CONTENTS

 INTRODUCTION
 DEFINTIONS RELATED TO STERILIZATION
 CLASSIFICATION
 STERILIZATION PARAMETERS
 METHODS OF CONTROL:-
o Control of Microorganisms by Physical Methods
o Control of Microorganisms by Chemical Methods
 FDA Guidance
 Conclusion
 References
 INTRODUCTION
 The Egyptians used fire to sterilize infectious materials and
disinfectants to embalm bodies, and the Greeks burned sulfur to
fumigate sick rooms (a gas or vapor that’s smells strongly or is
dangerous to breath).
 To day the ability to destroy microorganisms is No less important; it
makes possible the aseptic techniques used in microbiological research
, the preservation of food and the preservation of disease.
 These techniques are essential to personal safety in both the laboratory
and hospital.
 The goal is two fold
 1)To destroy pathogens and prevents their transmission .
 2)To reduce or eliminate microorganisms responsible for the
contamination of water, food and other substances.
 These two points are together referred as CONTROL
 Sterilization is an essential concept in the preparation of sterile
pharmaceutical products.
 It’s main aim is to provide a product that is safe and eliminates
the possibility of introducing infection.
 Definition :- sterilization is a process used to destroy or eliminate
viable microorganisms that may be present in or on a particular
product or package. (or)
 Is the process by which a product is rendered sterile i.e. the
destruction or removal of microorganisms.
 The term sterile indicates a probable condition of complete
freedom from viable microorganisms with the limitations.
 The currently accepted performance target for a sterilization
process is that it provides for a probability of finding a non sterile
unit less than 1 in 1 million
 DEFINTIONS
 STERILIZATION:- Sterilization is the process of killing all forms
of microbial life in or on the given object or preparation.
Microbiologically, sterile material is one that contains No living
organisms at all and the term sterile is therefore an absolute one.
Accordingly, an object is sterile or non sterile but it can never be
semi sterile or almost sterile.
 ANTISEPTIC:- A substance that arrests sepsis i.e. prevents the
growth or action of microorganisms by inhibiting their activity
without necessarily destroying them. These can be applied on
human body.
 BACTERIOSTATIC:-

 BACTERIOCIDAL:-

TIME
 VIABLE:- Live and growing bacteria or microorganisms +
spores.
 DISINFECTION:- A process that removes infection potential by
destroying microorganisms but not ordinarily bacterial spores.
 DISINFECTANTS:- These are generally meant for application on
inanimate objects.
 GERMICIDES:- A substance that kills disease microorganisms
i.e. pathogens/germs but not necessarily spores.
 STERILITY:- The absence of viable organisms.
 DEATH:- The term death as used in microbiology is defined as
the irreversible loss of the ability to reproduce
 CLASSIFICATION
 Sterilization process can be basically separated as Terminal and
Non Terminal process based on stage at which the preparation is
subjected to the process of sterilization.
 Terminal Sterilization: Different types of terminal sterilization
techniques employed are physical sterilization and chemical
sterilization.
 Non Terminal Sterilization: it includes the filtration procedure.
• Based on principle of mechanism involved in the process are
further classified as
 Physical sterilization: this class includes heat sterilization and
radiation sterilization as well.
Contd….
 Heat sterilization procedure is one in which the destruction of
microorganisms mainly occurs due to the high temperatures
employed includes
a) Moist heat sterilization
b) Dry heat sterilization
 Radiation sterilization: this process is accomplished by exposure
to UV light or high-energy ionizing radiation such as gamma rays
and accelerated electrons i.e. particulate radiation as well..
 Chemical sterilization: the procedures which involve treatment
of the preparations to be sterilized with certain chemicals in
either gaseous form or in liquid form are categorized under this
class.
 STERILIZATION PARAMETERS
 Definitions of terms
 Bioburden :- The population or number of living microorganisms
per defined unit.
 D value :- It is the parameter calculated as the time taken for one
log 90% reduction (Decimal reduction) in the number of
microorganisms.
 Z value :- It calculates the temperature or dose of radiation
sterilization required to produce a one log 90% reduction in D
value for a particular organism.
 F value :- This value is used to compare the lethality of different
heat sterilization procedures.
 Survival curves :- They are plots of the logarithm of the fraction
of survivals (microorganisms which retain viability following a
sterilization process) against the exposure time or dose.
Contd….
 Sterility Assurance Level :- An estimation of the effectiveness of a
sterilization process. It usually expressed in terms of negative
power of 10 (i.e. 1 in 1 million = 10-6).
 Validation :- The act of verifying that a procedure is capable of
producing the intended result under prescribed circumstances
and challenges to predefined specifications.
Control of M.O's by Physical Methods
 Includes :-
 Thermal methods (Heat)
 Radiation method
 Filtration method
o Thermal methods or Heat sterilization :-
 Heat sterilization is most widely used and reliable method of
sterilization, involving destruction of enzymes and other essential
cell constituents.
 This method of sterilization can be applied only to the thermo
stable products, but it can be used for
 Moisture – Sensitive materials –Dry heat sterilization
 Moisture – Resistance materials – Moist heat
Contd…
 The efficiency with which heat is able to inactivate
microorganisms is depends up on
 Degree of heat
 The exposure time &
 The presence of water
 This process is of two types
 Dry heat sterilization
 Moist heat sterilization
 Dry heat sterilization :-
 Examples of dry heat sterilization are
 1) Incineration
 2) Red heat
 3) flamming
 Hot air oven
 There are various temperatures and periods of treatment for dry
heat depending on the pharmacopeia.
 The U.S Pharmacopeia states that the dry heat sterilization
process for containers for sterile pharmaceutical products should
be at a temperature of 160-170 0C for a period of 2-4 hr.
 The British Pharmacopeia states that items sterilized by dry
heat should be kept at a temperature not less than 160 0C for at
least 1 hr.
 The benefit of dry heat includes good penetrability and non
corrosive nature which makes it applicable for sterilizing glass
ware and metal surgical instruments also non aqueous thermo
stable liquids and thermo stable powders.
 Hot Air Oven :-
 Dry heat sterilization is usually carried
out in a hot air oven , which consists
of the following..
 1) An insulated chamber surrounded
by an outer case containing electric
heater.
 2) A fan.
 3) Thermocouples.
 4) Temperature Sensor.
 5) Door locking controls.
 Operation :-
 Articles to be sterilized are first
wrapped or enclosed in a container of Hot Air Oven
card board paper or aluminium.
 Then the material are arranged to
ensure uninterrupted air flow.
 Moist heat sterilization :-
 Moist heat sterilization is otherwise referred as Steam
Sterilization Under Pressure.
 Moist heat may be used in three forms to achieve microbial
inactivation.
 Dry saturated steam – Autoclaving
 Boiling water / steam at atmospheric pressure
 Hot water below boiling point
 Conditions to be followed for the moist heat sterilization
 According to USP XXI and BP 1988 are given as
o Pressure : 15lb / square inch (psi)
o Temperature : 121 0C
o Time : 15 minutes.
 Autoclaves use pressurized steam to destroy microorganisms, and
are the most dependable systems available for the
decontamination of laboratory glass ware, media and reagents.
 For efficient heat transfer, steam must flush the air out of the
autoclave chamber.
 Before using the autoclave, check the drain screen at the bottom
of the chamber and clean if blocked.
 If the sieve is blocked with debris, a layer of air may form at the
bottom of the autoclave , preventing efficient operation.
 Autoclaves should be tested periodically with Biological
Indicators like of Bacillus sterothermophilus to ensure proper
function.
 Autoclaves or steam sterilizers essentially consists of :-
1) A cylindrical or rectangular chamber, with capacities ranging
from 400-800 lit.
2) Water heating systems or steam generating systems.
3) Steam outlet and inlet valves.
4) Single or double doors with locking mechanism.
5) Thermometer or temperature gauge.
6) Pressure gauge.
 Operation :
 For porous loads (dressings) sterilizers are generally operated at a
minimum temperature of 134 oC
 For bottled fluid, sterilizers employing a minimum temperature
of 121 oC are used
 Ensure that there should be
sufficient water in the
autoclave to produce the
steam.
 The stages of operation of
autoclaves includes air
removal, steam admission
and sterilization cycle
(including heating up,
holding / exposure &
cooling stages).

INTERNAL STRUCTURE OF AUTOCLAVE

 It sterilizes containers such as
1) LVPs in glass bottles
2) LVPs and SVPs in plastic
containers
3) Prefilled glass or plastic
syringes
4) Jars and similar containers with
press on or screw caps
5) Blisters containing various
materials, for example AUTOCLAVE
Disposable contact lenses.
Control of M.O's by chemical agents
 Also called as BIOCIDES
 Factors influencing on microbial chemical agent.
 The kinds of microorganisms potentially present
 The concentration and nature of the disinfectant to be used
 The disinfectant should be stable upon storage , odorless or
with odor, soluble in water and lipids for permeation into
microorganisms, and a low surface tension so that it can enter
cracks in surfaces
 The main factor is chemical must be toxic for infectious agents,
it should not be toxic to people or corrosive for common
materials in practice, this balance between effectiveness and low
toxicity for animals is hard to achieve.
PHENOLS
 Phenol is the first widely used antiseptic and disinfectant.
 In 1867 Joseph leister employed it to reduce the risk of infection
during operations.
 Today phenol and phenolics(phenol derivatives) such as
cresols,xylenols and ortho phenyl phenol are used as
disinfectants in laboratories and hospitals.
 Phenolics act by denaturing proteins and disrupting cell
membranes they have some advantages as disinfectants; phenolics
are tuberculocidal , effective in the presence of organic material
and remain active on surface long after application.
 Remarkable success has been achieved in modifying the phenol
molecule by the introduction of chlorine and methyl groups , as
in chlorocresol and chloroxylenol.
 Chlorocresol is used as a bactericide in injections and to preserve
oil-in-water creams, whereas chloroxylenol is employed as a
house hold and hospital antiseptic.
 Phenol may it self be rendered less caustic by dilution to 1% w/v
or less for lotions and gargles or by dissolving in glycerol for use
as ear drops.
 Bisphenols such as hexachlorophane useful in skin antiseptic
property but it restricted in UK because of brain damage.

Chlorocresol Chloroxylenol
ALCOHOLS
 Alcohols are among the most widely used disinfectant and
antiseptics they are bactericidal and fungicidal but not sporicidal;
some lipid containing viruses can also destroyed.
 The two most popular alcohols germicides are ethanol and
isopropanol usually used in about 70-80% concentration.
 They act by denaturing proteins and possibly by dissolving
membrane lipids.
 A 10-15 min soaking in sufficient to disinfectant thermometers
and small instruments.
 The effect of aromatic substitution is to produce a range of
compounds which are less volatile and less rapidly active and
final use as preservation.
HALOGENS
 The halogens Iodine and Chlorine are important antimicrobial
agents.
 Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and iodinating cell proteins.
 At high conc it may kill some spores .
 Iodine often has been applied as tincture of iodine(BP 1973), 2%
or more iodine in water – ethanol soln of potassium
iodide(lugol’s iodine BP 1973)
 Although it is an effective antiseptic the skin may be damaged a
stain is left and iodine allergies can result.
 The staining and irritant properties of iodine have resulted in the
development of IODOPHORES, mixtures of iodine with surface
active agents which hold the iodine in micellar combination from
which it released slowly.
 Some popular brands are Wescodyne for skin and Betadine
(polyvinyl pyrrolidone iodine formulated as 10% povidone
iodine) used as a non staining, non irritant antiseptic for wounds.
 Chlorine is usual disinfectant for municipal water supplies.
 One potential problem is that chorine reacts with organic
compounds to form carcinogenic trihalomethanes which must be
monitored in drinking water.
 Ozone some times has been used successfully as a alternative to
chlorination in Europe and Canada.
 Traditional chlorine containing pharmaceutical formulations are
Eusol- chlorinated lime and boric acid solution and Dakin’s
solution (surgical chlorinated soda solution BPC 1973) both of
which are designed to provide slow release of chlorine.
 Sometimes alternatively Chloramine T used for prolonged
release of chlorine.
 Halazone slowly released chloride when added to water and
disinfects.
HEAVY METALS
 Many metallic ions are toxic to essential enzyme systems
particularly those utilizing thiol (-SH) groups, but those used
medically are restricted to mercury, silver and aluminium.
 Phenyl mercuric nitrate(and acetate) as a bactericide in eye drops
and injections and Thomersal as a preservative in biological
products and certain eye drops.
 A 1% solution of silver nitrate is used into eyes of infants to
prevent ophthalmic gonorrhea
 Silver sulfadiazine is used on burns.
 Heavy metals combine with proteins often with their sulfhydryl
groups and inactivate them they may also precipitate cell
proteins.
 QUATERNARY AMMONIUM
COMPOUNDS
 The most popular of these disinfectant are quaternary
ammonium compounds characterized by a positively charged
quaternary nitrogen and a long hydrophobic aliphatic chain.
 They disrupt microbial membranes and may also denature
proteins.
 Cationic detergents like benzalkonium chloride and
cetylpyridinium chloride kill most bacteria but not
M.Tuberculosis or Endospores.
 Used as food utensils and skin antiseptics.
 Brands are Zephiran – Benzalkonium chloride
Ceepryn - Cetylpyridinium chloride
ALDEHYDES
 Commonly used aldehydes formaldehyde and glutaraldehyde are
highly reactive molecules that combine with nucleic acids and
proteins and inactivate them , probably by cross linking and
alkylating molecules.
 Formaldehyde is usually dissolved in water or alcohol before use.
 A 2% buffered solution of glutaraldehyde in an effective
disinfectant.
 It is less irritating than formaldehyde and is used to disinfect
hospital and laboratory equipment.
 Glutaraldehyde usually disinfectant objects within about 10 min
but may require as long as 12 hrs to destroy all spores.
ORGANIC ACIDS
 Benzoic acid , sorbic acid mechanism is these prevents the
uptake of substrates requiring a proton-motive force to enter the
cell.
 Preservation in oral products.
 Organic acid esters (parabens) – methyl, ethyl, butyl, propyl,
benzyl parabens and their salts.
 Preservation used principally in topical and oral products and in
some injections.
 Good activity against fungi.
BIGUANIDES AND AMIDINES
 Chlorhexidine is a widely used biocide which has activity against
gram +ve and gram –ve bacteria but little activity against
endospores or viruses.
 It is widely used in general surgery , both alone and in
combination with cetrimide and can also be used as a
preservative in eye drops.
 Biguanides act on the cytoplasmic membrane, causing leakage of
intracellular constituents.
 The aromatic diamidines , propamidines & di bromo
propamidine are non-toxic antiseptics mainly active against gram
+ve bacteria and fungi.
GASEOUS STERILIZATION
 The chemically reactive gases such as formaldehyde and ethylene
oxide (CH2)2O possess biocidal activity.
 Ethylene oxide is a liquid at temperature below 10.8 0C above
this temperature it vaporizes rapidly.
 Ethylene oxide is a colorless, odorless, & flammable gas.
 So this flammable nature overcome by preparing mixtures of
ethylene oxide in carbon dioxide which is now available
commercially.
 The CO2 – Ethylene oxide are non flammable and there is NO
alteration of the microbial activity of the ethylene oxide.
 The mechanism of Anti microbial action of these gases assumed
to be ALKYLATION of sulfhydryl , amino, hydroxyl and
carboxylic groups on proteins and amino groups of nucleic acids.
 Both of these gases being Alkylating agents are potentially
Mutagenic & Carcinogenic.
 They also produce acute toxicity including irradiation of the skin
, conjunctiva & nasal mucosa.
 It is used for sterilizing heat or moisture sensitive materials in
hospitals, industry & laboratories has become Universal.
Guidances as per FDA
 Guidance for the submission of documentation for sterilization
process validation in applications for Human and Veterinary drug
products .
 Additionally organizations like International Society For
Pharmaceutical Engineering (ISPE) and Parenteral Drug
Association have issued various documents which include all
facts of the International Regulatory Requirements.
 Guide to inspections of Lyophilization of parenterals.
 Guide to inspections of High Purity Water Systems.
 Guide to inspections of Microbiological Pharmaceutical Quality
Control Laboratories.
 Bacterial Endotoxins / Pyrogens; (Inspection Technical Guide).
Contd…
 Heat Exchangers to Avoid Contamination; (Inspection Technical
Guide).
 ICH Q5A, Guidance on Viral Safety Evaluation of Biotechnology
products derives from cell lines of Human or Animal Origin.
CONCLUSION
 Sterilization is a special process because it’s efficacy cannot be
verified by simple inspection and testing on the final product…..
For this reason , sterilization process have to be validated before
use, the performance of the process monitored routinely and
equipment regularly maintained.
References
 Prescott – Harley – Klein; Microbiology, Fifth Edition, Section
II , Microbial nutrition growth and control chapter 7; Control
of Microorganisms By Physical and Chemical agents, The
MEGRAW – HILL companies 2002 , Page. No ; 136 – 149.
 Microbiology by Michael J Pelczar , JR ; E.C.S Chan; Noel R .
Krieg, Fifth Edition , TATA MEGRAW – HILL Edition – 1993 ;
Page. No 469-507.
 Remington’s Theory and Practice of Pharmacy ; Sterilization
Vol I, chapter 40 ; 21st Edition, Page. No 776-801.
 Aulton’s Pharmaceutics ; The Design and Manufacturing of
Medicines ; Edited by ; Michael E Aulton ; Third edition;
Chrchill Livingston Elesevir ; Page. No 235-241.
 Pharmaceutical Microbiology and Biotechnology ; sterilization
methods and principles ; Dr Yashmin Sultana lecturer. Dept of
pharmaceutics, Jamia Hamdard New Delhi , Date 11-07-2007.
 Encyclopedia of PHARMACEUTICAL TECHNOLOGY
Third Edition VOLUME I Edited by James Swarbrick ;
Sterilization Dry heat, Page. No 3512 – 3517 and
Sterilization Moist heat ,Page. No 3529 – 3539.
 Sterilization; The Theory and Practice of Industrial
Pharmacy by Leon Lachman , Herbert A Lieberman,
special Indian edition 2009; CBS Publishers Page. No 619-
622.
 Sterilization (Microbiology)
http://wikipedia.org/wiki/sterilization_(microbiology)
 http://www.pharmainfo.net/siriki-praveen-
kumar/blog/principles-sterilization