Methods of Microbial Control

     With the advent of the germ theory of disease, it became obvious that disease could be spread by organisms too small for the eye to see.  Pioneers such as Ignaz Semmelweis and Joseph Lister utilized techniques such as the washing of hands and disinfecting of surfaces to decrease the likelihood of  infection.  In time, hospitals, clinics, and laboratories began to adopt these methods and improve upon them.

     Methods used to control the growth of microbial growth can be placed into two broad categories, physical and chemical.  Physical methods either exclude microbes, or reduce their numbers in a solution, or on the surface of a fomite (any nonliving material which might come into contact with the individual) .  Chemical methods involve the application of specific chemical agents which inhibit growth or kill microbes on fomites or the surface of skin.  The selection of an appropriate technique is important, since many physical and chemical agents can cause damage to the cells and tissues of the individual as well as the microbe.

     Agents of microbial control either sterilize or disinfect.  Sterilizing agents kill all living things, thus removing the living source of contagion.  Disinfecting agents kill some microbes, but inhibit the growth of others.  Most techniques only provide disinfection.  Also, several factors  influence the effectiveness of any method of microbial control.  These include population size, susceptability of the microorganism to the agent, concentration of the dose used, and the duration of treatment.

Physical Methods

     Filtration is the passing of either a solution or gasses through a device which traps microbes on one side of a container or space, preventing them from passing to the other.  Filters are materials which have pores (openings) of varying sizes.  Particulate matter larger than the pore size in a filter is excluded from passage and is this physically excluded.  The earliest form of filter used in microbiological was cotton, a fiberous material derived from plants.  Cotton fibers form a densely packed matrix which offers a torturous path for particulate matter containing microbes to pass, while still allowing air to do so.  This is only true however, as long as a cotton plug, filter, or bandage remains dry, since water clings to each fiber allowing microbes unrestricted access.

     In most cases, cotton has been replaced as a filter by ceramic filters and synthetic plastics such as nitrocellulose which offer very small pore sizes (0.2 m to 0.45 m) without taking up as much space.  Since these materials are not fiberous, all but the very smallest microbes can be removed from a solution passing through them.  This solution, called a filtrate, is generally free from contaminants so long as the original pre-filtered solution did not contain organisms such as mycoplasma bacteria or viruses, both of which are smaller than most filters.  As a consequence, filtration should be considered an agent of disinfection rather than sterilization.

     Dessication (drying) is the removal of moisture from the body of an organism.  Many bacteria are very sensitive to water loss and can be killed simply by removal of water.  For example, Treponema pallidum, the agent of syphillis, is so intolerant to water loss that it will die within twenty seconds on the surface of a dry fomite.  The physical preservation of foodstuffs by drying has been practiced by humans for thousands of years and in most cases does reduce the number of potentially pathogenic microbes.  One process, called lyophilization or freeze-drying, is used to rapidly remove water from the body of an organism under very cold temperatures in a partial vacuum.  This process does not kill organisms such as bacteria, but does inactivate their metabolic processes.  Lyophilization is used to preserve living bacterial cultures for storage and transport.  To restore the freeze-dried cells, an individual has only to rehydrate them in a nutrient broth solution and incubate the culture at the optimum temperature for growth of the microbe.

     It is important to note, however, that not all microbes are killed or inactivated by dessication.  Bacteria which form spores such as members of the genera Bacillus and Clostridium, cyst-forming protists, and viruses can withstand drying, simply becoming inactive until moisture becomes available.  For this reason, dessication can only be considered a form of disinfection.

