....

‫السالم عليكن ورحمة هللا وبركاته‬

cold sterilization
dry heat sterilization radiation
cold sterilization

1) Low temperature :

 Effect depends on microbe and treatment applied

 Types :

a) Refrigeration :

 Temperatures from 0- 7oc

 It is consider bacteriostatic method
 Reduces metabolic rate of most microbes so they cannot reproduce or produce
toxins

b) Freezing:

 Temperatures below 0oc

 Types :
o Flash freezing: does not kill most microbes ( -80oc)
o Slow freezing: more harmful because ice crystal disrupt cell structure ( -40oc)
 Over a third of vegetative bacteria may survive 1 year
 Most parasites are killed by few days of freezing
 Used for seeded agar

cold sterilization heat sterilization

low temperature method

Dr El-Attar Page 1
sterilization method 7
metabolic rate
freezing toxin
-80 flash
slow freezing
-4 cell structure disruption
parasites vegetative bacteria
freezing

2) Desiccation :

 Disrupts microbial metabolism

 Bacteriostatic by time becomes bactericidal method
 In the absence of water, microbes cannot grow or produce , but some may remain
viable for years . after water becomes available , they start growing again ,
Exceptions :
a) Neisseria gonorrhea , only survives about one hour
b) Mycobacterium tuberculosis , may survive several months
c) Viruses are fairly resistant to desiccation
d) Clostridium spp. and Bacillus spp. , may decades
e) Viruses and endospores can desiccation

desiccation
dosage forms
bacterial growth tablets contamination
Neisseria gonorrhea exception
viruses Mycobacterium tuberculosis
spores spores bacteria
bacillus clostridium

Dr El-Attar Page 2
 3) Osmotic pressure :

 The use of high concentrations of salts and sugars ( salting or sugar curing) in food is
used to increase the osmotic pressure and create a hypertonic environment
 Plasmolysis: as water leaves the cell, plasma membrane shrinks away from cell wall.
Cell may not die, but usually stops growing.
 In industries, preservative added to syrup must act on yeast and mold and
staphylococci because that:

o Yeast and molds: more resistant to high osmotic pressures

o Staphylococci spp. That live on skin fairly resistant to high pressure .e.g. mannitol
salt agar

osmotic pressures

plasma membrane hypertonic

molds yeast exceptions plasmolysis
mold yeast syrup preservative
staphylococci
mannitol salt agar

4) Filtration :

Liquid and aqueous solutions containing thermolabile ingredients may

be sterilized by filtration through, a bacterial filter

Advantages of filtration :

1. Rapid
2. Suitable for thermolabile
3. Conducted at any temperature
4. Remove both living and dead bacteria
5. Neutral " doesn't affect the physical or the chemical nature of the sterilized material "
6. No heat or humidity or pressure applied
7. Filtration is a unique process , it removes rather than destroys

pore size filter solution filtration

solution
suspension emulsion suspension

Dr El-Attar Page 3
 filterattion emulsion
filter

disadvantages of filtration :

1. Only for liquids or solutions or for air

2. Filling after sterilization requires good aseptic technique. In
aseptic area " must use HEPA filter (High efficiency particulate
air)"
3. No in – process control " sterility tests must be carried on the
final preparation
4. Not retain- viruses or bacterial toxins "pyrogen"
5. Need skills and highly trained staff

liquids filtration
air filter powder
toxins HEPA
sterilization heat sterilization
pores
filtration

Types of bacterial filters :

a) Depth filter :

Are thick filters which retain the bacteria inside sharp angled steaks

 Mechanism of action :

a) Trapping of microorganism
b) Adsorption

 Types :

a) Sintered ceramics : made from ground porcelain or diatomaceous melted as

candle
b) Sintered glass: made from borosilicate glass
c) Fibrous pads: containing asbestos and wood cellulose e.g. Seitz filter

Dr El-Attar Page 4
 depth filter
trapping pores
depth mechanism sterile solution
depth adsorption trapping filter
filter
Sintered ceramics 
Sintered glass borosilicate glass 
fibrous pads asbestos 
Seitz filter

b) Membrane filter :

 Thin filter consisting mainly of pores

 Apply vacuum
 Mechanism of action: Sieving "screening"
 Prepared from : Cellulose ester of "Acetate & Nitrate"
 Examples:
a) Oxoid ®
b) Millipore®

pores filter paper membrane filter

pore size pore size
viruses 0.01 0.22 0.45
screening sieving membrane filter
acetic acid cellulose ester
Millipores Oxoid membrane filter nitric acid
depth filter membrane filter

Dr El-Attar Page 5
 Difference between membrane & depth filter :

Membrane filter Depth filter

 Mechanism Screening "sieving" Trapping
 Use Disposable Durable
 Support Need support Not support ( except ; fibers)
Less easy ( streaks are larger so
Clogging is easy but can be
 Clogging that particles may be forced and
minimized by pre-filter
pass)
 Stability Stability to chemical is variable Good
Slower ( increased by vacuum
 Rate Faster filtration but rapid clogging
pump)
 Absorption & Low absorption and adsorption of
Higher
adsorption fluid or chemicals
Shedding of fibers and filter
 Shedding No shedding of particles
component

depth filter membrane 

membrane filter 
membrane filter sieving
support disposable
HCL caustic clogging
adsorption
filter paper

depth filter 
support trapping
particles clogging
rate stable pore size
depth filter vacuum
membrane filter adsorption adsorption
filtered solution

Dr El-Attar Page 6
 5) Radiation :

