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.
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.
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.
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.
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.
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
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:
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.
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.
Based on the calculated P.C. values, which is the most effective against