In this article we will discuss about Meaning and Routinely used Instruments for the Sterilizations of all Living Microorganisms.
Meaning of Sterilisation:
Sterilisation is the process of freeing of an article from all living microorganisms including their spores. Sterility is an absolute state, while the disinfection is a relative term indicating a mere removal of pathogenic microorganisms on the article.
Sterilisation of culture media, containers and instruments is essential in microbiological work for the isolation and maintenance of pure culture. In dental, nursing practice, surgery and medicine, the sterilisation of the instruments, drugs and other supplies is important for the prevention of the spread of diseases.
Sterilisation can be effected by the following methods:
Dry heat kills the microorganisms by destructive oxidation of essential cell constituents at 160°C for 60 minutes. Moist heat is more effective than dry heat, moist heat kills the microorganisms by coagulating and denaturing their enzymes and their structural proteins, a process in which the water participates. Heat resistant spores are killed at exposure to moist heat at 121°C for 10-30 minutes.
Chemical sterilising agents are widely used as disinfectants and antiseptics; disinfectants are potent, toxic and applied on in animate objects; antiseptics are non-toxic and used for superficial application on living tissues.
Routinely Used Instruments for Sterilisation:
a. Physical Methods – Dry Heat:
i. Hot air oven:
The oven is heated by electricity and has a thermostat to maintain the air temperature constant (160°C for 1 hour). It is the best method to sterilize dry glass water, cotton wool plugged test tubes, Petri dishes, flasks, pipettes, swabs, powder, fat or grease). All glassware’s should be dry, and wrapped in Kraft paper to soak any water drop on the glassware.
The oven should be loaded in such a way that the air can circulate through the load. The temperature should be raised slowly in the course of 1-2 hours to reach 160°C—which must be maintained for 1 hour. This is the holding period. Finally, the oven is allowed to cool gradually for 2 hours before the door is opened, otherwise the glassware’s may crack due to sudden atmospheric cooling.
ii. Infrared radiation:
The infrared rays are directed on the object to be sterilised at a temperature of 180°C as a means of sterilising surgical instruments.
b. Moist Heat:
i. Constant temperature water bath:
It is a metal box containing a heating element and a thermostat that keeps the water in the box at a constant temperature (56°C or 60°C). During the preparation of bacterial vaccine, the bacteria in suspension are killed in the water bath (vaccine bath) at a temperature of 60°C for 1 hour.
The sterilisation of serum or body fluids containing coagulable proteins is effected at 56°C for 1 hour in water bath. The temperature above 59°C may cause inspissation (coagulation).
2. Pasteurisation of milk can be done by two methods:
(a) Holder method (63°C for 30 minutes) or
(b) Flash method (72° for 20 seconds) in which tubercle bacilli, Brucella abortus and Salmonella are destroyed.
It consists of a water jacketed copper box. The temperature is between 75°-85°C in which the protein is completely solidified.
The serum (Loeffler serum) and egg (Dorset egg or Lowenstein Jensen medium) in screw-capped (MacCartney) bottles are placed in special racks so that the tubes are in slanting position to form slopes; water vapour can enter the interior of the inspissator, the medium is kept moist, sterilised at 75°C for 45 minutes intermittently for three days and solidified.
Exposure at 100°C for 20-45 minutes on each of the three successive days, sometimes referred to as Fractional sterilisation (Tyndallisation), is employed to sterilize sugar media, gelatin media etc.
Boiling water in fish kettle (100°C for 10 minutes) is sufficient to kill the non-spore forming bacilli. It does not ensure sterility and is used for rubber stoppers, instruments (scalpels, forceps, scissors), metal and glass type syringes.
Steam at 100°C is used for the sterilisation of culture media (nutrient broth and agar), but it is not as effective as autoclaving. Koch or Arnold steamer (sterilizer) heated by gas or electricity is employed. It is a vertical cylinder with a conical lid having a small opening for escaping steam, i.e. steam without pressure.
Water boils when its vapour pressure equals the pressure of the surrounding atmosphere. This occurs at 100°C for normal atmospheric pressure (i.e. 760 mm Hg = 14.7 lb. per square inch absolute pressure).
Thus, when vapour boils within a closed vessel at increased pressure, the temperature at which it boils will rise above 100°C.
This principle is employed in “Autoclave” which provides heat at temperatures above 100°C. Autoclaving is the reliable method most widely used for sterilisation of culture media and surgical supplies.