 Acts by destroying DNA or damaging it

 Its efficiency is dependent on:
a) Wavelength
b) Intensity
c) Duration

DNA radiation
penetration wavelength
duration radiation radiation intensity
sterilization

Classification of radiation :

1) Electromagnetic waves :
 IR radiation (dry heat sterilization)
 UV
 X-ray ( not absorbed by bacteria)
 Gamma radiation

2) minute particles :

 Alpha (α)
 Beta (β)

radiation 

IR radiation electromagnetic waves 

UV radiation sterilization dry heat sterilization IR
mutation thymidine dimer
X-ray
gamma radiation
beta alpha particles radiation 

Dr El-Attar Page 7
 Electromagnetic waves :
may be :
1. Ionizing radiation:
 High degree of penetration as , the low wave length , the high penetration
 Example:
a) Gamma rays
b) X-rays
c) High energy electron beams

2. Non ionizing radiation:

 Low degree of penetration " high wave length "
 Harmful to skin & eye
 Cell damage "thymidine dimer"
 Germicidal 260 nm
 Example:
a) UV " ultraviolet"

electromagnetic wave

high DNA ionized 

penetration penetration
X-ray Gamma ray
penetration power non-ionizing radiation 
cancer UV UV
260 nm thymidine dimer

Radiation expression :

 Radiating energy is effective only when absorbed

 it is expressed in no. of electron volts absorbed (eV) for UV radiation and millions of
electron volts (MeV) for ionizing radiation
 The energy content is higher in shorter wave length
 The low wave length , the high penetration

million electron volt electron volt radiation

penetration
radiation

Dr El-Attar Page 8
 Different radiations vary in their effects & mode of action :

a) IR radiation b) UV radiation c) Ionizing radiation

Wave length 700-950 nm 200 – 300 nm Very short
Excitation " thymidine
Mechanism Dry heat Ionization
dimer"

b) UV radiation :

 It is non- ionizing radiation

 Only narrow range suitable for sterilization (200-300 nm) Above and below ,
antimicrobial activity is very low

 Source : mercury lamps

 Uses :

 To maintain the microbial quality of air in aseptic area , hospitals,….. etc

 To improve microbial quality of water
 Sterilization of certain vaccines and antisera

dry heat sterilization IR

thymidine DNA excitation UV 700-950 nm
mechanism ionizing radiation dimer
ionization
non- ionizing radiation UV radiation
200-300 nm sterilization UV
mercury lamp UV
radiation source
2ry sterilization UV sterilization power
UV method
anti-sera vaccines bacterial growth

Dr El-Attar Page 9
 Mode of action :

 By excitation that is less damaging than ionization

 UV rays are mainly used for disinfection and not sterilization

Direct Indirect
 Excitation or intermolecular vibration
of vital molecules such as DNA & UV
enzymes causing death or mutation O2 + H2O O3 + H2O2
 Formation of thymidine dimer between
2 adjacent thymine bases in bacterial It forms Ozone , hydrogen peroxide & free
DNA leading to inhibition of DNA radicals are toxic and affect stability
replication
mutation DNA vibration mechanism 
radiation direct thymidine dimer
DNA
UV indirect 
stability free radical Hydrogen peroxides
DNA

Wave lengths 265 and 280 nm are more selective than the shorter
waves200 and 220 nm ?
Due to their selective absorption, by the vital targets:
 DNA at 265 nm
 Aromatic acids of enzymes at 280 nm

sterilization 

DNA 265 
280 nm aromatic acids 

 Disadvantages of UV radiation :

1. Low activity
2. Mutagenic
3. Poor penetration power through packaging materials
4. Activity is much affected by dust particles and distance
5. Damaged cells when exposed to visible light immediately after treatment that
may be reactivated "photo reactivation" , thus avoided by application in dark

Uv sterilization UV disadvantage
low penetration mutagenic

Dr El-Attar Page 10
 DNA visible light thymidine dimers dust

 Choose the correct answer :

1. Temperature range for 'pasteurization' is 2. the source of UV radiation is:

a) 60°C-70°C a) Na lamp
b) 62°C-72°C b) Tungsten lamp
c) 65°C-75°C c) Mercury lamp
d) 121°C-130°C d) fluorescent lamp
b c

Ionizing radiation :

Have very short wave length and hence very high energy content , act through the
following mode of energy

 Examples :

 Particles .e.g. alpha & beta particles

 Waves.e.g. gamma radiation

Mechanisms of action :

a) Direct effect b) Indirect effect

 Effect of free radicals:
-e- →H2O+→OH* +H+
H2O O2 O2
+e- →H2O-→OH- +H* HOO*
H2O2
Release electrons causing ionization
of DNA and vital molecules (  These are either:
excitation)
 Highly reducing (H* ).
 Oxidizing (HOO*,H2O2&OH* )

Therefore in presence of water the antimicrobial

activity is much Increased but the ability of the
product is much reduced

Dr El-Attar Page 11
 To minimize losing stability :

1. Freezing using iso-pentane (-1 40°C) to immobilize the free radical.

2. Using Quenching agents e.g., cysteine and thiourea.
3. Removal of O2." Prevent its reduction to prevent it converting to H2O2"

wave length ionizing radiation

alpha particles ionizing radiation
gamma radiation waves beta
mechanism 
DNA excitation direct 
DNA OH* H* free radical indirect 
H2O radiation
hydroxyl positive charge 
radical
hydroxyl gp negative charge 
hydroperoxyl hydrogen radical O2 H*
hydrogen peroxide O2 radical

oxidizing radical sterilization product

H* reducing radical OH* H2O2 HOO*
radicals radical
thiourea cysteine radical
hydrogen peroxide O2