     Radiation describes a physical phenomenon which occurs when matter releases either energy, atomic particules, or both.  Radiation can affect the chemical makeup of the cell by altering or disrupting the structure of biological molecules.  Ionizing radiation strips electrons away from biological molecules.  Both gamma and X-radiation are ionizing forms.  Ultraviolet radiation is absorbed by the pyrimidine bases cytosine and thymine in DNA.  When two thymine or cytosine molecules lie adjacent to one another on a nucleoside, ultraviolet radiation with wavelengths between 250 nm and 280 nm causes them to have a greater affinity for one another than for their complementary adenines on the opposite nucleoside.  The two bond together, forming a dimer, which disrupts the normal sequence of nucleotide bases. This kind of mutation prevents the cell from producing proteins which may be necessary for normal metabolism to occur.  Some cells can repair this damage if exposed to visibile light through a process called photoreactivation (light repair), wherein the dimer is nicked by a restriction endonuclease, then cut away and replaced by DNA polymerase.  The new thymine or cytosine bases are then bonded to their complementary adenines or guanines by DNA ligase.  Since light repair can occur, the use of ultraviolet radiation has only disinfecting activity and cannot be considered a sterilizing agent.
     Excess heat energy can cause proteins to become denatured, meaning that they lose their normal three-dimensional shape.  Effective temperature for the reduction of microbes is measured as the thermal death point (TDP) of each organism, which is the temperature at which all growth stops.  Thermal death time (TDT) is the amount of time it takes to kill all of the microbes in a sample, and the decimal reduction factor (DRF) is the amount of time at a specific heat necessary to reduce the population of microbes in a sample tenfold.

     The most common methods of applying excess heat energy are flaming and incineration, which completely destroy all life.  Flaming of inoculating loops and needles, as well as the tops of glass culture tubes and flasks insures that no contaminating microbes can infect sterile media.  Applying dry heat by forcing hot air onto the surface of an object can be used in a similar fashion, though many spore formers are capable of withstanding this.

     The application of moist heat, such as boiling, steaming, and pasteurization (application of high heat to a solution for a short period of time), is also commonly used.  These methods work well for most microbes, but are incapable of killing organisms which are thermoduric (capable of withstanding elevated temperatures), or are spore formers.  For example, the spores of Clostridium botulinum, the bacterium which causes botulism, can be boiled for up to five hours and still remain viable.  The most effective application of moist heat is through the use of a device called an autoclave.  The autoclave works on the principle of saturated steam.  The inner chamber is raised to an air  pressure of
15 lb/inch2, then steam at a temperature of 121o C is injected.  The steam strikes the surface of the object to be sterilized and condenses into water as its excess heat energy is released.  This condensation creates a partial vacuum which draws more steam to the object.  Saturated steam is extremely effective as a sterilizing agent, at least 1500 times more effective than the application of dry heat.  Autoclaves are usually operated in cycles between 15 and 90 minutes, and can be used to sterilize glassware, surgical implements, soil, water, and microbiological media such as broths and agars.  They cannot, however, be used to sterilize hydrophyllic powders which would clump, or hydrophobic oils since microbes suspended in oils would only be subjected to dry heat.  Also, while contaminated bandages can be placed in an autoclave, the toxins or exoenzymes left behind by killed microorganisms such as Clostridium perfringens (the agent of gas gangrene) may still be capable of causing host cell damage, so these should be rinsed thoroughly with sterile water prior to reuse.

     All of the above physical means of control can be checked for effectiveness utilizing various bacteria as quality control agents.  Devices which emit ionizing radiation can be tested with Micrococcus radiouridans, U.V. devices with Bacillus pumilis, and heat disinfecting and sterilizing units such as hot-air ovens, pressure cookers, and autoclaves with Bacillus stearothermophilus.  These organisms are generally supplied to laboratories live or in ampules or tape strips, which can be placed in the control device.  After a normal operating cycle, the organisms are incubated in microbiological media.  If growth occurs, the device is not operating properly and should be repaired.  Quality control checks and maintenance are vital to the effective microbiological laboratory or health-care facility, and should be performed on a regular basis to prevent contamination and the spread of disease.

