CHAPER 7: PHYSICAL AND CHEMICAL METHODS OF 5.

Type of material - the temperature required to

STERILIZATION sterilize materials depend on the sensitivity of the
material to heat. Heat-sensitive materials will
DEFINITION OF TERMS
require lower temperature than heat-resistant
1. Sterilization - the process of killing or removing all materials.
microbial forms, including spores. 6. Presence of organic material - the presence of
2. Disinfection - the process by which most microbial organic materials such as fats, proteins, and sugars
forms on inanimate objects are killed without may necessitate higher temperatures.
necessarily destroying saprophytes and bacterial
TYPES OF HEAT
endospores which leads to a reduction in the
number of organisms to a level that they cannot 1. Moist heat
produce infection.
 preferred over dry heat because of its more rapid
3. Antisepsis - use of chemical agents on living tissue
killing action.
(e.g., skin) to prevent the spread of
 Its main mechanism of action is to cause
microorganisms either by inhibiting their growth
coagulation and denaturation of proteins.
or destroying them.
 The various methods of moist heat may be
4. Bactericidal or germicidal agent - agent, physical
classified according to the temperature used.
or chemical, that kills bacteria.
5. Bacteriostatic agent - agent, physical or chemical, These include:
capable of inhibiting the growth of bacteria
without necessarily killing them. a. Temperature below 100°C
6. Sporicidal, fungicidal, viricidal - agents capable of
 Pasteurization
destroying spores, fungi, and viruses, respectively.
 This is the method of destroying disease-
PHYSICAL METHODS OF STERILIZATION producing organisms in milk and milk
products as well as other beverages.
HEATING  There are several variations of this method
based on the temperature utilized.
 Heating is the most common physical method of
 One method is called the conventional
sterilization.
method where the milk is heated at 60°C-65
 The rate of killing is expressed in thermal death
°C followed by rapid cooling.
time, i.e., the minimum time required to kill a
 The flash method involves heating at 72°C
suspension of an organism at a predetermined
for 15 seconds followed by quick cooling to
temperature and environment.
13°C.
 The mechanisms of action of heating include:
 A newer pasteurization method developed
 formation of single-strand breaks in the
is what they call ultra-high temperature
bacterial DNA;
(UHT) method where heating is done at
 coagulation and denaturation of proteins;
140°C for a period of 15 seconds and 149 °C
 accumulation of toxic levels of
for 0.5 seconds.
electrolytes; and
 Vaccine bath
 alteration of cell membrane structure.
 This is used to destroy contaminating
 Several factors can affect the process of
bacteria in vaccine preparations. The
sterilization through heating. These include:
vaccine preparation is heated in a water
1. Nature of the heat - moist heat has greater killing
bath at 60°C for one hour.
action than dry heat.
 This procedure is not sporicidal. Only the
2. Temperature and time - as temperature increases,
vegetative forms of the bacteria are
the time taken to sterilize decreases. In other
destroyed.
words, there is an inverse relationship between
 Serum bath
time and temperature.
 This is used to inactivate bacteria
contaminating serum preparations and is
done by heating at 56°C for several
3. Number of microorganisms - the more
successive days.
microorganisms there are, the higher the
 Similar to vaccine bath, only the vegetative
temperature and the longer the duration of the
forms are destroyed since higher
process required to destroy all of them.
temperatures will cause coagulation of
4. Nature of microorganisms - spore-forming
proteins present in the serum.
microorganisms are more difficult to destroy than
non-spore-forming ones.
  Inspissation
 This technique is used to solidify and
disinfect egg-containing and serum-
containing media.
 The culture medium is placed in the slopes of
a device called an inspissator and is heated at
80°C-85 °C for 30 minutes for three
successive days.
 The basis for the method is that on the first
day, vegetative forms will die and the spores
that will germinate the following day will also
die.