Radiation expression :

 Radiation is expressed in rad which is the amount of radiation that

delivers 100 ergs of energy/gm of the absorbing material
 Adequate close for sterilization is 2.5 million rad (Mrad) or 25 Kilogray (Kgy) are
measured by dosimeter

1Gy= 100 Rad = 1 joule per Kg = 6.24 x 1012 Mev per Kg

radiation gray rad radiation

dosimeter 25 kilogray 2.5million rad

Dr El-Attar Page 12
 100 rad Gy

Examples of ionizing radiation :

Ionizing radiation

particles e.g beta particles waves e.g gamma radiation

1) Beta particles " genetration of electron " :

 Electrons are generated and accelerated in a highly evacuated tube by a very high
potential difference and emitted, at a speed near to that of light, either:
 Types of electron accelerators :
linear accelerators Van de Graff accelerator
Send electrons as continuous stream of
Send electrons in pulses
high speed electrons

waves particle ionizing radiation

beta particles beta particles
accelerators

pulses linear accelerator 

Van de Graff accelerator 
Dr El-Attar Page 13
 2) Gamma radiation :

 Source by excitation of radioactive isotopes (60C0, 137Cs) held as pellets packed in

metal rods
 Gamma planet design & precautions :

 The rods are carefully arranged and replaced or

rearranged when the activity is decreased or
becomes uneven
 For safety and to prevent leakage: the source is
housed within reinforced concrete building with
walls 2m thick
 Source is raised from a sunken water-filled tank only when required

cesium cobalt isotopes gamma radiation

water bath
dosimeter

 Sterilization by gamma rays :

 Articles are packed in boxes of standard size
 Suspended from a monorail
 Sterilized by several slow passages around the source
 Initiation of radiation plant (radiation unit) is difficult &expensive but is cheap to run

sterilization
gamma radiation boxes
penetration power

radiation sterilization application

Dr El-Attar Page 14
 Difference between β-radiations and γ-radiations :

Gamma radiation Beta radiation

 Electromagnetic waves
 Radiatioactive isotopes 60CO (cobalt)
137CS (cesium)
 Particles
 Source: electron accelerators
 Higher penetration
 Lower penetration
 Lower absorption
 Higher absorption
 Could be deep but radiated several
 For small articles but irradiated only
time ( rotates slowly around the source
from one or two sides
and irradiated from all sides )
 Could be switched on and switched off
 Can't be switched off
 The half life is 5025 years

beta particles gamma radiation 

gamma radiation 

penetration waves
lower absorption beta particles power
sterilization

gamma radiation

beta radiation 
lower penetration accelerator
radiation gamma rays
gamma radiation

 Uses of ionizing radiations :

 For thermo-labile pharmaceutical and surgical products in large quality:

 Disposable rubber gloves
 Adhesive dressings
 Plastic disposable surgical equipment (catheters, syringes)
 Some antibiotics (penicillin, streptomycin)

thermolabile ionizing radiation

Dr El-Attar Page 15
 6) Gaseous sterilization :

 In cases where sterilization is necessary. and due to the frequent use of fragile
medical devices. hospitals have had 3 choose other methods instead of steam water
sterilization .
 Among the gaseous sterilization technologies . ethylene oxide is one of the best
alternatives possible.

chemical sterilization gaseous sterilization

spores disinfectant gaseous sterilization
gas penetration power liquid disinfectant
spores cortex

Characteristics of an ideal low temperature gaseous sterilizant :

1. High efficacy : should be bactericidal . tuberculocidal. virucidal. fungicidal and

sporicidal.
2. Rapid activity : able to achieve sterilization quickly and below 65 °C.
3. Organic material resistance : should withstand reasonable organic material
challenge without Ioss of efficacy.
4. Strong penetrability : able to penetrate common medical device packaging
materials and penetrate Into the interior of devices' lumens
5. Material compatibility : should be compatible with a wide range of products and
materials: no change in the appearance or function even after repeated processing.
6. Non-toxic (operation and patients):
a) should present no health risk to the operators , in case of toxic gas , it desirable for
the sterilizing agent to allow methods to prevent worker exposure .
b) should present no health risk to the patients . in particular leave no residues in
sterilized products and no toxic byproducts.
7. Environmental emission : should pose no hazard to the environment.
8. Monitoring capacity : should be monitored easily and accurately with physical.
chemical and biological process monitors.
9. Potential for misapplication : should not require extensive knowledge and operational
procedures to achieve effective sterilization routinely.
Dr El-Attar Page 16
10. Adaptability : should be suitable for large or small hospitals.
11. Cost-effectiveness : should have reasonable cost

gaseous sterilization
spores high efficacy
organic matter
strong penetrability spores tissues
non-toxic sterilization compatible
humidity sterilization
gas sterilization penetration power

Types of gaseous sterilizing agent and mechanisms of action :

There are two main categories of sterilizing agents . which are distinguished by their
antimicrobial action

gaseous sterilizing agent

alkylating agents oxidizing agents

gaseous
Ethylene oxide formaldehyde hydrogen peroxide gas
ozone

gas sterilization 

DNA alkyl alkylating agent 

formaldehyde Ethylene oxide
DNA oxidation oxidizing agent 
gaseous ozone hydrogen peroxide gas

Dr El-Attar Page 17
 1) alkylating agents :

 Alkylating gases ate highly reactive and interact with many cell structures.
 there are many possible sites of alkylation such as the amino . sulphydryl and
hydroxyl groups in proteins or the purine bases of nucleic acids.

