Sterilization.pptx

MODERN COLLEGE OF PHARMACY, PUNE
Savitribai Phule Pune University
B.PHARM
Semester- III
By
Mrs. Sneha K. Patil
Assistant Professor
Dept. of Pharmaceutics
Sterilization

UNIT OUTCOMES
 To understand
1. The different methods of sterilization with principle,
advantages, disadvantages and applications
2. Different equipments employed in large scale sterilization
3. The technique used for evaluation of efficiency of sterilization
methods
1

CONTENT
1. Introduction
2. Concepts related to controlling microorganism
3. Classification of sterilization methods
4. Physical methods
a. Dry heat sterilization
b. Moist heat/ steam sterilization
C. Radiation sterilization
D. Mechanical/filtration sterilization
5. Chemical methods
6. Definition of D value & Z value and its significance
7. Sterility indicators
8. Equipments employed in large scale sterilization
9. Reference
2

1. Introduction
3
 Sterilization is an essential stage in the processing of any product
used for parenteral administration, broken skin, mucosal surfaces or
internal organs
 Sterilization of microbiological materials, surgical dressings,
equipments and other contaminated items is necessary to minimize
the health hazard associated with these articles

Introduction continue…..
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 Importance
1. To prevent contamination in sterile products
2. To prevent transmission of pathogenic microorganisms which are
responsible for causing disease in plants, animals and human beings
3. To prevent decomposition and spoilage of food and food products
4. To prevent the contamination of unwanted microbes in pure cultures
and other microbiology experiments performed for research studies
5. To prevent unwanted microbial contamination in antibiotic, enzyme,
vitamins, fermentation and other industries process
6. To prevent contamination in aseptic areas/instruments which are
used for the preparation of sterile dosage forms and
sterility testing

2. Concepts related to controlling microorganism
5
Sterilization : It is a process by which an article, surface or medium is
free from all microorganisms either in vegetative or spore
form
Disinfection :
1. Destruction of all pathogens or organisms capable of producing
infections but not necessarily spores.
2. All organisms may not be killed but the number is reduced to a level
that is no longer harmful to health
Disinfectants : Disinfection is usually accompanied by chemical agents
is called disinfectants
It is applied to inanimate objects

Concepts related to controlling microorganism continue…..
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Sanitization : Sanitization is the process of disinfection including
cleansing action and sanitizers commonly applied to
inanimate objects
Antiseptic :
1. It is used to designate any substance which would prevent sepsis,
either by killing of microorganisms or by inhibiting their growth
2. It can be applied to body tissues without causing injury to the tissue
Germicide : It is an agent that kills vegetative cells but not necessarily
the resistant spore forms of germs

Concepts related to controlling microorganism continue…..
7
Microbiostasis: It is the process of preventing the growth, reproduction
and multiplication of microorganism but not of killing
them
Preservative :
1. It is a substance that prevents the growth of microorganisms
2. It is mainly added in food and pharmaceuticals to prevent microbial
growth
3. Preservatives are not harmful to living tissues

3. Classification of sterilization methods
8
1. Physical methods
a) Dry heat sterilization: e.g. Incineration, Direct flame, Red heat, Hot
air
b) Moist heat sterilization: e.g. Pasteurization, Tyndallisation, Autoclave
c) Radiation sterilization:
e.g. i. Use of Ultra-violet rays: UV light(Non-ionising),
ii. Ionising radiations: X-rays, Gamma rays, beta rays.
d) Filtration/mechanical method: e.g.
1. Asbestos filter(seitz)
2. sintered glass filter(morton)
3. filter candles(ceramic)
4. membrane filter(millipore/ ultra filter)

Classification of sterilization methods continue…
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2. Chemical methods
a) Gaseous sterilization: e.g. Ethylene oxide , Formaldehyde, Beta
propiolactone
b) By using disinfectant: e.g. Alcohols and Aldehydes, Phenols and
Halogens, Oxidizing agents and Salts

