In this article we will discuss about Sterilisation and Disinfection:- 1. Meaning of Sterilisation and Disinfection 2. Methods Affecting Sterilisation and Disinfection 3. High Pre-Vacuum Sterilizer 4. Sterilisation by Radiation 5. Sterilisation by Filtration 6. Chemical Agents of Disinfection 7. Sterilisation Methods in Dental Practice.
Contents:
- Meaning of Sterilisation and Disinfection
- Methods Affecting Sterilisation and Disinfection
- High Pre-Vacuum Sterilizer
- Sterilisation by Radiation
- Sterilisation by Filtration
- Chemical Agents of Disinfection
- Sterilisation Methods in Dental Practice
1. Meaning of Sterilisation and Disinfection:
These two terms are used to describe the killing or removal of microorganisms. Sterilisation is an absolute term which denotes the complete killing or removal of microorganisms of all kinds, while the disinfection is a relative term indicating a mere removal of burden of pathogenic microorganisms on the article.
Sterilisation: Sterilization is the process of freeing of an article from all living organisms, including viruses, bacteria and their spores, fungi and their spores, both pathogens and non-pathogens. Sterility is an absolute state. An article can never be “relatively sterile“.
Sterilisation of culture media, containers and instruments is essential in bacteriological work for the isolation and maintenance of pure culture.
In nursing practice, surgery and medicines, the sterilisation of the instruments, drug and other supplies is important for the prevention of the disease and it is also required for medical and surgical instruments and materials used in procedures that involve the penetration into the blood, tissues and other normally sterile parts of the body, e.g., surgical operation, intravenous transfusion, hypodermic injections and diagnostic aspirators.
Disinfection: Disinfection is a method of freeing an article or instrument from some or all living pathogens which may produce the infection during the use of the instruments contaminated with the pathogens.
This term is relative since the sterilisation procedures are unwanted and impracticable, the disinfection should be adopted, e.g., bedpans, baths, washbasins, furniture, eating utensils, bed clothes, fomites, which may spread the infection in the hospitals, cannot be sterilised, but they can be easily disinfected.
Similarly, it is not practicable to apply sterilising procedures to the skin. The non-spore forming bacteria present in the skin mostly infect the surgical wound, hence it is of great necessity to disinfect the operative site as a valuable preoperative precaution to kill the vegetative bacteria.
2. Methods Affecting Sterilisation and Disinfection:
I. Physical Methods:
(a) Heat:
(I) Dry heat (incineration, red hot, flaming, hot air oven, infrared radiation)
(II) Moist heat (pasteurization, boiling water, steam sterilizer, autoclave).
(b) Radiation:
(I) Ultraviolet radiation
(II) Ionising radiation (X-ray, gamma ray).
(c) Filtration (Berkefeld, Chamberland, Seitz, Sintered glass, Cellulose membrane filters).
II. Chemical Methods:
Basic Principles of Sterilisation by Heat:
Moist heat is more effective than dry heat by sterilising at a lower temperature in a given time, or in a shorter time at a given temperature. Moist heat kills the microorganisms by coagulating and denaturating their enzymes and their structural proteins — a process in which the water participates.
Sterilisation, e.g., Killing of the moist resistant spores requires exposure to moist heat at 121°C for 10-30 minutes. Dry heat is believed to kill the microorganisms by causing 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.
Factor influencing sterilisation by heat:
(1) The temperature and time of exposure.
(2) The number of microorganisms and spores.
(3) The species, strains and spore producing bacilli.
1. The Temperature and the Time of Exposure for Sterilisation are Inversely Related:
At higher temperature, shorter time exposure is sufficient, e.g., the heating must be hot enough. For surgical and bacteriological sterilisation, and exposure of the organisms to moist heat at 121°C for 10-20 minutes is sufficient for killing of all pathogenic spore forming organisms and all saprophytes except some strict thermophiles which grow best at more than 40°C.
Sterilising time (holding period) is the time during which the microbes themselves should be held at the given temperature and does not include the heating up time. The amount of time allowed for heating up is known as heat penetration time.
2. The Rapidity of the Sterilisation is Affected by the Number of Microbes and their Spores:
In practice, before applying heat for sterilisation, the cleaning of all the articles or instruments should be done to reduce the burden of contaminated bacteria for better and rapid sterilisation.
3. The Susceptibility to Heat is Affected by Species, Strains and Spore forming Ability of the Microorganisms:
The amount of heat required to kill a given strain or species of bacteria is related to the temperature and the time of exposure and it is stated that the thermal death point when the lowest temperature is required to kill completely the bacteria within 10 minutes or the thermal death time when the shortest time is required to kill completely the microbes at a particular temperature.