 The most commonly used alkylating agents are :

a) Ethylene oxide
b) Formaldehyde.

amino gp alkyl alkylating agents

nucleic acid purine hydroxyl gp sulphydryl gp
ethylene oxide guanine adenine
ethylene oxide formaldehyde

a) Ethylene oxide :

 properties of ethylene oxide:

 ethylene oxide occurs in gaseous form at room temperature
 the small size of the molecule as well as its very weak polarity give it a high
penetration power in narrow spaces
 it is very inflammable, can form an explosive mixture in air from concentration of
3%
 ethylene oxide is not corrosive for metals

 there are four parameters that must be maintained to ensure ethylene oxide
sterilization :

o Gas concentration 450 t0 1200 mg/L

o Temperature ranges 29o to 65oc
o Humidity 45% to 85%
o Exposure time two to five hours

gas ethylene oxide

very inflammable high penetration power
EO parameters

Dr El-Attar Page 18
 Microbiocidal activity :
 Ethylene oxide hos bactericidal, fungicidal, virucidal and Sporicidal properties.
 It reacts as an alkylating agent upon hydroxyl. sulphydryl.
 carboxyl and amino groups converting them to the hydroxyethyl adducts.
 these reactions lead to modifications of microbial metabolism and denaturation of
proteins. enzymes and nucleic acids.
 Apart from the prions , against which ethylene oxide has no activity. bacterial
spores are the most resistant microorganisms.
 bacillus atrophaeus spores are used for monitoring ethylene oxide

bacterial DNA alkyl EO

penetration power metabolism
prions sterilization spores
marker sterilization spores
markers bacillus atrophaeus spores

 Compatibility :
 Ethylene oxide is compatible with most medical devices
 Due to its small size, it is able to be adsorbed by plastic materials

 Toxicity :

 Ethylene oxide has been classified as a carcinogenic agent since I994

 the exposure in ethylene oxide vapors can generate small such as allergic
reaction headaches. dizziness. nausea. linked to irritation of ocular and
respiratory mucosa
 in high concentration , risks include severe caught and neurological damage
caused by a depressing effect on the central nervous system
 in the load state . ethylene oxide can induce skin irritations and burns.

compatible compatibility 
EO plastic
carcinogenic toxicity 
irritation burns allergy neurological damage

Dr El-Attar Page 19
 Indication :
 Used for heat sensitive instruments. Plastics, suture material. lenses and finely
sharpened instruments.
 Materials must be well aerated after sterilization.
 Material / instruments must be dry

EO indication
EO
gas penetration power humidity

b) Formaldehyde :

 Properties:
 formaldehyde is a strong bactericidal agent but it efficacy is dramatically
reduced at law temperature.

 Microbiocidal activity:
 the spectrum of formaldehyde activity is particularly wide. It is bactericidal,
fungicidal and virucidal it is also active on insects and other animal life .
 Its lethal effect are linked to the alkylation of nucleic acids, leading to inhibition of
the germination process.
 It interacts with proteins. RNA and DNA. the interaction with the proteins, RNA and
DNA , the interaction with the latter probably explaining its mutagenic activity

 Toxicity:
 Formaldehyde is toxic for humans when it penetrates the respiratory tract,
digestive tract or through the skin
 It is irritating for the eyes ,nose and throat

bactericidal formaldehyde
nucleic acid alkylation
formaldehyde WHO carcinogenic formaldehyde
formaldehyde hair product
throat nose eye irritation toxic fumes

Dr El-Attar Page 20
  Advantages and drawbacks :

a) Advantages :

 The main advantages are easy detection of low concentrations due to odor
and no ignition or explosion risk
 Its cost is lower than ethylene oxide .

b) Disadvantages :

 it exhibits very strong corrosion on certain materials

 Porous items must be aerated.
 It must not be applied either to cellulose-made materials ( diapers, bandages)
or to any material suspected of carrying prions
 It is also very unstable and therefore difficult to manage
 The sterilization cycle is very long (4h minimum)

So, it can used for big rooms , not items

formaldehyde
formaldehyde 
ethylene oxide
formaldehyde 
dressing cellulose –made material
sterilization unstable

 2) Oxidizing agents:

 Oxidation is characterized by the transfer of electrons from the target (oxidized)

molecule to the oxidizing molecule. which plays the role of the electron acceptor .
 Low temperature oxidizing agents used include chlorine compounds such as:
a) chlorine dioxide
b) superoxide
c) peroxides such as ozone & hydrogen peroxide
d) peracetic acid.
 The plasma phase of the last two compounds may also be used as oxidizing agents.

oxidizing agent
chlorine dioxide chlorine DNA

Dr El-Attar Page 21
hydrogen peroxide ozone peroxide superoxide
hydrogen ozone peroxides peracetic acid
peroxide

a) Hydrogen peroxide gas :

Hydrogen peroxide (HOOH) is a typical oxidizing agent

 Hydrogen peroxide is not actually an effective antiseptic or

disinfectant. this Is because bacteria and body tissues contain
Aerobes & human
enzymes (catalase & supra oxide dismutase) that inactivate
hydrogen peroxide.
 On the other hand, The oxygen released upon inactivation
Anaerobes can help oxygenate deep wounds and thus kill strict-
anaerobe contaminants.

