anti microbial substances neutralised prior to sterility testing

 Before conducting sterility testing, antimicrobial substances must be neutralized to prevent them from interfering with the growth of any viable microorganisms in the test sample. There are several approaches to neutralizing different antimicrobial substances, depending on their chemical properties and the formulation of the product. Here’s a breakdown of the methods used for different classes of antimicrobial agents:

1. Physical Neutralization

• Dilution: Reducing the concentration of the antimicrobial agent by diluting the sample with a neutralizing broth or diluent can lower its activity to non-inhibitory levels.
• Membrane Filtration: Involves filtering the product through a 0.45 µm or 0.22 µm filter to physically remove the antimicrobial agent. The filter is then rinsed to remove any residual antimicrobial before culturing.
• Centrifugation: For some suspensions, centrifugation can separate the microorganisms from the antimicrobial substance.

2. Chemical Neutralization

Specific neutralizing agents are used to inactivate different classes of antimicrobials. Here are some examples:

• Quaternary Ammonium Compounds (e.g., Benzalkonium Chloride):

  • Neutralizing agents: Lecithin, Polysorbate 80 (Tween 80), or a combination of both is often used to neutralize quaternary ammonium compounds.

• Aldehydes (e.g., Formaldehyde, Glutaraldehyde):

  • Neutralizing agents: Sodium bisulfite or glycine can inactivate aldehydes.

• Alcohols (e.g., Ethanol, Isopropyl Alcohol):

  • Neutralizing agents: Dilution is often sufficient, but sometimes inactivation can occur by dilution in culture media.

• Peroxides (e.g., Hydrogen Peroxide):

  • Neutralizing agents: Catalase enzyme or sodium thiosulfate can degrade hydrogen peroxide.

• Phenolic Compounds:

  • Neutralizing agents: Polysorbate 80 and lecithin are commonly used to neutralize phenols.

• Chlorine and Halogens (e.g., Hypochlorite, Iodine):

  • Neutralizing agents: Sodium thiosulfate is commonly used to neutralize chlorine and iodine-based disinfectants.

3. Enzymatic Neutralization

• For products that contain antimicrobial proteins or enzymes, specific enzymes may be used to degrade or neutralize the active agents. For example, lysozyme can be inactivated with proteolytic enzymes.

4. Neutralization Broth

• Specialized media such as Tryptic Soy Broth (TSB) with added neutralizers like lecithin, polysorbate, thioglycollate, and cysteine can be used to neutralize various antimicrobials during sterility testing.

5. Adsorption Techniques

• Activated charcoal or other adsorbent substances may be used to trap and neutralize antimicrobial agents. This method is particularly useful for certain types of disinfectants and antibiotics.

Considerations for Neutralization:

• The neutralizer must be validated for effectiveness without causing toxicity to any microorganisms present.
• Excess neutralizers should not inhibit microbial growth.
• If the antimicrobial agent cannot be neutralized or removed completely, alternative sterility test methods like dilution or membrane filtration are employed.

Neutralization is critical to ensuring that any viable microorganisms in a sample can grow during the sterility test, without being inhibited by residual antimicrobial substances.