STERILIZATION_AND_DISINFECTANT--1-1[1].ppt

STERILIZATION

1. WELCOME TO
STERILIZATION AND
DISINFECTANTS

2. INTRODUCTION
 Microorganisms are the agents of contamination, infection,
and decay. Hence it becomes necessary to remove them from
materials and areas.
 Early civilization practiced salting, smoking, and exposure to
sunlight .
In mid 1800s Lister developed Aseptic techniquesAseptic techniques to
prevent contamination of surgical wounds. Prior to this
development:
Nosocomial infections caused death in 10% of surgeries.
Up to 25% mothers delivering in hospitals died due to
infection.

3. DEFINITIONDEFINITION
1.1.STERILIZATIONSTERILIZATION
Sterilization is a complete removal of micro-organisms (bacteria,
fungi, viruses, parasites) both in their vegetative and non-
vegetative forms including their spores from an object or
environment.
 
2. DISINFECTION
Disinfection means reducing the number of viable microorganisms
present in a sample
3. DISINFECTANT
A disinfectant is a chemical or physical agent /substance that is
applied to inanimate objects to kill microorganisms.

4. 4. SEPSIS4. SEPSIS:
It Comes from Greek meaning “decay”. It indicates bacterial
contamination.
5. ASEPSIS5. ASEPSIS:
This means absence of significant contamination.
6. ASEPTIC TECHNIQUES
These are techniques used to prevent contamination of surgical
instruments, medical personnel, and the patient during surgery.

They are also used to prevent bacterial contamination in food
industry.

5. Importance
1.It prevents the spread of infections
2.Sterilization is useful when sterilizing media and glassware
which are used for obtaining bacteria in pure culture,
METHODS OF STERILISATION
There are three (3) types of sterilization
Physical sterilization
Mechanical sterilization
Chemical sterilization
The choice of method of sterilization or disinfection depends on:
The nature of the item to be treated.
The likely microbial contamination.
The risk of transmitting infection to patients or staff in
contact with the item.

6. Each methods has its own advantages and disadvantages
Nb
Physical method of sterilization is the best method of out of the
three.

7. Physical AgentsPhysical Agents
Heat Radiation
Dry Moist
Incineration
Dry Oven
Steam Under
Pressure
Boiling Water/Hot Water
Pasteurization
Sterilization
IonizingNon Ionizing
X Ray, Cathode,
Gamma
Disinfection
UV
SterilizationDisinfection
A. Physical sterilization
The main agents are; Radiation and Heat

8. 1. Radiation
This is a method that is usually employed in:
Industries for sterilization of single-use disposable items such
as needles, latex catheters and surgical gloves.
Food industry to reduce spoilage and remove pathogens.
Ultra-violet irradiation can be used to cut down the level of
contamination.
There are three types of rays. These are;
•Gamma rays
•Infra red rays
•U/V rays (ultra-violet rays)

9. a. U/V rays (ultra-violet rays)
The main source of U/V rays is sun and hood lamp. U/V rays
destroy microorganisms by interfering with hydrogen bonds that
binds double helix DNA in the bacterial cell.
Disadvantages
•They poorly penetrate
bacterial cell
•They can be absorbed
glass or drops of water

10. b. Gamma rays
Gamma rays destroys micro organism by interfering with the
bacterial intra-cellular or extra cellular structural formula thus
causing death of the bacterial cell.
Advantages
•They are very penetrative to the bacterial cell
•They are mostly used in sterilization of disposable materials
e.g. syringes, Needles.
Disadvantage
a. They are usually useless.
b. They cause chemical alteration in pharmaceutical products
hence they cannot be used.
c. Infra- red rays
•They are used in sterilization of metal instruments and
cylinders (metal cylinders and glass cylinders

11. 2. HEAT
This is the most reliable method of sterilization widely applied
under carefully controlled conditions at temperatures above 100
0
C
to ensure that bacterial spores are killed.
Factors influencing sterilization by heat
1.Temperature and time: The higher the temperatures the
shorter time taken and the lower the temperatures the longer
time taken in sterilization.
2. Number of organisms: The greater the number of organisms the
more time taken for sterilization and the fewer the organisms the
less time taken for sterilization.
3. Nature of material to be sterilized: These are the materials in
which the organism or spores forms are enclosed
4. Nature of organisms: Different species of organisms will resist
temperatures in different degrees e.g. Bacillus stearo-
thermophilus withstand temperature of up to 121
0
c.
5. Nature of the heat: Dry heat or Moist heat

