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

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Presentation on theme: "Sterilization & disinfection (control of microbial growth) Sterilization & disinfection (control of microbial growth) prepared by Dr.Ihsan Edan Alsaimary."— Presentation transcript:

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

2 Disinfection 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

3 Sterilization 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

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

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 washing Sepsis: Bacterial contamination Antisepsis: Reduction of or Inhibition of microbes found on living tissue Germincides, Fungicides, Virucides Physical versus Chemical disinfectants Static (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 (contd) 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 disinfectionin general vegetative cells in log phase are easiest to kill) Substrate effects (e.g., high organic content interferes with disinfectionstainless 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 Different Kinds of Bacteria Death 1. Bacteriostatic 2. Bacteriocidal 3. Bacteriolytic Log Cell # Time Total cell count Viable cell count

13 Resistance to Killing 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)

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 sterilizationthe steam must directly contact the material to be sterilized Pasteurization: destroys pathogens (Mycobacterium tuberculosis, Salmonella typhi, etc.) without altering the flavor of the fooddoes 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 171 o C, 60 minutes, dry heat 160 o C, 120 minutes, dry heat 149 o C, 150 minutes, dry heat 141 o C, 180 minutes, dry heat 121 o C, 12 hours, dry heat 121 o C, 15 minutes, moist heat (but dont start the clock until entire item is up to tempe.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 AgentMechanisms of ActionComments Moist Heat, boiling Denatures proteinsKills vegetative bacterial cells and viruses Endospores survive Moist Heat, Autoclaving Denatures proteins121°C at 15 p.s.i. for 30 min k ills everything Moist Heat, Pasteurization Denatures proteinsKills pathogens in food products Dry Heat, Flaming Incineration of contaminants Used for inoculating loop Dry Heat, Hot air oven Oxidation & Denatures proteins 170°C for 2 hours ; Used for glassware & instrument sterilization Filtration Separation of bacteria from liquid (HEPA: from air) Used for heat sensitive liquids Cold, Lyophilization (also desiccation) Desiccation and low temperature Used for food & drug preservation; Does not necessarily kill so used for Long-term storage of bacterial cultures Cold, Refrigeration Decreased chemical reaction rate Bacteriostatic Osmotic Pressure, Addition of salt or sugar Plasmolysis of contaminantsUsed in food preservation (less effective against fungi) Radiation, UV DNA damage (thymine dimers) Limited penetration Radiation, X-rays DNA damageUsed for sterilizing medical supplies Strong vis. LightLine-drying laundry

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 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

28 Heavy Metals Ag, Cu, Hg, Ni, Zn, Ag(NO 3 ) 2, CuSO 4, ZnCl 2, HgCl 2 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

29 Halogens 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

30 Chlorination 1744 discovered in Sweden 1810 identified as an element 1835 first used to control odors 1890s 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.

32 Alcohols 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.

33 Tincture 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.

34 Tincture II A tincture is a nonvolatile substance (medicine) presented as an alcohol solution, e.g., (for fun with numerous [sic])… 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.

35 Iodine & Iodophores

36 Phenol, Carbolic Acid, & Phenolics 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, dont 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

37 Oxidizing Agents 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.)

38 Gaseous Chemosterilizers Propylene oxide (C 3 H 6 O) Chlorine gas (Cl 2 ) Chlorine dioxide (ClO 2 ) Ozone (O 3 ) Ethylene oxide (C 2 H 4 O)… …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

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. (contd) After drying, place in porous package Expose package to gas [ ] of mg/L that is mixed with CO 2 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 Glutaraldehyde is capable of effectiving sterilizationat 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!

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 (contd) 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 days

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

45 Chemical Antimicrobials AgentMechanisms of ActionComments Surfactants Membrane Disruption; increased penetration Soaps; detergents Quats (cationic detergent) Denature proteins; Disrupts lipids Antiseptic - benzalconium chloride, Cepacol ; Disinfectant Organic acids and bases High/low pHMold and Fungi inhibitors; e.g., benzoate of soda Heavy MetalsDenature proteinAntiseptic & Disinfectant; Silver Nitrate HalogensOxidizing agent Disrupts cell membrane Antiseptic - Iodine (Betadine) Disinfectant - Chlorine (Chlorox) Alcohol s Denatures proteins ; Disrupts lipids Antiseptic & Disinfectant Ethanol and isopropyl PhenolicsDisrupts cell membraneDisinfectant Irritating odor AldehydesDenature proteins Gluteraldehyde - disinfectant (Cidex) ; Formaldehyde - disinfectant Ethylene OxideDenaturing proteinsUsed in a closed chamber to sterilize Oxidizing agents Denature proteinsHydrogen peroxide – antiseptic ; Hydrogen peroxide – disinfectan; Benzoyl peroxide – antiseptic

46 Evaluating Disinfectants 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

47 Disk Diffusion Method Hypochlorous acid Phenol Lysol Nisin Escherichia coli

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

49 Asepsis Sterilization Heat Steam Dry heat Chemical Gas Ethylene oxide Formalin Liquid Glutaric Aldehyde Radiation Gamma Disinfection Heat Boiling water Chemical Phenol 70% Alcohol Hexachlorophene Heat or chloramines solution Asepsis – Summary

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