The most reliable method of measurement of the thermal death point and the time of is the decimal reduction time or D value which is the time (in minutes) required to achieve a ten-fold reduction in viability of a bacterial suspension at a given temperature under standard conditions.
Effect of Moist Heat:
A temperature of 50°C to 65°C may kill within 10 minutes the vegetative forms of most bacteria, yeasts and fungi and animal viruses, Treponema pallidum is fragile and killed at 43°C in 10 minutes Coxiella burnetti causing Q fever in man is heat resistant. Bacillus stearothermophilus is extremely resistant to heat. Poliomyelitis virus is killed at 60°C for 30 minutes and serum hepatitis virus is killed at 60°C for 10 hours.
Bacteriophages are killed at the temperature of 65-80°C for 15-30 minutes. The most resistant forms of spores of actinomycetes, yeasts and fungi are killed at 80-90°C in 30 minutes. The spores of Clostridium tetani. CI. welchii are killed at 100°C for 10 minutes, whereas CI. botulinum in 8 hours.
Effect of Dry Heat:
Dry heat at 100°C for 60 minutes kill vegetative bacteria which are susceptible to moist heat at 60°C in 30 minutes. High content of organic substances protect spores and vegetative forms against the lethal action of heat.
The heat resistance of spores is diminished with increased acidity and alkalinity. The effect of alkali is used in the disinfection of metal instruments by boiling at 100°C in water containing 2 percent sodium carbonates but this method is not reliable as autoclaving.
Sterilisation by Moist Heat:
Microorganisms can be killed by the moist heat if they come in contact with water or steam; otherwise if the articles are protected from wetting, as by grease or in a relatively impervious container exposed at the same temperature; the moist heat will have the weaker effect than the dry heat.
Method of Sterilisation by Moist Heat:
In sterilisation, moist heat can be used:
(a) At temperatures below 100°C.
(b) At a temperature of 100°C (either boiling water or in free steam) or
(c) At a temperature above 100°C (in saturated steam under increased pressure) which ensures the complete sterilisation and killing of the most highly resistant spores.
Basic Principles of Moist heat Sterilization:
Because of its greatest lethal effect on microorganisms, quicker heating up effect on the exposed articles and its penetrating power through porous materials (paper, cloth wrapper, bundles of surgical instruments, wool stoppers, cotton), saturated steam is a more efficient sterilising agent than the hot air.
When the cooler surface of the articles comes in contact with the steam, the steam condenses into a small volume of water and liberates its large latent heat to that cooler surface of the article (e.g. 1,600 ml. of steam at 100°C condense, at atmospheric pressure into 1 ml of water at 100°C liberating 518 calories of heat).
The contraction in volume causes immediate suction of more steam to the same site and this process continues till the temperature of the article is raised to a temperature of the steam. The condensed water provides moist conditions of killing of the exposed microbes, since the penetration by the steam is hindered by the air, the air should not be trapped inside the autoclave; only pure steam is used.
The loss of water by evaporation by heating is prevented by the saturated steam.
Hence, culture media (both solid and liquid) can be sterilised by the steam under pressure. In the autoclave or steamer, the cotton wool stoppers can be drenched, this drenching can be avoided by covering the stopper with Kraft paper. If a wire basket containing test tube is used, it can be covered with a sheet of paper folded down the edges of the basket.
Moist Heat at Temperatures below 100°C:
The process which kills all non-spore forming pathogens (Mycobacterium tuberculosis, Brucella abortus, Salmonella) in milk by subjecting the milk to a temperature of 63°C for 30 minutes (the holder method) or 73°C for 20 seconds (the flash method) is known as “Pasteurization of milk“.
Serum or body fluids containing coagulable protein can be sterilised by heating for 1 hour at 56°C on several successive days in water bath. Care must be taken not to allow the temperature to rise above 59°C, which may cause inspissation. Vaccine can be prepared in a vaccine bath at a temperature of 60°C for 1 hour.
Moist Heat at a Temperature of 100°C:
Boiling at 100°C:
Boiling at 100°C for 5-10 minutes does not ensure the complete sterility, but it is enough to kill all non-sporing organisms and few spore-forming bacilli (e.g. strains of CI. tetani may survive boiling for 1-3 hours.
Sometimes in microbiology and medicine this boiling method is used, when the absolute sterility unwanted for pipettes, measuring cylinder, rubber stoppers, instruments (scalpels, forceps, scissors, metal or glass syringe) which do not withstand higher temperature.
If the supplied water is hard, the instruments, after removal from boiling water, may become covered with a film of calcium salts; to avoid this effect, distilled water should be used. Addition of 2 percent sodium bicarbonate may promote the sterilisation.
