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Dis-infection and sterilisation
Dr. J. Gitaka 28th September, 2012
Dis
infe
ctio
nDisinfection 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
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
Disinfectant Performance…Is dependent on Disinfectant concentrationsIs dependent on length (time) of
administrationIs 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
Cle
ansi
ngCleansing 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
Ste
riliz
atio
nSterilization 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
Oth
er T
erm
s 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)
Exp
onen
tial D
eath
Time.
Log
Cel
l #
10
9
80°C
60°C
50°C
8
010 20 30 40
Time.
Log
Cel
l #
10
9
60°C
50°C
8
010 20 30 40
60°C
Different Kinds of Bacteria “Death”
1. Bacteriostatic
2. Bacteriocidal
3. Bacteriolytic
Log
Cel
l #
Time
Total cell count
Viable cell count
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
Res
ista
nce
to K
illin
g 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)
Che
mic
al
Ant
imic
robi
als
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
Sur
fact
ants
Soa
p &
Det
erge
nts 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)
Qua
ts 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
Hea
vy M
etal
s 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
Hal
ogen
s 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
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
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
Alc
ohol
s 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.
Tin
ctur
e “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.
Tin
ctur
e 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.”
Iodi
ne &
Iodo
phor
es
Phe
nol,
Car
bolic
Aci
d, &
Phe
nolic
s 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
Oxi
dizi
ng A
gent
s 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.)
Glu
tara
ldeh
yde
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!
Gas
eous
Che
mos
teril
izer
s 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
Phy
sica
l A
ntim
icro
bial
s
Agent Mechanisms of Action Comments
Moist Heat, boiling Denatures proteins Kills vegetative bacterial cells and viruses Endospores survive
Moist Heat, Autoclaving
Denatures proteins 121°C at 15 p.s.i. for 30 min kills everything
Moist Heat, Pasteurization
Denatures proteins 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
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)
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
Ste
riliz
atio
n T
imes 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)
Filt
ratio
n: A
ir &
Flu
ids
Eva
luat
ing
Dis
infe
ctan
ts 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
Dis
k D
iffus
ion
Met
hod
Hypochlorous acid
Phenol
Lysol
NisinEscherichia coli
Hands Spread Disease
Ack
now
ledg
emen
ts
http://www.sw.vccs.edu/rth/RTH_132/decont.ppthttp://www.nt.gov.au/powerwater/html/pdf/tennant_creek_chlorination.ppthttp://www.bugcruncher.com/ws/docs/lekcii/D&S_INQUAL_eng.ppt (nice overview of specific antimicrobials)http://www.delmarhealthcare.com/olcs/white/ppts/chapter%2021.ppt (nice overview of microbilogy in nursing)http://www.countyofkings.com/health/ehs/forms/FDHNDLR.ppthttp://bioeng.ttu.edu.tw/~chunyi/microbes/Chapter7.ppthttp://microbiology.okstate.edu/courses/micro2124/babus/Chpt7_files/chapter7.ppt
Chemical Antimicrobials
* Type of Disinfectant: H = High level; I = Intermediate level; L = Low level