Sterilization and disinfection

1. Sterilization and disinfection Prepared by Anfal Mohammed M.Sc.

2. Introduction • Sterilization A physical or chemical process that completely destroys or removes all microbial life, including spores. • Disinfection It is killing or removing of harmful microorganisms • Disinfectant Products used to kill microorganisms on inanimate objects or surfaces. Disinfectants are not necessarily sporicidal, but may be sporostatic, inhibiting germination or outgrowth • Antiseptic A product that destroys or inhibits the growth of microorganisms in or on living tissue. • Aseptic Characterized by the absence of pathogenic microbes.

3. Methods of Sterilization 1. Physical methods • Heat (thermal steri.) • Dry • Moist • Radiation (cold sterilization) • U.V. light • Ionizing radiation • Filtration 2. Chemical Methods

4. Methods of Sterilization Radiation: • U.V. light- Has limited sterilizing power because of poor penetration into most materials. Generally used in irradiation of air in certain areas eg. Operating Rooms and T.B. laboratories. • Ionizing radiation- e.g. Gamma radiation: Source Cobalt60 has greater energy than U.V. light, therefore more effective. Used mainly in industrial facilities e.g. sterilization of disposable plastic syringes, gloves, specimens containers and Petri Dishes.

5. Methods of Sterilization • Filtration (for sol. And gases) • May be done under either negative or positive pressure. Best known example is the membrane filter made from cellulose acetate. Generally removes most bacteria, but viruses and some small bacteria e.g.Chlamydias & Mycoplasmas may pass through. Thus filtration does not technically sterilize items but it is adequate for circumstances under which it is used. • Main use: for heat labile substances e.g. sera, antibiotics. The recommended size filter that will exclude the smallest bacterial cells is 0.22 micron

6. Methods of Sterilization • Sterilization by Heat: Most common method • Dry Heat • Simplest method is exposing the item to be sterilized to the naked flame e.g. Bunsen burner- for sterilizing bacteriological loops, knives, blades. • Hot air oven expose items to 160°C for 1 hour. It has electric element in the chamber as source of heat plus a fan to circulate air for even distribution of heat in chamber. Oven without fan is dangerous. • Used for Metals, Glassware, Ointment, Oils, Waxes, Powders i.e. items that are lacking water

7. Methods of Sterilization Moist Heat: Uses hot water. Moist heat kills microorganisms by denaturing proteins. 1. Boiling – quite common especially in domestic circumstances. 2.Tyndallization named after John Tyndall • Lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method.

8. Methods of Sterilization Moist heat: Tyndallization • The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again(4). • The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. • The procedure only works for media that can support bacterial growth - it will not sterilize plain water.

9. Methods of Sterilization Moist heat: 3. Pasteurization • It aims to reduce the number of viable pathogens in liquids so they are unlikely to cause disease • It uses heat at temperatures sufficient to inactivate harmful organism in milk. Does not achieve sterilization (does not destroy pyrogens). • Temperature may be 138°C for a fraction of a second (flash method), 71.7°C for 15-20 seconds or 62°C for 30 minutes.

10. Methods of Sterilization Moist heat: 4. Autoclaving – Standard sterilization method in hospitals. • The Autoclave works under the same principle as the pressure cooker where water boils at increased atmospheric pressure i.e. because of increased pressure the boiling point of water is >100°C. • The autoclave is a tough double walled chamber in which air is replaced by pure saturated steam under pressure.

11. The air in the chamber is evacuated and filled with saturated steam. The chamber is closed tightly, the steam keeps on filling into it and the pressure gradually increases. • The items to be sterilized get completely surrounded by saturated steam (moist heat) which on contact with the surface of material to be sterilized, condenses to release its latent heat of condensation which adds to already raised temperature of steam so that eventually all the microorganisms in what ever form –are killed. • The usual temperature achieved is 121 °C at a pressure of 15 pps.i. at exposure time of only 15-20 mins. By increasing the temperature, the time for sterilizing is further reduced.

12. Advantages of Autoclave: • Temperature is > 100°C therefore spores are killed. • Condensation of steam generates extra heat (latent heat of condensation). • The condensation also allows the steam to penetrate rapidly into porous materials. • Note: that autoclavable items must be steam permeable. Can not be used for items that are lacking water.

