Sterilization • Killing or destroying All forms of living microorganisms from objects. (bacteria and their spores). • Sterilization is an absolute term means - germ free objects. • Disinfection: • Is removing or reducing the number of the pathogenic microorganisms from objects. (Not absolute) • Unable to destroy spores and some could not kill non enveloped viruses.
Methods used • Physical methods. • Chemical methods. • Physical methods. • Are more commonly used, simple, economical and reliable.
prevention of microbial growth: • important in: • Food industry. • Health care: nosocomial infections. • Drinking water distribution systems • otherindustrialprocesses: e.g., biofouling of oil pipeliness • The objective is to render the articles non infectious.
Physical methods • Heat: • Exposure of the objects to heat will kills microbes by coagulation of protein, denaturation of enzymes and oxidation. • Filtration: • Sterilization through removing of microbes from fluids by exposing them to small size filter. This is used for heat sensitive fluids like serum, sugars and urea. • Radiation: • Exposure to radiation: This causes denaturation of proteins and enzymes.
Red hot (Reddening) • Exposure of wires and forceps to the Bunsen flame until it becomes red hot, then cooled down and used. • Used for wire loop, straight wire, forceps, scalpels and metal rods.
Flaming • Slowly passing of objects to the Bunsen flame to reduce the number of microorganisms. • The Bunsen flame give partially sterile area around it. This is used for sterilization of the mouth of bottle, flasks, containers and test tubes. • it is also used for the preparation of smears, cultivation of bacteria ands. subculture
Hot air oven • This is a metal chamber (instruments) consists of a heater, thermometer, thermostat, perforated shelves, door and timer. • It kills microbes by oxidation-reduction. • There are certain thermal death points and Thermal death times for the articles to be sterilized: • 160C for 60 min. • 170C for 40 min. • 180C for 30 min.
Check the efficiency • Bowei Dick test (Adhesive tabe). • Browne’s tube No 3. (Red ----- green).
Incineration (Burnning) • Is treating of an objects to heating over 250 until they become black. • Done for used equipment.
Less than 100C • Pasteurization of milk: • Holding method (65C for 30 min) • Flash method (72C for 15 - 20 sec) • Inspeciation: • Heating at 80C for 30 - 1hrs until coagulation of protein. • Used for preparation of Dorset egg medium and L.J medium for TB. • Preparation of vaccine: • By heating at 56C for 30-60 min.
pasteurization: • 71ºC for 15 seconds (high temperature short time, HTST, method) • significantly reduces microbial population size • does not sterilize
At 100 C • Steaming (Koch steamer): • Single exposure of the microbe to steam at 100C for 90 min (Shelves over the boiler). • Tyndalization: • Steaming at 100C for 30 min for 3 successive days. • 1st day kill vegetative bacteria and germinate sporulated one ---- Put on the bench. • 2nd day kills all vegetative bacteria ---- Put on the bench. • 3rd day insure complete sterilization. • Boiling (Inside the boiler,no shelves) • At 100C for 30 min.
Above 100C (Autoclaving) • It is a double jacketed steal cylinder or chamber supplied with: • Heater, water and pressure. • Has a thermometer and pressure supplier. • Control valves for steam, water and pressure
Above 100C (Autoclaving) • Depends on steam and pressure. • Steam is a hot sticky air able to penetrate through things. • Pressure will rise the temperature from 100C to 121C • Kills microbes and their spores by coagulation of protein and denaturation of enzymes. • Make complete killing of bacteria, their spores, fungi and their spores, parasites and viruses including Envelop and non Envelop virus. • Thermal death point and thermal death time: • 121C (15 bound or 1.1 bar) for 15 min. • Flash autoclaving at 134C for 4-5min.
an autoclave: • steam under pressure enables temperature to reach 121ºC • sufficient to destroy endospores as well as vegetative cells
Filtration • Sterilization by mechanical removal of pathogenic microbes by passing through filter. • Used for sterilization of heat sensitive fluids like serum, glucose, urea, and Amino acids. • Different filtration units is used according to the amount of filtrate and pores of the filter: • Seitz filter ---- use asbestos • Chamberland filter ------ use ceramic • Sintered filter ----- use glass filter. • Milipore or membrane filter ---- use filter paper.
filter sterilization: • removal of particles from liquids • 0.2 μm pore size usual for sterilization 5 μm dia pores • filtration also used to separate or distinguish organisms based on size
depth filter: • fibrous sheet or mat of randomly overlapping fibers of different substances (paper, glass) • can use as pre-filter to remove suspended particles • “trapping action”
conventional membrane filter: • polymeric compounds such as cellulose acetate or cellulose nitrate • pore diameter variable during production • “sieve-like action”
Nucleopore filter: • thin, polycarbonate films (~10 mm thick) • pores formed by chemical “etching” • consistent pore size • useful for microscopy – filtered material is in a single plane on surface
disposable, presterilized, assembled membrane filter units syringe filter: small volumes system for larger volumes
Radiation • Sterilization by radiation kills microbes by causing mutation to the cellular protein and disrupting cellular elements. • Used for plastic syringes, disposable plastic and dental e • 2 types: • Ionizing radiation: • Depends on ionization of water which forms highly reactive hydroxyl group react with cellular components specially DNA causing mutation. • E.g.: X-rays, Gamma radiation and high energy electron beams. • Have a wavelength less than 1 nm. • Non ionizing radiation: • using short wave length rays like Ultraviolet with a wavelength of 260 nm. • Causing mutation by making thymine dimers. • Used for sterilization of plastic and surgical room
Disinfection Chemical methods of sterilization
Disinfection: • Is removing of pathogenic microorganism or reducing their number on the exposed area. • Unable to destroy spores and some could not kill non envelop viruses. • Factors affects disinfection action: • Type of disinfecant used. • Concentration of disinfectant. • Type of microorganism. • Number of microorganism. • Time of exposure. • Temperature. • Presence of organic compound.