Long handed forceps, kept immersed in 3 percent Lysol to a level approaching the finger grip, can be used to remove the articles and instruments from boiling water. If the instrument was taken into the hand, while it is wet, its working end (e.g. scalpel blade or syringe needle) may get contaminated with skin bacterium flowing from the fingers in the film of water.
However, boiling water can generally be used for contaminated dishes, bedding and bedpans, because these articles do require neither sterility nor the destruction of spores. Only disinfection is necessary for these articles. At altitude over 5,000 feet (1,500 metre), the boiling time should be increased by 50 percent, because the water boils at this altitude at a temperature of 95°C or below.
Boiling (at 100°C) for 10 minutes kills the vegetative and actively multiplying cells of pathogenic bacteria, rickettsiae, all viruses except hepatitis and poliomyelitis viruses, if these microorganisms are not protected by the blood, food, faeces, mucus, etc. It also kills or damages the cysts of protozoa, ova of helminths, ascospores of pathogenic yeasts, conidia of moulds.
Since the spores of Clostridium tetani, CI. welchii and Bacillus anthracis and hepatitis virus are resistant to boiling, even for 10 minutes boiling at 100°C for 10 minutes is not effective. Spores present in soil, faeces, dust on instruments and bandages contaminated with blood, dust may be a risk of infection. Whereas Anthrax spores occur only in areas where the infected animals died of anthrax.
Boiling at 100°C for 10 minutes is a sure means of disinfection (not sterility), if the spores and viruses are not present, of instruments and glassware’s, if they are not soiled with blood, faeces, food etc. The margin of safety can be increased by boiling for 30 minutes.
Summary:
(1) Vegetative forms of all bacteria and fungi pathogenic to man; rickettsiae pathogenic to man, helminths and their ova; many viruses (except hepatitis virus); bacterial toxin (except staphylococcal enterotoxin) are killed or inactivated by boiling at 100°C for 10 minutes.
(2) Bacterial spores; Hepatitis virus; ascospores of hingi, conidia of some fungi pathogenic to man; staphylococcal enterotoxin are not killed or inactivated by boiling at 100°C for 10 minutes.
Steaming at 100°C is commonly used in the microbiological laboratory to sterilize the culture media (both nutrient broth and nutrient agar). It is not as effective as autoclaving. Koch or Arnold steam sterilizer or “Steamer” is a vertical metal cylinder with a removable lid having a small outlet for the steam to escape and at its bottom it contains water which is heated up.
Above the water level, a tray bears the articles to be sterilised. Frequent opening of the steamer should be minimised, because the air permitted inside the steamer ultimately interferes with sterilisation.
Sterilization is carried out as follows:
(a) By a single exposure at 100°C for 90 minutes.
(b) By intermittent exposure at 100°C for 20-45 minutes on each three successive days.
(a) Single exposure at 100°C for 90 minutes-is not suitable as the spores of thermophilic and some mesophilic bacteria can survive this exposure.
(b) Intermittent exposure at 100°C for 20-45 minutes for three successive days is also known as “Tyndallisation“. In this method one exposure will destroy all the vegetative organisms between the heating’s, the spores in favourable nutrient medium become vegetative forms which are killed during the subsequent heating.
This method is suitable for the medium containing sugars which may be decomposed at higher temperature and for gelatin medium which after prolonged heating, may not solidify on cooling.
III. Moist Heat at Temperature above 100°C:
Sterilisation in the Autoclave:
Principle employed in the autoclave-water boils when its steam pressure equals atmospheric pressure or it can be interpreted the water boils at 100°C at normal atmospheric pressure (i.e. 76 mm Hg; 14 per square inch pressure). So when the water is boiled within a closed vessel at increased pressure, the temperature at which the water boils and that of its steam will rise above 100°C.
The most effective method of sterilisation of culture media (in the bacteriological laboratory) and surgical materials or supplies (in the hospital) is Autoclaving. The steam should penetrate all parts of the articles to be sterilised in the autoclave and should be hot saturated (i.e. point of condensing to liquid water) and dry (i.e. free from particles of liquid water).
This dry saturated steam affects the sterilisation because of its high temperature (latent heat). The ideal minimum holding time is 15-30 minutes at 121°C (15 lb. per square inch gauze pressure).
Air discharge:
To expose all articles in the autoclave to the pure steam during sterilisation, it is necessary to remove all the air from the autoclave, because:
(a) The mixture of steam and air results in a lower temperature at a particular pressure,
(b) The penetration of steam in the interstices of porous materials (e.g. surgical dressing), syringes, etc., may be interfered by the air, and the air, being denser than the steam from a cooler layer at the bottom of the autoclave, which prevents the sufficient heating of the articles at the lower level.
