(1) Specific Antimicrobials
(a) Methods by which live microorganisms may be removed from a surface, body, or solution, or at least prevented from replicating, include the application of heat, cold, desiccation, osmotic pressure, filtration, ionizing radiation, UV radiation, and chemical agents. By and large none of these methods, as discussed below, are effective in treating disease. Instead they are useful in preventing disease by preventing contamination.
(2) Specific antimicrobials (chemical agents, physical agents)
(a) Specific physical antimicrobials include:
(i) desiccation
(ii) filtration
(iii) freezing
(iv) heat
(v) moist heat
(vi) autoclaving
(vii) pasteurization
(viii) dry heat
(ix) ionizing radiation
(x) UV radiation
(b) Specific chemical antimicrobials include:
(i) alcohols
(ii) aldehydes
(iii) halogens
(iv) heavy metals
(v) organic acids
(vi) oxydizing agents
(vii) phenolic
(viii) quarternary ammonium compound
(ix) surfactants
(x) ultraviolet radiation
(xi) various gasses
(3) Death by heat
(a) Heat is:
(i) economical
(ii) easily controlled
(iii) kills by denaturing proteins
(iv) used either moist or dry
(b) In general, the greater the length of application of heat, the less heat required to do the same job, as can higher heats for shorter durations
(c) Heat sterilization is used in:
(i) canning food
(ii) autoclaving or baking laboratory supplies
(iii) autoclaving media
(iv) flaming inoculating instruments
(4) Moist heat
(a) Moist heat is:
(i) boiling water
(ii) unpressurized steam
(iii) pressurized steam
(iv) autoclaving
(v) pasteurization
(b) Unpressurized generally effective:
(i) Unpressurized moist heat is generally effective for killing most microorganisms
(ii) Exceptions include:
(1) extreme thermophiles
(2) endospores
(3) some viruses
(iii) Generally, though, boiling is effective for making food and water safe to eat or drink
(iv) An application of pressure is required to guarantee sterilization since the heat of steam generated by boiling water at normal atmospheric pressure is 100?C
(5) Autoclaving
(a) Pressurized moist heat:
(i) Autoclaving is a method of sterilization employing moist heat at temperatures in excess of that of boiling water at sea level (i.e., 100?C)
(ii) This is done by using high pressure steam
(b) Autoclaving tends to be used for sterilizing under all circumstances where the high temperatures employed (typically 121?C and higher) will not damage the item or substance being sterilized
(c) Achieving sterilization:
(i) Under circumstances where the item or substance is in direct contact with the steam, sterilization will occur after 15 minutes at 121?C
(ii) Higher temperatures may be employed for faster sterilization but only if the item or substance will not break down at these higher temperatures
(d) Efficacy requires penetration by steam:
(i) When heating larger, bulky items or high volume liquids it is necessary to autoclave for longer; Generally, though, once a moist item is up to temperature it takes only 15 minutes for complete sterilization to occur at 121?C
(ii) Anything that impedes the flow of moist heat (trapped air, materials impervious to moisture but not microorganisms, etc.) will not necessarily be sterilized by autoclaving
(6) Pasteurization
(a) Pasteurization is a much-less extreme use of moist heat than autoclaving
(b) Pasteurization does not sterilize nor even disinfect
(c) Thermodurics are resistant:
(i) This is because of the existence of thermoduric microorganisms, ones that are resistant to the application of modest amounts of moist heat for modest lengths of time
(ii) Fortunately, thermoduric microorganisms tend to be neither pathogenic nor excessively psychrotrophic
(7) Dry heat
(a) Dry heat kills by oxidizing organic compounds (i.e., it burns things)
(b) Higher temperatures required:
(i) Sterilization employing dry heat requires much higher temperatures then moist heat
(ii) Nevertheless, application of dry heat is very useful when sterilizing materials that are resistant to these high temperatures
(c) Direct flaming employs dry heat (e.g., such as the flaming of a loop)
(d) Example: baking in oven:
(i) Hot-air sterilization is also known as baking in an oven
(ii) Effective sterilization typically conservatively occurs by two hours at 170?C
(8) Filtration (filter sterilization)
(a) Filtration is generally employed when sterilizing or disinfecting a solution that is heat-labile
(b) Alternative if inconvenient to heat:
(i) Some substances, such as some biological molecules or air, are difficult or inconvenient to heat
(ii) These substances may, instead, be filtered
(c) Membrane filters are routinely used for filtering liquids
(d) Various filters (especially HEPA or High-Efficiency Particular Air Filters) are used to remove microbes from the air ventilating especially clean environments such as tissue culture hoods
(e) Note that employing filtration does not necessarily guarantee sterility unless filters which trap even the smallest of microorganisms are employed
(f) Small pores = low flow rate:
