Microbial populations should be
effectively controlled for the health condition of the general public. One can
remove, inhibit or kill microorganism by physical agents, physical processes or
chemical agents. There are various techniques and agents available, they act in
many different ways and each has its own limitations in practical applications.
There are terms used to describe the
physical processes and chemical agents and these terms are important in labeling
drugs and chemicals used against microorganisms.
Sterilization – the process
of destroying all forms of microbial life. A sterile object, therefore, is free
of living microorganisms.
Bactericidal – having the
property of killing the bacteria. The action is irreversible, i.e., the killed
organisms can no longer reproduce even after being removed from contact with
the agent.
Bacteriostatic – having the
property of inhibiting bacterial multiplication. When the agent is removed, the
bacteria resume multiplication.
Antiseptic – a substance
that prevents the growth of microorganisms either by destroying cells or
inhibiting their growth and activity. It is usually associated with substances
applied to the body.
Disinfectant – usually a
chemical agent that kills the growing forms, but not the resistant spore forms.
This term is applied to inanimate objects. These chemicals may be toxic to
tissues.
Septic – is
characterized by the presence of pathogenic microbes while aseptic is the
absence of pathogenic microbes.
Sanitizer – an agent
that reduces the bacterial population to a safe level. This is commonly applied
to inanimate objects and generally employed in the daily care of equipment and
utensil in food and plants, restaurants and other eating places.
Antimicrobial agents – a chemical
or biological agent that destroys or inhibits growth of microorganisms.
Conditions influencing
antimicrobial action
There are many factors that must be
considered in the application of any physical or chemical agent. Among these
are:
1.
Temperature – an increase
in temperature in the presence of a chemical agent, hastens the destruction of
microorganisms.
2.
Kind of microorganisms – species of
microorganisms have different susceptibility pattern to physical and chemical
agents. In spore–forming bacteria, the growing, vegetative forms are more
susceptible than the spore forms.
3.
Physiologic
state of the cells – young, actively metabolizing cells are more
easily destroyed than old. Dormant cells in the case of an agent which causes
damage through interference with metabolism.
4.
Environment – the
consistency of the material (aqueous or viscous) in which the microorganism is
grown markedly influence the penetration of the agent.
Mode of action antimicrobial
agents
1. Damage
to the cell wall – e.g. enzymes lysozyme, penicillin
2. Alteration
of cell permeability – e.g. phenolic compounds, synthetic detergents, soaps and
quaternary ammonium compounds.
3. Alteration
of protein and nucleic acid molecules – e.g. high temperatures and high
concentration of chemicals.
4. Inhibition
of enzyme action – e.g. cyanide inhibits cytochrome oxidase, fluoride inhibits
glycolysis, trivalent arsenic compounds block TCA cycle and strong oxidizing
agents such as halogens and hydrogen peroxide.
5. Inhibition
of nucleic acid synthesis – e.g. certain synthetic chemicals and some naturally
occurring substances.
Antimetabolite
or metabolic analogue – is a substance that is structurally related to
but slightly different from, the natural metabolite. Metabolite
is a chemical substance participating in metabolism.
Physical agents
A. High
Temperature – when combined with high humidity, it is considered to be the most
effective method of killing microorganisms.
These terms
are used to indicate the degree of heat resistance of bacteria:
1. Thermal
death point (TDP) – refers to the lowest temperature at which suspension of
bacteria is killed in 10 minutes.
2. Thermal
death time (TDT) – refers to the shortest period of time required to kill a
suspension of bacteria at a prescribed temperature and under specific
conditions.
Spore–formers have
2 TDP and TDT:
a. Vegetative
state – 100oC; 10 – 15 minutes
b. Spore
state – 121oC, 20 – 30 minutes
3. Decimal
reduction time – the time in minutes to reduce the population by 90%
Moist heat – kills
microorganisms by coagulating their proteins and is much more rapid and
effective than dry heat.
1. Steam
under pressure – the most practical and dependable agent for sterilization. It
has the advantage of rapid heating, penetration and moisture in abundance which
facilitate coagulation of proteins.
The autoclave
is the apparatus used and the temperature and pressure is under control. The
most important in operating this apparatus is that the temperature is
maintained and the air should be completely be replaced with saturated steam.
