Sterilization – Definition, Chemical and Physical Methods, Types, Advantages

Sterilization: What is sterilization? Methods of Sterilization. 

Definition of Sterilization:

  • Sterilization is the process of removal of unwanted microorganisms like bacteria, fungi, etc. from the fermentation medium.
  • Sterilization is a process by which all living microorganisms, including viable spores, are either remove or kill from an object, body surface or medium.

  • Sterilization is the process by which all forms of microbial life from surface,medium and instruments are remove or kill by various physical and chemical methods.

Sterilization - Definition, Chemical and Physical Methods, Types, Advantages

What is Sterilization?

  • Sterilization is the process of removal of unwanted organisms like bacteria, fungi, etc. from the fermentation medium.
  • It includes both pathogenic and non-pathogenic microorganisms, as well as bacterial spores and fungal spores.
  • Sterilization is a measure that uses strong physical and chemical factors to make all microorganisms inside and outside any object lose their ability to grow and reproduce forever. 
  • Sterilization is a necessary condition to obtain pure culture, and it is also a necessary technology in the field of food industry and medicine. 
  • Different methods can be used according to different needs, such as medium sterilization generally using moist heat sterilization, and air filter sterilization.
  • Commonly used methods of sterilization include chemical reagent sterilization, radiation sterilization, dry heat sterilization, moist heat sterilization, and filter sterilization.
  • The contamination can be avoided by sterilizing the fermenter, using pure inoculum to start the fermentation, sterilizing the medium, sterilizing all the components of the fermenter, and maintaining the aseptic conditions.

Factors affecting sterilization
There are different types of factors that can affect the sterilization process. Few are given  below : 

1. Different microorganisms or different ages of the same microorganism have different sensitivity to high temperature. The vegetative bodies and viruses of most microorganisms will be killed in 10 minutes at 50~65°C; however, various spores are resistant to heat such as Bacillus stearothermophilus. At 121°C, it took 15 minutes to kill. For the same microorganism, young bacteria are more sensitive to heat than old bacteria.

2. The number of microorganisms will obviously affect the effect of sterilization. The greater the number of microorganisms, the longer the heat time.

3. The composition of the medium will also affect the sterilization effect. Generally speaking, the presence of protein, sugar or fat will improve the heat resistance. When the pH is around 7, the heat resistance is strongest. If it is biased towards the poles, the heat resistance will decrease, and different salts may have different effects on sterilization. Solid medium takes longer to sterilize than liquid medium.

Methods of sterilization / Types of Sterilization :

1. Physical Methods of Sterilization

i) Heat / Temperature Sterilization 
Heat Sterilization is the process of removal of unwanted microorganisms like bacteria, fungi from the object with the help of heat.
Sterilization or disinfection by temperature is the most common, convenient and effective method. The two common  temperatures for sterilization are 121°C (250°F) and 132°C (270°F). High temperature can denature and inactivate proteins and enzymes in microbial cells, thereby playing a sterilizing effect, and low temperature usually plays a bacteriostatic effect. Heat sterilization are of two types Dry heat sterilization and Moist heat sterilization.

1) Dry heat sterilization method :
  • Dry heat sterilization is the most effective and widely used method of sterilization
  • Dry heat sterilization uses very high temperatures to kill microorganisms such as  bacterial spores, fungus, viruses etc.
  • Dry heat sterilization is used for the sterilization of materials which are difficult to sterilize by moist heat sterilization.
  • Hot air oven and Incineration are work on the principle of dry heat sterilization which generates very hot moisture-free air.
  • Dry heat sterilization is used on items that cannot get wet such as Paper, oils, powders, metal instruments, glassware and items wrapped in paper.
  • Dry heat sterilization are of different types such as burning sterilization method, dry hot air sterilization method.

(a) Burning sterilization method: use flame to directly burn microorganisms to death. This method is thorough and reliable, and sterilizes quickly, but it is easy to incinerate items, so the scope of use is limited, and it is only suitable for the sterilization of inoculation needles, loops, test tube ports and unusable contaminated items or the corpses of experimental animals.

(a) Dry hot air sterilization method: This is a commonly used method in the laboratory, that is, the items to be sterilized are evenly placed in an oven, heated to 160°C, and kept at a constant temperature for 1 hour. This method is suitable for sterilization of glass dishes, metal utensils, etc.