(i) Note that the down side of using very fine filters is that this impedes flow through
(ii) Consequently, there often is a trade-off between rate of flow through the filture and degree of filtration achieved
(9) Freezing
(a) Ice crystal disruption:
(i) Freezing kills by ice crystal disruption of cells and molecules
(ii) Freezing slowly is generally more harmful than rapid (or flash) freezing
(b) There are a number of methods by which microorganism viability is maintained through freezing such as suspension in glycerol or 10% DMSO
(10) Desiccation
(a) Loss of water:
(i) Desiccation is the loss of water, i.e., drying
(ii) The verb, to desiccate, means to remove or lose water
(b) Variation in resistance to:
(i) Not all microbes in all settings are equally resistant to desiccation
(ii) Some bacteria are much more resistant that others (e.g., Mycobacterium tuberculosis)
(iii) Many viruses and endospores are very resistant to desiccation
(iv) The presence of capsules can increase resistance to desiccation
(c) Within various body substances (e.g., pus, mucus, feces) microbes tend to be much more resistant to desiccation
(11) Ionizing radiation
(a) Forms free radicals from water:
(i) Ionizing radiation, generally, is high energy radiation
(ii) Ionizing radiation acts by ionizing water, which forms toxins (radicals) that are highly reactive with and destructive of biological molecules such as DNA
(b) It is important to keep in mind when selecting a source that the ionizing (and therefore killing) power of a given form of radiation may be offset by a lack of tissue penetrating power
(12) UV radiation (ultraviolet radiation)
(a) Ultraviolet (UV) light (or radiation, same thing) is damaging to DNA (especially at the 260 nm wavelength)
(b) UV light is not very penetrating; It is, however, useful for keeping UV resistant surfaces disinfected
(13) Phenolics
(a) Derivatives of phenol (a.k.a., carbolic acid)
(b) Phenolics act by disrupting membranes and denaturing proteins
(c) Phenolics are especially good at disinfecting in the presence of biological fluids such as pus, saliva, and feces
(d) They are long lasting and stable
(e) Cresols are an important group of phenolics and are the active ingredient in Lysol?
(f) Chlorhexidine is a related compound that, when combined with detergent or alcohol, is used as a surgical scrub
(14) Halogens
(a) Two halogens, i.e., the second from right on the chemical periodic table, serve as effective, commonly employed antiseptics or disinfectants
(b) These are iodine and chlorine
(15) Iodine [tincture, iodophore]
(a) Molecular iodine damages microorganisms via protein denaturation
(b) One means by which iodine is employed is as a tincture, an aqueous alcohol solution (a tincture)
(c) An iodophor is a tincture of iodine which has been combined with an organic compound that causes its (iodine's) slow release
(d) Betadine? and Isodine? are brand names of iodophors
(16) Chlorine [hypochlorite ion]
(a) Hypochlorite ion:
(i) The hypochlorite ion (OCl-) is made by bubbling chlorine gas in water as well upon the dissolving of various salts (Ca(OCl)2 and NaOCl, for example)
(ii) The hypochlorite ion is a strong oxidizing agent
(b) Hypochlorite ion is the active ingredient of Clorox? bleach
(17) Halogen-based water purification
(a) Gaseous chlorine is used extensively to purify water
(b) Bleach water purification:
(i) Adding two to four drops of Clorox? bleach, or equivalent, to a liter of water (8 drops per gallon or 16 drops per gallon of cloudy water) and letting it sit for 30 minutes is considered a safe emergency water treatment
(ii) Let the water sit for longer if temperatures are cold
(iii) Note, however, be sure to use unscented Clorox? because the scent of scented Clorox? is poisonous (p. 5 of Jan & Wong, 1999, Y2K Emergency Preparedness Handbook, Ten Speed Press, Berkeley, California)
(c) Iodine, too, can serve as a water treatment product
(18) Alcohols
(a) Alcohols act by disrupting lipid bilayers and denaturing proteins
(b) Alcohols act rapidly
(c) Alcohols easily remove themselves from the site of use via evaporation
(d) Alcohols have a cleansing effect by dissolving lipids so that they may be picked up by wiping
(e) Alcohols should not be applied to wounds since they can cause tissue damage
(f) Water solutions work best:
(i) Alcohols work best as 60 to 95% solutions with water
(ii) "Some water must be present for alcohols to disinfect because they act by coagulating (permanently denaturing) proteins, and water is needed for the coagulation reactions. Also, a 70 percent alcohol-water mixture penetrates more deeply than pure alcohol into most materials to be disinfected." (p. 334, Black, 1996)
(iii) Isopropanol (rubbing alcohol) is superior to ethanol for disinfecting
(g) Typical spectrum of activity:
(i) Like disinfectants in general, alcohols are ineffective against endospores or many viruses
(ii) "Alcohol disinfects but does not sterlize skin because it evaporates quickly and stays in contact with microbes for only a few seconds. It also does not penetrate deeply enough into pores in the skin. It kills vegatative microorganisms on the skin surface but does not kill endospores, resistant cells, or cells deep in skin pores." (p. 340, Black, 1996) (emphasis mine)
