1. Sterilization & Disinfection Physical Agents Hugh B. Fackrell PhysSter.ppt

2. 10/27/2015 2 Sterilization & Disinfection 4 Definitions 4 Bacterial Cell Death 4 Physical methods –Heat –Radiation –Filtration

3. 10/27/2015 3 Definitions 4 Sterilization –Complete Destruction of ALL microbes 4 Disinfection –Killing of pathogens on inanimate objects –physical or chemical 4 Antiseptic –A chemical agent for disinfection of living tissue –External skin, mouth vagina

4. 10/27/2015 4 Definitions 4 Bacteriocide –kill microbes –also germicide, fungicide, virucide 4 Bacteriostatic –Prevents or stops microbial growth –also fungistatic, virustatic 4 Aseptic(Asepsis) –Prevent contamination of person or object by microbes

5. 10/27/2015 5 Definitions 4 Sanitize –Removal of pathogens from inanimate objects –Mechanical or chemical cleaning –need not sterilize of disinfect 4 Contamination –Presence of living microbes on object

6. 10/27/2015 6 Definitions 4 Infection –Presence of living multiplying microbes in host tissues –often pathogenic

7. 10/27/2015 7 Bacterial Cell Death Time (min) Survivors (log) 0 1 2 3 4 5 1 log decrease = 90% kill 1 log Death rate (D) varies with bacteriocide D Time(min) to kill 90%

8. 10/27/2015 8 Physical Methods 4 Heat 4 Radiation 4 Filtration

9. 10/27/2015 9 Heat Sterilization 4 Factors –length of exposure –temperature 4 “Moist Heat” 4 “Dry Heat” Method of choice

10. 10/27/2015 1010 “Moist” heat 4 Effective quickly against most pathogens –low thermal death point –60-70% in 10 min 4 Pasteurization 60-70 o C Sanitizes 4 Hot water 60-100 o C Disinfects 4 Steam 15 psi 121 o C Sterilizes Endospores resist boiling for hours!!

11. 10/27/2015 1 Heat: Mechanism of Action 4 Inactivation of enzymes 4 Structural changes – coagulation of proteins 4 Oxidation –membrane disruption

12. 10/27/2015 1212 Pasteurization 4 Liquids 4 Minimal heat –145 o F (62.9 o C) - 30 min –162 o F (71.6 o C) - 15-30 sec 4 Flavour of drink preserved 4 Kills non spore forming pathogens – TB. Brucella, Salmonella, Streptococcus 4 Kills spoilage bacteria –Lactobacillus, Streptococcus

13. 10/27/2015 1313 “Flash” Pasteurization 4 72 o C - 15 sec 4 88 o C - 1 sec 4 90 o C 0.5 sec 4 96 o C 0.05 sec Heating coilCooling coil Raw milk

14. 10/27/2015 1414 Boiling Water 4 100 o C (212 o F) 10- 30 min 4 Disinfectant –spores & some viruses are resistant 4 Safe for clean instruments that DO NOT penetrate the tissue 4 Soiled items -add detergent

15. 10/27/2015 1515 Steam Under Pressure 4 Autoclave 4 15 psi 121 o C 250 o F 4 Time 15 min - dependent on volume 4 Steam displaces air & penetrates –Packing of autoclave critical! 4 Airlocks in packing –reduce effective temp & pressure

16. 10/27/2015 1616 Dry Heat 4 Longer & hotter 4 Oxidation 4 Carbonization – cotton plugs

17. 10/27/2015 1717 Types of Dry Heat 4 Oven –160-165 o C 2 hours –170-175 o C 1 hr –Glass, oils, waxes, glycerin 4 Incineration –destruction of pathological material 4 Flaming –used as aseptic techniques –scissors, scalpels, loops, needles

18. 10/27/2015 1818 Radiation Sterilization *Ionizing Radiation *Ultraviolet Radiation Point mutations involve changes in only one or a few nucleotides *

19. 10/27/2015 1919 Radiant Energy spectrum Ionizing Radiation UV light Increased energy

20. 10/27/2015 2020 Sterilization by Ionizing Radiation 4 Gamma Rays – Food –Plastics 4 Electron Beams –pharmaceuticals

21. 10/27/2015 2121 Ionizing Radiation 4 Creates free radicals 4 Causes breaks in two DNA strands –Results in deletions –Results in translocations

22. 10/27/2015 2 Free Radicals 4 High Energy Radiation –energy absorbed by atoms –energy added to electrons –electrons ejected from atoms –leaves ionized atoms & unpaired electrons Free radicals can breaks both phosphodiester bonds of a DNA helix

23. 10/27/2015 2323 Sterilization by Ultraviolet light 4 Circulating air 4 Surfaces –benchtops 4 Small samples of liquid Surfaces only poor penetration Photoreactivation white light reverses effect of ultraviolet

24. 10/27/2015 2424 Ultraviolet Radiation 4 Pyrimidine dimers –errors during repair 4 Peroxides, Free radicals –See ionizing radiation 4 Direct molecular damage –intense exposure

25. 10/27/2015 2525 Repair of thymine dimer GTA T GTC CATAACAG GTA TTGTC CATAACAG GTA GTC TT GT GTC CATAACAG A GT GTC CATAACAG TT A G

26. 10/27/2015 2626 Filtration Sterilization 4 HEPA filters –High Efficiency Particulate Air filter –Remove airborne microbes Filters particles >0.3  m –Operating rooms –“Clean rooms” instrument assembly –Burn patients –Immunocompromized

27. 10/27/2015 2727 Filter sterilization 4 Membrane filters –cellulose nitrate or synthetic polymers 4 Pore size –bacteria 0.22  m 0.45  m –viruses 0.01 mm 4 Sterilize heat or energy sensitive materials – bacterial media – drug solutions