There are two types of Autoclaves:
(a) The simple non-jacketed laboratory autoclave.
(b) Steam-jacketed autoclave with automatic air and condensate discharge.
(a) The Simple Non-Jacketed Laboratory Autoclave:
The simple non-jacketed laboratory autoclave is also called “pressure cooker type“. It is made up of a vertical or horizontal gun metal or stainless steel cylinder of about 18 inches in diameter and 30 inches in length with a supporting stand. The lid (door) is tightened by screw clamps and is rendered airtight by means of an asbestos washer.
The water level inside the cylinder should be up to 31/2 inches for a vertical cylinder of 19 inches internal length. The autoclave is heated by gas burner or electric heater from below the cylinder.
The articles to be sterilised are placed on a perforated tray situated above the water level. The lid of the autoclave is provided with a discharge tap for the air and steam, a pressure gauze and a safety valve which is adjusted to blow off automatically at any desired pressure.
Operating of the Simple Autoclave:
Inside the autoclave, the water should be up to required, level i.e., below the perforated tray. The materials to be sterilised are placed on the tray and the heater should be turned on. The lid should be placed in position and fastened by screw clamps, the discharge tap should remain open and the safety valve is to be adjusted to the required pressure.
In some autoclaves, the pressure has been predetermined and adjusted by the manufacturers. When water boils inside the cylinder of the autoclave, the steam rises, mixes with the air in the chamber and expels out this air through the discharge tap.
The operator should allow the steam and air mixture to escape freely till all the air is completely removed from the autoclave, which can be adjusted by connecting a rubber tube from the discharge tap into a container of cold water into which the steam condenses and the air rises as bubbles to the surface.
This procedure can be followed by an inexperienced operator for his own satisfaction, when there is no trained nurse.
When the air has completely escaped, the discharge tap should be closed. The steam pressure rises till it reaches the desired level, e.g. 15 lb. per square inch for 121°C, when the safety valve opens automatically and allows the excess steam to escape. From this point, the holding time begins and continues for 15 minutes.
The heater should be turned off, the discharge tap should be opened gradually and the autoclave should be allowed to cool till the inside pressure equals to the atmospheric pressure (0 lb. per square inch) which may take about 1 hour; when the chamber pressure is still high, if the discharge tap is opened, there is possibility of danger of explosion of autoclave lid, culture media will boil and spill from the container.
An excess amount of water will be evaporated and lost from the media if the discharge tap is not opened until the pressure has fallen much below the atmospheric pressure. It is necessary to have surgical linen and dressing wrapped in paper or cloth for quick drying of the materials after sterilisation.
These wrapped articles are moistened by the condensation of the steam; when damp even several layers of paper and cloth wrappings could not check the entry of contaminating bacteria into the sterilised articles. Therefore, the sterilised articles should not be kept in contact with unsterilized objects until their wrappings are dry.
(b) Steam-Jacketed Autoclave:
(Fig. 83.1) is mostly used in the hospital to sterilize the linen, surgical materials etc. It is a horizontal cylinder. At the front, there is a swing door which is tightened by screw clamps or by a “capstan head” with its radial bolts and it locks automatically while the chamber pressure is raised.
This safety door locked automatically by pressure can prevent the possibilities of a dangerous explosion, if the operator opens early the autoclave by mistake.
The Autoclave consists of:
(1) A steam supply from outside (e.g. an independent boiler),
(2) A steam jacket which heats the side walls of the autoclave,
(3) A channel discharging air from the bottom of the chamber,
(4) A thermometer in the discharge channel, indicating the temperature above the lowest and coolest part of the chamber, vacuum system to assist drying the load.
(6) An air intake with a self-sterilising filter for introducing warm sterile air in the chamber,
(7) A cooling system to hasten the cooling of the liquids without violent boiling,
(8) An automatic control system for heating up, holding, cooling and drying.
Steam Supply:
The steam supplied to the autoclave should be dry, i.e., free from excess of water, and saturated, i.e., not superheated.
Wet Steam:
The steam released from the boiler at a long distance may become “wet” and inefficient sterilising agent because of
(a) Cooling and condensation,
(b) Soaking of porous materials which prevents further penetration and
(c) The particles of water possess no latent heat.
This, the pressure of the main steam supply should be usually 55 lb. per square inch. The reduction in pressure dries the steam. Super-heated steam is not satisfactory because it abstracts the water from the exposed materials, may cause the less lethal effect and is still more destructive as dry heat.