2) Moist heat sterilization method:
  • Moist heat sterilization is effective and efficient methods of sterilization where the steam remove microorganisms.
  • In Moist heat sterilization  steam temperatures ranges from 121°C to 134°C.
  • An autoclave is a device that works on the principle of moist heat sterilization.
  • The advantages of moist steam sterilization is easy to control and monitor, microbicidal it has Rapid cycle time.
  • At the same temperature, the effect of moist heat sterilization is better than that of dry heat sterilization, because on the one hand, the protein in the cell has a high water content and is easy to denature. 
  • On the other hand, high-temperature water vapor has a high penetrating power to proteins, thereby accelerating protein denaturation and rapid death.
  • Moist heat sterilization are different types : Pasteurization method, Boiling disinfection method, Intermittent sterilization method, pressure steam sterilization method, 

(a) Pasteurization method: Some foods will destroy or loose nutritional quality due to high temperature, such as milk, soy sauce, beer, etc. To maintain the nutrition and quality of the food only lower temperature can be used to kill the pathogenic microorganisms. The method is generally 62 ° C, 30 minutes to achieve the purpose of disinfection. This method was pioneered by the French microbiologist Pasteur, hence the name pasteurization.

(b) Boiling disinfection method: directly put the items to be disinfected into clean water and boil for 15 minutes to kill all microorganisms and some spores of bacteria. If you add 1% sodium carbonate or 2% carbolic acid to the water, the effect is better. This method is suitable for the disinfection of syringes, towels and anatomical utensils.

(c) Intermittent sterilization method: The above two methods can only play a role in disinfection under normal pressure, and it is difficult to achieve complete sterilization. If batch sterilization is used, all microorganisms in the article can be killed. The specific method is: heat the item to be sterilized to 100°C for 15-30 minutes to kill the nutrients in it. It was then cooled and placed in incubator overnight at 37°C to allow residual spores to germinate into vegetative bodies. Repeat the above steps on the second day for about three times to achieve the purpose of sterilization. This method does not require a pressure sterilizer and is suitable for promotion, but it is troublesome to operate and takes a long time.

(d) Pressure steam sterilization: This is one of the most commonly used sterilization methods in fermentation industry, healthcare, food testing and microbiology laboratories. It is suitable for the sterilization of various heat-resistant and bulky culture media, as well as for the sterilization of glassware, work clothes and other items. Pressure steam sterilization is carried out by placing the items to be sterilized in a sealable pressure steam sterilizer, and the pressure is raised with a large amount of steam. As the vapor pressure rises, the boiling point of water also rises. When the steam pressure reaches 1.055 kg/cm2, the temperature in the autoclave can reach 121°C. In this case, microorganisms (including spores) will be killed within 15 to 20 minutes to achieve the purpose of sterilization. If the object of sterilization is sand, paraffin oil and other items

ii) Filter Sterilization

  • Filter Sterilization is the process of removal of unwanted organisms like bacteria, fungi, etc. from the liquid medium or from air with the help of filters.
  • It is divided into liquid filtration and air filtration
  • Filter sterilization method that uses a membrane filter with a certain pore size to prevent the passage of microorganisms and remove microorganisms in solution or in the air.
  • This method is suitable for the sterilization of thermally unstable liquid medium (such as animal serum, protein, enzyme, vitamin, etc.) and gas in small volume. 
  • Commonly used bacteria filters include membrane filter (0.45μm and 0.22μm pore size), HEPA filters, depth filters, ceramic filter, asbestos filter (Seitz filter), sintered glass filter, etc.
  • Commonly used filters are made of Cellulose, nitrocellulose, asbestos, ceramic soil, Porcelain, Borosilicate, Diatomaceous earth etc.
  • Mainly used for sterilization of heat-labile biological products such as serum, toxins, antibiotics and air.
  • The biggest advantage of filter sterilization is that it does not destroy the chemical composition of various substances in the medium.

iii) irradiation

Light disinfection also known as radiation disinfection is mainly achieved through the bactericidal effect of ultraviolet rays, which causes photolysis and denaturation of bacterial proteins, resulting in bacterial death. Irradiation method of sterilization different types such as sun exposure method, ultraviolet lamp, disinfection method and Ionizing radiation sterilization method.

(a) Sun exposure method: use the heat, drying, and ultraviolet rays of the sun to sterilize, put mattresses, blankets, books, clothes, etc. in direct sunlight, and expose to the sun for 6 hours to achieve the disinfection effect, and turn it regularly in the middle.

