PROTECTION AGAINST INFECTIONS / STERILIZATION PROCESS

The control of microbial growth is necessary in many practical situations, and

significant advances in agriculture, medicine, and food science have been made through study
of this area of microbiology. The microorganisms are ubiquitous in nature. In order to study
the nature and characteristics of a particular microbe, it is essential to isolate it from other
contaminating microorganisms. This can be achieved by maintaining a completely sterile
environment in which the microbe of interest is selectively grown. It is necessary that not
only the place you are working with microorganisms should be free from contamination
(other living organisms) but, the media and the materials you are using to handle and grow
specific microorganisms should be free from other microbial contaminants. For this purpose,
‘sterilization’ of the place of work materials and media have to be done.
“Control of growth” as used here means to prevent the growth of microorganisms.
This control is affected in two basic ways:
(1) by killing microorganisms or
(2) by inhibiting the growth of microorganisms. Control of growth usually involves the use
of physical or chemical agents which either kill or prevent the growth of microorganisms.
Agents which kill cells are called cidal agents; agents which inhibit the growth of cells
(without killing them) are referred to as static agents. Thus the term bactericidal refers to
killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A
bactericide kills bacteria; a fungicide kills fungi, and so on.
Sterilization is a process of complete removal or killing of all forms of microbial life
including spores from an object, surface, medium or environment without spoiling its nature.
Methods
There are various sterilization techniques available. However, several factors
influence the effectiveness of sterilization process like, the concentration of antimicrobial
agents, time and temperature of exposure, size of population, type of contaminating microbes
etc. Sterilization is brought about by a combination of physical and chemical agents that
adversely affect the microorganisms either by causing damage to the cell wall or cell
membrane or by inactivating the enzymes or by interfering with the synthesis of nucleic acids
and protein.
I. PHYSICAL AGENTS
There are different types of physical agents.
1. Heat:
The heat employed for removal of micro-organisms varied with the nature of object and
also depend on the purpose. Based on these different processes are employed.
(a) Moist heat
It is the widely used effective means of sterilization process. In this, steam under high
pressure is employed which imparts high penetration power resulting in the hydration of cells
and coagulation of protein leading to the death of the microorganism. Autoclave is the
apparatus used for sterilization by moist heat. The autoclave is a double-jacketed steam
chamber. The chamber is with equipped a device for generating saturated steam. It can be
maintained at a particular temperature and pressure for any period of time. During operation
of autoclave the air in the chamber is evacuated by steam since presence of air will reduce the
temperature in the chamber. The time required for sterilization will depend upon the materials
to be sterilized. Solid materials must be heated for a longer time (1-2 hours) while liquid
media can be sterilized within 15-30 minutes. Also, acidic materials require shorter period
than alkali materials. A temperature of 121°C for 15 min at a pressure of 15 lbs/ sq.inch is the
sterilizing condition in the autoclave.

Advantages
Steam can penetrate through materials and sterilization is achieved by the coagulation or
denaturation of proteins and other cell constituents. Liquid media, solid media, laboratory
equipments (cloth, glasswares, etc.,) can be sterilized. The temperature and pressure are high
enough to kill spores, vegetative cells and viruses.
Disadvantages
Temperature sensitive media, animal tissue culture media, antibiotics, amino acids, cannot
be sterilized. Sometimes water may get inside incase of improper packing.
(b) Dry heat
This process is accomplished in a hot-air oven. Hot air or dry heat is employed for
sterilization. The dry heat penetrates substances more slowly than the moist heat. Hence, the
time required for effective sterilization is long (2 to 3 hours) and also the temperature
required is too high (160°C -180°C). Microbial death results from the oxidation of cell
constituents.
Advantages
 Dry heat does not corrode glassware and metal instruments as moist heat does. All
glassware’s can be sterilized.
Disadvantages
The sterilization process is slow. It is not suitable for heat sensitive materials like many
plastic and rubber items.
(c) Boiling at 100°C for 30 minutes. Kills everything except some endospores (Actually, for
the purposes of purifying drinking water 100°C for five minutes is probably adequate though
there have been some reports that Giardia cysts can survive this process). To kill endospores,
and therefore sterilize the solution, very long or intermittent boiling is required.
(d) Pasteurization is the use of mild heat to reduce the number of microorganisms in a
product or food. In the case of pasteurization of milk, the time and temperature depend on
killing potential pathogens that are transmitted in milk, i.e., staphylococci, streptococci,
Brucella abortus and Mycobacterium tuberculosis.
