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Sterilization Process

27 March 2012 - Microbiology - Javed Shaik - No Comments

Definition
Sterilization is the freeing of an article from all living organisms, including bacteria and their spores.

Sterilization of culture media, containers and instruments is essential in microbiological work for isolation and maintenance of microbes. In surgery and medicine, the sterilization of instruments, drugs and other supplies is important for the prevention of infection. Sterilization can be effected in a variety of ways, which can be conveniently categorized as follows:

I. PHYSICAL METHODS
A.Heat :
1. Dry heat
2. Moist heat
B.Radiations
1. Ultraviolet radiations
2. Ionizing radiations
C.Filtration
II. CHEMICAL METHODS

STERILIZATION BY HEAT

Heat can be applied in two forms.
1. The dry heat
2. Moist Heat

Mechanism of killing by dry heat

  • Dry heat kills the organisms by destructive oxidation of essential cell constituents
  • Killing of the most resistant spores by dry heat requires a temperature of about 160 °C for 60 minutes
  • Dry heat is employed for glassware; syringes, metal instruments and paper wrapped goods, which are not spoiled by high temperatures.
  • It is also used for anhydrous fats, oils and powders that are impermeable to moisture.

Mechanism of killing by moist heat

  • Moist heat kills the organisms by coagulating and denaturing their enzymes and structural protein.
  • Sterilization by moist heat of the most resistant spores generally requires 121 °C for 15-30 minutes.
  • Moist heat is used for the sterilization of culture media, and all other materials through which steam can penetrate
  • Moist heat is more effective than dry heat
  • Sterilization can be done at lower temperatures in a given time at a shorter duration at the same temperature.

 

FACTORS INFLUENZING STERILIZATION BY HEAT

1. The temperature and time: they are inversely related, shorter time is sufficient at high temperatures.
2. Number of microorganisms and spores: The number of survivors diminished exponentially with the duration of heating
3. Depends on the species, strains and spore forming ability of the microbes.
4. Thermal death point is the lowest temperature to give complete killing in aqueous suspension within 10 minutes
5. Depends on the nature of material: a high content of organic substances generally tends to protect spores and vegetative organisms against heat.
6. Presence of organic or inorganic disinfectants facilitates killing by heat
7. pH also plays an important role in the killing of microorganisms

METHODS OF STERILIZATION BY DRY HEAT

1. RED HEAT

Inoculating wires, points of forceps and searing spatulas are sterilized by holding them in the flame of Bunsen burner until they are seen to be red-hot.

2. FLAMING

This method is used for sterilizing scalpel, mouth of culture tubes, glass slides etc. It involves passing of an article through Bunsen flame with-out allowing it to become red-hot.

3. HOT AIR OVEN

This is the main means of sterilization by dry heat. Exposure at a temperature of 160 °C for 1 hour is generally employed.

4. INFRARED RADIATIONS

Source employed is an electrically heated element, the infra red rays are directed on to the object to be sterilized and temperature of 180 °C can be obtained.

METHODS OF STERILIZATION BY MOIST HEAT
Moist heat can be employed at
1. Temperature below 100 °C

2. Temperature of 100 °C

3. Temperature above 100 °C

MOIST HEAT BELOW 100 °C

EXAMPLES

1. Pasteurization of milk
In Pasteurization of milk the temperature employed is either 63 °C for 30 minutes or 72 °C for 20 seconds. All non spore forming pathogens in milk like Salmonellae, M.tuberculosis are killed.

MOIST HEAT ABOVE 100°C
1. Sterilization in an autoclave

Autoclaving is the most reliable method It is the method most widely used for sterilization of culture media and surgical supplies

  • Autoclaving is the most reliable method
  • It is the method most widely used for sterilization of culture media and surgical supplies
  • When water is boiled within a closed vessel at an increased pressure, the temperature at which it boils and the steam it forms will rise above 100 °C
  • This principle is used in the autoclave
  • Normally autoclaving is done at 15 lbs. (pounds per sq. inch pressure) and 115 °C for 15 minutes

STERILIZATION BY FILTRATION

When fluids are passed through bacteria stopping filters, they are made free from bacteria.

  • It is useful for making preparations of soluble products of bacterial growth such as toxins
  • Liquids that would be damaged by heat such as serum and antibiotic solutions can be sterilized by filtration
  • Efficient filters should be able to retain Serratia marcescens

TYPES OF FILTERS

There are different kinds of filters
1. Earthenware candles – called Berkfield & Chamberland filters
2. Asbestos and asbestos-paper discs filters – called Seitz filters
3. Sintered glass filters
4. Cellulose membrane filters
5. Fibre glass filters.

Berkfield Filters

  • Made from Kieselguhr, a fossil diatomaceous earth
  • Three grades of porosity are available:
    1. Veil – coarsest one
    2. N – normal one
    3. W- wenig the finest one

Chamberland Filter

  • Made from unglazed porcelain
  • Four grades are available
  • L1- clarifying filters
  • 1a-Big
  • L2 – normal
  • L3- Finest

Seitz filter

  • Made up of asbestos pads
  • Three grades are available
    • K- clarifying filters
    • Normal
    • Special EK bacteria stopping filters

Sintered glass filters

  • Made from sintered glass
  • Different grades available
    • Grades 1 to 5
    • Grades 1-2 are for clarifying purpose
    • Grades3-5 is for sterilization purpose

Membrane filters

  • Made up of nitro-cellulose membranes
  • Made with different grades of porosity by adjusting the concentration of constituent

MERITS AND DEMERITS OF HEAT STERILIZATION

Advantages of heat sterilization

1. Sterilization is very effective
2. Instruments are standardized to deliver the required effective heat
3. Heat deliver system can be monitored effectively with various controls like pressure gauge, temperature meters etc
4. Established quality control methods available

Disadvantages

1. Steam impermeable materials like fats, oils and powders can not be sterilized by autoclaving.

2. Heat sensitive materials can not be sterilized by heat

Examples:

  • Serum can not be sterilized
  • Antibiotics
  • Plastic materials
  • Vaccines
  • Rubbers

3. Presence of organic matters interfere with effective sterilization

4. Dangers of explosion when high pressure is used

 

 

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