Bulletin | Microbiological Compliance Testing – Yeast and Mold Enumeration, Method Suitability, and Sample Size | November 13, 2026

As cannabis science evolves, a greater understanding continues to develop regarding the influence of cannabis and cannabis-derived matrices on analytical and microbiological testing. The Massachusetts Cannabis Control Commission (“Commission”) issues this bulletin to clarify expectations for Independent Testing Laboratories (“ITLs”) conducting microbiological testing and to reinforce the importance of method suitability, validated procedures, and consistent testing practices for every sample received and tested.

1. Regulatory Requirements and Method Suitability

The current Protocol for the Sampling and Analysis of Finished Marijuana Products and Marijuana Products for Marijuana Establishments, Medical Marijuana Treatment Centers, and Colocated Marijuana Operations (the “Sampling and Analysis Protocol”), Section 7.4, Microbiological Contaminants and Mycotoxins, states that:

“Analytical methods for enumerating and identifying specific microbiological contaminants must be consistent with the following USP chapters: USP Chapter <61>… USP 36, Chapter <61>; USP Chapter <62>… USP 36, Chapter <62>.”

These references establish the applicable framework for microbiological enumeration and specified microorganism testing.

USP Chapter <61>, under Growth Promotion Test, Suitability of the Counting Method and Negative Controls, states:

“Suitability must be confirmed if a change in testing performance or change in the product may affect the outcome of the test, is introduced.”

Consistent with USP Chapter <61>, the Commission expects ITLs to ensure that microbiological methods are suitable for the matrices being tested and that testing is conducted in accordance with the applicable validated method, including the specified sample preparation, sample size, incubation conditions, and enumeration procedures.

2. Yeast and Mold Enumeration in Cannabis Matrices

The Commission recognizes that cannabis-specific method validation is important when establishing appropriate conditions for microbiological enumeration. AOAC INTERNATIONAL (“AOAC”), through its Cannabis Analytical Science Program, has developed performance requirements and supported matrix-extension validation studies to evaluate analytical methods for cannabis testing.

AOAC has published three relevant cannabis-flower matrix-extension studies evaluating culture-based methods for yeast and mold enumeration:

• 3M Petrifilm Rapid Yeast and Mold Count Plate, AOAC Official Method 2014.05. The 2023 study compared the method with Dichloran Rose Bengal Chloramphenicol (“DRBC”) agar as the reference method in dried cannabis flower. The study reported equivalent average log counts under the evaluated conditions, with incubation and enumeration performed within a 60-to-72-hour window.
• CompactDry YMR. The cannabis-flower matrix-extension study compared the method with DRBC agar and reported equivalent performance under the evaluated conditions, including at 72 hours.
• 3M Petrifilm Yeast and Mold Count Plate, AOAC Official Method 997.02. The cannabis-flower matrix-extension study compared the method with DRBC agar and reported equivalent average log counts under the evaluated conditions, with incubation for five days.

The Commission also acknowledges the associated manufacturer matrix-extension validation documentation for the 3M Petrifilm Rapid Yeast and Mold Count Plate. This documentation provides additional support for considering the cannabis matrix and the potential presence of slow-growing yeasts and molds when establishing appropriate incubation and enumeration conditions.

These studies provide cannabis-specific comparative evidence for the evaluated methods and dried cannabis flower matrix. The studies used different methods and incubation conditions; they do not establish a single incubation period that is universally applicable to every culture-based method or every cannabis product matrix.

The Commission has considered this evidence in conjunction with the method-suitability principles of USP Chapter <61> and the applicable AOAC performance requirements. To promote consistent testing practices and account for the available cannabis-specific evidence, the Commission now establishes the following expectation for yeast and mold enumeration.

Effective immediately, ITLs should incubate and enumerate culture-based yeast and mold tests within a window of 60 to 72 hours. Please note, this bulletin establishes the Commission’s expected incubation window for culture-based yeast and mold enumeration and is not intended to represent a universal incubation requirement prescribed by USP or an identical finding across all published validation studies.

ITLs should ensure that their validated procedures and laboratory practices support compliance with this requirement. Where an ITL’s current method or validation does not support the required incubation window, the ITL must resolve the discrepancy through appropriate method validation or other action necessary to demonstrate suitability before applying the method under this requirement.

3. Validated Methods and Sample Size

The Commission expects ITLs to follow their validated methods for every sample prepared and tested, consistent with applicable Standard Operating Procedures and ISO 17025:2017. This includes adherence to the sample size, sample preparation, dilution, incubation, and enumeration procedures established by the applicable method.

Validated methods must specify the sample size to be tested. Where a method is based on a published reference method, ITLs are expected to use the sample size specified in that method unless an alternative sample size has been appropriately validated.

Any deviation from the published method’s specified sample size must be supported by a validation study demonstrating that the alternative sample size produces equivalent and suitable results for the intended application and matrix. A laboratory shall not change the sample size solely on the basis of convenience, routine practice, or an assumption that the change will not affect the test outcome. Test results obtained utilizing an unvalidated method may be deemed invalid and subject to further administrative action by the Commission, as appropriate.

4. Notice to ITLs

In sum, effective immediately, ITLs must ensure that:

1. Culture-based yeast and mold enumeration is incubated and enumerated within the Commission’s required 60-to-72-hour window.
2. Each microbiological method is suitable for the matrix being tested and is performed in accordance with its validated procedures.
3. The sample size specified by the applicable published method is used unless an alternative has been appropriately validated.
4. Testing records demonstrate that the applicable method requirements and the expectations established in this bulletin have been followed.

The Commission expects ITLs to implement these requirements consistently for all applicable samples and to maintain documentation demonstrating method suitability and compliance.

Test results originating from ITLs that do not comply with the above may be subject to further scrutiny consistent with the Commission’s authority pursuant to G.L. c. 94G and applicable regulations. The Commission may also take administrative action as necessary to protect the public health, safety, and welfare.

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