Understanding Low-Temperature Sterilization and the Role of Biological Indicators in Food Testing Microbiology Laboratories

Introduction

Sterilization is a fundamental process in food testing microbiology laboratories to ensure that media and materials remain free from viable microorganisms. The standard autoclaving temperature for sterilization is 121°C, typically maintained for 15 minutes at a pressure of 15 psi. However, certain heat-sensitive materials necessitate sterilization at lower temperatures, such as 115°C, to prevent degradation of essential nutrients in culture media. This process, known as Low-Temperature Sterilization, requires careful validation to ensure microbial elimination, often relying on biological indicators like Bacillus subtilis (ATCC 5230).

Importance of Low-Temperature Sterilization in Food Testing Laboratories

While sterilization at 121°C is effective for most materials, certain culture media used in food microbiology contain heat-sensitive ingredients that degrade at higher temperatures, leading to a loss of their nutritive properties. This can result in media that fail to support microbial growth or inhibit the development of characteristic colonies, which is critical for accurate food pathogen detection. Some commonly used media requiring sterilization at Low-Temperature include:

• Violet Red Bile Glucose Agar

• Rappaport Vassiliadis Medium

• Bismuth Sulphite Agar Medium

To maintain the efficacy of these media, they are sterilized at 115°C and 10 psi pressure or simple boiling  for an extended period. Ensuring proper sterilization is essential for reliable microbiological analysis of food samples, particularly in detecting foodborne pathogens such as Salmonella, E. coli, and Listeria monocytogenes.

Adjusting Time for Effective Sterilization

A decrease in sterilization temperature necessitates an increase in exposure time to achieve effective microbial elimination. The duration is calculated based on the D-value, which represents the time required to reduce the microbial population by one log cycle at a given temperature. For instance, if the D-value at 115°C is 3.75 minutes and an 8-log reduction is required, the sterilization cycle must run for:

D-value (3.75 minutes) x Log reduction (8) = 30 minutes

This ensures the effective killing of microorganisms, compensating for the reduced temperature. This step is especially crucial in food microbiology laboratories, where accurate detection of pathogens and spoilage organisms depends on the sterility and effectiveness of the culture media.

Validation of Low-Temperature Sterilization

All sterilization processes must be validated to confirm their effectiveness. The validation of low-temperature sterilization is performed using biological indicators (BIs), which contain spores of heat-resistant bacteria such as Bacillus subtilis (Bacillus atrophaeus) ATCC 5230. The validation process involves:

      Using Spore Strips

• Each strip contains Bacillus subtilis spores at a concentration of 1x10^6.

• The strips are placed within the sterilization load and subjected to the 115°C sterilization cycle.

• After sterilization, the strips are incubated in Soybean Casein Digest Medium at 35°C for seven days.

• Absence of turbidity in the medium indicates proper sterilization, while turbidity signals sterilization failure.

  Using Spore Ampoules

• Ampoules containing Bacillus subtilis spores are subjected to the sterilization cycle.

• After sterilization, the media inside the ampoules is released for incubation at 35°C for seven days.

• A color change in the ampoule indicates microbial growth, signifying inadequate sterilization.

Interpretation of Results

• No Turbidity/No Color Change: Proper sterilization achieved, and media is safe for use.

• Turbidity/Color Change Observed: Sterilization was ineffective, indicating a need for process optimization.

Role of Biological Indicators in Food Testing

Biological indicators are essential in food microbiology laboratories to verify the effectiveness of sterilization processes. The presence of viable microorganisms in sterilized culture media can lead to false-positive or false-negative test results, impacting food safety assessments. Using biological indicators helps ensure that:

• Culture media are free from contaminants before testing food samples.

• The sterilization cycle effectively eliminates pathogens.

• Microbiological results are accurate and reliable.

• Laboratory compliance with food safety regulations (e.g., FSSAI) is maintained.