(19) Heavy metals [heavy metal compounds]
(a) Metallic silver, mercury, copper, and zinc as well as salts of these metals (i.e., silver nitrate, mercuric chloride, copper sulfate, zinc chloride, and zinc oxide) have antimicrobial activities and are used for various purposes
(b) Silver nitrate/anti-eye gonorrheal:
(i) Only silver nitrate has a recent history of use in humans as an anti-gonorrheal eye treatment of newborns
(ii) "A few drops of silver nitrate solution were placed in the baby's eyes at the time of delivery to protect against infection by gonocci entering the eyes during passage through the birth canal. For a time, many hospitals replaced silver nitrate with antibiotics such as erythromycin. However, the development of antibiotic-resistant strains of gonococci has led some localities to require the use of silver nitrate, to which gonococci do not develop resistance." (p. 339, Black, 1996)
(20) Surfactants [surface-active agents, soaps, detergents]
(a) Soaps are Na+ and K+ salts of fatty acids
(b) Soaps typically are good degerming agents
(c) Not germicidal:
(i) Soaps, however, are also typically not very germicidal
(ii) However, free fatty acids are considered, in some circumstances, to be natural antimicrobials and are found, for example, on the surface of the skin
(d) Detergents are synthetic, soap-like organic compounds
(e) Varying germicidalness:
(i) Detergents tend to vary in terms of their germicidal activity
(ii) That is, they are not universally as poor germicides as are soaps
(21) Quats [quaternary ammonium compounds]
(a) Cationic detergents:
(i) Quats are cationic detergents
(ii) They are ammonium ion derivatives
(b) Mostly effective killers:
(i) Quats kill just about everything except:
(ii) endospores
(iii) Mycobacterium tuberculosis
(iv) Pseudomonas spp. (some Pseudomonas spp. can even grow in solutions of quats, subsisting on them)
(c) Neutralized by numerous substances:
(i) In contrast to phenolics, quats are not very effective in the presence of organic compounds
(ii) Quats are neutralized by:
(1) soaps
(2) various other (anionic) detergents
(3) the fibers of cotton, gauzes, and bandages
(d) Brand names quats
(i) Zephiran? is a brand name for the quat benzalkonium chloride
(ii) Cepacol? is a brand name for the quat cetylpyridinium chloride
(22) Organic acids
(a) Various organic acids and their salts are common antimicrobials used in foods
(b) These include:
(i) sorbic acid
(ii) benzoic acid
(iii) calcium propionate
(23) Aldehydes [formaldehyde, formalin, glutaraldehyde]
(a) Aldehydes employed to control microbial growth include formaldehyde and glutaraldehyde
(b) These are very potent antimicrobials, especially glutaraldehyde
(c) Aldehydes kill microorganisms by inactivating proteins
(d) Formalin:
(i) Formalin is what a 37% solution of formaldehyde in water is called
(ii) Formalin is used for:
(1) preserving dead tissues
(2) making toxoids from toxins
(3) creating whole-killed viral vaccines
(e) Sterlization:
(i) Glutaraldehyde in a 2% solution can actually sterilize
(ii) Sporicidal activity, however, occurs only after many hours of contact
(24) Gaseous chemosterilizers [ethylene oxide, propylene oxide, beta-propiolactone]
(a) Gaseous chemosterilizers include such gasses such as:
(i) ethylene oxide
(ii) propylene oxide
(iii) beta-propiolactone
(b) Gaseous chemosterilizers are very penetrating
(c) Given sufficient length of exposure ethylene oxide can be sterilizing
(d) Ethylene oxide is used to sterilize disposable lab plasticware
(25) Oxidizing agents [hydrogen peroxide, ozone]
(a) Ozone and hydrogen peroxide are examples of compounds whose antimicrobial activity stems from their oxidation of cellular components
(b) Ozone is a gas which is used in some water purification systems
(c) Hydrogen peroxide is not a very effective open-wound antiseptic because the host catalase freed in the wound quickly inactivates hydrogen peroxide
(d) Deep wound oxygenation:
(i) Oxidizing agents are effective at oxygenating deep wounds (host catalase catalyzes the conversion of hydrogen peroxide to water and molecular oxygen)
(ii) This poisons the growth of obligate anaerobes such as Clostridium tetani
(26) Measures of thermal stability
(a) Measures of a microorganism's thermal stability include:
(i) thermal death point
(ii) thermal death time
(iii) decimal reduction time
(b) Thermal death point (TDP) is equal to the lowest temperature at which all the microorganisms in a liquid culture will be killed in 10 minutes
(c) Thermal death time (TDT) is a less arbitrary measure of time (than TDP is of temperature) equal to the length of time at a given temperature before all microorganisms in a liquid culture will be killed
(d) Decimal reduction time (DRT, D value) is the time in minutes at a given temperature until a culture is reduced to 10% of its initial viability
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