Sterilization
Sterilization: Total removal or destruction of organisms from any surface article or medium, in either vegetative form or spore form.
Disinfection: Removal of microorganism or reducing the no.of microorganism from any surface. Here the spores are not killed are destroyed.
The sensitivity of microorganism to sterilization varies, some microorganisms are more sensitive, eg: E.coli; and some microorganisms are moderately sensitive, eg: mycobacterium tuberculosis & some are resistant eg: spores.
Classification of methods of Sterilization: It is broadly classified into 2 types:
1) Physical method
2) Chemical method
Physical method is again divided into 3 types
a) Heat: dry and moist
b) Radiation: ionizing and non-ionizing.
c) Filtration.
Heat: This is the most reliable method of Sterilization. The factors that influence heat Sterilization are:
- Time and temperature, which are inversely proportional to each other.
- The no. of organisms & types those are present on the article or the medium.
- The nature of material to be sterilized.
- The nature of heat to be used for Sterilization, whether dry heat or moist heat.
Dry heat Sterilization:
Mechanism: The microorganisms are destroyed or get killed due to denaturation of proteins, oxidative damage & lethal effect of increased level of electrolytes.
Types of dry heat:
1) Red heat: The article to be sterilized is held in the flame till it becomes red-hot. Eg: inoculation wire, inoculation loop.
2) Flaming: The articles that need to be sterilized are passed through the flame for several times for eg: the mouth of the test tube, scissors, scalpels.
3) By hot air Owen: The temperature required here is 160 oC for I hour or 180 oC for half an hour. This is the most reliable method of dry heat Sterilization. The article to be sterilized is kept inside the Owen. It is heated up by electricity & the heating element is laid on the walls of the chamber. It has also got an in-built fan for even distribution of air.
Uses: Sterilization of glassware like petridish, test tubes, all-glass syringes; cotton swabs, mineral oils can be done.
4) Precaution: The article should be wrapped in brown paper. The hot air Owen should not be over-loaded & the article to be sterilized should be dry. The door of the hot air Owen should not be opened till the temperature inside does not fall below 60 oC.
Incineration: Is a process where soil dressings, pathogenic specimen, infected animal part are completely burned.
Moist heat Sterilization:
Mechanism: The lethal effect is due to denaturation and coagulation of proteins.
Types of moist heat Sterilization:
1) Moist heat Sterilization below 100 oC
2) Moist heat Sterilization at 100 oC
3) Moist heat Sterilization above 100 oC: Steam under pressure.
(a) and (b) are physical type of disinfection.
Moist heat Sterilization below 100 oC
a) Pasteurization
Holder method: temp 63 oC for 30 minutes.
Flash method: temp 72 oC for 15-20 seconds with immediate cooling to 13 oC
b) Vaccine bath: Vaccines are sterilized using vaccine bath. The temperature required is 60 oC for one hour. The serum & protein also can be sterilized but the temperature required is 56 oC for half an hour.
c) Inspissator: It is the instrument that is used to sterilize culture medium which contains egg or serum. Temperature is 80-85 oC for half an hour for 3 consecutive days.
Moist heat Sterilization 100 oC
a) Boiling: Which only removes the vegetative form.
b) Tantalization: Fractional Sterilization. The temperature required is 100 oC for 20-45 minutes for 3 consecutive days.
Principle: during the first exposure to 100 oC for 20-45 minutes, the vegetative forms get killed. The spores that are present germinate & then get killed on the 2nd & 3rd exposure. The instrument used is Cock-Arnold steamer or simple lab autoclave.
Uses:
Sterilization of sugar solutions.
Sterilization of gelatin.
Moist heat Sterilization above 100 oC: By using autoclave.
Principle: When the water boils in the closed container under high pressure, the temperature at which it boils rises. The steam that is formed will have a high penetrating power & when it comes in contact with a cooler surface, it condenses to water giving out its latent heat to the surface, there by killing the bacteria on the surface by denaturation of proteins.