4. Physical methods
10
 These may involve the utilisation of heat in the presence or absence
of moisture or the applications of radiations or mechanical filtration
a) Dry heat sterilization
 Heat is the most reliable and rapid method of objects that can
withstand heat
 It is carried out by two ways i.e. Dry heat and moist heat
 Mechanism/ Principle
1. Protein denaturation
2. Oxidative damage
3. The toxic effect of elevated levels of electrolytes

Physical methods of sterilization continue….
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 Factors influencing sterilization
1. Nature of heat
2. Number of microorganisms present
3. Temperature and time
4. Characteristics of microorganisms
 Time required for sterilization is inversely proportional to the
temperature of exposure and this can be expressed as thermal
death time, which is the minimum time required to kill a
suspension of microorganisms at a prescribed temperature and
under specific conditions

12
 Sterilization time depend upon following factors,
1. Presence and nature of spores
2. Number of microorganisms present
3. Strain and characteristics of microorganisms
 Microbes are more resistant to dry heat as compared to moist heat,
therefore this process requires higher temperatures and long
exposure time

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1. Sunlight and drying
 Sunlight possess UV rays along with heat are responsible for
germicidal action, e.g. Natural method for sterilization of water in
tanks, reservoir, lakes
2. Heat
 Most reliable method of sterilization and should be the method of
choice
 Sterilization of metallic objects by holding them on a flame till they
are hot, e.g. Inoculating wire, needles, forceps

14
3. Flaming
 Passed over flame without allowing it to become red hot, e.g.
Mouth of culture tube, glass slides, scalpels, needles, cover slips, etc.
 It destroys only vegetative microorganisms
4. Incineration
 Excellent method for rapid destroying materials, e.g. Pathological
material, contaminated cloth, animals carcasses, bedding, soiled
dressing

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5. Hot air oven
 Hot air ovens are electrical devices and rapidly used in sterilization.
 The oven uses dry heat to sterilize articles
 Generally, they can be operated from 50 to 300 C (122 to 572 F)
 There is a thermostat controlling the temperature
Table 1. Temperature and time relationship for hot air oven
Temperature (°C) Time (minutes)
170 60
160 120
150 150
140 180

Hot air oven continue…..
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Fig. 1 Hot air oven

17
 Construction
1. Oven consist of a double walled chamber of aluminium or stainless
steel separated from the outer case by a thick layer of insulation
made of fibreglass
2. Insulation is filled in the hollow flanged door, which carries an
asbestos jacket that provides a tight seal
3. Heating is affected by electrical heating elements and thermostat
4. Material should be arranged in a manner which allows free
circulation of air between objects and not be overloaded
5. Glass ware should be dry and wrapped in kraft paper
before placed in oven

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 Construction
6. Oven must be allowed to cool for 2 hrs. before door is opened, to
avoid cracking of glasswares by sudden cooling
7. Substances that are not heat labile and can tolerate temp. upto
250 C are sterilized by hot air oven
8. Spores and vegetative forms of microbes are killed in two hrs, at a
temp. of 160 C

Hot air oven continue…..….
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 Advantages
1. It is suitable method for sterilization of substances destroyed by
moisture
2. They do not require water and there is not much pressure build up
within the oven, unlike an autoclave, making them safer to work with
3. Suitable and easy to be use in a laboratory environment and
inexpensive
4. They are much smaller than autoclaves but can still be as effective
5. It does not cause metals to corrode or rust
6. It does not release any harmful or hazardous fumes or pollutants

20
 Disdvantages
1. long heating time and high temperature is required for sterilization
2. As they use dry heat instead of moist heat, some organisms like
prions, may not be killed by them every time
3. Many objects cannot withstand the very high temperatures required
for dry heat sterilization (e.g. some plastics would melt)

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 Applications
1. To sterilize glassware, forceps, scalpels, scissors, spatula, swabs,
some pharmaceutical substances
2. To sterilize oils, fats and oily injections
3. To sterilize powders e.g. Talc, starch, zinc oxide

b) Moist heat sterilization
22
 Moist means killing of microorganism with hot water/steam
 Mechanism/principle - Denaturation and coagulation of proteins
 Divided into three forms in terms of temperature:
1. Temperature below 100°C
2. Temperature at 100°C
3. Temperature above 100°C