12.  1. DRY HEAT
Death of micro-organism is by destructive oxidation effects of
essential cell constituents especially protein denaturation. Most
resistant spores require temperatures of 160
0
C for 2hrs. There
are three type of dry heat.
•Hot air oven
•Flames
•Incineration

13. 1a. Hot air oven
This method utilizes dry heat to sterilize materials that can
withstand high temperatures. It consists of;
A thermostat for controlling the temperature.
Double walled insulation that keeps the heat in and conserves energy
It is operated between 50
o
C to 250/300
o
C
in two ways:
At 180
0
c for 30minutes
At 160
o
c for 60minutes
It is used to be sterilized Forceps,
Scissors, Scalpels, Swabs,
Pharmaceuticals products like Liquid
paraffin, dusting powder, fats and grease.
In this method, all vegetative micro-organisms and some spore formers
will be killed.
 

14. 1b. Flames
This is direct exposure of materials to naked flame.
Inoculation loop or Wire, the tip of Forceps
and spatulas are held in a bunsen flame
till they are red hot.
1c. Incineration
This is an excellent method of destroying materials such as
contaminated cloth, animal carcasses and pathological
materials, surgical dressings, sharp needles and other clinical
waste at high temperatures
(1500
°C).

15. 2.MOIST HEAT
This is a much more effective method compared to dry heat.
The method kills microorganisms by coagulatingcoagulating their proteins.
It is done in four (4) ways.
•Temperature at 100
o
C: Boiling.Boiling.
•Steam at 100
0
c (at atmospheric pressure)
•Temp below 100
o
C: “Pasteurisation”, Inspissator.“Pasteurisation”, Inspissator.
•Steam under pressure: Autoclave.Autoclave.

16. 2a. Boiling water at 100
0
c
This method kills vegetative forms of bacterial pathogens e.g.
Vegetative micro-organisms will be killed within 10min in
boiling water at 100
0
c
Hepatitis virus: Can survive up to 30 minutes of boiling.
Endospores: Can survive up to 20 hours or more of boiling.
Spore will be killed within 30min
Sometimes you can have bactericidal effects e.g. when 2%
NaCo
3 sodium carbonate is added to the boiling water at 100
0
c
to increases the killing power.
•Material sterilized are Slides, syringes, needles etc surgical
instruments e.g. scissors, forceps, rubber stoppers and
corks.

17. 2b. Steam at 100
0
C
This method is
employed in sterilization of media whose ingredients can be
destroyed in temperatures above 100
0
c. It is done in two (2)
ways;
•Temp. of 100
0
c for 90 minutes (single exposure): This is
known as single exposure in which spores of some
thermophillic and some mesophilic bacteria can survive this
type of temperatures.
•Temp. of 100
o
c for 30 minutes
This is done for 3 (three)
successive days. It is referred to as Tyndallisation Tyndallisation
/Intermittent /Intermittent or fractional sterilization.

18. Principle of TyndallisationTyndallisation
This method is used when
dealing with media containing sugar and gelatin. Single exposure
kills vegetative organism then in between the heating, the spores
being in favorable medium becomes vegetative form, which are
killed in the subsequent heating.
Procedure.
•First day: Sterilize at 100
0
c for 30 minutes and incubate overnight
at room temperature. This exposure kills vegetative forms.

•Second day: Re-sterilize at 100
0
c for 30 minutes, incubate
overnight at room temperature. This 2
nd
exposure kills spore
formers.

•Third day: Re-sterilizes at 100
0
c for 30 minutes. 3
rd
exposure kills
spores that could not have been killed in the 2
nd
exposure, and at
the end, vegetative organism and most of the spores will be killed.

