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Reference Culture in Microbiology: Why Limiting Passages is Crucial

Reference cultures, like those from ATCC, are vital for accurate microbiological testing. Each time a culture is transferred to fresh medium and grown, it's called a passage. Too many passages can lead to genetic or phenotypic changes, affecting test results. To maintain integrity, the USP recommends using cultures with no more than five passages from the original strain. The seed lot system is a best practice to control subculturing. Proper storage (e.g., −80°C for frozen cultures) also helps preserve strain characteristics. Following these guidelines ensures reliability and compliance in laboratory testing.

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In microbiological laboratories, the use of reference strains is critical to ensure the reliability and reproducibility of results. These strains, often sourced from recognized organizations like ATCC (American Type Culture Collection), are utilized for quality control, method validation, and as positive or negative controls in a variety of tests. However, one common and important concern persists across laboratories:

How many passages or subcultures are acceptable from the original reference strain? This question becomes essential when maintaining strain integrity and avoiding genetic drift, contamination, or phenotypic changes.

To address this, ATCC published a technical bulletin clarifying the concept of passages, how reference strains should be maintained, and what global standards like the USP (United States Pharmacopeia) and CLSI (Clinical and Laboratory Standards Institute) recommend regarding subculturing limits.

What is a Passage?

In simple terms, a passage is defined as the transfer of a viable microorganism from one growth medium to another, allowing it to grow under new conditions. According to USP <1117>, “any form of subculturing is considered a passage.” This includes both solid media (agar) and liquid media (broth). However, merely rehydrating or thawing a freeze-dried or frozen ATCC strain does not count as a passage because no actual growth takes place. The first time the organism is transferred from the vial and allowed to grow is considered Passage 1.

Why Do Passages Matter?

Each passage carries the risk of:

Genetic drift: small changes in the DNA of the microorganism that can accumulate over time.
Phenotypic variation: changes in physical traits or behavior of the organism, which could affect test outcomes.
Loss of characteristics: such as antibiotic resistance or biochemical traits, especially in plasmid-carrying organisms.
Contamination risk: with repeated handling, contamination from other microbes increases.

For these reasons, both the number of passages and the storage conditions play a vital role in preserving the authenticity and reproducibility of reference strains.

Seed Lot System: A Recommended Practice

To maintain quality and minimize passages, a seed lot system is highly recommended. This involves:

Obtaining a reference strain directly from ATCC (Passage 0).
Creating multiple stock cultures from the original vial (Passage 1).
Using these stock cultures to prepare working cultures as needed for routine laboratory use (Passage 2).

This type of system allows laboratories to limit the number of passages and ensure consistency across tests. Laboratories can freeze seed stocks and use them to regenerate fresh working cultures within the allowed passage limits.

What Do the Standards Say?

Both the USP and CLSI provide guidance on the maximum permissible passages of reference strains. While there are slight differences, both aim to minimize variability.

USP Guidelines

The USP is clear in its recommendation: microorganisms used in testing must not exceed five passages from the original ATCC strain. This includes passages to stock and working cultures. Beyond five, the reliability of the culture may be compromised, leading to inaccurate test results. USP <51> and <1117> both emphasize the importance of tracking the number of transfers and maintaining documentation to ensure compliance.

CLSI Guidelines

CLSI also emphasizes limiting subcultures but has varied over the years in specificity. Some guidelines suggest no maximum, while others recommend up to three subcultures of stock cultures and three of working cultures, which can result in a total of up to seven passages. However, this can vary depending on the document referred to (e.g., M02, M100, M27, M07).

Despite minor differences, both standards agree on one thing: excessive subculturing increases the risk of loss of strain characteristics.

Cold Storage Guidelines

Proper storage helps extend the usability of strains without needing frequent subculturing. Here are the key recommendations:

Frozen cultures: Store at −80°C in a mechanical freezer or in the vapor phase of liquid nitrogen. Avoid immersion in liquid nitrogen.
Freeze-dried cultures: Store at 2°C to 8°C.
Slants: Can be stored at 2°C to 8°C for up to one week.
Avoid storing in defrost-cycle freezers as fluctuating temperatures can damage cultures.

References:

U.S. Pharmacopeia <51> Antimicrobial Effectiveness Testing
U.S. Pharmacopeia <1117> Microbiological Best Laboratory Practices
CLSI M22-A3, M100, M02, M07, M27 Standards
ATCC Technical Bulletin: Reference Strains: How Many Passages Are Too Many?
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