Sterilisation by Physical method

Sterilization by physical methods

Sterilization is the process of destroying or removing all forms of microbial life from an object or the specimen . Thus a sterile item is one free of all living microbes. Antimicrobial agents are the substances that are used to kill the microbes or prevent their growth . An antimicrobial agent can be antibacterial,antiviral,anti fungal and anti protozone. Depending upon the kind of microbes affected, antimicrobial agents used for sterilization may be either done by chemical agents or by physical agents.

By Physically agents

Physical method which are used for sterilisation are high temperature, low temperature ,radiation ,filtration and desiccation

• High temperature

The use of high temperature is one of the most effective and widely used means of killing the microbes. Heat maybe apply in anyone of the following ways.

1. Moist heat

Moist heat is much more effective than dry heat. This is because moist heat causes denaturation and coagulation of vital proteins such as enzymes whereas dry heat causes oxidation of the organic constituents of the cell i.e causes them to grow slowly .Denaturation of cell protein occurs with lower temperature and shorter exposure times than those required for oxidation

Advantages of moist heat

• moist heat kills bacterial endospores into 2-15 minutes at 100 degree Celsius.

• Bacterial vegetative cells in 5 to 10 minutes at 60 to 70 degree Celsius

• Vegetative cells of yeast and fungi in 5 to 10 minutes at 50 to 60 degree Celsius

• Fungus spores in 5 to 10 minutes at 72 to 80 degree Celsius

• For protozones and viruses is same as most of vegetative cells.

Moist heat can be in the form of steam ,boiling water, water heated to sub boiling temperature

• Steam

Steam under pressure is very effective because it provides temperature higher than those possible with nonpressurised steam or boiling water. It also causes rapid heating and greater penetration .

Example: Autoclave

The lab operators design to sterilised with pressurised steam is known as autoclave. It developed in 19th century. Autoclave is an essential unit of equipment in every microbiology lab. It is used for sterilization of culture media, solutions, discarded cultures and contaminated materials

Structure of autoclave

Autoclave is an apparatus used in the microbiological labs as well as in the hospitals to sterilise the things. It resembles with home pressure cooker .The temperature and pressure inside the autoclave can be controlled which is not possible in pressure cooker.

It has double jacketed chamber which gets steam supply from below. The things to be sterilised ,example media solutions etc can be kept on the plate provided at the bottom of the chamber and for releasing extra , a safety lock is present on a chamber. Pressure gauge is to show the pressure inside the chamber and a vant to release the extra steam is present too.

Working of Autoclave

The double jacketed Chamber of the autoclave is flushed with free flowing steam. It is then filled with the pure steam and maintain at a particular temperature and pressure . All the air initially present in the chamber is released by opening the knob because if air is present it will reduce the temperature . An autoclave is usually operated at a pressure of 15 lbs per inch square , at which the temperature of pure steam is 121 degrees Celsius. The length of time needed to sterilise at this temperature depends on the material to be sterilised. Generally keeping for 15 -20minutes at this temperature and pressure is sufficient.

• Boiling water

Boiling water has a temperature of 100 degrees Celsius. When water is boiled , the vegetative microbes are killed. However endo spores of bacteria will survive therefore contaminated materials or objects exposed to boiling water for several minutes cannot be sterilised with certainly.

• Sub boiling temperature

Sterilization have adverse affect on many foods hence the sub boiling temperature are used in many cases to kill the microbes. One such method is pasteurization

Example: Pasteurization

In 1860, Louise pasture used slow heating at low temperature i.e less than 100 degree Celsius, to destroy unwanted microorganisms that was spoiling French wines.This controlled heat treatment is now called pasteurization. It kills the vegetative cells but does not sterilise completely. Two methods of pasteurization are in used today

• Batch method

In this, milk is held at 62.8 degrees Celsius for 30 minutes and then cool down. In this, sterilization is done by flowing milk through a heat exchanger where it is heated to 71.7 degrees Celsius for 15 minutes and then cool down quickly.

2. Dry heat method

In dry heat sterilization, we need much higher temperature as compared to moist heat and longer exposure time. This is because dry heat is not as effective as moist heat. Dry heat is used to sterilise petri plates, test tubes and other equipments, used in the lab like forceps and scissor

Example: Hot air oven

It is an instrument that is used in most of the labs for sterilising the things by dry heat

Structure

It is a box with thick walls and inside the chamber there are provided metal sheets to keep the things which are to be sterilised. There is a knob for setting the temperature. The oven is usually heated with electricity and has a thermostat that maintains the chamber constantly at a chosen temperature

Working

Hot air over is used in situation where a material should not be exposed to moisture. Petri plates , pipette etc are kept in the chamber and temperature is set for the required time. Commonly a temperature of 162 -180 degrees Celsius is maintained for 2 hours

Infrared radiations

These are used in some cases for sterilization by dry heat. Infrared rays are directed from an electrically heated element onto the objects to be sterilised and temperature of 180 degree Celsius can be obtained. Heating at or above 200 degrees Celsius by infrared ray, in vacuum, has been employed as a means of sterilising surgical instruments.

Incineration

Destruction of microbes by burning or incinerate, is a routine practice in the microbiology lab. Transfer loops or needles are regularly placed into the flame of bunsen burner or into specially designed incinerators.

Such flaming of loops is an integral part of isolating microbial colonies onto solid medium. Advantages by using specially designed incinerators is , in this transfer loop is inserted into electrically heated cone. This avoids scattering of heated method.

• Low temperature

Sub zero temperature inhibit the metabolism of microbes in general. In daily routine freezing is commonly used to preserve foods ,drugs and lab specimens.

Low temperature effectively stop microbial growth. A standard home refrigerator maintain an approximate temperature of -20 degrees Celsius

Limitations of low temperature

Low temperature is mainly microbio static instead of microbicidal . So complete sterilization cannot be achieved.

• Filtration

In 1884 the scientist Charles Chamberland ,developer of autoclave , describe the use of filter to remove bacteria from drinking water . Today filtration is common method to remove microbes from liquid and air . Filters are used in industries and in the labs to sterilise the material that cannot be sterilised by autoclaving such as heat sensitive vitamins and proteins

Membrane filters are cellulose esters are made into extremely then above 150 micrometer discs with pores small enough to prevent the passage of microbes. These filters are superior to the older type filters which were made from earthen wood or asbestose Because

• These have pores of uniform diameter

• the filters can be manufactured by only desired pore sizes

• they absorb very little of purine being filtered

• filtration process is very rapid

• these are disposable

HEPA filters

These are called as high efficiency particulate air chambers and are used in special biological safety cabinets. These are cabinets which provides protections to the researchers and create aseptic conditions inside the chamber. This cabinet has an open front through which air is drawn in and away from the workers. This air then exit the cabinet through a filter which traps particular matters such as microbes. It consists of cellulose acetate plated around aluminium foil. It captures 99% of the solid particles from the existing air.

Desiccation

It means drying of vegetative microbial cells by stopping their microbial activities. This physical process goes widely used to protect the eatable from microbes. This is mainly microbiostatic instead of microbicidal