For pasteurization of milk: batch method (Low temperature holding): 62.8°C for 30 minutes
flash method (High temperature short time): 71.7°C for 15 seconds
(e) Intermittent sterilization or Tyndallization is the process of boiling the materials at
100°C for 30 min. successively for three consecutive days. Destroys vegetative cells and
spores; germinated spores.
(f) Incineration burns organisms and physically destroys them. Incineration is the complete
burning of the material in to ashes. Used for needles, inoculating wires, glassware, etc. and
objects not destroyed in the incineration process. This is the direct and ultimate method of
destroying cells. It is achieved by keeping the materials directly in contact with the flame of
Bunsen burner as a result all the microorganisms in the surface are destroyed completely.
Inoculating loops, needles and spreading rods are sterilized by this method.
Advantages: Immediate and quick.
Disadvantages: Cannot be used to sterilize heat labile material, material is lost by
incineration.
Recommended use of heat to control bacterial growth:
Treatment Temperature Effectiveness
Incineration 500℃ Vaporizes organic material
on non-flammable Surfaces
but may destroy many
substances in the process
Boiling 100℃ 30 minutes of boiling kills
microbial pathogens and
vegetative forms of bacteria
but may not kill bacteria
Intermittent boiling 100℃ Three 30-minute intervals of
boiling, followed by Periods
of cooling kills bacterial
endospores.
 Autoclave and pressure 121℃ for 15 min. at 15 lbs/ Kills all forms of life
cooker (steam under sq. Inch pressure including bacterial
pressure) endospores. The substance
being sterilized must be
maintained at the effective T
for the full time
Dry heat (hot air oven) 160℃/2 hours For materials that must
remain dry and which are
not destroyed at the between
121℃ and 170℃. Good for
glassware, metal, not plastic
or rubber items
Dry heat (hot air oven) 180℃/1 hour Same as above. Note
increasing T by 10 degrees
shortens the sterilizing time
by 50 percent
Pasteurization 62.8℃/30 min. Kills most vegetative
bacterial cells including
pathogens such as
Streptococci, Staphylococci
and Mycobacterium
tuberculosis
Pasteurization (flash 71.7 ℃/15 seconds Effect on bacterial cells
method) similar to batch method; for
milk, this method is more
conducive to industry and
has fewer undesirable effects
on quality

II) Radiation Energy transmitted through space in a variety of forms is generally called
radiation. It is also known as "cold sterilization" as only little heat is produced during the
process. The most significant of this is electromagnetic radiation. The energy content and
radiation wavelength are inversely proportional to each other. Radiation may be ionizing or
non-ionizing.
Ionizing radiation
High-energy electron beams (Gamma, X-rays, alpha and beta particles) have sufficient
energy to cause ionization of molecules. They drive away electrons and split the molecules
into ions. Water molecules are split into hydroxyl radicals (OH-), electrons and hydrogen ions
(H+). OH- ions are highly reactive and destructive to normal cellular compounds such as
DNA and proteins. Thus ionizing radiations are used in sterilization.
e.g. 36Cs, 60Co
Advantages: X-rays and Gamma rays have high penetrating power. Packed food and medical
equipments are sterilized by using x-rays and gamma rays.
Disadvantages: Generating and controlling X-rays for sterilization is highly expensive.
Non-ionizing radiation This includes ultraviolet (UV) rays. UV at a wavelength of 265 nm
is most bactericidal. Absorption of UV radiation produces chemical modification of
nucleoproteins i.e., thymine dimer formation that leads to misleading of genetic codes. This
mutation impairs the total functions of the organism, consequently causing its death.
Advantages: It is used to maintain aseptic conditions in laminar air flow chamber, lab,
hospitals, pharmaceuticals, industries etc., and also in the sterilization of water and air.
Disadvantages: UV radiation has very little ability to penetrate matter and hence the
microorganisms on the surface of an object are destroyed.
III) Filtration
Filtration involves the passage of liquid or gas through a screen like material that has spores
small enough to retain the microorganism of certain size. It is used to sterilize heat sensitive
substance like enzyme solutions, bacterial toxins, certain biological media, cell extract and
some sugars. Various types of filters are available in different grades of porosity. Vacuum or
pressure is required to move the solutes through the filter. Involves the physical removal of
all cells in a liquid or gas, especially important to sterilize solutions which would be
denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins etc.)