(b) Ultraviolet lamp disinfection method: Ultraviolet rays belong to electromagnetic wave radiation, and are often used for disinfection of air and surfaces of objects. The wavelength range of the strongest sterilization is 250-270nm, and the timing starts from 5-7 minutes after the lamp is on. When the indoor temperature is 20-40 degrees, and the relative humidity is 40%-60%, ultraviolet disinfection is the most suitable.

① Air disinfection: The effective distance is no more than 2m, and the irradiation time is 20-30 minutes. 

② Item disinfection: The effective distance is no more than 25-60cm, and the irradiation time is 20-30 minutes.

(c) Ionizing radiation sterilization method: (also known as cold sterilization) is suitable for sterilization of heat-labile items, such as rubber, plastic, macromolecular polymers (disposable syringes, infusion sets, etc.), compact medical instruments, biomedical products, birth control Utensils and metals, etc.

2. Chemical Sterilization Methods 

  • Chemical Sterilization is the process of removal or killing of microorganisms by the use of chemical agents.
  • The killing mechanism of chemicals mainly depends on the type and quantity of microorganisms, the smoothness or surface of the object, and the properties of the chemical.
  • Verious types Chemicals Used for Sterilization or Disinfection such as Ethylene Oxide, Ozone, Bleach, Glutaraldehyde and Formaldehyde, Phthalaldehyde, Hydrogen Peroxide, Peracetic Acid, Silver.
  • The purpose of chemical sterilization is to reduce the number of microorganisms to control a certain sterile state.
  • Chemical sterilization used for sterilizing plastic containers, heat-sensitive solid drugs, drugs in paper or plastic packaging, rubber products, syringes, injection needles, clothing/dressings and instruments, etc.
  • The ideal chemical disinfectant should have the following conditions: wide bactericidal spectrum; low effective concentration; fast action speed; stable nature; easily soluble in water; can be used at low temperature; Influence; non-corrosive to articles; colorless, tasteless, odorless, easy to remove residual drugs after disinfection; low toxicity, not easy to burn and explode, non-hazardous to use; low price; easy to transport, can be supplied in large quantities.
  • Chemical sterilization can be divided into gas sterilization and liquid sterilization.

(a) Gas sterilization method: refers to the method of sterilization using chemical gaseous  (such as ozone, ethylene oxide, formaldehyde, propylene glycol, glycerol and peracetic acid steam, etc.). This method is suitable for the sterilization of items that are stable in gas succh as medical instruments, equipment and facilities that are not resistant to heat sterilization. It can also be used for powder injections, but it is not suitable for occasions where product quality is compromised. When using the gas sterilization method, attention should be paid to the flammable and explosive properties, teratogenicity and residual toxicity of the sterilization gas. The most commonly used gas in this method is ethylene oxide, which is generally mixed with 80% to 90% inert gas. This method can be used to sterilize medical devices, plastic products and other items that cannot be sterilized by high temperature. 

(b) Liquid sterilization method: a method of disinfection by using liquid chemicals (such as 75% ethanol, 1% povidone-iodine solution, 0.1% to 0.2% benzalkonium bromide, about 2% phenol or cresol soap solution, etc.). This method is often used as an auxiliary measure for other sterilization methods and is suitable for the disinfection of skin, sterile instruments and equipment. Common liquid chemical disinfection and sterilization methods are Wiping, Immersion. Wipe the surface of contaminated objects with chemical liquid, which is often used for disinfection of floors, furniture and display items. Immerse the items to be disinfected in the disinfectant, which is often used for household utensils that cannot or are inconvenient to sterilize by physical methods.

Advantages of sterilization:

  • With the help of sterilization we can kill all microorganisms, including spores, Mycobacterium tuberculosis, bacteria, viruses, fungi, etc. from the object.
  • With the help of sterilization methods we can utilize a broad range of products and equipment including different devices, media, medical instruments and products.
  • The contamination can be avoided by sterilizing the object, fermenter,  medium,  equipment etc. 
  • Sterilization is necessary to maintaining the aseptic conditions.
  • Chemical sterilization used for sterilizing plastic containers, drugs, rubber products, syringes, injection needles etc.
  • Filtration mainly used for sterilization of heat-labile biological products such as serum, toxins, antibiotics and air.

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