b. Temperature of 100 °C

 Boiling
 This method involves utilizing water at 2. Dry heat
boiling temperature of 100°C.
 the effectiveness of dry heat depends on the
 It is not sporicidal and will destroy only the
penetration of heat through the material to be
vegetative forms. The killing action can be
sterilized.
enhanced by the addition of 2% sodium
 It is used to sterilize materials in enclosed tubes,
bicarbonate.
oils, jellies, powders, and glasswares such as test
 Certain metal articles and glasswares can be
tubes and Petri dishes.
disinfected using this method for 10-20
minutes without opening the lid of the boiler. a. Red flame
 Fractional sterilization (Tyndallization)
 This method is used to sterilize articles like
 This method is also known as intermittent
bacteriological wire loops, straight wires, tips of
sterilization and involves exposing the
forceps, and searing spatulas.
material to be sterilized to live steam at
 The materials are held over the flame of a Bunsen
100°C for 30-90 minutes for three
burner until they become red hot. It is limited only
consecutive days, depending on the material
to articles that can be heated to redness in flame.
to be sterilized.
 This sterilization method can be used to b. Open flame (Flaming)
sterilize culture media such as TCBS and
selenite broth.  This method also makes use of the Bunsen burner
 The vegetative forms are killed on the first or alcohol lamp.
day and the spores that will germinate will be  The material to be sterilized is passed over the
destroyed on the next successive days. Only flame several times but is not heated to redness.
vegetative forms of the bacteria are  It is aimed at burning the organism into ashes and
destroyed with this method. is used to sterilize such articles as mouths of test
tubes, scalpels, glass slides, and cover slips.
c. Temperature above 100 °C  Only vegetative forms are destroyed. In addition,
 Autoclave (Steam under pressure)
 This is the most efficient method of
sterilization because it can destroy all
microbial forms.
 The temperature for sterilizing is
dependent on the pressure of the steam.
When the pressure reaches 15 pounds per
square inch (psi), the temperature inside
cracking of the glassware may occur.
the vessel reaches 121 °C.
 Because of the high temperature and
pressure, it would take only 15-20 minutes
to sterilize the material. c. Incineration
 This method is used to sterilize  This method is aimed at burning the organism into
instruments, surgical bandages, culture ashes.
media, and other contaminated materials  The contaminated material is burned using an
that can withstand high temperature and incinerator.
high pressure.
  Articles that must be incinerated include soiled  A cellulose ester filter with a pore size of 0.22 µm-
dressings and beddings, animal carcasses, and 0.45 µm is used which can filter all microorganisms
pathological material. except viruses and the three smallest bacteria-
 This will result in loss of the article and hence must Mycoplasma, Rickettsia, and Chlamydia.
be used only for articles that have to be disposed.  It is used for liquid solutions that will be destroyed
 Some materials such as polystyrene emit dense by heat or freezing such as serum, antibiotic
smoke and must not be incinerated. solutions, sugar solutions, or urea solution.
 This method can be used to remove bacteria from
d. Hot air oven
culture media or to prepare suspensions of viruses
 The use of the hot air oven was first introduced by and phages.
Louis Pasteur.
RADIATION
 Articles to be sterilized are placed in the oven with
a temperature of 160°C for a period of one hour. 1. Ultraviolet Light (UVL)/Non-ionizing radiation
 This can be used to sterilize metallic instruments
 the effective UVL wavelength is in the range of
such as forceps, scalpels, and scissors.
200 nm-280 nm, with 260 nm as the most
 It can also be used to sterilize certain glasswares
effective.
(e.g., petri dishes, pipettes, flasks) and it is the only
 This corresponds with the maximum absorption of
method used to sterilize powders and ointments.
bacterial DNA. UVL acts by inducing formation of
 The disadvantage of using this method is that
thymine-thymine dimers resulting in lethal
because air is a poor conductor of heat, then hot
frameshift mutations.
air will have poor penetration of the materials to
 Microorganisms such as bacteria, viruses, and
be sterilized.
yeasts can be inactivated within seconds.
 In addition, cotton wool and paper may get
However, UVL is not sporicidal and is more
slightly charred and glasses can become smoky.
frequently used for surface disinfection.
e. Infrared rays  It is used to disinfect hospital wards, operating
rooms, laboratories, and other rooms in the
 In this method, the articles to be sterilized are
hospital that need to be sterilized.
placed in a conveyor belt and passed through a
 The disadvantage of UV ray is that it has low
tunnel that is heated by infrared radiators.
penetrance. It is also limited by the lifespan of the
 The temperature to which the materials are
UV bulb.
subjected to is 180°C for a period of 7.5 minutes. It
 In addition, there are some bacteria that have
