture.
4. Frozen cultures should be stored in the vapor phase of liquid
USP standards have been more specific. USP General nitrogen or in a mechanical freezer at −80°C or below. Freeze-
Chapter: <1117> Microbiological Best Laboratory Practices states dried cultures should be stored at 2 to 8°C. Slants may be stored
“The number of transfers of working control cultures should be at 2 to 8°C for up to a week.
tracked to prevent excessive subculturing that increases the risk
of phenotypic alteration or mutation. The number of transfers
allowable for specific compendial tests may be specified in that
test.”5 USP clearly states that the working cultures used for
testing should not be more than five passages from the ATCC
reference culture. The USP 36-NF 31 <51> states: “The viable
microorganisms used in the test must not be more than five
passages removed from the original ATCC culture.”4

The USP 36-NF 31 General notices: 8, Terms and Definitions also

contains the following definition: “Microbial Strains – A microbial
strain cited and identified by its ATCC catalog number shall be
used directly or, if subcultured, shall be used not more than five
passages removed from the original strain.”7

The recommendation of five passages or less from the ATCC

reference culture has been broadly accepted in the healthcare
community and the pharmaceutical and biopharmaceutical
industries. ATCC agrees with this recommendation.

2 www.atcc.org
References
1. Friedel B. Culture passage guidelines. In: PMFList Archives
[Internet]. [The Pharmaceutical Microbiology Mail List] 2003
Jan 2. Available from: http://peach.ease.lsoft.com/archives/
PMFList.html
2. Kramer K. Seed stock culture. In: PMFList Archives [Internet].
[The Pharmaceutical Microbiology Mail List] 2003 June 10.
Available from: http://peach.ease.lsoft.com/archives/PMFList.
html
3. Anger C. Seed stock culture. In: PMFList Archives [Internet].
[The Pharmaceutical Microbiology Mail List] 2003 June 11.
Available from: http://peach.ease.lsoft.com/archives/PMFList.
html
4. U.S. Pharmacopeia. Antimicrobial Effectiveness Testing. In:
U.S. Pharmacopeia 36-NF 31, 2013 <51>. Rockville, MD: U.S.
Pharmacopeia; 2013.
5. U.S. Pharmacopeia. Microbiological Best Laboratory Practices,
<1117>. USP 36-NF 31, 2013.
6. National Committee for Clinical Laboratory Standards (CLSI).
Quality assurance for commercially prepared microbioogical
culture media. 2nd ed. Wayne, PA: CLSI; M22-A2, 1996.
7. U.S. Pharmacopeia. General Notices. In: USP 36-NF 31. Rockville,
MD: U.S. Pharmacopeia; 2013.
8. National Committee for Clinical Laboratory Standards (CLSI).
Performance standards for antimicrobial disk susceptibility
tests. 8th ed. Wayne, PA: CLSI; M2–A8, 2003.
9. National Committee for Clinical Laboratory Standards (CLSI).
Performance standards for antimicrobial disk and dilution
susceptibility tests for bacteria isolated from animals; approved
standard. 2nd ed. Wayne, PA: CLSI; 2002.
10. National Committee for Clinical Laboratory Standards (CLSI).
Reference method for broth dilution antifungal susceptibility
testing of yeasts; approved standard. Wayne, PA: CLSI; M27-A,
1997.

Reprinted from ATCC Connection 23(2): 6-7, 2003 and subsequently

updated.

www.atcc.org 3
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