 As It seemed very safe. it was used as a disinfectant in the load industry.

 the sterilizer chamber are simple in design. but the process of the sterilization cycle
differs depending on the method used to convert hydrogen peroxide into plasma or
vapor.

oxidizing agent hydrogen peroxide gas

supra oxide catalase
oxygen free radicals dismutase
inactivation hydrogen peroxide free radicals
oxygen anaerobic infection
anaerobes

hydrogen peroxide

anaerobes

sterilizing agent safe

pentration power plasma form gas state

Dr El-Attar Page 22
 Microbiological activity :

 Hydrogen peroxide is active against bacteria, fungi , yeast. spores and viruses.
Activity is greater Gram negative than against Gram-positive bacteria
 Its sporicidal activity is greatly improved by increased temperature and
concentration and is not affected by organic matter or salts.

bactericidal microbiological activity

gram gram negative vegetative spores
salts organic matter positive

 The main mechanism of action :

by formation of hydroxyl radicals (OH*) ,these radical could be produced under the
action of superoxide ions by the reaction :

O2 + H2O2 → OH* +OH* + O2

Such highly active hydroxyl radicals react with membrane lipids, DNA. and double
bonds at essential cell components

radicals hydroxyl radical mechanism

nucleic acid outer membrane lipids
double bond

 Hydrogen Peroxide Plasma sterilization :

 Basically. plasma is composed of gas molecules which have been dissociated by

an energy input.
 Gases can be convened Into the plasma form using a radiofrequency
electromagnetic field.

radiofrequency radiofrequency
Liquid electromagnetic vapor electromagnetic plasma form

Dr El-Attar Page 23
 The SterradTM process :

 The Sterrad® process is based on hydrogen peroxide. first in gas phase and then in
plasma form .
 A small quantity of liquid hydrogen peroxide is Injected into the chamber and
immediately evaporated.
 The vapor diffuses through the load to be sterilized in the second step.
 Hydrogen peroxide vapor is energized to plasma leading to production of hydroxyl
and hydroperoxyl free radicals.

hydrogen peroxide plasma form

sterilization free radical gaseous state plasma form
Sterrad spores penetration power
vapor electromagnetic waves liquid hydrogen peroxide
hydrogen peroxide plasma form state
hydroperoxyl hydroxyl free radical
Sterrad process
 The basic mechanism involved In the plasma inactivation of microbes are:
a) Direct destruction by UV radiation at the genetic material of microorganisms.
b) Erosions of the microorganisms through slow combustion using oxygen atoms or
radicals emanating from the plasma

We must add that no activity against prions by hydrogen peroxide or plasma has yet
been demonstrated

mechanism 

UV electromagnetic waves direct

hydrogen peroxide oxidation
prion hydrogen peroxide

 Microbiocidal activity:
 Plasma sterilization is active against vegetative bacteria as well as spores,
mycobacteria, coated or naked viruses, yeasts and fungi.
 Bacillus stearothermophilus spores were the most resistant species.

Dr El-Attar Page 24
 Compatibility:

 Plasma is compatible with fewer materials than ethylene oxide.

 There is a good compatibility with metals such as aluminum. Stainless steel and
alloys at polymers such as polypropylene , polyethylene , polyvinyl chloride,
Silicone , polyurethane. vinyl acetate. polycarbonate and Teflon .
 compatibility with glass Is good. Including optical fibers
 the process had a good compatibility with electronic devices and microsurgical
instruments , cellulose, viscose , cotton , liquid. powders and foam devices
 High weight orthopedics instruments must not be processed

prions activity
sterilization marker sterilization
sterilization spores Bacillus stearothermophilus spores
compatible moist heat sterilization
compatible ethylene oxide stainless steel
EO hydrogen peroxide medical device
supra oxide dismutase catalase safe
tissues hydrogen peroxide

b) Gaseous ozone :

 The composition of this gas. A triatomic allotropes oxygen.

 Ozone's was used for the first time in a dunking water treatment plant in 1893.

 Application :
 Many application using ozone in the liquid phase were developed. such as paper
pulp bleaching.
 the treatment of waste water.
 the disinfection of swimming-pools or aquaculture waters
 sterilization of mineral water homes of blood transfusion pouches.
 It is now also used In the treatment of water for industrial heat- exchangers.

ozone
ozone

heat exchanger

Dr El-Attar Page 25
 Mechanism of action of ozone :

 Ozone sterilizes by oxidation. it penetrates membrane of cells causing them to

explode Ozone induces modification of the microbial cell's chemical
components .
 the main mechanism involved in antimicrobial activity is
the formation of free radicals (O and OH) from ozone
decomposition.
 The formula is usually written as O3 but in fact the third
oxygen atom orbits around the molecular oxygen di-
atomic structure
 This means that the bond linking the third atom to the molecule structure is very
weak one and explains why the molecule is so highly reactive
 It can be obtained at low concentration by UV radiation "mercury lamp"

free oxidation Ozone mechanism

structure structure radical
double bond ozone
double di-atomic structure
single bond -R -I bond
UV reactivity
ozone

 Microbiocidal activity :
 Ozone is bactericidal mycobactericidal and sporicidal.
 It Is also active against yeast, fungi and amoebae.
 spores are far more resist to ozone than vegetative bacteria
 Gram-positive bacteria more sensitive to ozone than Gram negatives and
Candida albicans was more resistant than bacteria.
 methicillin-resistannt Staphylococcus aureus (MRSA) are more ozone – resistant
than methicillin – sensitive staph. aureus (MSSA).
 Most studies dealing with viricidal activity of ozone have been performed in water.
They coveted many species of naked or cooled viruses Including human
immunodeficiency type I "HIV type1" & HBV.