Types:
1) Simple lab autoclave
2) Steam Jacketed autoclave
3) Pre-vacuum Sterilizers.
Simple lab autoclave:
It can be of two types, horizontal or vertical. It is made up of stainless steel. It consists of heating coil. The lid of the autoclave is fitted with safety valve pressure gauged steamed outlets.
Principle: The article to be sterilized is kept on the perforated tray inside the autoclave. Heating is done by electricity. The lid of the autoclave is closed tightly. When the water boils, steam starts getting accumulated & this leads to the discharge of air through the steam outlet. The steam outlet is then closed. The steam starts getting accumulated in the autoclave. It increases pressure. When pressure reaches 15 pound, the temperature will be 121 oC. The holding time is 15 minutes. After which it is switched off. It can be open only after the pressure reaches 0.
Uses:
Sterilization of culture media. The media containing egg & serum cannot be sterilized. Sterilization & disposal of infected specimen, like sputum, blood etc.
Demerits: The article sterilized are not dry, air discharge may not be complete. Air discharge is am important step because if air is present it will reduce the temperature & it also prevents penetration of steam.
Efficacy of Sterilization: It is done by using sterilizing indicators. They are of 3 types.
a) Physical method: instruments like thermometers, pressure gauge & thermostats are used.
b) Chemical methods: By using Brownie’s tube & Bowie-dick test.
Brownie’s tube contains a red coloured solution that turns into green after complete Sterilization.
Bowie-dick test: a colourless tube is used. Colour is changed to black.
c) Biological indicators: By using spores of Bacillus Stereothermophilis. Spores are impregnated onto paper strip. It is dried & kept in an autoclave. After Sterilization these paper strips are inoculated into a suitable medium.
Stem jacketed autoclave:
This functions better than the simple autoclave. The article remains dry after Sterilization.
Pre-vacuum Sterilization:
This is most commonly used in hospitals. Here the air is removed by suction to create vacuum and then the autoclave is heated up. The temperature required is 134 oC for 3 minutes.
Central Sterile Supply Department
(C.S.S.D.)
Definition: It is services that fulfill the needs of hospitals or a group of hospitals for the supply of sterilized materials.
Objective for C.S.S.D.:
1) To supply sterile article towards casualty & O.T.
2) To reduce the workload on the nursing staff
3) To ensure quality control
4) To Sterilize used articles & to destroy infected materials.
Significance: It minimizes the risk of infection in hospital and efficient working in seen because; Sterilization is performed in a single unit.
Radiation:
Ionizing Radiation: Here g-rays, x-rays and accelerated electrons are used. These are lethal to all cells.
Uses: To sterilize plastic catheter, disposable syringes.
Non-Ionizing radiation: Which is a method of disinfection, which is achieved using UV, IR rays. Optimum bactericidal effect is seen at a wavelength of 240-280 nm; which is produced by mercury lamps.
Uses: In the treatment of air, water, thin films & surfaces such as Lab safety cabinet.
Filtration:
Here Sterilization in achieved by removal of organism & not by destruction or killing. The factors effecting filtration are, the no. of microorganisms, the types present, the composition & pore size of filters.
Types of filters:
a) Earthenware-candle filters
b) Asbestos Seitz filters
c) Sintered glass filters
d) Membrane filters
Membrane filters are most commonly used. This is of two types
1) Old membrane filters: Gradacol
2) Modern membrane filters: Millipore
These are more advantageous, because they are available in different pore sizes, less absorptive & filtration rate is high.
Filtration in achieved by using
1) Positive pressure: Here the solution is pushed through the filter paper & the device used is known as syringe filter.
2) Negative pressure: Where a suction apparatus is used for negative pressure.
Uses: It is used for filtration of sugar solutions, serum, toxin & it is also used for bacterial counting.
Disinfection by filtration: It is done by using air filters, known as HEPA [High Efficiency Particulate Air] filters, which is used in hospitals, in O.T. & I.C.U. so that air is free from pathogenic organisms.
