Ensuring the Presence of Anaerobic Bacteria in Cleanrooms

Monitoring anaerobic bacteria in cleanrooms is crucial as they can compromise product sterility, especially in pharmaceutical manufacturing. Since anaerobes require oxygen-deprived environments to grow, specific methods and tools are used for their detection.

1. Sampling for Anaerobes in Cleanrooms

a) Air Sampling

Use anaerobic-compatible settle plates (pre-reduced anaerobic agar) in areas prone to contamination.

b) Surface Sampling

Swab samples are collected using sterile swabs, which are immediately placed in anaerobic transport media to preserve viability.

RODAC plates with anaerobic-specific agar can also be used.

c) Personnel Monitoring

Collect samples from gloves or garments using anaerobic media to check contamination sources from operators.

2. Incubation for Identifying Anaerobic Bacteria

Anaerobes require a specific incubation environment:

a) Anaerobic Incubators

Create an oxygen-free atmosphere by purging oxygen and introducing gases like nitrogen (N₂), hydrogen (H₂), and carbon dioxide (CO₂).

Maintain temperature typically at 35-37°C depending on the organism.

b) Anaerobic Chambers/Glove Boxes

Fully sealed units where culture plates are handled and incubated under oxygen-free conditions.

c) Anaerobic Gas Pack Systems

These are widely used for small-scale anaerobic incubation.

3. How Anaerobic Gas Packs Work

Anaerobic gas packs create an oxygen-free environment in sealed containers or jars.

a) Components of an Anaerobic Gas Pack

Chemical Composition:

1. Sodium borohydride or sodium bicarbonate.

2.Citric acid.

3.Activated carbon.

4.A palladium catalyst for oxygen absorption.

Indicator Strips: Methylene blue or resazurin is used to confirm anaerobic conditions. These strips change color when oxygen is absent (blue to colorless for methylene blue).

b) Working Principle

1. Activation:

The gas pack is placed in an anaerobic jar along with culture plates.

Upon activation, the pack releases hydrogen and carbon dioxide gases.

2. Oxygen Removal:

The hydrogen reacts with oxygen in the presence of the palladium catalyst, forming water and depleting oxygen.

Carbon dioxide maintains a stable anaerobic atmosphere.

3. Sealing:

The jar is sealed to prevent external air from entering.

4. Media for Cultivating Anaerobes

Pre-reduced Anaerobic Agar: Media that is prepared and stored in oxygen-free conditions.

Thioglycollate Broth: Contains reducing agents like thioglycollate to support anaerobic growth.

5. Challenges in Anaerobe Monitoring

Transport: Samples must reach the laboratory quickly to prevent oxygen exposure.

Detection: Anaerobes grow slower than aerobes, requiring prolonged incubation (up to 7 days for some species).

Identification: Automated systems like Vitek 2 Compact or molecular methods like PCR are useful for accurate identification.