Unseen microbial aggression puts thousands of lives at risk every year. In microbiology, sterilisation is the first line of defence to avoid contamination in cultures, surgical instruments, and pharmaceutical products. This fact becomes even more worrying when considering that hospital-acquired infections (HAIs) affect 10-20% of admitted patients in India, as per a study in 2023. 

The knowledge of proper sterilisation techniques is essential for researchers and medical practitioners to maintain an aseptic environment, decrease HAIs, and ensure safety for patients and healthcare personnel. 

This guide explains the microbiology of sterilisation, emphasising its critical role in infection prevention and patient safety.

What is Sterilisation: An Overview

Sterilisation is the complete process of killing or removing every type of microorganism, including bacteria, viruses, etc. In medical, pharmaceutical, and laboratory settings, this is a critical process to avoid contamination and infections. 

Physical sterilisation and chemical sterilisation are some of the few methods of sterilisation used depending on the type of material that needs to be sterilised.

Physical Methods of Sterilisation

There are various physical methods of sterilisation, like heat, filtration, radiation, and inspissation. Here is a detailed overview:

1. Heat Sterilisation

One of the best physical procedures for sterilisation is heat. It acts by denaturing microbial proteins and enzymes and ultimately killing the cell. There are two types of heat sterilisation: dry heat sterilisation and moist heat sterilisation.

Dry Heat Sterilisation: 

• In this type, materials are subjected to high temperatures using instruments like hot air ovens. 
• It is used for the sterilisation of glassware, metal instruments, and powders. 
• The sterilisation technique usually differs based on temperature, like 146°C for 2 hours or 180°C for 30 minutes. 
• The benefits of this technique include the lack of moisture-related corrosion and material penetration.

Autoclave: 

• In this process, steam is used under pressure in an autoclave. 
• The steam seeps into materials and kills microorganisms and spores. 
• It is commonly used in clinical and microbiological laboratories, where it is crucial for sterilising surgical equipment, culture media, and infected waste. 
• Due to higher heat transfer, moist heat sterilisation is a more efficient and effective method than dry heat sterilisation in a shorter amount of time.

2. Filtration Sterilisation

Filtration is a physical method of sterilisation used to remove microbes from liquids and air. Membrane filters with specific pore sizes can trap bacteria and viruses, making this method ideal for sterilising heat-sensitive solutions, such as vaccines and antibiotics. 

HEPA (High-Efficiency Particulate Air) filters are commonly used in laboratory environments to maintain sterile airflow. The primary benefit of filtration sterilisation is that it does not expose sensitive substances to high temperatures, preserving their integrity.

3. Radiation Sterilisation

Another efficient method of physical sterilisation is radiation. It includes:

• Ultraviolet (UV) Radiation: This is a common means of disinfecting surfaces and air in laboratory hoods and hospital rooms. UV radiation is effective because it causes damage to microbial DNA and stops it from being able to replicate. Its penetration is limited but useful for surface sterilisation. 
• Gamma Radiation: This ionising radiation is applied to sterilise medical equipment, food, and pharmaceuticals by dismantling the DNA of microorganisms, thus making them inactive. 

This approach has substantial applications when sterilising large numbers of unsterilised cells in industrial environments. The penetration power of gamma radiation is high and thus efficient even in the case of dense materials.

4. Inspissation Sterilisation

This is a specific heat-based sterilisation technique, particularly used for media rich in proteins (subject for sterilisation) like the medium for tuberculosis (Lowenstein-Jensen medium). It is a repeated treatment with moist heat at 75–80°C that eradicates non-spore-forming bacteria. 

It is one of the most essential techniques of microbiology laboratories for the preparation of some specific culture media.

Chemical Methods of Sterilisation

Chemical sterilisation methods include gas and liquid sterilisation and chemical fumigation. Find a detailed overview below:

1. Gas Sterilisation

Gas sterilisation is a chemical method of sterilisation using gaseous agents to destroy microorganisms. Common gases used include:

• Ethylene Oxide (EtO): A common gas used to sterilise heat-sensitive medical equipment, including catheters, syringes, and surgical instruments. It is a broad-spectrum germicide, effective against all microorganisms, including spores, but must be aerated to remove toxic residues. 

Ethylene oxide sterilisation is widely used in the world of medical device contract manufacturing.

• Formaldehyde Gas: It is another disinfectant effective on a wide variety of microorganisms and is used in hospitals and laboratories to sterilise both surfaces and instruments. 

Often, it is used at low-temperature steam to achieve deep penetration. Good ventilation is required to minimise toxic exposure.