stetrilizing agent activity

MSSA MRSA G- G+
HIV type HBV ozone
1

Dr El-Attar Page 26
  Advantages & disadvantages of ozone :

Advantages Disadvantages
 It is safe  can be corrosive " it will oxidize steel
 It is simple and inexpensive to and aluminum".
operate (Cost is only Electric Charge)
 An alternative for Ethylene oxide  It destroys natural gum rubber. such as
sterilization of many heat-and latex. and some plastics.
moisture sensitive items.
 It does not affect titanium. chromium,
silicone and teflon.
 Aeration is not necessary: ozone
leaves no residue and converts to
oxygen in a short time.
 No Chemical material is required.

safe ozone 
sterilization silicone chromium titanium
aeration product
steel ozone 
sterilization

 Validation methods may be:


1) In-process:

a) Physical indicator
b) chemical indicator
c) biological indicator

2) After manufacturing, by subjecting the product to sterility assurance test.

validation sterilization sterilization 

parameters sterilization
marker chemical indicators chemical physical
biological indicator
sterility assurance test sterilization 

Dr El-Attar Page 27
 a) Physical indicator :

 Thermocouples: temperature
 Pressure and vacuum gauge: pressure
 Timer: time
 Hygrometer: humidity
 Dosimeter: for radiation
 Bubble point pressure: the pore size of bacterial filter

Bubble point pressure :

A liquid of known surface tension is used. Pressure is gradually

increased and when the first gas bubble pass pressure is
recorded then:

D=4Ø/P
 D : pore size
 Ø : surface tension
 P: pressure

physical parameter physical indicator

pressure gauge pressure thermocouples temperature
pore size dosimeter radiation hygrometer humidity
filter bubble point pressure bacterial filter
buble filter surface tension liquid
filter pore size
pressure p surface tension D=4Ø/P

b) Chemical indicators :

Using chemicals which me" or change color at temperature or radiation dose. These are:

1) Browne's tube : an ester is hydrolyzed at specified temperature producing

acid which change the color of an indicator
2) Witness tube : chemical melts in sealed tube at a specified temperate,
e.g. sulfur at 115oc and benzoic at 120oc
3) Hour glass : a chemical melts and flow down in certain time
4) Steri tapes : a chemical change color writing the word " sterile"

Dr El-Attar Page 28
parameters chemical indicator
sterilization
ester tube Browne 's tube
indicator acid sterilization
sulfur witness tube
benzoic acid
hour glass
sterilization
steri tapes
sterile

Choose the correct answer :

Microbial kill in the ozone sterilizer is achieved through a process called :

a) Condensation
b) Alkylation
c) Cavitation
d) Oxidation

:D

c) biological indicators :

1. The spores of marker organisms :

 are used to impregnate paper or metal foil ships, placed with the
sterilized load and then tested for viability after sterilization

2. The efficiency of bacterial filter :

 could be validated by filtering culture of small Gram-negative microorganisms as

serratia marcescens or Pseudomonas diminuta.

Dr El-Attar Page 29
3. Mathematical methods:

 An increase in D-value and Z-value indicates:

a) leakage.
b) Presence of highly resistant organisms. which may necessities re-evaluation of
the sterilization cycle and equipment

biological indicators 

sterilization
product sterilization sterilization spores
incubation
pore size filter
serratia filter filter
Pseudomonas diminuta marcescens
D-value Z-value

 Choose the correct answer :

At the end of the sterilization cycle the EO cartridges and the gloves used to handle
them should be

a) Aerated
b) Disinfected
c) Washed
d) Irradiated

:A

Dr El-Attar Page 30
The main aim of aseptic technique is to prevent the access of
microorganism during preparation e.g. aseptic filling, and during
testing of pharmaceutical products e.g. in sterility testing

 Aseptic room is a special area the design of which greatly reduces contamination
because of the following :

1) Air sterilized by HEPA filter

2) Positive pressure
3) Air in the room is changed 10 to 20 times per hour
4) Analysis of air
5) UV
6) Doors are double with air lock
7) Windows are double with air lock
8) Corners are curved and surfaces (walls,floors and ceiling) are smooth
9) Furniture and equipment are minimum and sample
10)Workers are free from infections and wear sterile clothes (overheads , over shoes,
face mask and cloves)

aseptic area sterile product

HEPA filter
particles positive pressure
conrners UV

contamination sterile

 The entry to the area is through the following sequence:

black area white area

grey area

 Black area (non-sterile)

 Gray area (supplied with sterile at and used for washing and changing clothes) .
 White area (aseptic area)
Chemical disinfections (aerosols and fumigation).

Dr El-Attar Page 31
 
black area 
grey area 
aspetic area white area 

 laminar flow cabinet :

 It is considered to be a small scale aseptic area.

 Air is forced through HEPA filter and comes out in parallel streams
without turbulence to replace non-sterile air
 Disinfection and UV also minimize contamination when the cabinet
is out of work.

 The air streams may be:

a) Horizontal
b) vertical but vertical type is preferable when
dealing with pathogenic microorganism or
carcinogenic substance

aseptic area laminar flow cabinet 

contamination UV radiation HEPA filter

horizontal 

carcinogenic
pathogenic organism

 Sterilization of air :

Sterile air (<2CFU/cubic feet) is required in aseptic area. aeration of fermentor. .. ..etc.

 Different sterilization methods are available including:

a) chemical agents e.g. formaldehyde
b) physical agents e.g. UV & heat.