Chemical Methods

     Chemical agents for the control of microbial growth are either microbiocidal or microbiostatic.  Microbiocidal agents are sterilants which kill all living cells.  Microbiostatic agents kill some cells and inhibit the growth of others. The spectrum of activity exhibited by any microbiocidal or microbiostatic agent is an important factor in choice, and should be considered, along with potential harmful effects on the user.  An agent which kills staphylococci may be totally ineffective against mycobacteria, and would be useless in a tuberculosis ward.  Also, a broadly killing sterilant may release gasses which are toxic to patients and staff.  Most often, chemical agents which disinfect are utilized by clinics, hospitals, and laboratories.  While these agents do not sterilize, their toxicity is usually much less than that of a sterilant, and prevention of infection is stressed.

      There are four large categories for agents of chemical control.  Antibiotics are produced by microorganisms to kill or inhibit the growth of other microbes.   These agents are generally selectively toxic, and can be naturally produced, synthesized, or or semisynthetic.  Antiseptics are synthetic compounds which kill or inhibt the growth of microbes on the surface of the skin. Disinfectants are chemical compounds which kill or inhibit microbes on the surface of fomites.  Preservatives, such as sugars, salt, nitrates, nitrites, sulfate, and sulfites inhibit microbial growth in food, usually by producing osmotic environments which are unfavorable to microbial growth.  These can be further subdivided as high-, intermediate, or low level agents.  High-level germicides sterilize fomites, but are toxic to skin and mucus membranes.  Intermediate-level disinfectants and antiseptics kill and inhibit on fomites and skin, but can be toxic to the user at medium to high concentrations.  Examples include phenolics and halogens.  Low-level disinfectants, such as alcohols, hydrogen peroxide, heavy metals, and soaps kill some microbes but inhibit the growth of most.

High-Level Germicides
     These are called agents of cold sterilization, since no heat needs to be applied to increase their activity.  These are generally alkylating agents, which kill by adding ethyl or methyl groups to nucleic acids or proteins.  While the agents are capable of killing vegetative cells, spores, and inactivating viruses, some take up to several hours to complete their germicidal activity.

     Aldehydes, such as formaldehyde and gluteraldehyde, fix tissues by alkylating and forming cross-links between adjacent proteins.  They are commonly used as fixitive compounds for electron microscopy, preservatives of specimens and cadavers, in some synthetic plastic compounds, and can be used to sterilize anesthesia tubing and surgical implements. Aldehydes can fix living tissues such as mucus membranes and have the ability to vaporize or outgas from compounds containing them, so they should be handled with caution.  b-propiolactone is a liquid alkylating sterilant with a high boiling point (155oC).  It is generally used to sterilize bone used in grafts.  It quickly breaks down into nontoxic compounds when it comes into contact with organic matter, but can burn skin.

     Ethylene oxide (carboxide) kills vegetative cells and spores.  It is a liquid at temperatures below 10.8o C, but rapidly sublimates into a highly inflammable gaseous state above this temperature.  It is generally used in a chamber similar to an autoclave at 60o C for 1-10 hours, where it is mixed in a 9:1 ratio with carbon dioxide (90% CO2, 10% ethylene oxide), which reduces its toxicity, but also its inflammability.  Carboxide can be used to sterilize surgical implements and glassware, but these fomites must be allowed to degass before use, since residues can stimulate mutations in bacteria.

    Ozone (O3) occurs naturally in the upper atmosphere, where it serves to shield the surface of the earth from solar radiation, and is produced as an exhaust gas by vehicles and industry, acting as a pollutant in the lower atmosphere.  Applied properly in a chamber, ozone is a powerful oxidizing agent which kills cells and spores on the surface of glassware, surgical implements, and bandages.  An advantage of sterilization with this compound is that it outgasses quickly, leaving no toxic residues as can ethylene oxide and b-propiolactone.

