Introduction to common methods of sterilization

Introduction to common methods of sterilization

     The concept of sterilization for making the materials free from any contamination was given by Louis Pasteur. Thus, sterilization is a process of making an article, surface or medium free from any type of microorganisms. Sterilization is one of the most important steps for culturing, isolation and study of microorganisms in laboratory.

There are two methods of sterilization:

1)   Physical method

2)   Chemical method

Physical Methods of Sterilization

There are many kinds of approaches done to sterilize the materials.

1)   Heat

Heat is the most reliable method of sterilization. Either dry heat or moist heat could be used.

i) Moist Heat

Culture media and water are sterilized using moist heat i.e., steam under pressure. It is done through autoclaving and also by using a pressure cooker. Moist heat kills by degrading nucleic acids and by denaturing enzymes and other essential proteins. It also may disrupt cell membranes. Steam liberates latent heat when it condenses on a surface and thereby raises the temperature of that surface. 

Autoclave

1)   Autoclave is a device like a pressure cooker used for steam sterilization. Water is boiled to produce steam, which is released into the autoclave’s chamber. The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed.

2)   Autoclaving is performed at 121°C and 15 lb per inch² pressure for a period of 20 minutes. At this temperature saturated steam destroys all vegetative cells and endospores. Treatment is continued for about 15- 20 minutes to ensure sterility.

For determining the efficiency of moist heat sterilization, spores of Bacillus stearothermophilus or Clostridium PA3679 is used as test organism.  The spores of this organism need an exposure of 12 minutes at 121^oC to be killed. 

ii) Dry heat

Dry heat kills microorganisms due to protein denaturation and oxidative damage. Flaming of inoculating loop or wire, forceps tip, spatula, the mouths of culture tubes or flasks etc. where they are held in Bunsen burner flame till red hot is dry heat sterilization. Incineration is also dry heat sterilization where contaminated cloth, animal carcasses etc., are burned to destroy microorganisms present. Another common equipment used in laboratories for dry heat sterilization is hot air oven. Glass wares, glass syringes, scalpels, pipettes, flasks, petridishes etc. are sterilized in an oven at 160^oC for 2 hours and 180^oC for 1 hour. 

Dry heat sterilization is slow. Care should be taken to remove the apparatus and instruments only after the temperature cools down, otherwise the glass wares may crack due to sudden or uneven cooling.  Dry heat takes more time to kill microorganisms than moist heat, but it has certain advantages such as it does not corrode glassware and metal instruments, and can be used to sterilize powders, oils, liquid paraffin, fats, grease, etc. It is not useful for heat sensitive materials such as plastic and rubber items.

Spores of nontoxigenic strain of Clostridium tetani are used a microbiological test of dry heat efficiency. 

2) Radiation

There are 2 general types of radiation used for sterilization, ionizing radiation and non-ionizing radiation. Ionizing radiation is of short wavelength, high-intensity and react with DNA and cause cell killing. Ionizing radiation including gamma rays, X-rays, and high-speed electron beams penetrates deep into objects, destroying bacterial endospores and vegetative cells, but not effective against viruses. Gamma radiation from a cobalt 60 source is used in the cold sterilization of antibiotics, hormones, sutures, and plastic disposable supplies such as syringes.

Non-ionizing radiation uses longer wavelength and lower energy. As a result, non-ionizing radiation loses the ability to penetrate substances, and can only be used for sterilizing surfaces. The most common form of non-ionizing radiation is ultraviolet light. Ultraviolet radiation is used to sterilize workspaces such as laminar airflow and tools used in microbiology laboratories and health care facilities. Expose the working area under ultraviolet radiation before half an hour or prior to start of work. The source of ultraviolet radiation is generally ultraviolet lamps or ultraviolet tubes enclosed in quartz, because glass will not transmit ultraviolet radiation. UV light at germicidal wavelengths (185 nm and 265 nm) causes adjacent thymine molecules on DNA to dimerize, thereby inhibiting DNA replication. It damages the cell by hydration also. Care should be taken not to view ultraviolet radiation with naked eyes.

3)   Filtration

Filtration is used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics, and other heat-sensitive solutions which cannot be autoclaved. The filters retain the bacteria thus removing them.

Millipore membrane filters are commonly used. They are porous circular membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, or other synthetic materials of pore size 0.22 µm. Millipore membrane filter is placed inside the filtration assembly and the entire unit sterilized by autoclaving before use. The solution is pulled or forced through the filter with a vacuum or with pressure from a syringe or peristaltic pump and collected in previously sterilized containers. Membrane filters remove microorganisms by screening them out much as a sieve separates large sand particles from small ones. They can’t filter viruses.

Chemical methods of sterilization

     There are several chemicals used for sterilization of glass wares, working tubes, hands etc., for microbiological work.

1)   Alcohol

Alcohols are bactericidal and fungicidal but not sporicidal; some lipid-containing viruses are also destroyed. Ethanol and isopropanol are usually used in about 70 to 80% concentration to sterilize working table tops, inoculation chamber etc. They act by denaturing proteins and by dissolving membrane lipids.

2) Aldehydes

Formaldehyde and glutaraldehyde are sporicidal and are used as chemical sterilants. They combine with nucleic acids and proteins and inactivate them, probably by crosslinking and alkylating molecules. Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of glutaraldehyde is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipment.

3) Inorganic chemicals

There are certain heavy metals which are toxic to any organisms such as mercury, silver, zinc, and copper were used as germicides. Silver chloride (1%) solution is used as disinfectant for seeds, explants or any other materials. Heavy metals combine with proteins, often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins. Sodium hypochlorite (NaOCl₂) (10% soultion) or Calcium hypochlorite (CaOCl₂) maybe similarly used.

Other methods

They include,

1)   Disinfection: Disinfection is the killing, inhibition, or removal of microorganisms that may cause disease. The goal is to destroy potential pathogens from objects but not the culture media. Disinfectants are agents, usually chemical, used to carry out disinfection and are normally used only on inanimate objects. Disinfection of table tops, equipments and other surfaces are usually done using formaldehyde, glycolic acid or carbonic acid. A disinfectant does not necessarily sterilize an object because viable spores and a few microorganisms may remain.

2)   Sanitization: Sanitization is closely related to disinfection. In sanitization, the microbial population is reduced to levels that are considered safe by public health standards. The inanimate object is usually cleaned as well as partially disinfected. For example, sanitizers are used to clean eating utensils in restaurants.