1. Temperature below 100°C
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 Heat labile fluids disinfected by heating at temp. below 100°C
 Pasteurization – Temp. employed is either 63°C for 30 min.(holder
method) or 72°C for 20 seconds (flash method) followed by rapid
cooling
 It is applied in dairy products e.g. milk and butter
 By this method non sporing microorganisms such as mycobacteria,
brucella and salmonellae are destroyed
 Heat labile fluids e.g. serum may be disinfected by heating at 56°C
for one hr.
 Vaccines prepared from nonsporing bacteria may be
inactivated in water bath at 60°C for one hr.

2. Temperature at 100°C
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 Boiling at 100°C for 10 – 30 minutes may kill most of vegetative
bacteria and some bacterial spores
 It is not suitable for sterilization of surgical instruments
 Addition of small quantity of acid, alkali or washing soda to increase
penetration power of boiling water
 Tyndallisation/intermittent/fractional sterilisation
1. Single exposure to steam at 100°C for 20 minutes on three
successive days e.g. used for egg , serum and sugar containing media
2. First exposure to steam kills all vegetative bacteria, second exposure
all spores germinate in a favourable medium and are
killled on subsequent occasions

Temperature at 100°C continue…
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 An atmosphere of free steam is used to sterilize culture media which
may decompose if subjected to higher temp.
 Steam sterilizer : Steam at 100°C for 90 minutes and used for media
which are decomposed at high temperature e.g. A koch or arnold
steam sterilizer

3. Temperature above 100°C
26
 Heat in the form of saturated steam under pressure is used
 Laboratory apparatus designed to use steam under regulated
pressure is called an autoclave
 Mode of action :
1. Moist heat is responsible for disruption of cell components
2. Coagulation and denaturation of proteins ( proteins are denatured
more rapidly at lower temp. if moisture is present)
3. Other cell components – cell membrane, ribosome, DNA and RNA
also denatured by moist heat

Temperature above 100°C continue….
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 Saturated steam is more efficient sterilizing agent than hot air,
because,
1. Moist heat provides greater lethal action
2. It is quicker in heating up the exposed articles
3. It can penetrate easily porous material such as cotton wool
stoppers, paper and cloth wrappers
 Autoclave consists of vertical or horizontal cylinder of gunmetal or
stainless steel
 Lid is fastened by screw clamps and rendered air tight by an
asbestos gasket
 Lid bears a discharge tap for air and steam, a pressure
gauge and a safety valve

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Fig. 2 Vertical autoclave

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Fig. 3 Horizontal autoclave

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 Working of autoclave
1. Water is added on the bottom of the autoclave and articles to be
sterilized are placed in a perforated shelf
2. The lid is closed, discharge tap is opened and safety valve is
adjusted to the required pressure
3. When the air bubbles stop emitting from the discharge tap it
indicates all the air from inside the autoclave has been removed
4. At this stage, the discharge tap is closed
5. Steam pressure rises inside and when it reaches the desire set
level(15p.s.i) the safety valve opens and excess steam escapes

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Working of autoclave
6. From this point the holding time(15mins) is counted
7. When the holding time is over, the heating is stopped and autoclave
is allowed to cool till pressure gauze indicates that the inside
pressure has reached to the atmospheric pressure
8. The discharge tap is opened slowly and air is allowed to removed
from the autoclave
9. The lid is opened and the sterilized articles are removed

Temperature at 100°C continue…
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Temperature (°C) Steam pressure
(lb/sq.inch)
Holding time (Minutes)
115-118 10 30
121-124 15 15
126-129 20 10
135-138 30 3
Table 2. Autoclaving conditions (temperature/time/pressure
relationships)

Temperature above 100°C continue…
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 Advantages
1. It is rapid and effective.
2. It destroys microorganisms more efficiently than dry heat and
therefore a shorter exposure at a lower temperature is possible.
3. It can be used for a large proportion of the official injections.
4. It is supplied with dry saturated steam porous materials and can be
sterilized without damage.
5. Equipment or components of rubber and certain plastics such as
nylon and P.V.C will withstand the conditions.