13. Monitoring of autoclaves • Physical- use of thermocouple to measure accurately the temperature. • Chemical- it consists of heat sensitive chemical that changes color at the right temperature and exposure time. • Autoclave tape • Browne’s tube. • Biological – where a spore-bearing organism is added during the sterilization process and then cultured later to ensure that it has been killed.

14. Sterilization by Chemical Methods • Useful for heat sensitive materials e.g. plastics and lensed instruments endoscopes. • Ethylene Oxide (EO) Chamber: • Ethylene oxide alkylates DNA molecules and thereby inactivates microorganisms. • Ethylene oxide may cause explosion if used pure so it is mixed with an inert gas e.g. Neon, Freon at a ratio of 10:90 • It requires high humidity and is used at relative humidity 50-60%, Temperature : 55-60°C and exposure period 4-6 hours (to reduce the sterilization time). • Activated alkaline Glutaraldehyde 2%: • Immerse item in solution for about 20 minutes if organism is TB. In case of spores, the immersion period is extended to 2-3 hours.

15. DISINFECTANTS •  Factors influencing activity of Disinfectants • Directly proportional to temperature. • Directly proportional to concentration up to a point – optimum concentration. After this level no advantage in further increases in concentration. • Time: Disinfectants need time to work. • Range of Action : Disinfectants are not equally effective against the whole spectrum of microbes. e.g. Chlorhexidine is less active against GNB than Gram Positive Cocci. • May be inactivated by • Dirt, organic matter • Proteins, Pus, Blood, Mucus, Faeces • Cork and some plastics. • Hypochlorites and Glutaraldehydeare more active against hepatitis viruses than most other disinfectants.

16. DISINFECTANTS Types of Disinfectants Phenol and phenolics • Phenol (carbolic acid) is seldom used today. Derivatives of the phenol molecule, however, are widely used. • Phenolics injure plasma membrane, inactivate enzymes, or denature proteins. They are stable, persistent, and are not sensitive to organic matter. O-Phenylphenol • It is the main ingredient in most formulations of Lysol. Hexachlorophene • It is main ingredient of a prescription lotion, pHisoHex, used in nurseries and for surgical and hospital microbial control procedures to control gram positive skin bacteria such as staphylococci and streptococci. • Excessive use can cause neurological damage. Triclosan • It is a widely used found in many household products. It has broad spectrum of activity, especially against gram positive bacteria. It is also effective against gram negative bacteria and fungi.

17. DISINFECTANTS Biguanides • Chlorhexidine, a member of the biguanide group, is not a phenol, but its structure and applications resemble hexachlorophene. It is frequently used for surgical skin preparation and surgical hand scrubs. Halogens • Iodine: is effective against all kinds of bacteria, many endospores, fungi, and some viruses. Its mechanism of activity may be its combination with the amino acid tyrosine in enzyme and cellular proteins. • An iodophore is a combination of iodine and an organic molecule. Iodophores do not stain and are less irritating than iodine. Examples are Isodine and Betadine. • Chlorine: is used as a gas or in combination with other chemicals. Chlorine gas is used for disinfecting local water supplies, swimming pools, and sewage. Sodium hypochlorite – ordinary household bleach- is good disinfectant. • Chloramines consist of chlorine and ammonia. They are more stable than most chlorine. The U.S. military uses tablets for field disinfection of water. • Chlorine dioxide in gaseous form is used for area disinfection, most commonly to kill endospores of anthrax bacteria.

18. DISINFECTANTS Alcohols • Both ethanol and isopropanol (rubbing alcohol) are widely used, normally at a concentration of about 70%. • Concentrations of 60% to 95% are effective. • They are bactericidal and fungicidal but are not effective against endospores or non-enveloped viruses. • Alcohols enhance the effectiveness of other chemical agents.

19. Heavy metals and their compounds • Tiny amount of heavy metals (e.g. silver and copper) are effective antimicrobials. A silver coin on an inoculated nutrient medium will inhibit growth for some distance. • 1% silver nitrate solution has been used to prevent gonorrheal ophthalmianeonatorum, which the infants might have contracted as they passed through the birth canal (recently been replaced by antibiotics). • Silver-sulfadiazine is used in wound dressings. Available as topical cream for use on burns. • Mercuric chloride is highly bactericidal, but is toxic and corrosive and is inactivated by organic matter. Organic mercury compounds such as Mercurochrome are less irritating and less toxic than inorganic mercury. • Copper sulfate is often used to destroy green algae in reservoirs or other water. • Zinc chloride is used in mouthwashes, and zinc oxide is used in paints as antifungal.