19. Disadvantages
Fails to kill thermophillic anaerobic
bacteria whose spores will not germinate in a particular medium
and under the same condition of storage in between the heating.
2c. Steam below 100
0
C
This is used for sterilization of fluids whose protein value can be
destroyed by steam at 100
0
c e.g. milk. There are 3- methods.
Pasteurization.
 Inspization
 Vaccine bath

20. . Pasteurization.
There are two methods under pasteurization. These are;
•Holder method at 63
o
c for 30 minutes.
•Flash method at 72
0
c for 15 minutes rapid cooling to
13
o
C
•Ultrahigh-temperature pasteurization at 134ºC for 1
second then rapid cooling
In this method, all vegetative bacteria
are killed except spores of the spore
formers organisms.
Water BathWater Bath

21. . Inspization.
This is employed in sterilization of media e.g. Lowenstein
Jensen medium (LJ) for micro-bacterium Tubercle bacilli
(Medium containing eggs or serum). Sterilizes by heating at 80-
85
o
C for half an hour for 3 successive days





. Vaccine bath
This is used for sterilization of vaccine at 60
0
c for 1 hour in
water bath.
Steam Sterilizer

22. 4. Steam under pressure.
This employs use of high temperatures of 121
o
c for 15 minutes
under pressure of 20lbs.
Invented by Charles Chamberland in 1879.

It is the most effective type of sterilization e.g. autoclaving by
use of an autoclave.
The method kills bacteria in the following ways:
Coagulation of bacterial cell protein.
Denaturization of the bacterial cell wall.

Disrupting the bacterial enzymes.
Oxidation.
 It is used to sterilize media such as p
3
broth, urea broth, Petri-
dishes (glass type), swabs and apparatus used in incineration
(incinerators).

23. AUTOCLAVE
T
e
m
p
e
r
a
t
u
r
e

(
C
)
Flowing
steam
Begin
pressure
Autoclave time
Stop
steam
Sterilization time
Temperature
of object being
sterilized
Temperature
of autoclave
Total cycle time (min)

24. Autoclave
Autoclave

25. •Autoclave consists of a vertical or a horizontal cylinder.
•One end has an opening which is meant for keeping
materials to be sterilized.
•The lid is provided with a Pressure gauge, to measure the
pressure
•A safety valve is present to permit the escape of steam from
the chamber
•Articles to be sterilized are placed in the basket provided
•Sterilization is carried out under pressure at 121º for 15
minutes

26. Controls used in sterilization using Autoclaves
The efficiency of an auto-clave should be tested to be sure of
complete sterilization. The apparatus and devices used for this
purpose are called sterilization control.
1.Temperature recorder (thermometer) to make sure that the required
temperature is attained.
2.Pressure recorder (barometer) to make sure that the required
pressure is attained.
3.Browne’s tubes. These are tube impregnated with chemicals which
are:
•Red to green meaning complete sterilization.
•Red to brown meaning incomplete sterilization.
This is done under the following:
•At 115
0
c for 25 minutes.
•At 145
0
c for 15 minutes.
•At 160
0
c for 1 minutes.

27. 4). Bowio-dick tapes (autoclave types)
This consists of invisible lines at room temperatures. At certain
temperatures, they will turn red to black and this will mean
complete sterilization of the material.

5). Spore indicator (biological control).
T.D.P- (Thermal death point) –means the lowest temperatures
at which a given organism will die at a given time.
They are biological controls (indicators) includes:

•Spores used are of high resistance bacilli called Bacillus
stearo-thermophilus. The thermal death point of these spores
is 121
0
c for 12-15 minutes. If after sterilization at these
temperatures the spores grow, this means incomplete
sterilization.

28. B. MECHANICAL METHODS OF STERILIZATION:
This is a sub-class of physical sterilization whereby
microorganisms are removed without destroying them. This can
be fastened by applying negative pressure. Filtration helps to
remove bacteria from heat labile liquids such as sera and
solutions of sugar, Antibiotics. These semi-permeable agents
includes filter-like agents such as:
Candle filters Candle filters
Asbestos filtersAsbestos filters
Sintered glass filterSintered glass filter
Membrane filtersMembrane filters
Porcelain,

29. Mechanical Removal Mechanical Removal
MethodsMethods
Filtration
Air Liquids
Disinfection Sterilization

30. Candle filters: Candle filters:
These are filter widely used for purification of water. They are of
Two types:
 Diamatoceous earth filter such as:
»German ‘Berkefeld filters.
»American ‘Mandler filters
Ceramic filter such as: Chamberland filter
German Berkefeld filters consists of Asbestos and organic
matter. They are made up of three grades:
V-viel (coarse),
N-normal,
W-wenig (fine).
These grades are assessed by the rate of passage of water
at a standard pressure. V-grade filters are used mainly for
clarifying liquids but they are not fine enough to sterilize liquids
by holding back bacteria.