can be used to sterilize metallic equipment and
DNA repair systems that can counteract the
glassware.
damage done by UV rays.
DESSICATION  Care should also be observed by the handler
because UV rays can be harmful to the skin and
 This method is based on the principle of depriving eyes.
the microorganism of moisture.
 It is used mainly for food preservation, such as in 2. Ionizing radiation
the preparation of dried fish and fruits.
 ionizing rays have greater penetrance than UV
 It may destroy vegetative forms. Endospores are
rays.
resistant to drying.
 It exerts its effect by causing formation of free
FREEZING radicals that chemically interact with proteins and
nucleic acids, resulting in cell death.
 Freezing is not a reliable method of sterilization  It is not routinely used because of its potential to
because most pathogenic organisms are resistant harm human tissues.
to low temperatures.  There are two types of ionizing radiation used for
 Its main use in the laboratory is for the sterilization purposes: electron beams and
preservation of microorganisms in a process called electromagnetic rays.
lyophilization or freeze-drying where the
organism is rapidly frozen then dehydrated in high a. Electron beams
vacuum and stored in a vacuum-sealed container.
 Electron beams are particulate in nature. A linear
FILTRATION accelerator from a heated cathode is used to
generate high speed electrons.
 This is a form of mechanical sieving that does not  It can be used to sterilize syringes, gloves,
kill microorganisms but merely separates them dressing packs, food, and some pharmaceuticals. It
from the fluid. has lower penetrance and requires sophisticated
instruments.
b. Electromagnetic rays (Gamma rays) lower the concentration of the chemical agent or
may bind the chemical agent to its surface.
 Electromagnetic rays are produced from nuclear
5. Nature of the organism. This refers to the innate
disintegration of selected radioactive isotopes.
resistance of the microorganism to disinfectants.
 They have greater penetrance than electron
Microorganisms vary in their resistance to
beams but require longer exposure time.
disinfectants. Bacteria that produce endospores
 The high energy radiation produced cause damage
may be resistant to most chemical agents.
to the microorganism's nucleic acid.
Mycobacterial cell wall is lipid-rich that makes it
 It is bactericidal, fungicidal, viricidal, and
difficult for the chemicals to penetrate it. Gram-
sporicidal.
negative bacteria have an outer membrane that
 It is used commercially to sterilize disposable Petri
confers resistance to disinfectants.
dishes, plastic syringes, vitamins, antibiotics,
6. Number of organisms/Size of inoculum. The
hormones, fabrics and glassware.
larger the number of microorganism present, the
SONIC AND ULTRASONIC VIBRATIONS more time needed for a disinfectant to destroy all
of them.
 Some bacteria can be killed after exposure to  A chemical agent, to be effective as a disinfectant
certain frequency of sound waves. or antiseptic, must be chosen carefully based on
 Exposure to sound waves at a frequency of the specific purpose, pathogen, and environment.
approximately 20,000 cycles/second for one hour A good chemical agent must possess the following
can kill some bacteria and viruses. characteristics:
 High frequency sound waves act by disrupting 1. It should be broad spectrum, able to destroy a
cells. wide variety of microorganisms.
 They are used to disinfect and clean instruments 2. It should be fast-acting, able to destroy microbes
and to reduce microbial load, within a short period of time.
3. It should be active in the presence of organic
OSMOTIC PRESSURE
matter.
 This method is based on the principle of osmosis, 4. It should be active in any pH.
so that when the concentration of the fluid 5. It should be stable.
surrounding the organism is altered, this will cause 6. It should be non-toxic, non-allergenic, non-
the bacterial cell to collapse. irritative, and non-corrosive.
 This is used for preservation of fruits in syrup and 7. It should be soluble in water and easy to apply.
meats in brine. 8. It should leave a residual antimicrobial film on the
treated surface.
9. It should have high penetrating power.
CHEMICAL METHODS OF STERILIZATION 10. It should not be expensive and must be easily
available.
 Chemicals can inhibit the growth of pathogenic 11. It should be safe under storage and shipping for
organisms, either temporarily or permanently. reasonable periods of time.
 Several factors can affect the efficacy of a 12. It should not have a bad odor.
chemical agent. These include:
1. Concentration and potency of the chemical agent.
In general, a higher concentration is bactericidal CLASSIFICATION OF CHEMICAL DISINFECTANTS
whereas a lower concentration may only be
bacteriostatic. This is not true for alcohol. For  Chemical disinfectants may be classified based on
alcohol, the effective bactericidal concentration is the following:
at 50% to 80%.  (1) consistency (liquid or gaseous);
2. Duration of exposure. The longer the time of  (2) spectrum of activity (high level, intermediate