20. FILTRATION AND CLARIFICATION

21. DEFINITIONS • Filtration: The separation of solid from a fluid by means of a porous medium that retains the solid but allows the fluid to pass. • Clarification: This term is applied when solid do not exceed 1.0% and filtrate is the primary product. • Ultra-Filtration: Separation of intermicellar liquid from solid by the use of pressure on a semi permeable membrane. • Cake Filtration: If recovery of solid is desired, the process is called cake filtration.

22. Feed or Slurry: The suspension of solid and liquid to be filtered is known as the slurry or feed. • Filter Medium: The porous medium used to retain the solids is described as the filter medium. • Filter Cake: The accumulation of solids on the filter is referred to as the filter cake. • Filtrate: The clear liquid passing through the filter is the filtrate.

23. MECHANISM OF FILTRATION The flow of solid is resisted by the filter medium while the liquid is allow to pass. As the filtration proceeds, the retention of the solid on the filter media goes on increasing which acts as a secondary and some times more efficient filtering media.

24. FACTORS AFFECTING RATE OF FILTRATION • Area of filter surface. • Particle size of cake solids. • Pore size of filter media. • The resistant of the filter cake and filter media. • Viscosity of liquid to be filtered. • Temperature. • Pressure difference across the filter

25. EQUATION FOR DETERMINE THE RATE OF FILTRATIONRate = Driving force / Resistance Where: V= Volume of filtrate T= Time A= Filter area P= Total pressure drop through cake and filter media. µ= Filtrate viscosity α= Average specific Cake resistant W= Weight of dry cake solids. R=Resistant of filter medium.

26. FILTER MEDIA “The surface upon which solids are deposited in a filter is called the Filter medium”. PROPERTIES OF IDEAL FILTER MEDIA: Chemically inert. High retention power. Sufficient mechanical strength. Absorbs negligible amount of material. Resistant to the corrosive action of liquid. Selection of filter media depends on followings: Size of particle to be filtered. Amount of liquid to be filtered. Nature of product to be filtered. Purpose of filter.

27. TYPES OF FILTER MEDIA • Filter paper: Filter paper is a common filter medium since it offers controlled porosity, limited absorption characteristic, and low cost • It has different grades and qualities, different pore size such as coarse medium and fine. Disadvantages: • They shed very fine particle to the filtrate. • Absorb small quantity of liquid.

28. Woven Material: Cotton, silk, wool, nylon & glass etc. • Nylon cloth: • Superior to the cotton cloth. • Not affected by molds, fungus and bacteria. • Has negligible absorption properties. • It is extremely strong as compared to cotton cloth. • Woven wire cloth: • Made from stainless steel. • Easily cleaned. • Long lasting. • Resistant to the chemicals.

29. Cotton Wool: • Commonly used. • Small tough of cotton wool placed in the neck of funnel. • Glass wool: • Use for filtering highly corrosive chemicals. • May contaminate the filtrate with glass fibers

30. Asbestos: • Also used for filtering the corrosive liquid. • They impart alkalinity to the filtrate. • Alkaloids may get absorbed. • May contaminate the filtrate.

31. Membrane Filter: • These are very common among the ultra filtration methods. • Made up of cellulose, Polyvinylchloride, Nylon and other cellulose derivatives. • They are very fine having a very wide range of pore size from 8µ down to 0.22µ.

32. Advantages: • Bacteria are removed by sieving • Absorption of medicament is negligible • In every new operation, a new disc is used • Filtration is quite rapid • Don’t liberate particles to the filtrate. • Disadvantages: • Fine pores may get clogged easily • Soluble in certain organic solutions e.g. ketones and esters • Very brittle when dry.

33. Craft Paper: • Mainly used in plate and frame filters. • They offer controlled porosity • Limited absorption. • Quite cheap.  • Sintered Glass: • Consist of Pyrex glass powder. • Used for filtering parenteral preparations. • Useful for filtering the corrosive liquid and oxidizing agent. • Don’t shed particles. • Don’t absorbs any liquid. • Can be easily washed.

