Sterilization Techniques and Contamination Control

Sterilization Techniques

• Sterilization is the complete elimination of all viable organisms using heat, radiation, chemicals, or physical removal.

Fermentation Contamination

• Contamination by foreign microbes in fermentation results in reduced productivity, possible contamination of final desired bioproduct , and degradation of the desired product.

• Recovery procedures may also be hindered, and extraction of the desired bioproduct can become difficult.

Avoiding Contamination

• Contamination can be prevented by using pure inoculums, sterilizing all materials in contact with the medium and fermenter, and maintaining aseptic conditions throughout the process.

Sterilization Techniques Used in Experiments

• Common sterilization techniques are:
  • Moist heat (steam): Autoclaving at 121°C for 15-20 minutes is a common method for sterilizing liquids and other heat-resistant materials.
  • Dry heat: Heating at 160°C for 2 hours (or 170°C for 1 hour) is used for glassware, metals and other heat-resistant materials.
  • Chemicals:** Various chemicals are used for sterilization, such as formaldehyde, phenolics (Lysol), and hypochlorite (bleach).
  • Radiation: UV and gamma radiation are used to sterilize surfaces and materials. X-rays and electron beams are also employed.
  • Filter sterilization: Used when heat is not suitable because it does not destroy the product, involves filtering out microorganisms/contaminants using filters.

Moist Heat Sterilization (Steam)

• Autoclaving is the most reliable and economical approach for liquid sterilization

• Autoclaving generally uses steam at a high temperature (121°C) for a specified duration (15-20 minutes) to eliminate all living cells.

• Moist heat sterilization, in particular autoclaving, is beneficial for sterilizing liquids because it's easy, dependable, and cost-efficient.

• Drawbacks: Heat-sensitive components like vitamins, amino acids, and growth factors (e.g., EGF, NGF) might be damaged by this process.

Dry Heat Sterilization

• A method for sterilization that employs elevated temperatures without water; it's useful for materials sensitive to moisture
• A common application of this method is the sterilization of glassware and other lab equipment.

Chemical Sterilization

• Disinfectants like formaldehyde, phenolics (Lysol), hypochlorite (bleach), mercuric chloride, and ethanol are widely used in laboratory settings to sterilize equipment and surfaces.

Radiation Sterilization

• UV, X-rays, gamma radiation and electron beams are used for sterilizing certain lab equipment and materials.

Sterilization Using Filtration

• Filtration methods can be used to remove microbes from heat-sensitive liquids/solutions by passing the material through a filtration apparatus with a filter membrane with varying pore sizes.

• Using filters to separate microorganisms and suspended particles from liquids

• Two types are commonly used:

  • Depth filtration: Used as pre-filters to remove large particles.
  • Membrane filtration: used for heat-sensitive products, microorganisms are trapped in the membrane.

Membrane Filters

• Types of membrane filters include depth and membrane/surface filters
• Membrane filters with small pore sizes remove microorganisms more effectively.

Considerations in Filter Sterilization

• Specific conditions for filtration:* Filtration is best suited for heat-sensitive biological materials; pore size considerations are needed for efficiency.

Additional Filter Sterilization Techniques

• Different filter types are used, each with different pore sizes for separating various particles.

• The throughput (amount passing through) increases if the same pore size filters are used.

• Different pore sizes allow for separation of different microorganisms.

Types of Membrane Filtration

• Different types of membrane filtrations (MF) exist, each with a specific purpose: -Microfiltration (MF): For removing larger particles. -Ultrafiltration (UF): Separates particles based on molecular weight. -Nanofiltration (NF): Used to separate molecules with specific size, and charge. -Reverse Osmosis (RO): Used to remove small molecules.

Microfiltration

• Used when there is a need to remove particles of size 0.1-10µm
• Microfiltration is the process of separating smaller microbes from heat-sensitive fermentation media.

Ultrafiltration

• Smaller pore sizes to separate components based on molecular weights are used in ultra-filtration
• It's used for fractionating components, and its success is based on correct pore size specifications.

Nanofiltration

• It is a sterilisation method where components are separated based on pore sizes ranging from 0.1 to 10 nanometers.
• It works best with materials that are heat-sensitive.

Reverse Osmosis

• Reverse osmosis is a method of filtering water to remove dissolved salts and other impurities.

• In this process, hydrostatic pressure is used to push water through a membrane that selectively allows water molecules to pass while retaining dissolved solids.

This quiz covers sterilization methods and the control of contamination in fermentation processes. It highlights the importance of eliminating viable organisms and the procedures to maintain aseptic conditions. Understand the techniques and their applications in laboratory experiments.