Operating of Steam Jacketed Autoclave:
A space between the double side walls of the chamber (jacket) is filled throughout the day with the steam at 121 °C introduced from the boiler. Water of condensation from the steam is drained off through the jacket discharge channel which is thermostatically controlled. The operator should ensure that there is no obstruction of this discharge channel.
Loading of the Chamber:
The articles to be sterilised should be arranged loosely so that the steam circulates freely and the air is displaced.
Heating Up and Air Replacement Method:
The swing door should be closed systematically. The steam from the jacket should enter through a baffle at the back of the chamber. The pressure and temperature should be maintained in the chamber as in the jacket.
The cooler and denser air should be displaced by the steam under pressure. If more steam is introduced under pressure, it displaces the air downwards through the articles to be sterilised and the air is discharged out through the chamber discharge channel situated at the bottom of the chamber.
At the same time the water of condensation on the cool load and the door of the chamber should be drained off through the same channel. As soon as all free air is eliminated, the inflow of pure steam raises the trap’s temperature to 121°C, it closes automatically and it prevents further escape. The steam replaces the air in about 5-10 minutes.
Holding Period of Sterilisation:
Holding period will start when the thermometer in the discharge channel reaches the temperature of 121°C. The exact duration of the holding period depends upon the nature of the load. A “near to steam” trap is essential as it opens when the temperature falls by 1°C below that of the pure steam.
Cooling and drying period:
The steam supply to the chamber is dropped at the end of the holding period. The steam inside the chamber starts to cool by loss of heat through the door and pressure falls slowly.
3. High Pre-Vacuum Sterilizer:
High vacuum surgical sterilizer is the only method of sterilisation that can overcome the effects of bad packing or overloading of the sterilizer. Tightly packed load is heated rapidly and uniformly to the sterilising temperature for a shorter time, e.g., 35°C for 3 minutes. The damage to heat sensitive materials is avoided by greatly shortening the time.
The steam, admitted to the chamber, heats the whole load to 135°C within 2-3 minutes. The holding period is maintained for 3 minutes, the temperature reaches 135°C. The load is then dried within a few minutes by exhaustion of the chamber to a high vacuum.
Temperature Record:
Recording thermometer can help the operator to avoid the errors in timing the holding period.
The overall efficiency of the autoclave can be judged by the following two methods:
Chemical Indicator and Spore Indicators:
(a) Chemical Indicators:
Chemical Indicators, which show a change of colour or shape after exposure to a sterilising temperature, may be placed under the load, for example,
(i) A sulphur pellet kept in a small glass tube will show a change of shape by melting when exposed to 120°C for a few minutes,
(ii) Browne’s control tubes contain a red solution which turns green when heated at 115°C for 25 minutes (type 1); or 15 minutes (type 2) or at 160°C for 60 minutes. These tubes should be stored at less than 20°C to avoid the deterioration and premature change in colour,
(iii) Browne-Dick tape, applied to packs and articles in the autoclave develop diagonal lines when exposed to sterilising temperature for the correct time,
(iv) Sterilisation in a cellulose tape having on it a chemical indicator which changes its colour when properly heated in the autoclave.
(b) Spore Indicator:
Bacillus stearothermophilus grows best at 55°C-60°C. But a preparation of its dried spores is killed at 121°C in 12 minutes when placed within the load in the autoclave. After autoclaving, it can be tested for its viability on the culture media.
Nurse using the autoclaving should be sure that
i) All the air is allowed to escape and is replaced by the steam.
ii) The pressure of the steam reaches at least 15 pounds to the square inch and remains there;
iii) The thermometer reaches at least 121°C for 20 minutes.
4. Sterilisation by Radiation:
Non-Ionising Radiation:
Ultraviolet rays of sunlight have the bactericidal effects which depends upon the wave length. It is bactericidal when it reaches a wave length of 330µ (3300A0 or Angstrom units). Angstrom is unit of measurement of wavelength. Thus, as the wavelength decreases, the effectiveness of the ultraviolet light increases as sterilising agent.
The shortest ultraviolet rays of sunlight reaching the surface of the earth have a wavelength of more than 20µ, however most commonly used mercury vapours can emit even more effective radiations of 240-280 µ. The intensity of ultraviolet radiations applied with precautions to protect the skin to the side of operations to check out operative sepsis.
Ray from ultraviolet lamps can reduce the bacterial load in the atmospheric dust. Pathogenic microorganisms on the floor and furniture’s in rooms can be destroyed by the daylight passing through ordinary glass windows.
Ionising Radiation:
In practice, sterilisation by radiation is achieved by the use of high speed cathode rays (electrons), X-rays and short X-rays (gamma rays) from an apparatus (linear accelerator) or gamma rays from an isotope source (cobalt 60) This method is very costly for hospital use, but it is used commercially only for the sterilisation of pre-packed disposable plastic syringes, transfusion sets and catheters which cannot withstand the heat.
