Disinfection And Sterlization

DISINFECTION AND STERLIZATION

PHYSICAL AGENTS Physical agents are usually preferred over chemical agents for performing sterilization. Heat is the method of choice wherever possible. Both moist as well as dry heat can be applied. Moist Heat This is heating in the presence of water. The best and widely used examples of this technique are holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and the 'flash' modification of the same wherein objects are subjected to a temperature of 71.1°C for 15 seconds. Tyndallization is another methodology in which steaming of the object is done for 30 minutes on each of the 3 consecutive days. The principle is that spores which survived the heating process would germinate before the next thermal exposure and would then be killed. Dry saturated steam acts as an excellent agent for sterilization because of: · High temperature · Wealth of latent heat · Ability to form water of condensation · Instantaneous contraction in volume that occurs during condensation. Superheated steam is not that effective because it is hotter than dry saturated steam and the process is akin to dry heat which is not as efficient as moist heat. Autoclaves are designed upon the principles of moist heat. The ideal time-temperature relationship in moist heat and dry sterilization processes are as follows: Process Moist heat (Autoclaving) Dry Heat

Temperature 121°C 126°C 134°C 160°C 170°C IS0°C

Steps of functioning of an Autoclave are: ¾ Put sufficient quantity of water in the cylinder ¾ Put the materials to be sterilized on the tray ¾ Start the heating

Holding period 15 minutes 10 minutes 3 minutes 120 minutes 60 minutes 30 minutes

¾ Screw tight the lid, keeping the discharge tap open ¾ Adjust the safety valve to the required pressure ¾ Allow the steam-air mixture to escape freely till all the air has been displaced. This can be tested by leading the escaping steam into a pail of water through a rubber tubing close the discharge tap when no more air bubbles come through ¾ Count the holding time from the time when the safety valve opens and the excess steam escapes ¾ Turn off the heater when the holding time is complete ¾ Allow it to cool, tilt the pressure gauge indicates that the pressure inside is equal to the atmospheric pressure ¾ Open the discharge tap slowly and let the air into autoclone. Low Temperature Steam with Formaldehyde Low temperature steam at 80°C was found to be more effective than water at the same temperature. The addition of formaldehyde to low temperature steam achieves a sporocidal effect and it has been found to be suitable for thermolabile equipment. Mechanism of Microbial Inactivation by Moist Heat Bacterial spores Non-sporulating bacteria . Denaturation of spore enzyme .Damage to cytoplasmic membrane · Impairment of germination · Breakdown of RNA · Damage to membrane · Coagulation of proteins · Increased sensitivity to inhibitory agents · Damage to bacterial chromosome · Structural damage · Damage to chromosome Dry Heat Dry heat is less efficient process than moist heat and bacterial spores are most resistant to it. Spores may require a temperature of 140°C for three hours to get killed. Dry heat can be used by following means: • Incineration • Red Heat • Flaming • Hot Air Sterilizers (Ovens) Mechanism of Inactivation: Microbial inactivation by dry heat is primarily an oxidation process. However, the possibility of DNA damage is also now incriminated as one of the mechanisms. Dry heat is employed for sterilization of glassware, glass syringes, oil and oily injections and metal instruments. The thermal death point is the time required to kill all the bacteria in a particular culture at a specified temperature. The decimal reduction time, also known as the DRT or D value, is the length of time needed to kill 90 per cent of the organisms in a given population at a specified temperature. Ionizing Radiations Ionizing radiations include X-rays, gamma rays and beta rays. These can induce single stranded and sometimes double stranded breakdown of DNA. Mechanism of Microbial Inactivation: These radiations induce defects in the microbial DNA. If no repair takes place, DNA synthesis is inhibited and errors in the protein synthesis manifest resulting into cell death. Spores are more resistant to ionizing radiations than non-sporulating bacteria. The ionizing radiations are used for the sterilization of single use disposable medical items. Ultraviolet Radiations Bacterial spores are more resistant to UV rays than the vegetative cells. Even viruses are often more resistant than vegetative bacteria. Whereas Sarcina lutea is highly sensitive to UV rays, Salmonella typhimurium is moderately sensitive and organisms such as Escherichia coli, Staphylococcus and Proteus vulgaris are mildly sensitive to the action of these rays.

Mechanism of Inactivation: Because of the presence of UV rays, the sunlight has got bactericidal activity. Sunlight can inactivate even spores provided the exposure is of prolonged duration. The desert surfaces are found to be sterile not only because of immense dry heat but also because of prolonged exposure to UV rays present in the sunlight Sterilization by Filtration Several types of filters available are: Unglazed ceramic filters: These are manufactured in different grades of porosity and used for large scale clarification of water. Asbestos filters: These filters have high adsorbing capacity but suffer from disadvantages of alkalinizing the solutions and possible carcinogenesis. Sintered glass filters: These contain finely powdered glass particles of different sizes-the size being adjusted according to the required pore size. These are widely used nowadays and consist of cellulose ester. Indicators of Sterilization Process The commonly employed methods are chemical indicators, autoclave tapes and thermocouples. Process Dry heat Moist heat Ionizing Radiations Filtration

Physical methods -Temperature recording chart -Temperature recording chart -Dosimeters -Bubble point pressure test

Chemical methods -Colour change indicators -Colour change indicators -Radiochromic chemicals -None

Biological test organism used B. subtilis var niger B. stearothermophilus B. pumilus Ser. marcescens, Ps. diminuta

CHEMICAL AGENTS chemical disinfectants are highly toxic for all types of the cells. Their efficacy, however, also depends upon the conditions in which they are made to work like: Concentration of bactericidal agents, Time of exposure, pH of the medium, Temperature of the medium, Nature of the targetted organisms and Presence of extraneous materials etc. These include: - Agents that Interfere with Membrane Function - Surface Acting Agents o Cationic Surface Acting Agents o Anionic Surface Acting Agents o Nonionic Surface Acting Agents - Agents that Denature Proteins - Agents that Destroy or Modify Functional Groups of Proteins o Heavy Metals o Oxidizing Agents o Dyes o Alkylating Agents - Sterilants IDEAL DISINFECTANT Properties of an ideal disinfectant: • Broad spectrum: Should always have the widest possible antimicrobial spectrum. • Fast acting: should have a rapidly lethal action on all vegetative forms and spores of bacteria and fungi, protozoa and viruses. • Not affected by physical factors: • Nontoxic • Surface compatibility • Residual effect on treated surfaces • Easy to use • Odorless: an inoffensive odor would facilitate its routine use. • Economical: cost should not be prohibitively high.

Evaluation of Disinfectants Phenol Coefficient Test: A phenol coefficient of 1.0 means that the disinfectant being compared has the same effectiveness as phenol. A coefficient less than 1.0 means it is less effective; a coefficient greater than 1.0 means it is more effective than phenol. Kelsey-Sykes Test: This test measures the capacity of a disinfectant to retain its activity when repeatedly used microbiologically. Filter Paper Method: This method uses small filter paper discs, each soaked with a different chemical agent. It is simpler than the determination of phenol coefficient. In Use Dilution Test: It uses standard preparations of certain test bacteria. These bacteria are added to tubes containing different dilutions of a chemical agent, the tubes are incubated, and then the tubes are observed for the presence or absence of growth. Agents that prevent growth at the greatest dilutions are considered most effective. COMBINED TREATMENTS Enhanced sterilizing activity can take place if two or more processes, chemical or physical, are employed together. There are various kinds of such treatments. • Thermochemical treatment • Chemical treatment and irradiation • Thermoradiation