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 Disadvantages
1. Items sensitive to heat cannot be sterilized
2. It is unsuitable for anhydrous materials such as powders and oils
3. It cannot be used for injections and articles such as plastics that
deteriorate at 115°C

35
 Applications
1. This method is most essential biocidal agent
2. It is used for surgical dressings, sheets, surgical and diagnostic
equipments, containers, closures, aqueous injections, ophthalmic
preparations, glassware etc.
3. To sterilize aq. Solutions e.g. broth and media

C) Radiation sterilization
36
 Energy transmitted through space in a variety of forms is called
radiation
 Radiation sterilization also called as cold sterilization because
ionizing radiation produce little heat in the material irradiated
 It is suitable for sterilization of heat sensitive substances
 Based on wavelength and penetration power, can be divided into
two categories
1. Non ionizing radiation - Less energy and do not disturb the
atomic configuration of the target molecules
2. Ionizing radiations - High energy and ionize target
molecules

1. Non ionizing radiation
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 UV radiations having wavelength 2537 A shows greatest activity in
destroying microbes
 Penetration power is negligible, so effectiveness is limited to surfaces
only
 Source of artificial radiation is UV lamps, called as sterilizing
lamps/germicidal lamps
 Vegetative bacteria is susceptible but spores are resistant to UV light
 UV rays can damage eyes and are known to cause sun burns and skin
cancers in humans

Non ionizing radiation continue……
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 Mechanism –
1. UV light is absorbed by the nucleic acids of the cell where it does
greatest damage
2. These rays induce the production of abnormal nucleotides such as
thymine dimers
3. These interface in the process of DNA replication
Fig. 3 Mode of action of
non-ionizing radiations

Non ionizing radiation continue……
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 Applications
1. UV rays are used extensively in hospital rooms, in aseptic filling
rooms, in the pharmaceutical industry (sterile product preparation),
food and dairy industries for treatment of contaminated surfaces
2. Sterilizing biological fluids such as blood plasma and vaccines
3. Purification of liquid including milk, fruit, juice, wine and beer
 Disadvantages - UV rays can damage eyes and are known to cause
sun burns and skin cancers in humans

2. Ionizing radiation (cold sterilization)
40
 X-rays, gamma rays and cathode rays are highly lethal to DNA and
other vital cell constituents
 They have very high penetration power and considerable energy
 The factors that effect the lethal activity of ionizing radiations are as
follows;
a. Oxygen
b. Protective compounds
c. Sensitizing agents
d. pH of culture
e. Freezing
f. Moisture and recovery conditions

Ionizing radiation continue…….
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A. X-rays
1. X-rays have considerable energy and penetration ability that is used
to produce lethal effect on microorganisms
2. Disadvantage – Impractical for purposes of controlling microbial
populations because they are very expensive and difficult to utilize
efficiently
3. It is widely employed experimentally to produce microbial mutants

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B. Gamma rays
1. Gamma rays are similar to X-rays but have higher energy and
shorter wavelength
2. It is obtained using radioactive isotopes of 60Co
3. Two gamma rays are emitted in a succession as a result of
disintegration of almost all of the unstable atoms of this isotope
4. The radiant energy particle makes a ‘direct hit’ on some essential
substances such as DNA within bacterial cell, causing ionization
which results in the death of cell

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B. Gamma rays
 Application- Because of high penetrating ability and microcidal
effect, these rays are ideal for sterilization of bulk material i.e.
packaged food, medical instruments

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c. Cathode rays (electron-beam radiation)
1. A high- voltage potential is established between a cathode and an
anode in an evacuated tube, the cathode emits beams of electrons,
called cathode rays or electron beams
2. Special instruments to produce electron beams of high intensity and
velocity
3. Electron accelerator, which is extensively used for sterilization of
drugs, surgical and other materials