It is the high temperature that kills the microorganisms and not the high
pressure. The material to be sterilized should be at 121oC
(115 lbs/sq.in.) for 15 to 20 minutes.
2. Fractional
or Intermittent sterilization (Tyndallization) – Arnold sterilizer is used
employing free flowing steam at 100oC for 30 minutes on 3 successive
days with incubation period in between sterilization to allow the spore forming
bacteria to germinate and be killed or destroyed the next day.
3. Boiling
– exposure of the materials at 100oC for 2 to 3 minutes will kill
all vegetative forms but not the spore forms even for many hours.
4. Pasteurization
– a controlled heat treatment which kills certain types of bacteria but will
not kill all microorganisms.
Two methods:
a. LTH
(Low Temperature Holding) or Batch method – exposed at 62oC for 30
minutes
b. HTST
(High Temperature, Short time) or Flush process – exposed at 72oC
for 15 minutes.
5. Inspissation
– thickening through evaporization. This is used in the sterilization of high
protein media that cannot withstand high temperatures of the autoclave. Usual
application is at 75 – 80oC for 2 hours or successive days.
Dry heat –
kills microorganism by oxidizing their chemical constituents
1. Hot–air
sterilization – recommended to sterilize laboratory glasswares using a special
electric or gas oven at temperature of 160oC for 2 hours. Oven is
the apparatus used.
2. Incineration
– actual burning of materials at 300–400oC. It is used for the
destruction of carcasses, infected laboratory animals and other infected
material to be disposed of.
B. Low
temperature – considered to be a microbiostatic because it reduces the rate of
metabolism. Low temperatures are useful for preservation of cultures and cannot
be depended upon for disinfection or sterilization.
C. Dessication
and lyophilization – the mechanism of microbial inhibition is dehydration. The
effect is chiefly bacteriostatic. They are recommended for the preservation of
foods, bacterial cultures and viruses.
D. Osmotic
pressure – the mechanism of action is plasmolysis; the cells are dehydrated
therefore, they are unable to metabolize and grow. Most of the microorganisms
are inhibited by high concentrations of salts and sugars. This fact forms the
basis of preserving foods by “salting” or by concentrated sugar solution.
E. Sonic
titration and vibration – there is a breaking of the cell wall of the bacteria.
These are used in research work of cellular constituents.
F. Radiation
– when it passes through the cells, it creates free hydrogen and hydroxyl
radicals and some peroxides which in turn cause different kinds of
intracellular damage. There have been many developments in the application of
ionizing radiation to sterilize biological materials and this is called cold
sterilization which is being developed in the food and pharmaceutical industries.
1. X–rays
– are lethal to microorganisms and they have considerable energy and
penetration ability but they are impractical for purposes of controlling
microbial population because they are very expensive in large amount and they
are difficult to utilize efficiently since they are given off in all directions
from their point of origin.
2. Ultraviolet
light – has very little ability to penetrate matter, only those microorganisms
that are directly exposed to ultraviolet light are susceptible to destruction.
They are widely used in hospital operating rooms and aseptic filling rooms in
pharmaceutical industry and for treatment of contaminated surfaces in food and
dairy industry.
3. Gamma
rays – are practically used in sterilization of materials of considerable
thickness or volume because of great penetrating power and their microbicidal
effect.
The “target”
theory of action was made based on the effect of ionizing radiations on the
cells and this means that the radiant–energy particle makes a “direct hit” on some
substances within the cell, causing ionization which results in bacterial
death.
·
If ultraviolet radiation is not
too extensive, the cell can repair the damage by excision of dimers which is
accomplished by:
a. Photoreactivation
– light induced mechanism
b. Excision
repair – dark reaction
G. Titration
– is used to remove microorganism in some biological fluid like animal serum or
enzymes and some vitamins or antibiotics that are thermolabile, i.e., destroyed
by heat.
A variety of bacteriological
filters have been available and they are made up different material like
diatomaceous earth like in Berkefield filter, asbestos pad in Seitz filter,
unglazed porcelain in Chamberland–Pasteur filter, sintered glass in Morton
filter and cellulose ester in membrane or molecular filter. Most filters are
available in several grades, based on the average size of the pores. It is not
porosity alone as the only factor preventing the passage of organisms. Other factors
contributing the efficiency of filtration include electric charge of the
filters and carried by the organisms and the nature of the fluid being
filtered.