2. Liquid Chemical Sterilisation

Liquid chemical agents are common in chemical methods of sterilisation. These include:

Hydrogen Peroxide: 

• Used as a vapour for sterilising closed spaces and in liquid form for antiseptic treatment of surgical materials. 
• Hydrogen peroxide vapour helps in decontaminating indoor spaces, especially in hospitals. 
• It decomposes into water and oxygen and is environment-friendly.

Glutaraldehyde:

• Glutaraldehyde is a high-level disinfectant used to sterilise medical and dental equipment. 
• It is homologous to bacteria, viruses, fungi, and spores. However, it has a long exposure time. 
• Thus, it needs to be handled and ventilated properly because of its toxicity.

Hypochlorous Acid:

• Hypochlorous acid is a natural disinfectant produced by the immune system. 
• It is very effective against a broad range of pathogens and is also a potent oxidising agent, biodegradable, and environment-friendly. 
• This disinfectant is commonly used for the sterilisation of endoscopes.

3. Chemical Fumigation

It is another method of chemical sterilisation used in microbiology and hospital settings. Decontaminating entire rooms or biological safety cabinets usually involves the use of formaldehyde and hydrogen peroxide vapours as biocidal agents. 

This technique is used to ensure full disinfection of enclosed spaces.

Techniques and Controls for Sterilisation in Microbiology

There are several sterilisation techniques in microbiology that ensure aseptic conditions in laboratories:

1. Flaming and Incineration: These are commonly used to sterilise inoculating loops, needles, and other small metal instruments in microbiology. Similarly, microbial contaminants are immediately incinerated by oxidation when they come into direct contact with the flame of a Bunsen burner. 

This process destroys pathogens and is effective in disposing of biohazardous waste that needs to be addressed by incineration.

2. Autoclaving: This is considered the gold standard in sterilisation, which uses pressurised steam at elevated temperatures (usually 121°C at 15 psi for 15-20 minutes) to kill all microbes, including heat-resistant spores.

It finds extensive use in the sterilisation of culture media, surgical instruments, and biohazardous waste to maintain aseptic conditions in microbiology and medical environments.

3. Chemical Fumigation: Gaseous disinfectants like formaldehyde, hydrogen peroxide vapour, or chlorine dioxide are used to sterilise entire workplaces. This is especially useful when working in biosafety labs and cleanrooms where surface and airborne contamination need to be reduced. 

Such practices serve to maintain aseptic conditions and minimise the chance of migrating microbes.

Sterilisation Controls and Validation

Control measures must be implemented to confirm the efficacy of sterilisation methods in microbiology. These include:

• Biological Indicators: Biological indicators involve the use of highly resistant bacterial spores, such as Geobacillus stearothermophilus, to assess sterilisation effectiveness. If these spores survive after the sterilisation process, it indicates a failure, necessitating a review and corrective actions to improve sterilisation procedures.
• Chemical Indicators: Chemical indicators, including heat-sensitive tapes, strips, and special inks, visibly change colour when exposed to specific sterilisation conditions. 

They provide an immediate, non-biological confirmation that sterilisation parameters, such as temperature and time, were met, ensuring a preliminary assessment of sterilisation success.

• Mechanical Indicators: Mechanical indicators involve monitoring key sterilisation parameters such as pressure, temperature, and exposure time, particularly in autoclaves. 

These indicators ensure that proper sterilisation conditions are consistently maintained, reducing errors and helping validate the overall reliability of the sterilisation process in microbiological settings.

FAQs About Sterilisation

1. What is the difference between sterilisation and disinfection?

Sterilisation destroys all forms of microbial life, spores included, while disinfection lowers microbial loads without destroying spores.

2. Which sterilisation method is best for heat-sensitive materials?

For heat-sensitive materials, the most appropriate methods are filtration and gas sterilisation (i.e., using ethylene oxide).

3. How does autoclaving work in sterilisation?

Autoclaving utilises pressurised steam at temperatures above 121 °C for 15–20 minutes to sterilise. This method kills all existing microorganisms and spores.

4. What is the role of inspissation sterilisation in microbiology?

It is mainly used to sterilise culture media by incremental heating at low temperatures for several days.

5. Are chemical methods of sterilisation effective against spores?

Yes, some chemical disinfectants (e.g., peracetic acid and glutaraldehyde) exhibit sporicidal activity.

Conclusion

Sterilisation plays a vital role in microbiology, medicine, and a wide range of industries by promoting safety and preventing contamination. Thorough knowledge of sterilisation microbiology is critical for individuals in clinical, research, and industrial settings. It is also essential for medical students who are preparing for exams like MBBS and NEET PG. 

For them, DocTutorials offers a wide range of topics covering medicine, disease, maternity, and more. They also provide top-quality video lectures, mock tests, question banks, mentorship, and a quick revision program that can help boost exam preparation. Join our NEET PG courses today and take a step closer to your dream medical career!