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Mode of action
 Ionizing radiations disrupts the atomic structure of molecule by
dislodging/ejecting the electron and thus results in an electrical
imbalance and formation of ions
 One of the most sensitive targets id DNA molecule
 Ionizing radiations induce mutations which generally do not get
repaired
 Radiations dosage is measured in rads (radiation absorbed dose)
 Depending on application, exposure ranges from 0.5 to 5 megarads

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Mode of action
 All ionizing radiation can penetrate solids and liquids but gamma rays
have the highest penetrating power, whereas x-rays have
intermediate and cathode rays have least penetrating power

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 Mode of action
Fig. 4 Mode of action of ionizing radiations

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Applications
 Ionizing radiations is a satisfactory method of sterilization of
antibiotics e.g. benzyl penicillin, streptomycin sulphate, polymyxin
sulphate and vitamins e.g. ascorbic acid. Sulphonamides, lactose, talc
 Irradiations have also been applied to inactivate suspensions of
influenza, vaccinia, rabies and poliomyelitis viruses for use as
vaccines
 Sterilizing medical products including drugs, syringes, surgical gloves
and tissues like bone, cartilage, skin and heart valves
 Foods like, meat can be sterilized

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Disadvantages
 Radiation irradiated food are altered in flavour
 Main risk is to the machine operator from exposure of radiation

d) Filtration/mechanical methods of sterilization
50
 To sterilize fluids (liquid/gas) , they are passed through various
bacteriological filters
 This method used specifically for heat sensitive fluids
 Steps,
1. Passage of the solution through a previously sterilized bacteria
proof filter unit
2. Aseptic transfer of the filtrate to sterile containers which are then
sealed aseptically
3. Testing of sample for sterility

Filtration sterilization continue……
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 Filter efficiencies depend on the following factors,
1. Pore size
2. Wall thickness
3. Filtration rate
4. Positive or negative pressures
5. Nature of liquid to be filtered
 During filtration sterilization of pharmaceutical product, a sterile
technique must be maintained throughout the operation and filters
together with all of the assembly must be sterilized before use

52
 The various types of bacteria proof filters used are as follows
1. Asbestos filter/Seitz filter
 Disposable, single use discs made up of asbestos (magnesium
trisilicate)
Fig. 5 Seitz filter

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1. Asbestos filter/Seitz filter
 It consists of two parts
 The lower part holds a perforated disc and the upper part is
compressed asbestos sheet, two parts are joined together with the
help of nuts
 Fluid to be sterilized is put into the funnel and flask is connected to
the exhaust pump
 After completion of filtration, filter is discarded and entire unit is
sterilized
 Pore size of filters range from 0.01 to 5 microns

54
2. Sintered glass filters (fritted glass filters/morton filters)
 Borosilicate glass is finely powdered in a ball mill and packed into
disc moulds and heated until suitable adhesion takes place between
granules
Fig. 6 Sintered glass filter

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2. Sintered glass filters (fritted glass filters/morton filters)
 Sintered glass filters are available in different pore sizes and are
numbered accordingly, i.e. grade 5 / 5 on 3 must be used
 They have a low adsorptive property and can be cleaned easily
 They are brittle and expensive and have a small area of filtration

56
3. Filter candles (ceramic / Berkefield filters)
 They are made of either porous porcelain or kieselghur and are
available in a range of pore size
Fig. 7 Filter candles

57
3. Filter candles (ceramic / Berkefield filters)
 The candle is placed in the solution to be sterilized and its opening is
attached to the vacuum system
 When vacuum is applied the pressure inside the candle is decrease
and due to difference in pressure between the outside and inside of
the candle, the solution moves into the candle
 The filtrate is collected in sterile container
 These filters are inexpensive
 The main disadvantage of ceramic filter is its tendency to absorb
materials from aqueous solutions