exposure to the chemical agent, the better the level, low level);
killing action.  (3) mechanism of action.
3. Temperature. A higher temperature speeds up the
rate of a chemical reaction and thus accelerates
killing action. However, there are also certain MECHANISM OF ACTION
chemical agents that exert optimal effect at lower
temperatures. Damage to the cell membrane
4. Nature of the surrounding medium. The pH of the
 Damage to the cell membrane can cause smaller
medium and the presence of extraneous materials
molecules to leak out of the bacterial cell and
like pus or blood decreases the efficiency of the
interfere with the active transport and energy
chemical agent. These materials may inactivate or
 metabolism within the cell. Chemicals under this d. Chloroxylenols are used for topical
include the following: purposes. They are effective against
gram-positive bacteria.
1. Surface active agents
e. Hexachlorophene is a chlorinated
 compounds have long chain hydrocarbons that are diphenyl which has greater activity
fat-soluble and charged ions that are water- against gram-positive bacteria similar to
soluble. chloroxylenols.
 They concentrate on the surface of membranes f. Triclosan, an organic phenyl ether, has
and disrupt membrane resulting in leakage of cell good activity against gram-positive
components. bacteria and a number of gram-negative
 These agents are active against vegetative bacteria including Pseudomonas. It has
microbial forms including Mycobacteria as well as some activity on fungi and viruses.
enveloped viruses.
3. Alcohols
 They are widely used as disinfectants in homes
and hospitals but their activity is reduced in the  disorganize the lipid structure of the cell
presence of hard water and organic matter. membrane, dehydrate cells, and cause
denaturation and coagulation of cellular proteins.
a. Cationic agents
 The microbial killing property of alcohol is seen
 These are detergents where the fat-soluble better in a 70% aqueous solution compared to
portion is positively charged due to combination absolute alcohol.
with a quaternary nitrogen atom.  The disadvantage of using alcohols is that they are
 These are called quaternary ammonium skin irritants and are also flammable.
compounds and are effective at alkaline pH. a. Ethyl alcohol - used as skin antiseptic, it is
Examples are cetrimide and benzalkonium bactericidal and removes lipids from skin
chloride. surfaces.
b. Isopropyl alcohol - it has greater
b. Anionic agents bactericidal activity than ethyl alcohol and
is less volatile. It can be used to disinfect
 These are negatively charged agents that contain
surfaces. Inhalation of its fumes can cause
long chain hydrocarbons.
narcosis.
 Examples are soaps and bile salts. They remove
c. Benzyl alcohol - it is used mainly as a
dirt through the process of emulsification and are
preservative.
most effective at acidic pH.
d. Methyl alcohol - it is fungicidal and
2. Phenolic compounds sporicidal used in disinfecting inoculation
hoods.
 these act by disrupting cell membranes as well as
causing precipitation of proteins and inactivation
of enzymes.
DENATURATION OF CELLULAR PROTEINS
 These are coal-tar derivatives that act as
disinfectants at high concentration and as  Substances that cause denaturation or loss of the
antiseptic at low concentrations. normal structure of proteins pave the way for the
 Phenols are bactericidal and fungicidal with good eventual destruction of the bacterial cell.
activity against Mycobacteria but have poor Denaturing agents include:
activity against spores and most viruses. (1) acids and alkalis,
a. Phenol is no longer used as a disinfectant (2) alcohol and acetone, and
because it is toxic to human cells. It is (3) phenol and cresol.
used as a gold standard in the chemical
evaluation of new chemical agents using MODIFICATION OF THE FUNCTIONAL GROUPS OF PROTEINS
the phenol coefficient test. AND NUCLEIC ACIDS
b. Cresols are phenol derivatives more
1. Heavy metals
potent and safer than phenol. An example
is Lysol®.  cause damage to the enzyme activity of bacteria.
c. Chlorhexidine is used as a skin  They also cause precipitation of proteins and
disinfectant if in isopropanol solution. The oxidation of sulfhydryl groups.
aqueous preparation is used for wound  Heavy metals are mostly bacteriostatic than
irrigation. Its main use is as antiseptic bactericidal.
hand wash.
a. Mercurials
  (e.g., mercurochrome and merthiolate) are b. Ethylene oxide
biocidal and are used as antiseptics. These are
 is also sporicidal and is used in the gaseous
active against viruses at dilution of 1:500 to
sterilization of heat-sensitive materials or
1:1000.
equipment like heart-lung machine, respiratory
b. Silver compounds and dental equipment, and polyethylene tubes in
anesthesia machines. It is more potent than
 (e.g., silver nitrate) are bactericidal. 1% silver
glutaraldehyde but slower-acting. It is highly
nitrate solution is used clinically as treatment for
flammable and is usually combined with 10% CO2.
ophthalmia neonatorum (Crede's prophylaxis).
It causes eye irritation and is mutagenic and
Silver sulfadiazine is used topically in the
carcinogenic.
treatment of burn wounds.

