Sterilization & disinfection (control of microbial growth) prepared by Dr.Ihsan Edan Alsaimary

1. Sterilization & disinfection(control of microbial growth)prepared byDr.IhsanEdanAlsaimary

2. Disinfection is the elimination of pathogens, except spores, from inanimate objects • Disinfectants are chemical solutions used to clean inanimate objects (physical processes, e.g., UV radiation, may also be employed to effect disinfection) • Germicides are chemicals that can be applied to both animate (living) and inanimate objects for the purpose of eliminating pathogens • Antiseptics are formulated for application to living tissue Disinfection

3. Sterilization is the total elimination of all microorganisms including spores • Typically the last things to die are the highly heat- and chemical-resistant bacterial endospores • Instruments used for invasive procedures must be sterilized prior to use • Moist heat or steam, radiation, chemicals (e.g., glutaraldehyde), and ethylene oxide (a gas) are employed for sterilization • Sterilization by autoclaving, which uses moist heat, is used in most hospital and microbiology laboratory settings Sterilization

4. STERILIZATION Process of removing microbes inclusive ofbacterial endospores DISINFECTION process of removing microbes except bacterial endospores DISINFECTION & STERILIZATION

5. Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)Degerming: Mechanical removal of microbes, e.g., from hands with washingSepsis: Bacterial contaminationAntisepsis: Reduction of or Inhibition of microbes found on living tissueGermincides, Fungicides, VirucidesPhysical versus Chemical disinfectantsStatic (stasis) versus Cidal (e.g., bacteriostatic versus bacteriocidal)

6. Pasteurization • Disinfection - not sterilization (removes unwanted organisms) • Mycobacterium tuberculosis • 63 C for 30 minutes • 72 C for 15 seconds (HTST) • Thermodurics • able to survive high temps.

7. Pasteurization • Involves heating a liquid to temps. sufficient to destroy vegetative organsims • Equipment is immersed in hot water bath at 63˚C for 30 min. • Is disinfection, not sterilization. The moist heat coagulates the cellular protein of microbes. Spores are not destroyed d/t being more resistant

8. Pasteurization (cont’d) • Prepare equip. by disassembling, washing in detergent, and then rinsing • After pasteurization remove equip. and place in drying cabinet • When dry, assemble, package and date equipment • Advantages of pasteur. include: safe for plastics, less expensive than chemicals, no employee exposure to chemicals

9. The Ideal Disinfectant • Resistant to inactviation • Broadly active (killing pathogens) • Not poisonous (or otherwise harmful) • Penetrating (to pathogens) • Not damaging to non-living materials • Stable • Easy to work with • Otherwise not unpleasant

10. Disinfectant Performance… • Is dependent on Disinfectant concentrations • Is dependent on length (time) of administration • Is dependent on temperature during administration (usual chemical reaction 2x increase in rate with each 10°C increase in temperature) • Microbe type (e.g., mycobacteria, spores, and certain viruses can be very resistant to disinfection—in general vegetative cells in log phase are easiest to kill) • Substrate effects (e.g., high organic content interferes with disinfection—stainless steel bench easier to disinfect than turd) • It is easier (and faster) to kill fewer microbes than many microbes

11. Cleansing Cleansing is the removal of soil or organic material from instruments and equipment & may be done, clinically, in four steps: • Rinsing the object under cold water • Applying detergent and scrubbing object • Rinsing the object under warm water • Drying the object prior to sterilization or disinfection

12. Total cell count Viable cell count Log Cell # Time Different Kinds of Bacteria “Death” 1. Bacteriostatic 2. Bacteriocidal 3. Bacteriolytic

13. Gram-negative bacteria (with their outer membrane) are generally more resistant than gram-positive bacteria to disinfectants and antiseptics • Stationary-phase (I.e., non-growing) bacteria generally are more resistant than log-phase (I.e., growing) bacteria • Mycobacteria, endospores, and protozoan cysts and oocysts are very resistant to disinfectants and antiseptics • Nonenveloped viruses are generally more resistant than enveloped viruses to disinfectants and antiseptics • Organic matter (such as vomit and feces) frequently affects the actions of chemical control agent • Disinfectant activity is inhibited by cold temperatures • Longer application times are preferable to shorter • Higher concentrations, though, are not always preferable to lower concentration (e.g., alcohols) Resistance to Killing