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4. Membrane filter (Millipore/ultra filter)
 They are made up of cellulose and cellulose esters
 They are 150 m thick and contains millions of microscopic pores
ranging from 0.01 to 10 m in diameter
Fig. 8 Membrane filters

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Membrane filter (Millipore/ultra filter)
 Pore size is 0.45 m ± 0.02 m (millipore, HA) or 0.22 m ± 0.02 m
(millipore, GS) , particularly for very small bacterial contaminants
 They are sterilized by autoclaving, in the holder or packed between
thick filter pads to prevent curling
 They are available at ready sterilised form(by ethylene oxide/
ionizing radiation)
 Membrane filters are supported on a rigid base of perforated metal,
plastic or coarse sintered glass (fig. 8)
 HA grade filters are approximately 65 ml/min./sq.cm.
(GS- 22 ml/min./sq.cm. ) with a differential pressure of
70 cm mercury across the membrane

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Advantages
1. All microorganisms are separated by process of sieving
2. Membranes have a high and uniform porosity permitting a rapid
rate of filtration
3. Membranes are disposable, so no cross contamination between
filtered products
4. Adsorption is very less

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Disadvantages
1. Prefilter is used before the membrane filter to avoid clogging and
breaking
2. They have less chemical resistance to certain organic solvents such
as chloroform, ketones and esters
3. It cannot remove viruses and toxins

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Applications
 Membrane filters are routinely used in water purification and
analysis, sterilization, sterility testing and for the preparation of
solutions for parenteral use
 They also used for identification and enumeration of microbes from
water samples and other materials
 To sterilize liquids that cannot withstand heat e.g. serum, other
blood products, enzymes and culture media

2. Chemical methods
63
A. Gaseous sterilization
 Gaseous sterilization is defined as the destruction of all living
microorganism with a chemical in a gaseous or vapour state
 Material adversely affected by dry and moist heat are then sterilised
this method
 All these gases are toxic to human being above certain
concentrations and exhibit other unpleasant or undesirable side
effects
 Ethylene oxide is most widely used than formaldehyde and  -
propiolactone, in addition to these, various glycols, methyl bromide
and alcohol have been used for room sterilization

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1. Formaldehyde (HCHO)
 This gas is generated by heating a concentrated solution of
formaldehyde
 Formaldehyde in aq. Solution is known as formalin and contain 37 to
40 % formaldehyde
 Vaporisation of formaldehyde, either from formalin or
paraformaldehyde, is used to sterilize n enclosed area
 Formaldehyde gas is generated by adding 150 gm KMnO4 to 280 ml
formalin for every 1,000 cu. ft. of room volume

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 After start of generation of formaldehyde vapour, the doors should
be sealed and left unopened for 48 hrs. and about 70% RH and 22C
temp. gives best sterilization results
 It is an extremely reactive material
 Mode of action –
a. It is also a group of alkylating agent
b. It inactivates microorganisms by alkylating the amino acid and
sulfhydryl groups of proteins and ring nitrogen atoms of purine
bases
 It is bactericidal agent with poor penetration power

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 Applications
1. Disinfection and sterilization of enclosed area such as operation
theatres, hospital rooms, aseptic area and microbiology laboratories
2. It kills both vegetative cells and spores

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2.  - propiolactone
 Heterocyclic ring compound, colourless liquid, at room temp. with
high boiling point 155C
 It capable of killing all microorganisms and very active against
viruses
 BPL vapour is 25 times more active as disinfectant than
formaldehyde, 4000 times more active than ethylene oxide and
50000 times more active than methyl bromide

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2.  - propiolactone
 Bactericidal agent conc. is 2 to 5 mg/litre
 Low penetration power
 It shows irritation and carcinogenic properties, so not recommended
for pharmaceutical applications

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3. Ethylene oxide
 It is colourless liquid with a boiling point of 10.8C
 Highly inflammable and may be explosive when mixed with air in
concentrations greater then 3%
 Its mixtures with carbon dioxide or fluorinated hydrocarbons
(freons) in certain proportions makes ethylene oxide non-flammable
 The carbon dioxide and freons act as inert diluents which prevent
flammability

