Techniques of Sterilization in Bacteria

In this article we will discuss about the physical, chemical and physiochemical techniques of sterilization in bacteria.

Physical Methods of Sterilization:

(i) Sun-Light:

Ultraviolet rays present in the sun-light are responsible for spontaneous sterilization in natural conditions. In tropical countries the sun light is more effective in killing bacteria due to combination of ultraviolet rays and heat. By killing bacteria in suspended water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes.

Those articles which cannot withstand high temperature can still be sterilised at lower temperature by prolonging the duration of exposure.

(ii) Heat:

It is considered to be the most reliable method of sterilization of articles—that withstand heat. There are two methods of the sterilization: dry heat and moist heat.

(A) Dry Heat:

It acts by protein denaturation and oxidative damage. Sterilization by dry heat is as follows:

(a) Red Heat:

Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are sterilised by holding them in a Bunsen flame, till they become hot red.

(b) Flaming:

Articles are passed over a Bunsen flame but not heating it to redness.

(c) Incineration:

Contaminated, materials are destroyed by burning them in incinerator.

(d) Hot Air Oven:

Articles are exposed to high temperature (160°C) for duration of one hour in an electrically heated oven (method was introduced by Louis Pasteur).

(B) Moist Heat:

It acts by coagulation and denaturation of proteins.

(i) At Temperature below 100°C:  

This process is developed by Louis Pasteur (1822-95). Articles to be sterilized are heated at 65°C for 30 minutes (holder method) or heated at 72°C for 15 seconds (flash method) followed by quick cooling to below 10°C. This method is suitable to destroy most milk born pathogenic bacteria e.g. Salmonella, Staphylococci and Brucella.

Certain other methods of sterilization at below 60°C temperature are:

Vaccine bath (contaminating bacteria in a vaccine preparation can be inactivated by heating in a water bath at 60°C for one hour), Serum bath (the contaminating bacteria in a serum preparation can be inactivated by heating in a water bath at 56°C for one hour) and inspissation (to disinfect egg and serum containing media by keeping then in the slopes of an inspissator heated at 80-85 °C for 30 minutes on three successive days).

(ii) At Temperature 100°C:

(a) Boiling:

Boiling water (100C°) kills most vegetative bacteria.

(b) Steam at 100°C:

Passing the steam at 100°C over articles kills bacteria. Sugars and gelatin in medium may get decomposed by autoclaving. So, these can be sterilised by exposing them to free steaming for 20 minutes for three successive days. This process is known as tyndallisation (after John Tyndall).

(iii) At Temperature above 100°C:

(a) Autoclave:

Sterilization can be effectively achieved at a temperature above 100°C using an autoclave.

(b) Structure of Autoclave:

A simple autoclave has vertical or horizontal cylindrical body with a heating element, a per-forted tray to keep the articles, a lid that can be fastened by screw clamps, a pressure gauge, a safety valve and a discharge tap (Fig. 1). The lid is closed but the discharge tap is kept open and the water is heated.

As the water starts boiling the steam drives air out of the discharge tap, when all the air is displaced and steam starts appearing through the discharge tap, the tap is closed. The pressure inside is allowed to rise up to 15 lbs. per square inch. At this pressure the articles are heated for 15 minutes, after which the heating is stopped and the autoclave is allowed to cool.

Once the pressure gauge shows the pressure equal to atmospheric pressure, the discharged tap is opened to let the air in. The lid is opened and articles are removed. Culture media, dressing, certain equipment’s can be sterilised by autoclave.

(iii) Sonic and Ultrasonic Vibrations

Sound waves of frequency 720,000 cycle/second kills bacteria and some viruses exposing for one hour.

(iv) Radiation:

Two types of rays are used for sterilization:

Non-ionizing and ionizing.

(a) Non-Ionizing Rays:

These are low-energy rays with poor penetrative power, e.g., U.V. rays (wavelength 200-280 nms, effective 260 nm).

(b) Ionizing Rays:

These are high-energy rays with good penetration power.

These are of two types:

Particulate and electromagnetic. Electron beams are particulate while gamma rays are electromagnetic in nature. High speed electrons are produced by a linear accelerator from a heated cathode. Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain radioactive isotopes (Co⁶⁰, Cs¹³⁷).

A degree of 2.5 megabrands of electromagnetic rays kills all bacteria, fungi, virus and spores. In some parts of Europe, fruits and vegetable are irradiated to increase their shelf life up to 500 percent. Since radiation does not generate heat, it is called Cold sterilization. 

Chemical Methods of Sterilization:

Chemicals destroy pathogenic bacteria from inanimate surfaces and are all also called disinfectants.

Liquid:

Alcohols:

E.g., Ethyl alcohol, Isopropyl alcohol and methyl alcohol. (A 70% solution kills bacteria).

Aldehydeles:

E.g., Fomaldehyele, Gluteraldehydele (40% formaldehyde is used for surface disinfection).

Phenol:

E.g., 50% phenol, 1-5% cresol, 5% lysol, chloroxylenol (Dettol).

Halogens:

E.g., chlorine compounds (chlorine bleach, hydrochloride) and iodine compounds (tincture, iodine, iodophores). Tincture of iodine (2% iodine in 70% alcohol) is antiseptic.

Heavy metals:

E.g., Mercuric chloride, silver nitrate, copper sulfate, organic mercuric salts. Surface active agents: e.g., soaps or detergents.

Dyes:

Acridin dyes e.g., acriflavin and aminacrine are bactericidal (interact with bacterial nucleic acids).

Gaseous:

E.g. Ethylene oxide, formaldehyde gas, highly effective, killing of spores.

Physiochemical Methods of Sterilization:

A physiochemical method adapts both physical and chemical method. Use of steam-formaldehyde is a physiochemical method of sterilization.