14. cidal vs. static • Bactericidal - kills bacteria • Bacteristatic - inhibits bacterial growth • Fungicidal • Fungistatic • Algacidal • Algastatic

15. Factors that effect Antimicrobial Activity • 1. Temp • 2. Time • 3. Concentration of Antimicrobial agent • 4. Type of Microbe • 5. Activity of Microbe • 6. Presence of organic matter

16. Targets of Antimicrobial Agents • 1. Cell membrane • 2. Enzymes & Proteins • 3. DNA & RNA

17. Application of Heat • Heat is frequently used to kill microorganisms • Thermal death point (TDP) is the lowest temperature at which all bacteria in a liquid culture will be killed in 10 minutes • Thermal death time (TDT) is the length of time required to kill all bacteria in a liquid culture at a given temperature • Decimal reduction time (DRT) is the length of time in which 90% of a bacterial population will be killed at a given temperature (especially useful in canning industry) • Dry heat kills by oxidation (slow, uneven penetration) • Moist heat kills by protein coagulation (denaturation) so requires lower temperatures or shorter times, but the moisture must penetrate to pathogens to be effective (grease & oil can block)

18. Moist Heat • Moist heat kills microbes by denaturing enzymes (coagulation of proteins) • Boiling (at 100°C, I.e., at sea level) kills many vegetative cells and viruses within 10 minutes • Autoclaving: steam applied under pressure (121°C for 15 min) is the most effective method of moist heat sterilization—the steam must directly contact the material to be sterilized • Pasteurization: destroys pathogens (Mycobacterium tuberculosis, Salmonella typhi, etc.) without altering the flavor of the food—does not sterilize (63°C for 30 seconds) • Higher temperature short time pasteurization applies higher heat for a much shorter time (72°C for 15 seconds) • An ultra-high-temperature, very short duration treatment (140°C for 3 sec.) is used to sterilize dairy products

19. Sterilization Times • 171o C, 60 minutes, dry heat • 160o C, 120 minutes, dry heat • 149o C, 150 minutes, dry heat • 141o C, 180 minutes, dry heat • 121o C, 12 hours, dry heat • 121o C, 15 minutes, moist heat (but don’t start the clock until entire item is up to temp—e.g., large volumes fluid)

20. Radiation • 1. Ionizing Radiation • gamma rays & x-rays • penetrates most substances • Used on substances that could be damaged by heat • plastic petri dishes • plastic syringes • catheters • surgical gloves

21. Radiation • 2. Non-Ionizing Radiation • UV Light • does not penetrate plastic, glass or proteinaceous matter • Used to reduce microbial populations • hospital rooms • nurseries • operating rooms Thymine Dimers

22. Filtration • Removes microorganisms from solutions that might be damaged by heat • culture media • enzymes • vaccines • antibiotics

23. Filtration: Air & Fluids

24. Physical Antimicrobials

25. Surfactants

26. Soap & Detergents • Soaps are sodium or potassium salts of fatty acids, a natural product • Detergents, instead, are artificial surfactants • While soaps are always negatively charged, some detergents are negatively charged while others are positively charged • One example of a positively charged detergent are quaternary ammonium compounds (a.k.a., quats)

27. Quats are cationic detergents that act by disrupting lipid bilayers • Quats are bactericidal, fungicidal, viricidal (enveloped), and amoebicidal • Quats are most effective against Gram-positive bacteria • Quats do not kill endospores, Mycobacteria spp., nor non-enveloped viruses • Quats are rapidly inactivated by organics including cotton and soap • Zephiran  Benzalkonium chloride • Cepacol Cetylpyridinium chloride Quats

28. Ag, Cu, Hg, Ni, Zn, Ag(NO3)2, CuSO4, ZnCl2, HgCl2 • These metals (and metal ions) react with sulfhydral (–SH) groups of proteins, denaturing proteins • Silver nitrate is used to treat Ophthalmia neonatorum in newborns as caused by Neisseria gonorrhoeae • Oligodynamic action: the ability of very small amounts of heavy metals (especially silver and copper) to exert antimicrobial activity Heavy Metals

