Causes of Mold Contamination in Upright Incubation: Analysis and Solutions

News 9 10 月, 2024

Since the implementation of GB4789.15-2016 for mold and yeast counting, mold contamination during upright incubation has been a common issue for many labs. The method introduced in this standard, compared to the previous GB4789.15-2010, has led to concerns about environmental contamination, especially during upright incubation. This article will analyze the causes and provide solutions to help address this issue.

Key Differences in GB4789.15-2016

The most significant change in the 2016 version of the standard is the way plates are incubated. Section 5.2 of GB4789.15-2016 specifies that after the agar solidifies, plates should be incubated upright at 28°C ± 1°C and observed on the 5th day for results. There are two critical points to note:

1. Frequent Observation: Unlike bacterial cultures, molds produce spores that can spread rapidly across the plate. Instead of waiting until day 5, it is essential to start observing and counting colonies from day 2 onwards and continue daily until the 5th day.
2. Upright Incubation: This is the major change from the 2010 version, which recommended inverted incubation. With upright incubation, many have encountered mold contamination issues, especially in the plates. Below are the common causes and corresponding solutions.

Common Causes of Contamination and Solutions

1. Incubator Environmental Contamination

Mold incubators often have high spore levels due to the presence of mold in the air inside the incubator. The gap between the lid and the base of the plate allows spores to enter, especially with glass plates, which have larger gaps. These spores land on the agar surface, leading to contamination. The contamination is usually found around the edges of the plate. One solution is to use disposable plastic plates, as they tend to have smaller gaps, reducing the chances of contamination.

Solution: Regular sterilization of the incubator is crucial. Disinfect the incubator with 75% alcohol and New Germinal disinfectant. After disinfection, leave the incubator unused for a few days before starting any new cultures. Regular cleaning is recommended, ideally twice a week, as mold spores are notoriously difficult to eliminate.

2. Condensation in Plates

Condensation is a common issue during upright incubation. If water droplets fall from the lid onto the agar surface, they can cause colonies to spread and plates to become contaminated.

Solution: Ensure the plates are dry before incubation. Plates can be dried in a 37°C environment to eliminate condensation before use, which helps prevent this type of contamination.

3. Contaminated Work Environment and Culture Media

An insufficiently sterile environment in the lab or contaminated culture media can also lead to mold contamination. Even after using UV light for sterilization, spores might still be present, especially after performing mold-related tests.

Solution: Extend the sterilization time after mold testing. To further improve sterility, consider installing an ozone generator in the lab, which, when used alongside UV light, provides an effective double-layer of disinfection.

How to Identify Contamination Source: To identify the source of contamination, a simple experiment can be done: Take two groups of empty plates (Group A sealed with sealing film, and Group B unsealed) and incubate them upright in the incubator. If both groups show mold growth, the contamination likely comes from the media or the lab environment. If only Group B shows contamination, the source is the incubator environment. Based on this, you can take appropriate corrective measures.