 However. the most common methods are:

a) Electrostatic precipitation : Retain 95% of particles and can be used as a pre filter.
b) Filtration : is the most efficient method and it retain up to 99.99 % of particles.
Dr El-Attar Page 32
 sterile sterilization of air
chemical 2CFU
UV heat physical formaldehyde
filtration electrostatic precipitation

a) Electrostatic precipitation :

Air particles are charged either +ve and -ve by passing air
through high voltage plates then separated by passing
through alternating + ve and -ve plates which attract
particles to opposite charge
The plates can be removed from time to time be cleaned

b) filtration :

 Types of filter :

1) Cotton, glass wool, cellulose paper and fibrous pads (can be

used as pre-filters)
2) Cartilage multitude membrane filter
3) High efficiency particulate air (HEPA) filters.

electrostatic precipitation 

filtration filtration 
HEPA filter

 Sterilization is a final step for quality assurance of parental and eye preparations.
 it is not an alteration to GMP and its efficacy is controlled by its inactivation factor (IF)
where:
IF=NO / N
No Is the initial number & N is the final number at contaminant.

Dr El-Attar Page 33
 the degree of sterility (DS) is controlled by:
Ds = IF/ X

Where X is the number of contaminant (bioburden).

So. the lower is the initial contaminant. the higher is the DS and sterility assurance which
is defined as the numerical value denoting the probability of funding a single surviving
organism in a product (It depends on the bioburden)

toxins sterile parenteral

IF sterilization sterilization
final number initial number inactivation factor
gram negative bacteria bioburdens parenteral

sterilization
DS bioburden
sterilization number of bioburden IF

 Sterility testing :

A general control test performed for all sterile products to assure their freedom from
ordinary bacteria, fungi but not viruses or partially damaged cells .

 General procedure :

1) Sampling :

 Depending upon :
 the number of units
 volume per units
 sterilization method.
 the use of biological indicator during sterilization , application of GMP etc.

Dr El-Attar Page 34
 sterilization sterility testing
viruses bioburdens fungi
agar intracelluar viruses
sterility testing
product sampling
biological indicator sterilization

 The sample size may be :

 2% of the batch to maximum 20 units.
 2% from the first 1000 units and 0.2% from the remaining units.
 0.4 √ where N is the number of articles.
 the test is destructive as samples are not reused.

20 unit sample size

0.4 √
destructive sampling

2) Quantity :

the membrane filtration technique should be employed. It depends on the volume or

weight/unit. e.g.

a) For liquid:

 The whole contents (if less than 1 ml).

 Half the contents (for units containing 1-4ml)
 2ml (if 4-20ml).
 10% ( if 20-100ml).
 20% for volumes greater than 100ml.

b) For powder:

 The whole contents ( if less than 50 mg / unit)

 Half the contents ( if 50 - 200 mg)
 100 mg ( if 200 mg or more)

Dr El-Attar Page 35
 

1 ml 1ml 
20 ml 2ml 20ml 4ml 4ml
100ml 10 % 100 ml
pharmacopeia

powder
200mg 200mg 50 50mg 
100 mg

3) Media :

a. Fluid thioglycolate media ( FTM):

support growth of aerobes and anaerobes. the main

contents are:

 Nutrient: yeast extract. digest of coseine.,dextrose.

 Antioxidant ( Na thioglycolate ,glucose. L-cysteine)
 Agar at 0.75%
 Resazuarin (pH indicator; pink if oxidized and yellow if not)
 Na Cl & water.
 pH after autoclaving is 7-7.2.

fluid thioglycolate media media

yeast dextrose coseine nutrients anerobes aerobes
L-cysteine glucose Na thioglycolate antioxidant
yellow pink Resazuarin indicator

b. Trypticase soya broth (TSB):

It supports not only fungi but also highly aerobic bacteria e.g.
Bacillus, Pseudomonas species.
 Nutrients (digest of soya bean. casein and dextrose) .
 H2O, buffer
 (pH after autoclaving 7.2-7.4).

Dr El-Attar Page 36
 highly aerobic bacteria fungi Trypticase soya broth
soya bean Pseudomonas Bacillus
Trypticase soya broth

c. Sabouraud's media:

It supports mainly fungi

Trypticase soya broth Sabouraud's media

fungi

4) Methods of transfer:

a) Direct transfer method (DT) :

 the special volume or weight each unit is transferred to FTM and TSB tubes.

b) Membrane filtration method (MF) :

 the whole contents of all test units one aseptically filtered through membrane
filter (0.45 µm) with hydrophobic edge. washed several
 times with sterile H2O to remove the antimicrobial agent. cut into 2 pieces. one
to FIM and one to TSB.

product 

TSB FTM ampoule direct 

sterilization
filtration membrane bacterial filter 
TSB FTM bacterial filter

 Advantages of MF method :

 MF method eliminates antibiotic or preservative effect efficiently

 Uses less media and concentrates the contaminant
 Ease of separation of contaminants from oily preparation

Dr El-Attar Page 37
 disadvantages of MF method :

 High probability of contamination during the test

 The level of contamination in different units is not known

 Need equipment with validated assurance for sterility

direct transfer preservative 

medium
oily preparation contaminant
contamination 
membrane contamination

condition incubation

5) incubation and interpretation of result:

 At 30-35oC / 14 days for bacteria on (FTM)

 At 20 -25oC / 14 days for fungi (TSB)

FTM incubator agar

partial damage microorganism

TSB fungi disinfectant

positive negative

–ve +Ve

6) control:

a) Positive control :