Advantages: It si the best way to reduce microbial population in solutions of heat sensitive
materials and it is sued to sterilize liquid media, vitamin solutions, hormones, growth factors,
enzymes.
Disadvantages: Pleomorphic structures like mycoplasma cannot be effectively filtered by
this technique. It is applicable to sterilize only small quantities.
Commonly used filters in micro biology
The sintered glass filter is made of fused Jen or pyrex glass, manufactured in such a
way as to be porous, with a pore size and adsorptive charge sufficient to retain bacteria. The
seitz filters are compressed asbestos discs having porosity sufficiently small to retain bacteria.
Tye chamber land filters are made of porcelain. The mandler/berkfield filters are made of
diatomaceous earth. The membrane filter is a cellulose or nitrocellulose membrane with a
pore size sufficiently small (0.01mm to 10 mm) to trap and thereby remove bacterial from a
liquid. The membrane filters are also used to concentrate and trap the microorganisms in
water and other liquids. HEPA (High efficiency particulate air filters are of fibre galss filters
for sterilization of air.
Low temperature
Most organisms grow very little or not at all at 0℃. Store perishable foods at low
temperatures to slow rate of growth and consequent spoilage (eg: milk). Low temperatures
are not bactericidal. Psychrotrophs, rather tah true psychrophiles are the usual cause of food
spoilage in refrigerated foods.
Dessication / Drying (removal of H2O)
Most microorganisms cannot grow at reduced water activity (aw < 0.90). Often used
to preserve foods (eg: fruits, grains etc). methods involve removal of water from product by
heat, evaporation, freeze drying, addition of salt or sugar.
Surface tension is a property of the surface of a liquid that allows it to resist an external
force. It is revealed, for example, in floating of some objects on the surface of water, even
though they are denser than water, and in the ability of some insects (e.g. water striders) and
even reptiles (basilisk) to run on the water surface. This property is caused by cohesion of
like molecules, and is responsible for many of the behaviors of liquids.
Surface tension has the dimension of force per unit length, or of energy per unit area. The two
are equivalent—but when referring to energy per unit of area, people use the term surface
energy—which is a more general term in the sense that it applies also to solids and not just
liquids. In materials science, surface tension is used for either surface stress or surface free
energy.
Osmotic pressure – plasmolysis/ plasmotysis
Is the process in plant cells where the plasma membrane pulls away from the cell wall
due to the loss of water through osmosis. The reverse process, cytolysis, can occur if the cell
is in a hypotonic solution resulting in a higher external osmotic pressure and a net flow of
water into the cell. Through observation of plasmolysis and deplasmolysis it is possible to
determine the tonicity of the cell's environment as well as the rate solute molecules cross the
cellular membrane.
Chemical agents
Chemical that is used to kill or inhibit the growth and development of microorganisms
are called antimicrobial agents. Disinfectants and antiseptics come under antimicrobial agents
and are usually used on inanimate materials. The mechanism of action is complex and
nonspecific. It may act on lipid portion of cell membrane, oxidize or reduce an important
functional group of an enzyme, prevent certain bio synthesis or cause extensive breakdown of
DNA.
Types of microbial agents
Chemical sterilants
Chemical sterilants are chemical antimicrobial agents that are used for sterilization of
heat sensitive substance/ materials. Normally plastic petriplates and medical supplies such as
blood transfusion sets, plastic syringes, lenses etc. could be sterilized even in packets or
bundles using ethylene oxide, formaldehyde or formalin is effectively used to sterilize
enclosed areas/a septic chambers at 22 O C with a relative humidity of 60 – 80 %.
Antispetics
Microbicidal agents harmless enough to be applied to the skin and mucous membrane,
should not be taken internally. Eg: mercurails, silver nitrate, iodine solution, alcohols,
detergents.
Disinfectants
Agents that kill microorganisms, but not necessary their spores, not safe for
application to living tissues, they are used on inanimate objects such as tables, floors, utensils
etc. eg: Chlorine, hypochlorites, chlorine compounds, Lysol, copper sulfate, quaternary
ammonium compounds.
Phenol
Derivative of phenol like benzyl resorcinol, o-cresol, m-cresol, etc., are used as
effective disinfectants 5% aqueous solutions of phenols are used as disinfectant. It alters the
protein structure and leads to denaturation of proteins and enzymes. Also affects permeability
of cytoplasmic membrane. They readily kill vegetative cells of bacteria and fungi but for
spores.
