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

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. DISINFECTION & 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 (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 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
Cleansing

12. Different Kinds of Bacteria “Death”
Log Cell #
Time
Total cell count
Viable cell count
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
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)
Sterilization Times

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
Agent
Mechanisms of Action
Comments
Moist Heat, boiling
Denatures proteins
Kills vegetative bacterial cells and viruses Endospores survive
Moist Heat, Autoclaving
121°C at 15 p.s.i. for 30 min kills everything
Moist Heat, Pasteurization
Kills 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 contaminants
Used in food preservation (less effective against fungi)
Radiation, UV
DNA damage (thymine dimers)
Limited penetration
Radiation, X-rays
DNA damage
Used for sterilizing medical supplies
Strong vis. Light
Line-drying laundry
Physical Antimicrobials

25. Surfactants

26. 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)
Soap & Detergents

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. Heavy Metals 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.”

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

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, 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. 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.)
Oxidizing Agents

38. Gaseous Chemosterilizers
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 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. and even in the presence of organics,
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 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
Agent
Mechanisms of Action
Comments
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 pH
Mold and Fungi inhibitors; e.g., benzoate of soda
Heavy Metals
Denature protein
Antiseptic & Disinfectant; Silver Nitrate
Halogens
Oxidizing agent Disrupts cell membrane
Antiseptic - Iodine (Betadine) Disinfectant - Chlorine (Chlorox)
Alcohols
Denatures proteins; Disrupts lipids
Antiseptic & Disinfectant Ethanol and isopropyl
Phenolics
Disrupts cell membrane
Disinfectant Irritating odor
Aldehydes
Denature proteins 
Gluteraldehyde - disinfectant (Cidex); Formaldehyde - disinfectant
Ethylene Oxide
Denaturing proteins
Used in a closed chamber to sterilize
Oxidizing agents
Denature proteins
Hydrogen peroxide – antiseptic; Hydrogen peroxide – disinfectan; Benzoyl peroxide – antiseptic
Chemical Antimicrobials

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
Evaluating Disinfectants

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

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

49. Asepsis – Summary