5. Sterilisation by Filtration:
In this process, the fluids (bacterial fluid cultures) can be freed from bacteria by passing through special filters. This method of sterilisation by filtration is specially useful to prevent toxin from bacterial growth and to sterilize the liquids (sugars, serum, antibiotic solutions) which are liable to be damaged by heat.
These sterilising filters should be considered as rendering a liquid bacteria free, but not virus free and are satisfactory for laboratory purposes, but in clinical practice, such fluids (e.g. glucose, serum) sterilised by Seitz filtration are not safe.
Types of Filters:
For bacteriological work, the following types of filters are used:
Sinter ware candles (e.g. Berkefeld, Chamberland filters, Asbestos, Asbestos paper disk (e.g. Seitz filter) Fig. 4.2. Sintered glass filters; Cellulose membrane filters.
1. (a) Berkefeld Filters:
They are made from a fossil diatomaceous earth found in deposits in Germany and other parts of the world and are of three grades of porosity, e.g., V(Veil) the coarsest, W (Wening) the finest, and Normal (N) intermediate. Berkefeld V is often used. A small organism (Serratia marcescens) should pass through this filter.
(b) Chamberland Filters:
They are made of unglazed porcelain of various grades of porosity. The finer grades will pass only certain viruses of extreme minuteness (e.g. Foot and Mouth Disease virus). The most porous grade Ly clarifying filter, allows many microorganisms to pass. The next three L1a, L2, L3 are comparable with the Berkefeld V, N, and W candles, respectively. These filters can also be used to obtain bacterial toxin.
2. Seitz Filters:
It consist of asbestos disk through which (Fig 4.2) the fluid is passed. The disk is inserted into a metal holder which is jointed tightly by screw clamps. The whole filter is wrapped in Kraft paper and sterilised in the autoclave.
The asbestos disk is discarded after each filtration and is replaced by another new disk for fresh filtration. The disks are available in three grades : Clarifying (K), normal and “special Ek.” The normal and “special Ek” do not allow the ordinary test bacterium Serratia marcescens to pass through.
3. Sintered Glass Cylinder:
They are made of finely ground glass attached to the filtering apparatus and sterilised as in case of Seitz filter. After use they should be washed with running water in reverse direction and cleaned with warm sulfuric acid to which potassium nitrate is added.
4. Cellulose Membrane Filters:
There are two types of cellulose membrane filters:
The gradacol membrane (older type) consists of cellulose nitrate and the modern membrane filter is made of cellulose acetate. The size of many viruses can be determined by these cellulose membrane filters; these filters can retain Serratia marcescens.
Advantages of Cell Membrane Filters are :
(a) They are much less absorptive and the rate of filtration is much greater,
(b) Bacteria retained on the surface of this filter can be cultivated by placing this filter on culture media.
6. Chemical Agents of Disinfection:
Chemical sterilising agents are widely used as disinfectants and antiseptics.
Disinfectants are:
(a) Chemical agents which are capable of destroying pathogenic microorganisms, but not resistant spores;
(b) Potent and toxic;
(c) Suitable only for application to inanimate objects. Antiseptics are:
(a) Chemical agents which kill microorganisms or prevent their growth;
(b) Non-toxic;
(c) Suitable for superficial application to living tissues.
Volatile Antiseptic, e.g., Chloroform:
In the sterilisation and preservation of serum for culture media. Chloroform used in the proportion of 0.2 per cent and later it may be removed by heating at 56°C. Alcohol (Isopropyl alcohol, ethyl alcohol) are bacteriocidal in 70-75 per cent concentration Chlorhexidine or Iodine is superior to alcohol alone for skin disinfectant before venepuncture or hypodermic injection.
2. Antiseptics of the Phenol Group:
Lysol (liquor cresolis saponatus) and cresol (black and white fluid) are powerful disinfectants, they are used for sterilising surgical instruments, discarded cultures and killing cultures accidently spilt on the floor or table. Lysol is generally used in a 3 per cent solution and phenol in 0.5 per cent for preserving sera.
Sudol is less toxic substitute for Lysol. Dettol is less toxic, irritant and also less active. Hexachlorophene is an even blender agent and is incorporated in various antiseptic preparations. It is effective against Gram-positive and Gram-negative bacteria. It is potentially toxic and should be used with care.
3. Metallic Salts or Organic Compounds of Metals:
Mercuric chloride (1:1000) is used as disinfectant. Merthiolate, a proprietary name for sodium ethyl-mercuri-thio-salicylate, is used in a dilution of 1:10000 for preservation of antisera or sera, A drop of silver nitrate (1 per cent) solution is used for prophylaxis of gonococcal ophthalmia in newborn babies. It is replaced by chlorhexidine (modern antiseptic).