Alcohol
Alcohol at 70% concentration is more effective. It brings about denaturation and
coagulation of protein. Ethanol is routinely used in laboratories to surface sterilize worktables
and hands of the researcher/ experiment.
Halogens
Halogens such as hypocholrites, choramines and povidone- iodine are used to sanitize
utensils, surface sterilize in animate objects, table surfaces and other instruments.
Heavy metals
Heavy metals such as mercuric chloride are also used for surface sterilization
purposes. Heavy metals act as oxidizing agents and kill the microorganisms on the surface of
the object. Usually, 0.1 % mercuric chloride is used in the laboratories to sterilize the surface
of worktable and explants.
Detergents
Detergents are those compounds that make water repellent surfaces more wettable.
There are two types of detergents viz., ionic and non-ionic. Detergent soaps and other
synthetic detergents are used for washing/cleaning glass wares, table tops etc.,
Common antiseptics and disinfectants
Chemical Action Uses
Ethanol (50 -70 %) Denatures proteins and Anti septic used on skin
solubilizes lipids.
Isopropanol (50 – 70 %) Denatures proteins and Anti septic used on skin.
solubilizes lipids.
Formaldehyde (8%) Reacts with NH2, SH and Disinfectant, kills
COOH groups. endopsores.
Tincture of Iodine (2% in 70 Inactivates proteins Antiseptic used on skin
% alcohol)
Chlorine (Cl2) gas Forms hypochlorous acid Disinfect drinking water,
(HClO), a strong oxidizing general disinfectant.
agent.
Silver Nitrate (AgNo3) Precipitates proteins. General antiseptic and used
in the eyes of newborns.
Mercuric chloride Inactivates proteins by Disinfectant although
reacting with sulfide groups. occasionally used as an
antiseptic on skin.
Detergents (eg: Quaternary Disrupts cell membranes. Skin antiseptics and
ammonium compounds) disinfectants.
Chemotherapeutic agents
Antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease.
Examples: sulfonilamides, isoniazid, ethambutol, AZT, chloramphenicol.
Antibiotics
Antimicrobial agents produced by microorganisms that kill or inhibit other microorganisms.
This is the microbiologist’s definition. A more broadened definition of an antibiotic includes
many chemicals of natural origin which has the effect to kill pr inhibit the growth of other
types cells. Since most clinically useful antibiotics are produced by microorganisms and are
used to kill or inhibit infectious bacteria we follow classic definition. Antibiotics are low
molecular weight (non- protein) molecules produced as secondary metabolites, mainly by
microorganisms that live in the soil. Most of these microorganisms form some type of a spore
or other dormant cell, and there is thought to be some relationship between anti biotic
production and the process of sporulation. Among the Molds, the notable antibiotic producers
are penicillium and cephalosporium, which are the main source of the beta lactam antibiotics.
In the bacteria, the Actinomycetes, notable Streptomyces species, produce a variety of types of
antibiotics including the aminoglycosides (eg: streptomycin), macrolides (eg: erythromycin)
and the tetracycline. Endospore forming bacillus species produce polypeptide antibiotics such
as polymyxin and bacitracin.
Chemical class Examples Biological Spectrum Mode of action
source (effective
against)
Beta – lactams Penicillin G, Penicillium Gram positive Inhibits steps in
(Penicillins and Cephalothin notatum and bacteria cell wall
Cephalosporins) Cephalospori (peptidoglycan)
um sp synthesis and
murein
assembly.
Aminoglycoside Streptomycin Streptomyces Gram positive Inhibit
s griseus and gram- translation
negative (protein
bacteria synthesis)
Glycopeptides Vancomycin Streptomyces Gram positive Inhibits steps
orientales bacteria, esp. inn murein
Staphylococcu (peptidoglycan)
s aurues biosynthesis
and assembly
Macrolides Erythromycin Streptomyces Gram positive Inhibits
erythreus and gram- translation
negative (protein
bacteria not synthesis)
enteric,
Neisseria,
legionella,
mycoplasma
 Polypeptides Polymyxin Bacillus Gram- Damages
polymyxa negative cytoplasmic
bacteria membranes
Polyenes Amphotericin Streptomyces Fungi Inactivate
nodosus membranes
containing
sterols
Tetracyclines Tetracycline Streptomyces Gram positive Inhibit
sp and gram- translation
negative (protein
bacteria, synthesis)
rickettsias
Chloramphenicol Chloramphenicol Streptomyces Gram positive Inhibit
venezuelae and gram- translation
negative (protein
bacteria synthesis)