The ppt is all about the physical methods of Sterilization. it is simplified way and can be helpful. I made did for BSc MLT class.

1. STERILIZATION AND DISINFECTION I
Ms. M. Mwinga

2. CONTENTS
 Terminologies
 Applications
 Factors affecting efficacy of sterilant /disinfectant
 Methods of sterilization and disinfection
Physical methods
Chemical methods

3. • Sterilization: is the freeing of an article from all living organisms
including viable spores.
• Disinfection: Is a freeing of an article from harmful microorganisms
that are capable of causing infection but does not destroy spores.
• Antiseptics: chemical disinfectants used on skin or mucus membrane;
and prevents infection by inhibiting the growth of bacteria. Ex.
Alcohol, boric acid, hydrogen peroxide.

4. • Asepsis: is a process where the chemical agents (antiseptics) are
applied on to the body surface (skin), which kill or inhibit the
microorganisms present on skin.
• Decontamination: Is the reduction of pathogenic microbial population
to a level at which items are considered safe to handle without
protective attire.
• Bactericidal agents or germicides: they are chemicals that kill
bacteria.
• Bacteriostatic agents: agents that prevent the multiplication of
bacteria.

5. Applications of sterilization
Prevents contamination by extraneous organisms.
Used in surgery to maintain asepsis.
In food and drug industry; to ensure safety from contaminating
organisms.

6. Methods of sterilization (classification)
Physical methods. Chemical methods.
Sunlight drying heat filtration radiation
Dry heat Moist
Flaming
Incineration
Membrane
filters
Depth
filters
Hot air
oven
Below
100°C
At 100 °C
Above
100 °C
Non-ionizing
radiation
Ionizing radiation

7. Factors affecting the efficacy of sterilant/disinfectant
1. Organism load
2. Nature of organism
3. Concentration of chemical agents
4. Nature of the sterilant/disinfectant
5. Duration of exposure
6. Temperature
7. pH, organic matter and biofilm.

8. I. SUNLIGHT
Possesses bactericidal/germicidal activity.
Action is due to Ultraviolet rays and presence of the ozone layer in the
atmosphere.
II. DRYING
Drying has a deleterious effect on many bacteria.
Many bacteria requires moisture for their growth.
Spores are not affected by drying.
Solar-water purification

9. III. HEAT
Kills microorganisms by denaturing their enzymes and other proteins.
Factors influencing sterilization by heat
Nature of heat -dry or moist.
Temperature and time.
Number of organisms present.
Characteristics of the organisms, such as strain capacity.
Type of material from which the organisms must be eradicated.

10. • Mechanism of Action of Heat
1. Dry heat: used for glassware, instruments and paper-wrapped
articles, water impermeable oils, waxes and powders.
2. Moist heat: best at lower temperatures, used in autoclaves to
sterilize media and other water containing materials.
• The time of sterilization is inversely proportional to the temperature of
exposure.
• Thermal death time: is the minimum time required to kill a
suspension of organisms at a predetermined temperature in a specific
environment.

11. Dry heat methods
• Flaming: used to sterilize items in a flame of a Bunsen burner
-Red heat: applied to inoculation loops and wires, forceps and spatula;
for a longer time.
-Sterilization of the mouth of test tubes is applied for a shorter time.
Sterilizing an inoculating
loop by flaming

12. • Incineration: used to sterilize anatomical wastes and microbiological
wastes over high temperatures (870-1200°C) into ash and gas.
• Hot air oven: removes all microbes including spores
• used for glassware such as tubes, flasks, measuring cylinders, glass
syringes and glass pipettes, and metal instruments.
-Requires a temperature of 160-180°C for 120 minutes.
Insulated wall
Trays
Temperature regulator

13. Indicators of hot air oven
• Physical indicators: thermocouples- measures the temperature.
• Biological indicators: uses spores (106) of non-toxigenic strains of
Clostridium tetani or Bacillus subtilis.
• Chemical indicators: Browne’s tube- has a heat sensitive dye which
turns green after exposure to certain temperature for a period of time.

14. Adv: -Do not require water.
-less pressure build up.
-very efficient & smaller.
-more rapid and reaches higher temperatures.
-non corrosive.
Disad: -Time consuming (takes up to 30 to 120 minutes).
-high temperatures are not suitable for most materials.
-Ex. Prions.

15. Moist heat
• Moist heat kills microorganisms at a lower temperature than dry heat.
Temperature for moist heat
1. At a temperature below 100°C.
2. At a temperature of 100°C.
3. At a temperature above 100°C.

16. 1. At a temperature below 100°C
a)Pasteurization
-is a method to control microorganisms from beverages such as fruit and
vegetable juices, beer, and dairy products such as milk.
-it destroys all non-sporing pathogens. Ex: Mycobacteria, Brucellae, and
Salmonellae, except for heat resistant Coxiella burnetii.
Methods of pasteurization
• Holder method: done at 63°C for 30minutes.
- Ex: HIV in serum at 56°C for 25 minutes.
• Flash method: done at 72°C, followed by
rapid cooling to 13°C or lower.

17. b) Water bath
• Is a method used for disinfection of serum, body fluids (at 56°C) and
vaccines (at 60°C).

18. C) Inspissation /fractional sterilization
• Process of heating an article on three successive days at 80-85°C for
30minutes by an inspissator.
• The method kills all vegetative forms of spores.
• It is used to sterilize egg-based media such as Lowenstein-Jensen (LJ)
medium and Dorset’s egg medium, and serum-based media such
Loffler's serum slope.