examples of modes of action: • alcohol - lipid solvent, protein denaturant • hydrogen peroxide (H2O2) – oxidizing agent • triclosan (a phenolic) – disrupts cell membrane • chlorine gas – oxidizing agent • ethylene oxide – alkylating agent
1. Phenolic group of disinfectant: • E.g.: Phenol crystal, Dittol, Lysol, Cresol. • Injuring lipid containing plasma membrane leads to leakage of cellular contents. • Remain active in the presence of organic materials, stable and persist for long period of time. • Suitable for disinfecting pus, blood and sputum. • Active against G+ve, G-ve, Mycobacterium & viruses.
2. Bisphenol: • Contains 2 groups of phenol. • Hexaclorophenol used for surgical and microbial control, Excessive use for infant could leads to neurologic damage. • Triclosan found in antimicrobial soaps, inhibits an enzyme needed for synthesis of fatty acid which affects the integrity of plasma membrane.
3. Biguanides: • E.g.: Chlorohexidine. • Broad spectrum activity as disinfectant of skin and mucus membrane due to it is ability to bind to mucus membrane. • Cause injury to plasma membrane.
4. Haloges: • Iodine have broad spectrum activity to many bacteria, spores, fungi and some viruses, it binds to certain amino acids of enzyme and proteins. • Iodophore (Betadine and Isodine) is non pigmented iodine comprise from iodine + organic materials to release the iodine slowly. Used for skin disinfection and wounds. • Chlorine in shape of gas or solution combined with water to give HOCl (Oxidizing agents). Used for swimming pools, drinking water and sewage.
5. Alcohols: • Causes denaturation of proteins, disrupt membrane and dissolve lipids. • They able to act and Evaporate (Volatile). • Used as skin disinfectant and in vein puncture in a concentration of 70%.
6. Heavy metals: • Silver, mercury and copper • Causes denaturation of proteins when bind to it. • Silver nitrate is used as a droper to avoid opthalmia neontam by N. gonorrhoea. • Silver sulfadiazine (Flamazine) is used in treating infection associated with burns. • Copper sulfate is used to destroy green algae (effective in one part/million of water).
7. Dyes: • Gention violet, Crystal violet and Eosine are very effective antiseptic.
8. Surface active agents: • Soap and other detergent make mechanical remove of microbe by scrubbing of dead tissue so reduce their number. • Quaternary ammonium compound are positively charged molecules kills G+ve bacteria. Affects plasma membrane and changes cell permiability. E.g.: Citerimide.
9. Aldehyde: • E.g.: Formaldehyde and Gultraldehyde. • Inactivate proteins by covalent cross-links with COOH, NH, OH and SH. • Used for disinfect hospital instruments, benches and room. E.g.: Formaldehyde and Gultraldehyde. • Inactivate proteins by covalent cross-links with COOH, NH, OH and SH. • Used for disinfect hospital instruments, benches and room.
10. Ethylene oxide gas: • Used to sterilize plastics in closed chamber similar to autoclave. • Denaturate proteins. • Bacteriocidal, fungicidal and kills viruses. • Used at high temperature 60C for 4-24 hrs. • Carcinogenic.
11. Peroxygens: • H2O2 is an oxidizing agents kills many microbes especially who fail to produce catalase enzymes (Anaerobic bacteria). Used as disinfectant for gas gangrene. • Ozone (O3) also toxic for microbes and used instead of chlorine in treating drinking water.
Tests of Disinfectant activity: • In-use test: • Used to determine the proper disinfectant concentration to be used and to check if the used disinfectant is working properly or not: • Take 1ml from used disinfectant to 9 ml of nutrient broth. Immediately transfer 0.02ml into 10 different area of well dried nutrient agar. • Incubate one plate at 37C for 3 days and the other at R.T for 7 days. • Growth in more than5drops- Failure of Disinfectant • No growth ------- good disinfectant • Growth in less than 5 drop --- success but needs increasing the concentration.
Continue….. • Phenol co-efficient test: • Serial 2 fold dilution of both tested disinfectant and phenol are inoculated with Salmonella typhi and immediately re-subcultured on agar medium at 0 time, after 5 minutes and after 10 minutes. • Good disinfectant gives real number after dividing the MIC of the disinfectant against the phenol. • 4/2= 2 Good • 2/4= ½ Bad disinfectant.
Continue…… • Time killing assay: • Used to determine the efficiency of disinfectant against bacteria during the times. • Make serial dilution of disinfectant and subculture the commonly isolated bacteria and immediate culture on agar medium at 0 time, after 5 min and 10 min. • Determine the last dilution of disinfectant that able to kill M.O in a shortest time. • Use 4X this concentration in your lab.