31. CANDLE FILTER

32. Asbestos filters:
These are disposable single use discs with high adsorbing
tendency. They are carcinogenic e. g: Seitz filter

SEITZ FILTER
ASBESTOS DISCS

33. Sintered glass filters:
•These filters prepared by heat fusing powdered glass
particles of graded size (grade 1-5) filters.
•They are cleaned easily, brittle and expensive.

34. Membrane filters:
Membrane filters are made of cellulose esters or other
polymers and are commonly used in purification & analysis of
water, media such as Urea broth, plasma for Coagulase test .
They function more like a sieve and can be accomplished by
syringe, pump, or vacuum

36. Advantages of membrane filters:
Maintains both nutrients value and the structural formula
Disadvantages.
1.The filters may contain bacterial toxins.

2.Viruses might pass through these filters.

3.Mycoplasma can also pass through the filters.
Nb.
1. Bacterial -free filtrate may be obtained by use of filters with
maximum pore sizes not exceeding 1 nanometer e.g.
organism of Serratia mascescens.
2. The biological indicators to control this type of sterilization
(mechanical sterilization) is Serratia mascescens which
has a diameter of 0.75um.
3. The purpose of biological control is to tell whether the filter
paper in use is effective or not.

37. C. CHEMICAL TYPE OF STERILIZATION
Chemicals (disinfectants or
antiseptics) are used to free objects or environment or skin
from all living organism excluding spores but they cannot be
relied on to kill or inhibit all pathogenic micro-organisms. They
are either liquids or gaseous chemicals. These chemicals can
be in form of disinfectants or antiseptics. Some disinfectant
can be converted into antiseptics by dilution. The efficacy of
a particular method of chemical disinfectant depends on:
•Nature of the item to be disinfected
•Type and concentration of disinfectant
•Amount of organic material present
•Duration and temperature of exposure
Generally Gram positive bacteria are more sensitive to
disinfectants than Gram negative bacteria while Mycobacterium
and fungal/bacterial spores are highly resistant.

38. Chemical AgentChemical Agent
Gas Liquids
SterilizationDisinfection Animate Inanimate
ChemotherapyAntisepticsSterilizationDisinfection

39. Chemical agents.Chemical agents.
Chemical agents Chemical agents kill micro organism by either one of the
following ways.
•Oxidation.
•Interference with the bacterial enzymes activities.
•Disrupting the bacterial cell membranes.
•Coagulation of the bacterial protein synthesis.
•Interference with the bacterial nucleic acid synthesis.
Chemical agents Chemical agents can be classified as:
 bacteriostatic,
bacteriocidal,
bacteriolytic

40. Bacteriocidal effects: This is the ability of a chemical agent to
kill bacteria

Bacteriostatic
This is the ability of a chemical agent to inhibit growth and
multiplication of bacteria
L
o
g

c
e
l
l

n
u
m
b
e
r
Time
Time
L
o
g

c
e
l
l

n
u
m
b
e
r
L
o
g

c
e
l
l

n
u
m
b
e
r
Time
Bacteriostatic
Viable cell count
Total cell count Bacteriocidal
Total cell count
Viable
cell count
Bacteriolytic
Total cell count
Viable
cell count

41. Examples of chemical agents.
1.Alcohol:
60-90% alcohol in water or 60-90% methylated spirit in water
concentrations are recommended.
•They are used as antiseptics in disinfection of clinical
thermometer and the skin during Vein-puncture
2. Lysol:
This is a disinfectant applied on non-living materials like benches
3-5% Lysol is an antiseptic after dilution.
3. phenol:
1-2% phenol is used as a disinfectant and 0.5% as an antiseptic.
4. Hycolin: 1% hycolin as an antiseptic.

42. 5. Iodine:
2% Iodine in 70% alcohol is used as an antiseptic in surgical
operation while 5% in 50% alcohol used in laboratory when
dealing with organisms as tincture of Iodine.
6. Glycerol: 50% glycerol is used as a disinfectant.
7. Chloroform: 0.25% chloroform is used as a disinfectant.
8. Ethylene oxide in form of gas: This gas sterilizer is heat labile
(sensitive materials) and lethal (killing effect) to all kind of micro-
organism and spores.
9. Formaldehyde:- This is in form of water soluble gas that is lethal
to all kinds of micro organisms and spores e.g. woods furniture,
clothing etc.