19. 2. Moist heat at 100°C
• A) Boiling: kills vegetative bacteria, HBV, and some bacterial pores.
• B) Steaming: steamer is used to sterilize media which decomposes at
high temperature of autoclave.
• Method is carried out for 90minutes, killing most vegetative forms
except thermophiles and spores.
• C) Tyndallisation or Intermitted sterilization: sterilizes gelatine,
egg, serum or sugar containing media for 20 minutes.

20. 3. Moist heat above 100°C (Autoclave)
• Autoclave: is an equipment used to remove microorganisms and
spores using high pressure and high temperature.
Principle of Autoclave:
• When water boils, its vapour pressure equals that of the surrounding
atmosphere, so when the atm pressure is raised, the boiling
temperature is also raised (Boyle’s Law).
• Sterilization conditions: 121°C for 15 minutes at a pressure of 15lbs
per square inch (psi), 126°C for 10 minutes at pressure of 20 psi or
134°C at for 3 minutes.

21. Components of autoclave:
a. Pressure chamber: is a large cylinder (vertical or horizontal) in
which materials to be sterilized are placed.
b. Steam jacket: it is where water is placed
c. The lid: fastened by screw clamp, rendered airtight by an asbestos
washer.
• It has a discharge tap for air and steam discharge, a pressure gauge and
a safety valve (to remove excess steam).
d. An electrical heater: heats the water to produce steam.

22. Mechanism of autoclave
• Sufficient water is added to the cylinder, material to be sterilized is placed
on the tray and the autoclave is heated.
• The lid is screwed tight, discharge tap is left open until no more air bubbles
come out in the pail. Safety valve is adjusted to the required pressure.
• Steam pressure rises and penetrates objects inside the autoclave
• Condensation creates negative pressure and draws additional steam
• Moist heat kills microorganisms via coagulation of proteins.

23. A schematic diagram of laboratory autoclave

24. Types of autoclaves
a. Gravity displacement type.
• Cold air escapes through the bottom of the chamber as steam displaces
it from above.
• Ex. Vertical type-small volume capacity
• and horizontal autoclave-large volume capacity.
b. Positive pressure displacement type autoclave
c. Negative pressure (vacuum) displacement type.

25. Uses of autoclave
• Sterilization of surgical instruments
• Culture media containing water that cannot be sterilized by dry heat
• Autoclavable plastic containers
• Plastic tubes and pipette tips
• Solutions and water
• Biohazardous wastes
• Glassware

26. Advantages
• Destroys all forms of microbes, including spores.
Disadvantages
• Expensive.
• Uses more electricity.
• Unsuitable for powders and oils, injections and plastic which get
spoiled at autoclave temperatures.

27. Indicators of autoclave
• Biological indicators: uses spores of Geobacillus stearothermophilus.
• Browne's tube and thermocouple.
• Autoclave tapes.

28. IV. FILTRATION METHOD
• Filtration: is the removal of microbes by passing of a liquid or gas
through a screen like material with small pores.
• Method removes bacteria from heat labile liquids such as sera and
solutions of sugars or antibiotics used for preparation of culture media.
Types of filters
a. Depth filters.
b. Membrane filter.
A. Depth filter B. Membrane filter

29. a. Depth filters
• They are porous and retain particles throughout the depth of the filter.
• Made of random mats of metallic polymeric or inorganic material.
• They rely on the density and thickness of the filter to trap the particles.
Advantages:
• Retain large mass of particles before becoming clogged
• Have high flow rate of the fluid
• Have low cost
Disadvantage: not suitable for filtration of bacterial solutions as some
particles still come out in the filtrate.

30. Examples of Depth filters
i. Candle filters
ii. Asbestos filters:
iii. Sintered glass filters:
Applications: in industries for filtration
of food, beverages and chemicals.
Asbestos filterMembrane filters
Glass filter

31. b. Membrane filters
• Have uniform pore size, retain larger particles on the surface of their
pores.
• Made of cellulose acetate, cellulose nitrate, polycarbonate,
polyvinylidene fluoride or other synthetic materials.
• Pore size:
0.22 µm-removes most bacteria, allows viruses to pass through;
0.45 µm-retains coliforms in water microbiology and
0.8 µm-removes airborne microbes in clean rooms and for production
of bacteria free gases.

32. Filtration of liquid
• Is done to sterilize sera, sugar and antibiotic solutions.
• Separates toxins and bacteriophages from bacteria.
• Obtain bacteria free filtrates of clinical samples for virus isolation.
• Water purification.
Filtration of air
• Used to deliver bacteria-free air.
• Ex. HEPA (High-efficiency particulate air) filters: removes 99.97% of
particles with size of 0.3 µm or more.
• Used in ICU.
• ULPA (Ultra-low particulate/penetration air) filters: removes from air
atleast 99.999% dust, mould, bacteria and airborne particles with size of
0.12 µm or larger.

33. V. RADIATION
Two types
a. Ionizing radiation.
b. Non-ionizing radiation.
a. Ionizing radiation: includes x-rays, gamma rays and cosmic rays.
• Cause breakdown of DNA without high temperature (cold sterilization).
• Destroys bacterial endospores and vegetative cells, eukaryotic and prokaryotic
cells.
• Ex. Gamma radiation: for sterilization of plastics, syringes, swabs, catheters,
animal feeds, cardboards, oils, fabric and metal foils.
• Advantages: it has high penetrative power, rapidity of action and does not require
rise in temperature.

34. b. Non-ionizing radiation
• Includes infra-red and ultraviolet rays.
• They are lethal but do not penetrate glass, dirt films and water.
• Ex. Infrared radiation: used for rapid mass sterilization of prepacked
items. Ex. Syringes and catheters.
• Ultraviolet radiation: used for disinfecting enclosed areas, such as
entryways, operation theatres and laboratories.