The
development of high–efficiency particulate air (HEPA) filters has made it
possible to deliver clean air to an enclosure such as cubicle or room and
together with system of laminar flow, it is now used extensively to provide
dust and bacteria free air.
Chemical agents
Chemical agents do, however, have
value in disinfection, that is, reducing the numbers of and eliminating certain
dangerous microbes. Among the disinfectant used are:
1.
Acids
a. Acidic
disinfectants function by destroying the bonds of nucleic acids and
precipitating proteins. Acids also change the pH of the environment making it
detrimental to many microorganisms. Concentrated solutions of acids can be
caustic, cause chemical burns, and can be toxic at high concentrations in the
air. These characteristics limit their use. The antimicrobial activity of acids
is highly pH dependent. Acids have defined but limited use as disinfectants.
b. Acetic
acid is usually sold as glacial acetic acid (95% acetic acid) which is then
diluted with water to make a working solution concentration of 5%. The
concentrated form is corrosive to the skin and lungs, but the typical dilution
(5%) is considered non–toxic and non–irritating. Acetic acid is typically
applied by spraying, misting or immersing an item in a diluted solution. Household
vinegar is a 4–5% soluton of acetic acid (by volume). Acetic acid has poor
activity in organic material.
2.
Alcohols
a. Examples
are ethanol and isopropanol. Ethanol is considered virucidal; isopropanol is
not effective against non–enveloped viruses.
b. Alcohols
are broad spectrum antimicrobial agents that damage microorganisms by
denaturing proteins, causing membrane damage and cell lysis. Alcohols are used
for surface disinfection, topical antiseptic and hand sanitizing lotions.
Alcohols are considered fast–acting capable of killing most bacteria within
five minutes of exposure but are limited in virucidal activity and are ineffective against spores.
c. An
important consideration with alcohols is the concentration used, with 70–90%
being optimum. Higher concentrations (95%) are actually less effective because
some degree of water is required for efficacy (to denature proteins). Alcohols
evaporate quickly but leave behind no residue. The activity of alcohols is
limited in the presence of organic matter. Alcohols are highly flammable, can
cause damage to rubber and plastic, and can be very irritating to injured skin.
3.
Aldehydes
a. Acts
by denaturing proteins and disrupting nucleic acids. Aldehydes are non–corrosive
to metals, rubber, plastic and cement. These chemicals are highly irritating,
toxic to humans or animals with contact or inhalation, and are potentially
carcinogenic; therefore their use is limited.
b. Formaldehyde
is used as a surface disinfectant and a fumigant and has been used to
decontaminate wooden surfaces, bricks and crevices of electronic and mechanical
equipment. Its use must occur in air tight building, which must remain closed
for at least 24 hours after treatment. The efficacy of formaldehyde is
dependent on relative humidity and temperature; optimum being humidity close to
70% and a temperature close to 57oF
c. Formalin
is 37% solution of formaldehyde in water
d. Glutaraldehyde
is primarily used as a disinfectant for medical equipment (e.g. endoscopes),
but can provide sterilization at prolonged contact times. A 2% concentration is
used for high–level disinfection. Its efficacy is highly dependent on pH and
temperature, working best at a pH greater than 7 and high temperatures. It is
considered more efficacious in the presence of organic matter, soaps and hard
water than formaldehyde.
4.
Alkalis
a. Alkaline
agents work by saponifying lipids within the envelopes of microorganisms. The
activity of alkali compounds is slow but can be increased by raising the
temperature. Alkalis have good microbicidal properties, but are very corrosive
agents and personal protection should be observed.
b. Sodium
hydroxide (lye, caustic soda, soda ash) is a strong alkali used to disinfect
buildings but is highly caustic. Protective clothing, rubber gloves, and safety
glasses should be worn when mixing and applying the chemical. Lye should always
be carefully added to water. Never pour water into lye; a very violent reaction
will occur as well as the production of high heat that can melt plastic
containers. Sodium hydroxide is corrosive for metals.
c. Ammonium
hydroxide is effective disinfectant against coccidial oocysts however strong
solutions emit intense and pungent fumes. This substance is not considered
effective against most bacteria.
d. Sodium
carbonate (soda ash, washing soda) has been used in hot solution (180oF)
for disinfecting buildings. It is more effective as a cleanser than a
disinfectant since it lacks efficacy against some bacteria and most viruses. It
has poor activity in the presence of organic material and can be deactivated by
hard water. It can be irritating and requires protective clothing and is
harmful to aquatic life.
e. Calcium
oxide (quicklime) becomes lime when mixed with water. This has biocidal effects
on some bacteria and virus and is sometimes spread on the ground following
depopulation of infected premises and has also been used to retard putrefaction
of buried carcasses after depopulation.