29. Halogens are the seventh (VII) column of the periodic table of elements • Two halogens are regularly employed as antimicrobials: Iodine and Chloride • Iodine:commonly used as an antiseptic against all microbes, fungi, and viruses • Iodine:It inhibits protein synthesis and oxidizes –SH groups of amino acids • Chlorine: Used as a disinfectant (10% bleach) • Chlorine: Hypochlorous acid (HOCl) is a product, formed in water, that is the active form of the disinfectant • Chlorine: Applied in treatment of drinking water, swimming pool, and sewage Halogens

30. Chlorination • 1744 discovered in Sweden • 1810 identified as an element • 1835 first used to control odors • 1890’s started to be used as a disinfectant • 1896 earliest recorded use in experiments on water supplies • 1897 used in England to sterilize water mains following typhoid outbreak • 1902 first continuous use in water supplies in Belgium • 1909 liquid chlorine (compressed gas) became commercially available • Subsequent rapid spread in use of chlorine throughout the world • WWI: Chlorine gas used as chemical warfare agent

31. Chlorination • Hypochlorite may either be added directly (i.e., in the form of bleach) or created within water by bubbling chlorine gas through the water • Chlorine gas - preferred for medium to large disinfection systems • Sodium Hypochlorite (liquid) - typically used for small disinfection systems and large swimming pools • Calcium Hypochlorite (powder, tablet) - typically used for private swimming pools • For water purification, do not use scented bleach • Bromine sometimes used as a less-smelly alternative • Hypochlorite is less effective in the presence of significant organic compounds “What is known as modern chemical warfare began during World War I. The first chemical agent to be used was large amounts of chlorine gas, about one hundred sixty tons, which was released from 6,000 pressurized cylinders into the wind by the Germans against the Allies. The chlorine floated in a huge clouds toward the Allies until it reached the Allied lines causing men to die from the effects of the chlorine gas. Because of the large amounts of gas released the chlorine caused large amounts of yellowish fluid to form in the lungs of its victim, also causing eye, nose, and throat burning before causing death by choking.” http://www.geocities.com/CapeCanaveral/Lab/4239/chemweapons/history.html

32. Aqueous ethanol (60-95%) and isopropanol are used as disinfectants • Effectively kill bacteria and fungi but not endospores nor nonenveloped viruses • Fast acting, no residue (evaporate away), no staining • But not very penetrating and no residual activity (once gone gone) • Exert their action by denaturing proteins and dissolving lipids • In tinctures, they enhance the effectiveness of other antimicrobial chemicals • Flammable; also may damage rubber, plastic, etc. Alcohols

33. “Formulae: Fresh juice of Organic Habanero peppers, New Mexico Jalapeno, African Bird peppers and Hatch Chili peppers. • Dosage: Five to thirty drops, three times daily.  CAUTION ~ EXTREMELY HOT!! • Therapeutic Action: Cayenne is the greatest herbal aid to circulation and should be used on a regular basis.  The extract is very concentrated and gets into the bloodstream quickly and makes it a perfect first aid remedy for heart attacks, stroke, fainting, shock, dizziness, hemorrhage, internal and external bleeding.  Use a few drops to 10 droppers full.  It has saved many lives. Tincture

34. “Formulae: Fresh Garlic Juice, Goldenseal root, Usnea lichen, Myrrh gum, Pine resin, Echinacea root juice, Tea Tree oil, Kelp, Black Walnut inner hulls, Oak galls and Cayenne pepper in 80% grain alcohol. • Dosage: Generally for external use but can be used in the oral cavity.  Soak a cotton swab in the tincture and scrub into the infected area, let air dry.  It has a burning sensation. If the wound is tender, just flush it with multiple droppers full of the tincture but no need to scrub it in. • Therapeutic Action: There has never been an infected occur when this formula has been used.  It's excellent for treating any cut or wound and it is anti-bacterial, anti-viral and anti-fungal.  The tree resins in this formula leave an invisible protective, anti-bacterial coating over the wound.  This formula was use on a man in England who had the top of his knee torn off in an automobile accident.  In 24 hours it literally glued his knee back together.  A nurse from Ireland on the scene said in all the years in the hospital, she had never seen such a severe wound close right up and heal, and with no infections.” Tincture II A tincture is a nonvolatile substance (medicine) presented as an alcohol solution, e.g., (for fun with numerous [sic])…