4. Formaldehyde is an irritant water soluble gas which is highly lethal to all kinds of microbes and spores, killing bacterial spores as readily as the vegetative forms.
It is cheap and non-injurious to cloth, fabrics, wool, leather, rubber, paints and metals. So it can be used to disinfect the rooms, furniture, and a wide variety of articles which are liable to be damaged by heat (e.g. woolen blankets and clothing) shoes, gum elastic catheters). It can be applied as an aqueous solution or in gaseous forms.
Disinfection with Aaqueous Formaldehyde Solution:
A 40 percent (W/V) solution of formaldehyde in water containing 10 per cent methanol to inhibit polymerisation is known as “Commercial formalin.” Formaldehyde diluted in water (5-10 per cent) is a powerful and rapid disinfectant when applied directly to a contaminated surface.
Bacterial cultures and suspensions are also killed by this disinfectant. Cleaned metal instruments may be sterilised overnight immersion in a borax-formaldehyde solution.
Sodium tetra-borate 50 g Formaldehyde, 4 per cent in water, 1000 ml. Glutaraldehyde is bactericidal sporicidal, more effective and less toxic. It is used to sterilize cystoscope anesthetic equipment’s, plastic materials and thermometers.
Disinfection by Formaldehyde Gas:
The articles which can be damaged by wetting or cannot be wetted completely with formaldehyde solution can be disinfected by formaldehyde gas provided that the conditions suitable for the action of gas are maintained.
The atmosphere must have relatively high (80-90 per cent) humidity and the temperature should be 18°C. Since the penetration of the gas into the porous fabrics is slow, the materials should be arranged in such a way the gas can have access to all contaminated surfaces.
Spraying, heating formalin or heating solid of paraformaldehyde can liberate the gas, when spraying cold, formalin, its polymerisation can be prevented by adding in equal volume of industrial-spirit to cold formalin. Formalin diluted with sufficient water can be boiled to produce an adequate atmospheric humidity.
This is a best method, because of the tendency of the gas to polymerize to paraformaldehyde and a maximal vapour concentration attainable at 20°C is 2.0 mg per litre of air, which is a desirable concentration. Higher concentration obtained at high temperature may be explosive.
After disinfection, the articles may contain paraformaldehyde emitting irritant vapour for a long period which may be neutralized by exposure to ammonia vapour. Exposure for at least 3 hours to formaldehyde gas in an airtight metal or painted wooden cabinet is sufficient to disinfect small articles (instruments, shoes, a hair brush).
The gas is introduced inside the cabinet by boiling formalin in an electric boiler to the extent of 50 ml 40 percent of formaldehyde per 100 cu ft of air space.
Similarly, blankets and the surfaces of mattresses are disinfected in a large cabinet where they are hung unfolded so that the fabric can absorb formaldehyde gas (e.g. 500 ml of formalin can be used for 100 lb., fabrics). Finally, the vapour is vented to the open air, folded blankets and clothing’s can also be disinfected only if some heat is applied.
They are packed in the chamber of steam jacketed autoclave, then 100 ml formalin per 100 cu ft. is placed at the bottom of the autoclave and the chamber is heated at 100°C for 3 hours by passing free steam through the jacket. At last, the vapour is vented through the autoclave vacuum system.
5. Ethylene Oxide:
It is a colourless liquids boiling at 107°C. It is moderately toxic gas above this temperature and forms an explosive mixture when more than 3 percent is present in air. In non-explosive mixture (10 percent ethylene oxide in CO2 or in halogenated hydrocarbon) can be used for sterilisation. It destroys bacteria and viruses, and kills the spores almost as easily as vegetative forms.
This is a gaseous disinfectant killing microorganisms and spores. It can diffuse more rapidly into dry, porous materials and is of great importance to sterilize articles which are liable to be damaged by heat (e.g. plastic, rubber articles, blankets, pharmaceutical products, lung heart machine etc.).
7. Sterilisation Methods in Dental Practice:
A. Sterilisation of Bottled Fluids:
Hydrated fluids used for therapeutic intravenous infusions can be sterilised at 121°C for 12 minutes as the contaminating bacteria are already moist and can be easily destroyed at this condition. Similarly sterile water used in the operation theatre can be sterilised in the autoclave.
B. Sterilisation of Empty Bottles and Impervious Bottles:
Empty and dry bottles can be sterilised in the autoclave, if they are not tightly stoppered so that the steam can enter and displace the inside air and are arranged on their sides in the auto-calve to permit a horizontal pathway for the entry of steam and escape of air.