Inoculated media must support the growth of aerobes. anaerobes and fungi as
test organisms
 FTM supports B. subtilis (aerobes) and Bacteroid vulgaris (anaerobic)
 TSB supports B. subtilis and Candida albicans (yeast)

Dr El-Attar Page 38
 b) Negative control :

 uninoculated media must be sterile (no growth)

agar 
B. subtilis FTM agar
B. subtilis TSB anaerobic Bacteroid vulgaris aerobe
agar Candida albicans
positive control agar
incubator agar negative control 
agar contaminant

 Problems during sterility testing :

1) turbidity:

 if the original solution is turbid so subculture loopful to fresh media after incubation

2) Oily preparation:

e.g. progesterone, testosterone

 These preparations tend to float on the surface, so filtration is recommended offer
dissolving in sesame oil or isopropyl myristate
 Or Emulsify with sterile agents and test normally
 Or Centrifugation and test the deposit

turbid agar
loop bacterial growth positive result
bacterial growth agar loopful
isopropyl oil preparation
centrifugation emulsion myristate
bacteria

Dr El-Attar Page 39
 3) Surgical dressing:

 If small: whole article is transferred to media

 If large: the inner most part is transferred to 100 mL media

4) sutures:

 e.g. cat gut

 good neutralization of preservative and test the whole article

surgical dressing
inner most part media
medium

preservative
preservative

5) Devices:

 If small: e.g. needles; the whole article to media

 If large: wash with sterile H2O, test the wash or transfer the most suspected part to
media

sterile water media

 Neutralization of Antimicrobial activity :

1) Membrane filtration e.g. for most antibiotics and for heavy metals).
2) Dilution: for agent with high concentration exponent e.g. phenol and alcohol.
3) Antagonism:
a) By media e.g. thioglycolate for heavy metals as phenyI mercuric nirate
b) By antagonist e.g.
 penicillinase for penicillin
 cysteine for streptomycin
 lecithin for QACs " quaternary amino cpds".
 PABA "para amino benzoic acid" for sulfonamides
 tween 80 for parabens.

Dr El-Attar Page 40
 antimicrobial agent

dilution heavy metal antibiotic

n disinfectant
medium media disinfectant
phenyI mercuric nirate heavy metal thioglycolate
cysteine penicillinase penicillin
tween 80 sulfonamide PABPA QACs lecithin streptomycin
parabens

 limitations and significance of sterility testing :

Sterility test is limited in its scope because:

 It is conducted on a fraction of the total batch

 Doesn’t detect viruses, existing parasitic bacteria or the majority of thermophilic and
psychrophilic bacteria
 Doesn't detect organisms that have been shocked by sublethal heat treatment
 May not detect aged bacterial spores which have long a germination period
 Low degrees of contamination may be missed
 Therefore, sterility test detects relatively gross
 contamination in a product. if is more significant in case of failure
 It detects abnormally resistant microorganisms
 it is of a special significance for products sterilized by filtration which lack in-process
controls

product
parasites
ampole
biological indicator contamination
sterilization sterilization

Dr El-Attar Page 41
 Microbial analysis of air :

1) Settling plates

 Disadvantages: Slow, doesn't give an indication of the volume of

air tested, and fails to collect very small particles

2) Air sampler .

 Use agar support aerobes

bacterial growth agar

settling plate
aerobic agar air sampling

 Validation of Aseptic Area :

1) By system suitability test (SST):

carried by testing for microorganisms in everything: in air,

machines. persons. ‘ walls and floors by swabbing

2) Validation of HEPA filter " smoke test" :

Performed by using dioctylphthalate vapor that is forced through the filter

by a fan then tested on the other side

swabs SST test sterile air

smoke test
filter

 Tests for pyrogens:

Pyrogens are the metabolic products of bacteria. They are a group of bacterial
endotoxins which cause on immediate rise in temperature upon injection into humans
and could lead to shock
Certain pharmaceutical products and containers are required to be free from "Pyrogen"
or have a limit for bacterial endotoxins

Dr El-Attar Page 42
a) Rabbit pyrogen test

Measures the rise in temperature of rabbits upon Intravenous injection of a sterile

solution of the substance to be tested, under standardized conditions.

b) MAT test :

monocyte activation test: The test, which uses whole blood, is intended to be an
alternative to the rabbit pyrogen test. It is a determination of pyrogenic activity in o
solution or suspension by measuring the interleukin production of human monocytes

pyrogen metabolic products

pyrogen rabbit pyrogens test

monocyte interleukin monocyte
pyrogens interleukin

c) LAL Test :

 It is more precise test

 Limulus Amebocyte Lysote reagent. which is obtained
from aqueous extracts of the circulating amebocytes of
the horseshoe crab Limulus polyphemus. LAL reacts with bacterial endotoxin
lipopolysaccharide (LPS), which is a membrane
component of gram negative bacteria
 The contents of each tube are mixed and incubated,
with positive and negative controls for 60 minutes
 A positive reaction is obtained when a gel is observed
that remains firm when inverted 180°. Any other
reaction is negative. The test ‘may be carried out on
slide with incubation for 20 minutes, protected from loss
of moisture
 In the official LAL test, a series of dilutions of the
standard endotoxin and a series of dilutions of the
preparation being tested are placed in test tubes, and
to each, the LAL reagent is added

amebocytes reagent LAL test

endotoxin reagent Limulus polyphemus.
Dr El-Attar Page 43
 endotoxin test tube gelatin
moisture slide test tube

test tube serial dilution

LAL reagent

Dr El-Attar Page 44