All parts of the load to be sterilised should be permeated by the steam.
1. The simple form of laboratory autoclave, the so-called “pressure cooker type” (Fig. 4.1), consists of a vertical cylinder of gun metal or stainless steel. Its size is 18 inch (45 cm) in diameter and 30 inch (75 cm), in length. The lid (or door) is fastened by screw clamps and is rendered airtight by means of asbestos washer.
The cylinder contains water up to a certain level and is heated electrically below the cylinder. The bottles, tubes, culture media etc. to be sterilised are placed on a perforated tray situated above the water level. This apparatus is furnished on its lid with a discharge tap for air and steam, a pressure gauge, and a safety valve.
Direction for using the simple autoclave:
Water should be sufficient in the cylinder. Insert the material to be sterilised and turn on the heater. Place the lid in position, open the discharge tap and screw down the lid. Adjust the safety valve to the required pressure. Allow the steam and air mixture to escape freely until all the air has been eliminated from the autoclave.
Now close the discharge tap. The steam pressure up to 15 lb. pressure per sq. inch for 121°C should be maintained for 15 minutes. This is the holding period. Then turn off the heater and allow the autoclave to cool until the pressure is 0 lb. per sq. inch. To avoid a violent explosion, do not open the autoclave when its inside pressure is still high.
2. Steam jacketed autoclave (hospital type):
This autoclave is horizontal and is made of rust-less metal. At its front is a swing door fastened by a “capstan head” which operates radially and remains locked. A pressure locked safety door is a valuable guard against a dangerous explosion through premature opening by the operator.
The hospital type autoclave (Fig. 4.2) has:
(a) A supply of steam from external source;
(b) Steam jacket;
(c) A channel to discharge air and steam;
(d) Steam trap with thermometer to control the discharge;
(e) A vacuum system to assist the drying of the load;
(f) A cooling system to hasten the cooling.
To test the efficiency of autoclaving:
Two methods (chemical and spore indicators) are available to detect the efficiency of autoclaving.
(i) Chemical indicators:
It may be placed inside the load. Browne’s sterilizer control tubes contain a red solution that turns green when heated at 115°C for 25 minutes (type 1) or 15 minutes (type 2) or at 160°C for 60 minutes (type 3). Bowie Dick tape, applied to packs and articles in the autoclave, develops diagonal lines when exposed to the sterilising temperature for the correct time.
(ii) Spore indicators:
A preparation of dried spores of Bacillus stearothermophilus is placed within the load in the autoclave and after autoclaving it is tested for viability on transfer to culture media; its spores are killed at 121°C in about 12 minutes.
Non- Ionising Radiation
Ultraviolet rays from suitably shielded lamps have been used to reduce the number of bacteria in the atmosphere. Infrared radiation is used for rapid mass sterilisation of glass syringes. Ionising radiation includes high speed electrons; X-rays and gamma rays (short X-rays).
In sufficient dose, these radiations are lethal to all cells including bacteria by damaging DNA. Spores are generally resistant. It is employed commercially for sterilisation of plastic syringes, rubber and catheters that are unable to withstand the heat. It is too expensive for the hospital use. This method is called cold sterilisation. Plastic syringes can also be sterilised by ethylene oxide gas.
It is possible to render the fluids, bacterial cultures, sugar by passing through the special filters with pore size of less than 0.75 pm. In general, sterilising filters render liquid free from bacteria, but not from mycoplasma or virus. Thus, the serum “sterilised” by Seitz filtration must not be regarded as safe for clinical use.
Types of filter used in bacteriological work include:
(a) Earthenware candles (Berkefeld Chamberland);
(b) Asbestos disks (Seitz filter);
(c) Sintered glass filters;
(d) In recent years, cellulose membrane filters are used to separate viruses of different sizes.
It consists of a disk of an asbestos through which the fluid is filtered. The disk is inserted into the metal holder fitted to a filtering flask. Before use, the whole apparatus should be sterilised by autoclave (Fig. 4.3).
The development of high efficiency particulate air (HEPA) filters or fiberglass filters has made it possible to deliver clean air to an enclosure (cubicle or room). This type of air filtration together with a system of Laminar Airflow is now recommended as air disinfection and used very extensively to produce dust and bacteria-free air in vaccine sterility testing laboratory; but it is expensive.
e. Chemical Methods:
Common chemical disinfectants used: