This document provides an overview of sterilization including: - Definitions of key terms like sterilization, antiseptic, bacteriostatic, and viable - Classification of sterilization methods into terminal and non-terminal processes - Parameters used to measure sterilization effectiveness like D-value and z-value - Methods of controlling microorganisms through physical sterilization techniques like heat and radiation or chemical sterilization agents - Guidance on sterilization from regulatory bodies like the FDA - Conclusions on the importance of sterilization in various applications like pharmaceuticals and healthcare

2. 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

3. 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

4.  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

5. 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.

6.  BACTERIOSTATIC:-
 BACTERIOCIDAL:-
TIME

7.  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

8. 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.

9. 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.

10. 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.

11. 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.

12. 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

13. 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

14.  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

15.  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 card board paper
or aluminium.
 Then the material are arranged
to ensure uninterrupted air
flow.
Hot Air Oven

16. 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.

17.  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.

18.  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

19.  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

20.  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 Disposable
contact lenses.
AUTOCLAVE

21. 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

22. 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

23.  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

24. 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.

25. 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

26.  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.

27.  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.

28. 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.

29. 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

30. 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.

31. 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.

32. 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.

33. 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 ,

34.  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.

35. 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

36. 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.

37. 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.

38. 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.

39.  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