5.
Biguanides
a. Example
is chlorhexidine
b. Biguanidies
are detrimental to microorganisms by reacting with the negatively charged
groups on cell membranes which alters the permeability. Biguanidines have a
broad antibacterial spectrum; however, they are limited in their effectiveness
against viruses only. Biguanidines can only function in a limited pH range (5 –
7) and are easily inactivated by soaps and detergents. These products are toxic
to marine life.
6.
Halogens
a. Halogen
compound are broad spectrum compounds that are considered low toxicity, low
cost and easy to use. They do lose potency over time and are not active at
temperatures above 110oF or at high pH (>9). Since these
compounds lose activity quickly in the presence of organic debris, sunlight and
some metals, they must be applied to thoroughly cleaned surfaces for
disinfection.
b. Chlorine
compounds function through their electronegative nature to denature proteins
and are considered broad spectrum, being effective against bacteria, enveloped
and non–enveloped viruses, mycobacteria and fungi. Sodium hypochlorite is one
of the most widely used chlorine containing disinfectant. Hypochlorites
should never be mixed with acids or ammonia as this will result in the release
of toxic chlorine gas.
c. Iodine
compounds function by denaturing proteins to interfere with the enzymatic
systems of microorganisms. Iodines are often formulated with soaps and
considered relatively safe. Iodine agents are inactivated by QACs and organic
debris.
d. Iodophors
are iodine complexes that have increased solubility and sustained release of
iodine. One of the more commonly used iodophors is povidone–iodine. They are
good for general use and are less readily inactivated by organic matter than
elemental iodine compounds. The dilution of iodophors actually increases the
free iodine concentrations and antimicrobial activity.
7.
Oxidizing
agents
a. Function
by denaturing the proteins and lipid of microorganisms.
b. Hydrogen
peroxide at a concentration of 5 – 20% acts best with bacteria, virus and fungi
but has limited activity with Mycobacteria.
c. Peracetic
acid is a strong oxidizing agent and is a formulation of hydrogen peroxide and
acetic acid.
d. Potassium
peroxymonosulfate and sodium chloride.
8.
Phenols
a. Function
by denaturing proteins and inactivating membrane bound enzymes to alter the
cell wall permeability of microorganisms. They usually have a milky or cloudy
appearance when added to water with a strong pine odor.
9.
Quaternary
ammonium compounds (QACs)
a. QACs
are cationic detergents that are attracted to the negatively charged surfaces
of microorganism, where they irreversibly bind phospholipids in the cell
membrane and denature proteins impairing permeability.
Characteristics of a good
disinfectant
1. Should
attack all types of microorganisms
2. Be
rapid in its action
3. Should
not damage materials being disinfected
4. Should
not be retarded in its action by organic matter
5. Should
dissolve easily in or mix with water to form a stable solution or emulsion.
6. Should
not penetrate material being disinfected
7. Should
not decompose when exposed to heat, light rays or unfavorable weather
condition.
8. Should
not destroy body tissues or act as a poison when taken internally
9. Should
not have unpleasant odor or discolor the material being disinfected and be
easily obtained at a comparatively low cost.
Phenol coefficient
This is the standard method of testing
chemical agents. It is used for evaluating the effectiveness of disinfectant by
comparing with phenol under identical condition. The larger the resulting
value, phenol coefficient, the more active the agent under those conditions.
Coefficient is determined by taking
the highest dilution of the disinfectant that gives evidence of killing the
organism in 10 minutes (but not in 5 minutes). This is divided by the highest
dilution of phenol giving the same result.
Example:
1/1000
= disinfectant X
1/90
= phenol
Coefficient
= 1000/90
= 11.1 for disinfectant X
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