Intermediate-Level Disinfectants and Antiseptics
     Phenol (carbolic acid) is one of the earliest disinfectant compounds to be used in health care facilities and laboratories.  Joseph Lister used atomized phenolic compounds in the 1860's to disinfect his surgery during invasive procedures as a means of cutting down on postoperative septic infections.  Phenol kills microorganisms by denaturing proteins and destabilizing cell membranes, is bacteriocidal, fungicidal, and virucidal at high concentrations, but is not effective against bacterial endospores, and is effective against many potential pathogens, including mycobacteria, staphylocci, streptococci, and gram-negative coliforms, such as E. coli.  It can be used to disinfect garbage cans, surgical operating facilities, laboratory equipment, feces, urine, and sputum, but it is very corrosive at higher concentrations and its fumes can be lethal.  Because of its toxicity, this compound is generally used as a solution between 2% to 5% in concentration, and many less toxic derivitives have been produced. The most common of th phenolic derivatives are called cresols and bisphenols.   Cresols are formed by adding methyl groups to phenol, and are used for the preservation of wood products, as compounds such as creosote (para-cresol). Bisphenols are produced by combining two phenol molecules.  Lysol™ is a combination of cresol and soap, which has about the same spectrum of activity as phenol, but is much less toxic to skin.  Other cresols include resorcinol, hexylresorcinol, and hexachlorophene.  Hexachlorophene soaps such as pHizoHex were once widely utilized as antiseptic soaps by health-care personnel for hand washing and the bathing of newborn infants, but have been discarded, since it was found that this phenolic could be absorbed through the skin and potentially cause birth defects.

     Since phenol has such a broad-spectrum of activity, it is used as a standard by which to judge how well other disinfecting compounds work.  The phenol coefficient (P.C.) is a mathematical value used to compare the effectiveness of a test disinfectant to that of phenol, and is derived from the following formula:

  dilution of a test disinfectant necessary to kill a standard population of bacteria
P.C. =                            dilution of phenol which has the same effect

     For example, a 1:250 dilution of a test reagent kills a standard population of S. aureus.  A 1:60 dilution of phenol kills the same size population.  To derive the P.C., we divide 250/60:

P.C. = 250/60 =4.2  Therefore the test disinfectant is 4.2 times as effective as phenol.

     Halogens are a family of elements with a high affinity for electrons.  This affinity makes them very reactive with biological molecules, and they can serve to disrupt enzyme activity, break down lipid structure, and produce oxidizing agents such as singlet oxygen (O).  The halogens most commonly used as disinfectants are chlorine and iodine.  Chlorine is used as a disinfectant only, either as a gas or in liquid form which is effective against many vegetative forms of microbes as well as some viruses such as HIV and hepatitis.  Commonly, chlorine is supplied in sodium hypochlorite (NaOCl) bleach, which is a combination of 94.75% water and 5.25% NaOCl.  It is used as a bleaching compound as well as a disinfectant for used hypodermic needles, in swimming pools, toilets, water supplies, and in sewage treatment plants.  It is inactivated by organic matter, and produces toxic fumes (the "mustard gas" used in World War I because of its yellow color) which can cause considerable damage to skin and mucus membranes with direct contact.  Iodine is lethal to all vegetative forms of microorganisms, can inactivate viruses, and is fairly effective in higher concentrations against endospores.  Pure iodine is caustic to tissues, so it is diluted with other compounds.  Tincture of iodine is produced by dissolving crystaline iodine in alcohol.  This solution is a good antiseptic for most minor cuts and scrapes, but it excites pain receptors at the site of an injury, and as the alcohol component dries, the iodine may concentrate and damage exposed tissues.  Iodophore compounds, such as Betadine™ and Wescodine™ are composed of iodine dissolved in mild detergent and alcohol.  These do not excite pain receptors as readily and can be used to clean and disinfect large areas of skin prior to invasive surgical procedures.