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3. Ethylene oxide
 Effect of ethylene oxide as sterilizing agent depend on conc. of gas,
temperature, moisture, time, conditions and accessibility of the
microbes
 Concentration and time relationship commonly used for sterilization
is given in table
Table 3. Temperature and time relationship of ethylene oxide as a
sterilizing agent
Concentration (Mg/lit.) Exposure time (hours)
44 24
88 10
442 4
884 2

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Ethylene oxide
Mode of action
 Its power of alkylating the amino, carboxyl, hydroxyl and sulphydryl
groups in the enzymes and protein molecule
 It reacts with DNA and RNA
 In this reaction the ring in the ethylene oxide molecule splits and
attaches itself where the hydrogen is present

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3. Ethylene oxide
Applications
 Powerful sterilizing agent for heat and moisture sensitive materials
 Useful for sterilization of medical and biological preparations, catgut,
plastic equipments, books, clothing and soil

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B. By using disinfectants or antimicrobial agents
 Chemical agents most commonly used as disinfectants and
antiseptics are phenols, alcohols, halogens, dyes, aldehydes etc.

6. Definition of D value & Z value and its significance
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 D value / decimal reduction time
1. Time in minutes at any defined temp. to destroy 90% viable
microorganisms is called D- value
2. It usually has a subscript showing the temp. at which it was
measured, e.g. D115C or D121C
3. Significance – Indicate efficiency of sterilization process

Definition of D value & Z value and its significance continue….
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Fig. 9 Calculation of D value and Z value

Definition of D value & Z value and its significance continue….
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 Z value/ thermal destruction value
1. Number of degrees of temperature change required to produce a
10 fold change in D value called Z- value
2. Bacterial spore have a Z value in the range 10 to 15C while most
nonsporing organisms have Z value of 4 to 6C
3. Significance – This relates the heat resistance of a microorganism to
changes in temperature

7. Sterility indicators
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 It is essential that strict controls are carried out on products to be
called ‘sterile’, such controls must then ensure, the absence of viable
microorganism from these products
 Two types of controls
1. Controls on process of sterilization i.e. sterilization monitors or
sterilization indicators
2. Sterility testing of the products
 Monitoring of the sterilization process can be achieved by the use of
physical, chemical or biological indicators of the sterilization
performance

Sterility indicators continue….
78
1. Physical indicators
a. Moist heat
 A Master Process Record (MPR) is prepared as part of the validation
procedure for a particular autoclave and for each specified product
and load configuration
 This may be used as a reference for the process record obtained
from a single thermocouple placed in a strategic part of each load
(BPR)
 The MPR should be checked at annual intervals and whenever
significant changes occur in the BPR (Batch Production Records)
when compared with the MPR

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a. Moist heat
 Microprocessor-controlled sterilization cycles are now a part of
modern autoclaves
 Pressure is measured by pressure gauzes/ pressure transducers
b. Dry heat
 In dry heat sterilization processes, a temperature record chart is
made of each sterilization cycle and is compared against a master
temperature record

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c. Radio sterilization
 A plastic dosimeter gives an accurate measure of the radiation dose
absorbed and is considered to be the best technique currently
available for the radio sterilization process

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d. Gaseous methods
 For gaseous sterilization procedures, elevated temperatures are
monitored for each sterilization cycle by temperature probes and
routine leak test are performed to ensure gas-tight seals
 Gas concentration is measured independently of pressure rise,
often by reference to the weight of gas used
 Pressure and humidity measurements are record

82
e. Filtration
 Bubble point pressure test is a technique employed for determining
the pore size of filters and may also be used to check the integrity
of certain types of filter devices immediately after use
 The principle of the test is that the filter is soaked in an appropriate
fluid and pressure is applied to the filter
 The pressure difference when the first bubble of air breaks away
from the filter is equivalent to the maximum pore size

83
e. Filtration
 When the air pressure is further increased slowly, there is general
eruption of bubbles over the entire surface
 The pressure difference is equivalent to the mean pore size