35. Iodine & Iodophores

36. Phenol (carbolic acid) and derivatives • Affect plasma membrane, inactivates enzymes, and denature proteins • Stable, persistant, and especially effective when dealing with disinfecting materials contaminated with organics… • … but leave residual films, can irritate skin, don’t kill endospores, and are corrosive to rubber and plastics • Some phenolics are mild enough for use as antiseptics while others are too harsh or otherwise dangerous to be employed on living tissue • Hexachlorophene, Triclosan, Lysol, soap Phenol, Carbolic Acid, & Phenolics

37. HOOH, hydrogen peroxide, is most common • HOOH is not a terribly effective disinfectant or anticeptic • This is because bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen peroxide • However, the oxygen released upon inactivation can help oxygenate deep wounds and thus kill strict-anaerobe contaminants, e.g., Clostridium tetani • Ozone and peracetic acid are also oxidizing antimicrobial agents • They exert their effect by oxidizing cell macromolecules (e.g., proteins, DNA, etc.) Oxidizing Agents

38. Propylene oxide (C3H6O) • Chlorine gas (Cl2) • Chlorine dioxide (ClO2) • Ozone (O3) • Ethylene oxide (C2H4O)… • …is used to sterilize heat- or moisture-sensitive items • …is used for items damaged by heat or moisture • …is not corrosive, not damaging to delicate instruments, microscopes, disposable plastic instruments and materials • …permeates porous materials • …dissipates rapidly from material • …but is costly, toxic, carcinogenic, explosive, and relatively lengthy process Gaseous Chemosterilizers

39. Ethylene Oxide • Is a toxic gas that is combined with moisture and heat to sterilize equip. • Effectiveness depends on equip. prep., gas [ ], humidity, and temp. • Works by affecting the enzymes, reproduction, and metabolism of microbes. • Prepare equip. by washing in detergent, rinsing, and allow to dry(ethylene glycol)

40. Et. O. (cont’d) • After drying, place in porous package • Expose package to gas [ ] of 800-1000 mg/L that is mixed with CO2 or freon (decr. explosion hazard), 49-57˚C, 30-60% humidity, for 3-4 hours • After EtO, aerate equip. in special cabinet for 12 hr. - several days, depending on material

41. Glutaraldehyde is capable of effectiving sterilization—at room temperature, even against endospores, and even in the presence of organics, but achieving sterilization requries many hours of exposure… and it is nasty stuff to work with! Glutaraldehyde

42. Glutaraldehyde • Chemical used to cold sterilize or disinfect equip. by immersion • “Cidex” is most common example • Can be alkaline or acid glutaraldehyde • Disassemble equip., wash in detergent, rinse, shake dry, and immerse in glut. • Alkaline glut. - disinfection after 10 min., sterilization after 10 hours • Acid glut. - disinf. after 20 min.

43. Glutaraldehyde (cont’d) • After immersion, rinse with sterile water or bleach solution and allow to dry in a drying cabinet • Dermal sensitivity or allergy to fumes with some people • Good for rubber and plastic, not electronic equipment • Life of product varies from 14 - 30 days

44. 2% GLUTARLDEHYDE Sterilization Disinfection 8-10 Hrs used for :- Pneumatic circuits. i.e.- ventilator tubing O2 masks ventury devices nebulizer chamber 15-30 Mts used for dis infecting endoscopes Respiratory tubing's

45. Chemical Antimicrobials

46. Phenol Coefficient… • …compares efficacy to that of phenol, with greater efficacy indicated with coefficient >1 • …Salmonella typhi and Staphylococcus aureus commonly used to determine coefficients • Filter-Paper/Disk Diffusion method… • …placement of disinfectant impregnated filter paper on well-inocated agar • Use-Dilution test… • …drying bacteria to surface followed by exposure to disinfectant and subsequent washing and inoculation of sterile broth Evaluating Disinfectants

47. Hypochlorous acid Phenol Nisin Escherichia coli Lysol Disk Diffusion Method

48. Chemical Antimicrobials * Type of Disinfectant: H = High level; I = Intermediate level; L = Low level

49. Asepsis – Summary