2.  Killing or destroying A ll 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.

3.  Physical methods. ◦ Chemical methods. ◦ Physical methods. ◦ Are more commonly used, simple, economical and reliable.

4. prevention of microbial growth: important in: Food industry. Health care: nosocomial infections. Drinking water distribution systems other industrial processes : e.g., biofouling of oil pipeliness The objective is to render the articles non infectious.

5. Sterilization by Physical methods HeatRadiationFiltration

6.  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.

7. Heat Dry heat Moist heat

8. Dry heat Red hotFlaming Hot air oven Incineration

9.  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.

10.  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

12.  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.

13. oven for dry heat sterilization

14.  Bowei Dick test (Adhesive tabe).  Browne’s tube No 3. (Red ----- green).

15.  Is treating of an objects to heating over 250 until they become black.  Done for used equipment.

16. Moist heat Less than 100C At 100C Above 100C

17.  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.

18. pasteurization: 71ºC for 15 seconds (high temperature short time, HTST, method) significantly reduces microbial population size does not sterilize

19.  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.  1 st day kill vegetative bacteria and germinate sporulated one ---- Put on the bench.  2 nd day kills all vegetative bacteria ---- Put on the bench.  3 rd day insure complete sterilization.  Boiling (Inside the boiler,no shelves) ◦ At 100C for 30 min.

20.  It is a double jacketed steal cylinder or chamber supplied with: 1. Heater, water and pressure. 2. Has a thermometer and pressure supplier. 3. Control valves for steam, water and pressure

21. flow of steam through an autoclave:

22.  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.

23. an autoclave: steam under pressure enables temperature to reach 121ºC sufficient to destroy endospores as well as vegetative cells

24.  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.

25. removal of particles from liquids 0.2 μm pore size usual for sterilization filter sterilization: filtration also used to separate or distinguish organisms based on size 5 μm dia pores

26. 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”

27. conventional membrane filter: polymeric compounds such as cellulose acetate or cellulose nitrate pore diameter variable during production “sieve-like action”

28. thin, polycarbonate films (~10  m thick) pores formed by chemical “etching” consistent pore size useful for microscopy – filtered material is in a single plane on surface Nucleopore filter:

29. lab-scale membrane filtration:

30. syringe filter: small volumes system for larger volumes disposable, presterilized, assembled membrane filter units

31. ◦ 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

32. ultraviolet (UV) radiation:

33. Chemical methods of sterilization

34.  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.

35. examples of modes of action: alcohol - lipid solvent, protein denaturant hydrogen peroxide (H 2 O 2 ) – oxidizing agent triclosan (a phenolic) – disrupts cell membrane chlorine gas – oxidizing agent ethylene oxide – alkylating agent

36.  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.

37.  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.

38.  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.

39.  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.

40.  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%.

41.  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).

42.  Gention violet, Crystal violet and Eosine are very effective antiseptic.

43.  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.

44.  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.

45.  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.

46.  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.

47.  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.

48.  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.

49.  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.