What are Sterile Techniques?

In any laboratory setting, maintaining a sterile environment is essential for the success of experiments. Especially when you are working with cultures, tissues, or microbial samples. The primary goal of sterilization is to get rid of all microorganisms from surfaces and materials. This helps with reducing the risk of contamination and ensuring accurate results. 

Principles of Sterilization

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Sterilization vs. Disinfection

Sterilization is the complete removal of microorganisms from surface, material, or environment. Disinfection reduces harmful organisms, however, the method does not guarantee complete removal of microorganisms.

The distinction between sterilization and disinfection is important when working with pure cultures, tissues, or cells. These require clean and uncontaminated environment for accurate and reliable results. Even a small microbial presence, which disinfection might allow, can lead to contamination of cultures or false experimental outcomes. Understanding and applying the right method is an important part of laboratory practice.

Common Sterilization Methods

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Wet heat sterilization (Autoclaving)

This is one of the most widely used methods in laboratories. It uses pressurized steam to kill microbes through hydrolysis and coagulation of cellular proteins. The method ensures the destruction of all microorganisms, including spores and viruses. Wet heat sterilization is an ideal method to use when a researcher is sterilizing laboratory equipment, preparing culture media or decontaminating biohazardous waste.

Autoclaving works by using pressurized steam to sterilize materials, achieving high temperatures 121°C for 15 minutes. The process involves placing items in a sealed chamber where steam is introduced, increasing the pressure to 15 psi (or higher) to allow water to boil at a higher temperature. The combination of heat, moisture, and pressure disrupts microbial structures, ensuring complete sterilization after a specific duration. Stem flow in autoclave ensures uniform sterilization by penetrating all surfaces of the item being sterilized and involves three main steps:

1. Steam introduction: Pressurized steam produced by a heating system enters the autoclave, typically at the top of side of the chamber, depending on the design.
2. Air displacement: The steam displaces air from the chamber through the drain valve. This creates a steam-saturated environment.
3. Steam saturation: Once the air is pushed out, the steam maintains consistent pressure and temperature essential for the sterilization.

Dry heat sterilization

Sterilization by dry heat is another effective method. This process does not involve water, making it suitable for metallic instruments or other materials prone to corrosion. Common conditions include 160°C for 2 hours or 180°C for 30 minutes in a hot air oven. The high temperatures help sterilize the materials by oxidation of cellular components in microorganisms. Dry heat sterilization involves circulating hot air in an insulated chamber to ensure uniform heating of the materials. 

Filtration

Sterilization by filtration removes microorganisms from liquids or gases by passing them through a filter. Solutions are passed through filters with pore sizes too small for microbes, usually 0.2 micrometres. Filtration is ideal for sterilizing heat-sensitive solutions, such as culture media, enzymes and antibiotics. A vacuum pump or pressure system forces the liquid through the filter, ensuring efficient separation while maintaining the sterility of the filtrate. The sterile filtered solution is collected in a pre-sterilized container, ready for use in experiments or medical applications. 

Chemical solvents

Chemical solvents such as ethanol and isopropanol are frequently used for sterilizing surfaces and equipment. These solvents denature proteins. They are most effective when diluted to concentrations between 60% and 90%. While they are effective against microbial cells, they do not eliminate spores.

Radiation-based sterilization

This technique uses electromagnetic radiation, such as UV light, gamma rays, or X-rays, to damage microbial DNA. UV radiation is useful for sterilizing specific areas like laminar flow hoods. Gamma rays, electron beams, or X-rays are commonly used, with gamma rays being particularly effective because of their deep penetration into materials. This method is often used in sterilizing medical equipment, pharmaceuticals, and disposable materials. The items or surfaces are exposed to a controlled dose of radiation in a sterilization chamber, ensuring consistent microbial inactivation without leaving harmful residues. This type of sterilization is very efficient. 

Gas sterilization

Gas sterilization employs ethylene oxide. Researchers often use gas sterilization for heat- and moisture-sensitive equipment. This method works by alkylating microbial DNA to prevent cell metabolism and replication. Due to its toxic nature, aeration must be performed post-sterilization to remove remaining ethylene oxide.

Precautions in Sterilization

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Before beginning any sterilization procedure, prepare the workspace thoroughly. For example, in laminar flow hoods, the working surface should be sterilized with 70% ethanol and exposed to UV radiation for at least 30 minutes. Cultures must also be sterilized before disposal to prevent microbial growth. Microscopic monitoring can help detect unwanted microbes that may not be visible to the naked eye.

Summary

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Sterilization is a crucial technique for good laboratory work. Whether using wet heat, dry heat, filtration, radiation, or chemical methods, understanding the principles and applications of each is crucial. From preventing contamination to achieving reliable experimental outcomes, sterilization practices help with safe and effective laboratory operations. By following these methods, you can maintain a clean, contamination-free workspace and keep your cultures happy and healthy.

Learn More

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References

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1. K. Tennant & C. L. Rivers Sterile technique National Library of Medicine, 2022