Low-Level Disinfectants
    Hydrogen peroxide is a good low-level disinfectant agent when used in concentrations of 3% or lower.  Higher concentrations are caustic to human skin, and cannot be used.  This compound is used as an antiseptic for the treatment of minor cuts and scrapes and as a bleaching agent.  When placed on the surface of an injury, hydrogen peroxide bubbles due to the release of the enzyme catalase from tissues, which break it down into water and oxygen.  This breakdown also releases the peroxide ion (O2-2), a strong oxidizing agent, and that the water released provides hydroxide ions which strip hydrogen from biological molecules.  Obligate anaerobic microbes are especially sensitive to hydrogen peroxide, since they do not produce catalase and the rapid release of oxygen gas inhibits their growth.

     Alcohols such as ethanol and isopropanol are effective antiseptics and disinfectants when used in concentrations between 70% and 80%.  Alcohols kill microbes by denaturing proteins, dehydrating (100% concentration), and as nonpolar solvents which disrupt the phospholipid structure of the cell membrane, but are relatively ineffective against spores and viruses.  Also, dehydration may actually be beneficial to some microbes, enhancing their survival by extracting extracellular water, so alcohols such as ethanol are normally used in lower concentrations.  Isopropanol is used as an antiseptic and to clean the epidermis prior to syringe and I.V. needle use.

     Heavy metals such as mercury, silver, and copper tend to combine with sulfur groups in the proteins of microbes, causing them to denature.  This oligodynamic activity makes the heavy metals useful in small concentrations.  These are some of the earliest used agents for the control of microorganisms.  Mercury, in the form of mercuric chloride, was used by the Greeks and Romans to disinfect skin.  This element is toxic in high concentrations, so it is commonly blended with other compounds such as iodine and alcohol in mercurochrome.  Silver, applied as silver nitrate (AgNO3) in a 1% solution, is commonly used to inhibit the growth of Neisseria gonorrhoeae in the eyes of newborn infants, a condition called neonatorum opthamalia (though many hospitals now use antibiotic ointments such as erythromycin or tetracycline), which can lead to blindness and is acquired as the organisms are transmitted to the infant as it passes through the birth canal.  Copper, in the form of copper sulfate, is used to limit the growth of algaes in ponds and lakes, and as an antifungal compound for use on plants.

     Detergents and soaps are composed of lipids and compounds having basic pH, such as sodium hydroxide.  These break up surface tension, act as wetting agents which release particles attached to the surface of objects, and destabilize the phosphate portions of the plasma membrane of microorganisms.  Detergents are either anionic or cationic, releasing negatively or positively- charged ions into solution.  Anionic forms are weakly active against gram-positive bacteria but tend to repel negatively-charged cells, thus they are generally used in the production of iodophore compounds.  Cationic forms are attracted to bacterial cells and are bacteriostatic, while remaining relatively mild to the surface of skin.  Quartenary ammonium compounds (QUATS) are cationic detergents which contain one or more long-chain akyl groups.  Quats such as benzalkonium chloride (Zephiran™) have broad-spectrum inhibitory activity against bacteria, fungi, and protozoa, are mildly antiseptic and disinfecting when used as cleaning agents for laboratory fomites and on the surface of skin, and remain active after drying, but lose much of their activity when mixed with soaps.  Cetylpyridium chloride (ceepryn) is the quat in Cepacol™ which is mildly antiseptic and safe for use on the mucus membranes of the oral cavity.

     Some dyes can not only be used to stain microorganisms, but also have antimicrobial activity.  Crystal violet (used in very low concentrations as gentian violet) can be used to treat oral infections by bacteria such as Rochlaemia quintana, the agent of trenchmouth, and fungal infections such as Candida albicans, which causes oral thrush.

 Cooperative Activites

      The following disinfectants were tested against a known concentration of E. coli.  Given the
      concentrations of the test disinfectants, establish the phenol coefficient for each if a dilution of
     1:50 of phenol is necessary to achieve the same killing effect:

Disinfectant                                                 Concentration
       A                                                                1:250
       B                                                                1:75
       C                                                                1:500
       D                                                                1:45

Based on the calculated P.C. values, which is the most effective against E. coli?

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