It is important to know that the Certified Reference Materials contain the right strain. Biosisto guarantees that the correct strain is present in the CRM.
How do we do that, and what are the dangers when there are too many passages?

Transferring microorganisms from one agar plate to another, known as passages, can pose risks and disadvantages if done too frequently.
Here are some of the main dangers and problems associated with frequent subculturing:

1. Genetic Drift

Each time bacteria are subcultured, there is a chance of genetic changes occurring through mutations. Frequent subculturing can lead to:

Loss of specific traits: Characteristics originally present in the bacterial strain may be lost.
Increased genetic variability: This can lead to unwanted variations within the culture.

2. Phenotypic Changes

Microorganisms can undergo phenotypic changes as a result of repeated subculturing. These changes can affect the physiological and biochemical properties of the bacteria:

Loss of virulence: Pathogenic bacteria may lose their ability to cause disease.
Change in growth rate: The growth rate may increase or decrease, affecting experimental results.

3. Contamination

Frequent subculturing increases the risk of contamination with other microorganisms:

Cross-contamination: Using the same equipment or workspace can lead to the introduction of unwanted microorganisms.
Environmental contamination: Open plates and repeated handling of cultures can expose them to airborne or surface particles.

4. Culture Fatigue

Repeated subculturing can cause the culture to become “tired,” meaning the microorganisms become less viable:

Reduced viability: The number of viable cells may decrease.
Loss of vigor: The bacteria may grow more slowly or not respond well to growth media.

5. Loss of Authenticity

Frequent subculturing can lead to the loss of the original characteristics of the culture:

Loss of plasmids: Plasmids, which often contain genetic information for antibiotic resistance or metabolic functions, can be lost.
Loss of specific phenotypes: Bacteria may lose traits essential for certain studies or applications.

6. Other Errors

Each subculturing introduces a chance of user error, such as incorrect colony identification or improper handling techniques:

Human errors: Incorrect techniques or mixing up cultures can lead to unreliable results.
Incorrect selection: Selecting non-representative colonies can result in a non-representative culture.

What is the number of acceptable passages?

Both the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopoeia (USP) usually limit passages from the original culture received from a recognized institution to up to five. Biosisto also follows this guideline.

Biosisto policy

The source of the Biosisto strains is from recognized culture banks. These are banks that are mentioned at
When a new strain arrives at Biosisto, it will be passage 0. After 1 transfer in our laboratory, this will be passage 1.
This strain, with passage 1, will be stored in our cryobank. New batches of Certified Reference Materials will be produced from this source.
Biosisto’s policy is that the Certified Reference Materials contain a maximum of 4 passages.
This is sometimes mentioned on the Certificate for reference materials, particularly the CRM for Pharma.

If you want to know exactly how much passage has taken place in a particular CRM, please get in touch with





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