Chemical agents or chemicals that are used to control microorganisms on body surface & on inanimate objects are called as disinfectants.
Factors influencing the performance of chemicals used as disinfectants are,
a) Conc. Of disinfectant
b) No. of types and location of microorganism
c) Temperature & pH
d) The presence of organic & other interfering substances.
Qualities of an ideal disinfectant
a) It should be able to destroy all forms of microorganisms within a given practical period of time
b) It should be active in the presence of organic matter.
c) It should have high penetrating power & speedy in action.
d) It should be non-irritating, non-allergic & non-toxic.
e) It should be readily soluble.
f) It should be readily available and cheap.
Types:
Group 1: Alcohol group
Eg: Isopropanol, ethanol & methylated spirit.
The optimum bactericidal effect is seen at a con. of 70-90% care should be taken because it is highly inflammable.
Group 2: Aldehyde group.
Eg: Glutaraldehyde, formaldehyde.
Glutaraldehyde has a broad-spectrum action against vegetative bacteria, virus & fungi. It is used to disinfect equipment like endoscopes.
Disadvantage: It is highly irritant to the eyes, skin & respiratory mucosa.
Group 3: Biguanides.
Eg: Chlorhexidine
It has low irritancy & toxicity. It is commonly used as a disinfectant for skin & mucosa. It is often combined with a compatible detergent for hand washing or with an alcohol for hand rubbing.
Group 4: Halogens
Eg: Chlorine, Iodine, hypochlorite.
Hypochlorites are broad-spectrum, inexpensive, chlorine releasing disinfectants. These are the disinfectants of choice for viruses including Hepatitis B.
It is widely used as disinfectants in laboratories. Chlorine is used to disinfect drinking water. Iodine like chlorine gets inactivated in presence of organic matter. It has got additional disadvantage like staining hypersensitivity. Iodophore is used as disinfectant. It has got Iodine in combination with anionic detergents.
Group 5: Phenols-These are the coal tar derivatives after distillation.
Eg: Phenol, Lysol, dettol.
Mode of action: By protein denaturation.
Uses: It is used as a disinfectant.
3% Lysol & 5% phenol is used as disinfectant. 0.5% phenol is used as preservative in vaccine; Dettol is used as a skin disinfectant.
Group 6: Oxidizing agents.
Various agents including chlorine dioxide, per acetic acid, hydrogen peroxide are good anti-microbial agents but they are corrosive to skin & metals.
Heavy Metals:
Salts of heavy metals have a greater action. Salts of silver, copper, mercury are used as disinfectants.
Group 7: Surface-active agents.
They are widely used as wetting agents, detergents & emulsifiers. They are of 4 types.
1) Anionic; eg: soaps
2) Cationic; eg: quaternary ammonium compounds
3) Non toxic; eg: Tween 80 – These are used as emulsifiers
4) Amphoteric; eg: Tego compounds, which are rarely used.
Sterilization by gaseous process:
1) Using ethylene oxide. This is a highly penetrative, non-corrosive, microbicidal gas. Sterilization is usually carried out at temperature below 60 oC in a condition of high relative humidity. Care should be taken because of the toxicity to the personnel, flammability and explosive risk.
Uses: It is used in the industry for Sterilization of single use heat sensitive medical devices, such as prosthetic heart valve, plastic catheter & disposable syringes.
2) Low temperature steam and formaldehyde. Low temperature (73 – 80 oC) steam has a slow process of killing bacteria in a very slow rate. Formaldehyde at a high concentration has a sporicidal effect. Both together can add synergically and hence a low concentration of formaldehyde in the presence of steam can kill all microorganisms.
Uses: It is used for sterilizing heat sensitive instruments, which can withstand the temperature till 80 oC.
Disinfection by gasses:
This is achieved by using formaldehyde gas and it is used to disinfect rooms, heat sensitive equipment & a periodic decontamination of laboratory safety cabinet. Formalin vapor can be got by mixing of potassium permanganate with formalin. By using b-propiolactone gas has a very low penetrating power but it is very active against viruses.