34. FILTER AIDS “The substances which when added to the liquid to be filtered, reduce the resistance of the filter cake and increase the filtration”. • Properties of Filter Aids: • Chemically inert • Low specific gravity • Insoluble in liquids • Form a porous cake • Free from impurities • Suitable particle size with irregular shape • Able to remain suspended in liquids • Free from moisture

36. DIATOMACEOUS EARTH PERLITE ASBESTOSE CELLULOSE CARBON

37. Precoating: It requires suspending the filter aid in a liquid and circulating the feed until the filter aid is uniformly deposited on the filter septum. Quantity required: • 5-15 pounds / 1002 feet of filter area. • Or filter cake thickness=1/16 to 1/8 inch Body Mix: • Direct addition of filter aid to the filter feed. • Ratio of filter aid=0.1 to 0.5 % of total liquid. • OR 1 kg filter cake : 1-2kg of filter aid.

38. FILTRATION EQUIPMENTS Selection of method and equipment required for filtration of a liquid depends on nature of the material and quantity to be filtered as well as the object of the operation.

39. FILTRATION EQUIPMENTS: • FILTER FUNNEL: • Funnels are conical shaped devices • Made up of Glass, Aluminum, Polythene, Stainless steel OR any other suitable material. • Neutral Glass made funnel are most commonly used. • White filter paper of suitable pore size is folded in such a way that it fits in the funnel.

40. BUCHNER FUNNEL: • Made up of porcelain. • It has a perforated plate. • Used for filtration under reduced pressure. • HOT WATER FUNNEL: • These are doubled wall funnels made up of metals. • Viscous substances such as liquid paraffin, Glycerin, Castor oil and fatty substances like wool fat, bees wax, ointments and cream etc are filtered easily from this filter. • Boiling water or steam is circulated in jacketed funnel.

41. FILTER PRESS: • It consists of hollow frames and solid plates. • Plates have grooved surface to support the filter cloth. • Each plate has an outlet for filtrate. • Frames are opened with an inlet for the liquid to be filtered.

42. Advantages: • Construction is very simple. • Used for coarse to fine filtration. • Operation and maintenance is easy. • Filter cloth can be easily replaced. • Disadvantages: • Not economical for filtration of small quantities • Leakage between the plates may take place • Suitable when the slurry contain less than 5% solids.

43. ROTARY FILTERS: • They are used when the proportion of solid content in slurry is 15 to 30%. • It consists of perforated matter drum wrapped with filter cloth. • Drum is partially immersed in the tank containing the materials to be filtered. • Drum rotates at the slow speed and creates vacuum due to which filtrate enters into the drum. • Filter cake deposits on outer surface of the filter media. • Cake is removed by scrapping with a knife.

44. Advantages: • Labor costs are very low. • Area of filtration is very high. • Thickness of the filtration cake is controlled by altering with the speed of rotation of drum. • Disadvantages: • Very expensive. • Can’t be used for slurries containing low percentage of solids.

45. FILTER LEAF: • It consists of frame in which drainage screen is enclosed. • Whole unit is covered with a filter media. • Outlet is connected to the vacuum pump. • Frames may be shaped in round, square or rectangular.

46. Advantages: • Liquid can be filtered form any vessel. • Filter cake can be removed simply by washing or blowing air. • It is very economical. • Disadvantage: • It is not effective when solid content in the liquid is more than 5%.

47. VACUUM FILTRATION : • Vacuum filtration is used primarily to collect a desired solid (cake filteration). • Vacuum filtration uses a Buchner funnel and a side-arm flask. • Vacuum filtration is faster than gravity filtration, because the solvent or solution and air is forced through the filter paper by the application of reduced pressure.

48. To perform a vacuum filtration: • Clamp the flask securely to a ring stand. • Add a Buchner funnel with a rubber funnel adaptor. • Obtain a piece of filter paper in the funnel that is small enough to remain flat but large enough to cover all of the holes in the filter. If necessary, you can cut a larger piece of filter paper down to size.

49. Place the paper in the funnel. • Connect the side arm flask to a vacuum source. Always use thick-walled tubes. • Wet the paper with a small amount of the solvent to be used in the filtration. This causes the paper to adhere to the plate and keeps materials from passing under the paper during filtration.

50. Turn on the vacuum source. • Pour the mixture to be filtered onto the filter paper. The vacuum should rapidly pull the liquid through the funnel. • Rinse the cake with a small amount of cold fresh solvent to help remove impurities that were dissolved in the filtrate.