2. Halogens

 bactericidal oxidizing agents that cause oxidation

of essential sulfhydryl groups of enzymes causing
inactivation of the enzymes.

a. Iodine

 (tincture of iodine, iodophores) is considered the

best antiseptic because it is sporicidal, bactericidal,
fungicidal, viricidal, and amoebicidal. It can be
combined with neutral carrier polymers to
produce iodophores (e.g., povidone-iodine). A 10%
solution of povidone-iodine is used for pre-
operative and post-operative skin disinfection.

b. Chlorine

 is mainly used in the treatment of water (chlorine

gas). Hypochlorites are used for sanitizing dairy
and food processing equipment. It is also a
common household disinfectant. At higher
concentrations, it is used to disinfect swimming
pools.

c. Hydrogen peroxide

 is a weak antiseptic and used only for cleaning

wounds and in the disinfection of surgical devices
and soft plastic contact lenses.

3. Alkylating agents

a. Aldehydes

 damage nucleic acids by alkylation of amino-,

carboxyl-, or hydroxyl groups. It kills all
microorganisms including spores.
 Formaldehyde (formalin) is used for surface
disinfection. It can be used to sterilize bedding and
furniture. It is also used to kill Mycobacterium
tuberculosis in sputum and fungi in athlete's foot.
 Glutaraldehyde is sporicidal and used as a cold
sterilant in sterilizing medical equipment such as
respiratory therapy machines and other
equipment that can be damaged by heat. It is
more potent than aldehyde. It requires alkaline pH
for its action and exposure time of at least 3 hours
to be effective.