If un-stoppered, they can be sterilised quickly; if stoppered with cotton wool or loosened screw cap, the displacement of air is slow and the holding period is 121°C for 10 minutes.
Since the displacement of air in the stoppered container is not certain, it is always better to sterilize the stoppered empty container in hot air oven. The rubber liner inside the screw cap cannot withstand the high temperature (160°C) of hot air oven. This problem can be overcome by using silicone rubber liner.
C. Sterilisation of Wrapped Dry Porous Goods and Surgical Dressings:
When dry porous goods (e.g. paper), apparatus wrapped in cloth and surgical linen and dressings are to be sterilised by autoclave, it is essential that:
(i) The steam should displace by gravity completely the air from the autoclave,
(ii) The sterilised articles should be dry before they are removed from the autoclave.
In this method, the load should be carefully packed so that there will be adequate spaces for the circulation of the steam. Glass and metal containers are kept open or covered only loosely. Metal drums and caskets must be provided with ports which must not be obstructed by the contents packed against them and must always be fully open during sterilisation and the steam should flow freely from top to bottom.
A wrapping of two layers of good muslin it recommended for surgical packs. A single layer of coarse brown (Kraft) paper is also satisfactory.
Surgical dressing and other cloth articles should be arranged in packs no bigger than 12 x 12 x 20 inches and placed on edge in the autoclave so that the layers of cloth are vertical. Rubber gloves are powdered and packed loosely in muslin cloth to allow access of the steam to all parts.
It is customary to autoclave rubber gloves at 5 to 10 lbs. pressure for a short time to avoid the deterioration, but this exposure does not guarantee sterility, so that the gloves should be autoclaved at the temperature as few other goods, instruments and syringes must be free from oil and grease, jointed instruments open and syringes disassembled.
The chamber of the autoclave should not be overloaded and the perforated tray should not be removed.
D. Sterilisation of Surgical Instruments:
Boiling is effective when 2 per cent sodium bicarbonate or a germicide is added to the water. There is the risk of recontamination when the instruments are washed before use to remove these substances. Heat can blunt the sharp instrument, this effect is mainly due to oxidation, but the pure steam cannot damage.
Ethylene oxide and formaldehyde are the only established chemical sterilising agents and can be used to sterilize the instruments (scalpels). Pasteurization at 75°C or treatment with chlorhexidine in 75 per cent ethanol is recommended to sterilize the instruments (Cystoscopies).
Sterilisation of Syringes:
In hospital wards and in microbiological laboratories, the syringes are very important in day-to-day use, therefore much heed need be paid to their use, care and sterilisation; sterilisation by chemical agents are not satisfactory, but the sterilisation by heat is the best method.
All glass syringes have many advantages over the glass metal type of syringes; the glass metal syringe is more difficult to clean and is more likely to break on heating due to the differences of expansion of glass and metal, it cannot be sterilised when assembled and it is difficult to keep sterile until ready for use.
The solder uniting the glass and metal may melt in hot air oven and autoclave, syringes with cement at the glass metal junction which withstands 200°C are available. The mounts of the stainless steel needle of good quality must fit accurately the nozzle of the syringe.
All Glass Syringes:
All new syringes must be well washed in soap and water with test tube brush according to their sizes. After washing in clean warm water, barrel and piston should be dried. The syringes should be assembled, wrapped, sterilised and ready for use. If this facility is not available, all gas syringes should be sterilised just before use by boiling in a fish kettle saucepan. Distilled water is preferable if the tap water is hard.
The syringes should always be dismantled, the barrel and piston are placed in cold water which is brought to boil and kept boiling for 5-10 minutes. When cool and dry, the barrel and piston should be assembled with sterile forceps or clean, dry fingers, touching only outside of the barrel and the top of the piston.
The sterile syringes should be used immediately. The needle should also be boiled at the same time and affixed to the nozzle by means of sterile forceps.
It is always better to sterilize all glass syringes in the hot air oven after they are wrapped with Kraft paper or the syringe with fitted needle should be accommodated in a test tube with cotton wool plug and the whole unit should be wrapped in Kraft paper and sterilised in hot air oven at 160°C for 1 hour. It is sterile indefinitely and is ready for use and also sterilised in the test tubes plugged with cotton wool.
Syringes used for blood culture or aspiration etc. should be immediately washed in cold .solution of 2 per cent Lysol. If the blood is clotted in the syringe, it is difficult to remove the piston. Hot water should not be used as it will coagulate the protein and the piston will stick. The syringe is cleaned with soapy water with a brush, washed in clean warm water dried and sterilised.