84
2. Chemical indicators
 Chemical monitoring of a sterilization process is based on the ability
of heat, steam, sterility gases and ionizing radiation to alter the
chemical or physical characteristics of a variety of chemical
substances

85
a. Browne’s tubes
 Most commonly used chemical indicator for heat process
 Contains small sealed coloured tubes having a reaction mixture and
an indicator
 Expose to high temperature resulting in the change of colour of the
indicators (Red to green)

86
a. Browne’s tubes
 Four types of browne’s tube as follows
Browne’s tube Method of
sterilization
Temp. (C) Colour of
indicator
Type I Moist heat 126 Black spot
Type II High vacuum moist heat 130 or more Yellow spot
Type III Dry heat 160 Green spot
Type IV Dry heat Infra red
conveyor oven
180 Blue spot

87
b. Witness tubes
 Consist of single crystalline substances of known melting point
contained in glass tubes, e.g. Sulphur (115°C), Succinic anhydride
(120°C), Benzoic acid(121°C), etc
 A dye may be included to show more clearly that the crystals have
melted
 Indicates that a certain temperature has been reached

88
b. Witness tubes
 Exposure time can be calculated by putting the crystals in one end
of an ‘hour glass tube’, the volume of the crystals and diameter of
the constriction of the tube being adjusted so that the time for
transfer of the melt is same as that required for the sterilisation at
the required temperature

89
c. Heat sensitive tape
 It is used quantitatively in the Bowie Dick test
 This is test to determine that all air has been removed from
dressings and that subsequent steam penetration has been even
and rapid
 Tape is placed suitably wrapped at the centre of a test pack
 All the bars on the tape should change colour to demonstrate full
penetration of steam

90
d. Royce sachet
 It is used as chemical indicator for Ethylene oxide sterilization
 Consists of a polyethylene sachet containing magnesium chloride,
HCl and bromophenol blue indicator
 A given concentration- time exposure to ethylene oxide results in
formation of ethylene chlorhydrin and colour change from yellow to
purple

91
e. Chemical dosimeters
 Chemical dosimeter give an accurate measure of the radiation dose
absorbed and best technique currently available for controlling
radiation sterilization
 Qualitative indicators made of radiosensitive chemicals
impregnated in plastic are available
 The indicator changes from yellow to red during irradiation

92
3. Biological indicators
 It consist of suitable organism deposited on a carrier and are
distributed throughout the sterilizer load
 At the end of the sterilization process, the units are recovered and
cultured to determine the presence or absence of survivors
 It measures sterilization process directly and is able to integrate all
sterilization parameters

93
3. Biological indicators
Sterilization process Species D value
Autoclave at 121 C Bacillus stearothermophilus
Clostridium sporogenes
1.5 min
0.8 min
Dry heat at 160 C Bacillus subtilis var. niger 5 – 10 min
Ethylene oxide at 600 mg/lit
(Temperature - 54C, 60% - relative
humidity)
Bacillus subtilis var. niger 2.5 min
Ionizing Radiation Bacillus pumilus 3 kGy (0.3 M rad)
Membrane filter (0.45 m pore size) Serratia marcescens -
Membrane filter (0.22 m pore size) Pseudomonas diminuta -

8. Equipments employed in large scale sterilization
94
Fig. 10 Hot air oven

Equipments employed in large scale sterilization continue…
95
Fig. 11 Composite material large
scale autoclave equipment

96
Fig. 12 Non ionizing radiation

97
Fig. 13 Cold sterilization

98
Fig. 12 Ethylene oxide sterilization

5. References
99
 Patil U, Kulkarni JS, Chaudhari AB, Chincholkar SB. Foundations
in microbiology. 4th ed. Pune: Nirali prakashan; 2006. Pg. no.
1.1-1.4, 5.1 – 5.2
 Kokare C. Pharmaceutical microbiology. 2nd ed. Pune: Nirali
prakashan; 2019. Pg. no. 1.1 -1.18
 https://en.wikipedia.org/wiki/Staining

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