STERILIZATION BY

PHYSICAL METHODS
Dr.T.V.Rao MD

12/2/2012

Dr.T.V.Rao MD

Why we need Sterilization

Microorganisms capable of causing infection are constantly present in the external environment and on the human body. Microorganisms are responsible for contamination and infection. The aim of sterilisation is to remove or destroy them from materials or from surfaces.
12/2/2012 Dr.T.V.Rao MD

How can microorganisms be killed?

Denaturation of proteins (e.g. wet heat, ethylene oxide) Oxidation (e.g. dry heat, hydrogen peroxide) Filtration Interruption of DNA synthesis/repair (e.g. radiation) Interference with protein synthesis (e.g. bleach) Disruption of cell membranes (e.g. phenols)
12/2/2012 Dr.T.V.Rao MD 3

Classification
1. There are two types of sterilization: physical and chemical. Physical sterilization includes: heat radiation filtration 2. Chemical sterilization includes: Alcohols Aldehydes Phenolics Oxidizing agents Quaternary ammonium compounds ethylene oxide gas Others

12/2/2012

Dr.T.V.Rao MD

Definitions:
Sterilisation :
It is a process by which an article, surface or medium is made free of all microorganisms either in vegetative or spore form.

Disinfection :
Destruction of all pathogens or organisms capable of producing infections but not necessarily spores. All organisms may not be killed but the number is reduced to a level that is no longer harmful to health.
12/2/2012 Dr.T.V.Rao MD 5

Antiseptics :
Antiseptics :
Chemical disinfectants which can safely applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms.

Asepsis :
Technique by which the occurrence of infection into an uninfected tissue is prevented.
Dr.T.V.Rao MD

12/2/2012

Factors that influence efficacy of disinfection/sterilization

3 4 5 6 7 Contact time Physico-chemical environment (e.g. pH) Presence of organic material Temperature Type of microorganism Number of microorganisms Material composition
Dr.T.V.Rao MD 7

12/2/2012

Uses of sterilisation:
1. Sterilisation of materials, instruments used in surgical and diagnostic procedures. 2. Sterilisation of Media and reagents used in the microbiology laboratory. 3. Food and drug manufacturing to ensure safety from contaminating organisms.
12/2/2012 Dr.T.V.Rao MD 8

Understanding the Terminology

a suffix indicating that the antimicrobial agent will kill or destroy a certain group of microorganism suffix cide meaning to kill viricide destroys virus fungicide destroys fungi bactericide destroys bacteria Suffix static/stasis meaning to stand still a suffix indicating that the agent will prevent the growth or multiplication of the type of organism but are not killed outright bacteriostatic - prevents the growth of bacteria fungi static prevents the growth of fungi
12/2/2012 Dr.T.V.Rao MD 9

Relative Resistance of Microbial Forms

Highest resistance Moderate resistance Least resistance

bacterial endospore (Bacillus & Clostridium)

protozoan cyst some fungal spores some naked virus vegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas)

most bacterial vegetative cells ordinary fungal spores & hypae enveloped virus Yeasts Trophozoites

12/2/2012

Dr.T.V.Rao MD

10

What to sterilize?
It is mandatory to sterilize :
all instruments that penetrate soft tissues and bone. Instruments that are not intended to penetrate the tissues, but that may come into contact with oral tissues.

If the sterilization procedure may damage the instruments, then, sterilization can be replaced by Disinfection procedure

12/2/2012

Dr.T.V.Rao MD

11

Ideal sterilization/disinfection process

Highly efficacious Fast Good penetrability Compatible with all materials Non-toxic Effective despite presence of organic material Difficult to make significant mistakes in process Easily monitored
Dr.T.V.Rao MD 12

12/2/2012

Control of Microbial Growth: Rate of Microbial Death

When bacterial populations are heated or treated antimicrobial chemicals, they usually die at a constant rate.

12/2/2012

Dr.T.V.Rao MD

13

Figure 9.1 A plot of microbial death rate

Number of living microbes

90% die 1 min

Constant percentage of the extant population is killed each minute

90% die 1 min

Time (min)
12/2/2012 Dr.T.V.Rao MD 14

Methods 1.Physical methods 2.Chemical methods

12/2/2012 Dr.T.V.Rao MD 15

Physical methods:
Physical methods: 1.Sunlight 2.Heat 1.Dry heat 2.Moist heat 3.Filtration 4.Radiation
12/2/2012 Dr.T.V.Rao MD 16

Chemical methods
Chemical methods:
1. 2. 3. 4. 5. 6. 7. 8. 9.
12/2/2012

Alcohols Aldehydes Phenols Halogens Oxidizing agents Salts Surface active agents Dyes Vapor phase disinfectants
Dr.T.V.Rao MD 17

Physical Methods

12/2/2012

Dr.T.V.Rao MD

18

How to Sterilize
Materials 1 2 3 4 5 Inoculating wires and loops Glass ware- syringes, petridishes, testtubes, flasks etc. Disposable syringes, and other disposable items Culture media Culture media containing serum and egg Method Red heat Hot air oven Gamma radiation Autoclaving Tyndallisation

6
7 8

Toxin , serum, sugar, and antibiotic solutions

Cystoscope and endoscope Infected soiled dressings

Filtration
Glutaraldehyde Incineration

9
10
12/2/2012

Skin
Milk
Dr.T.V.Rao MD

Iodine, alcohol
Pasteurisation
19

Physical Methods of Microbial Control Dry Heat:

Direct Flaming: Used to sterilize inoculating
loops and needles. Heat metal until it has a red glow.

Incineration: Effective way to sterilize disposable

items (paper cups, dressings) and biological waste. Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat. 12/2/2012 Dr.T.V.Rao MD 20

Physical Methods of Microbial Control

Heat-Related Methods
Moist heat Pasteurization
Used for milk, ice cream, yogurt, and fruit juices Not sterilization Heat-tolerant microbes survive Pasteurization of milk Batch method Flash pasteurization Ultrahigh-temperature pasteurization
12/2/2012
2012 Pearson Education Inc.

Dr.T.V.Rao MD

21

Physical Methods of Microbial Control

Moist Heat (Continued): Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.
Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes.

High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds.
12/2/2012 Dr.T.V.Rao MD 22

Inspissation:
1. Inspissation: Heating at 80-85C for half an hour daily on three consecutive days Serum or egg media are sterilised 2. Vaccine bath: Heating at 60C for an hour daily in vaccine bath for several successive days. Serum or body fluids can be sterilised by heating at 56C for an hour daily for several successive days.
12/2/2012 Dr.T.V.Rao MD 23

Sun light:
Sun light:
Active germicidal effect due to its content of ultraviolet rays . Natural method of sterilisation of water in tanks, rivers and lakes.
12/2/2012 Dr.T.V.Rao MD 24

Heat :
Factors influencing:
Nature of heat Temperature and duration Characteristic of organism and spores Type of material
12/2/2012 Dr.T.V.Rao MD 25

Heat effectively kills Majority of Microbes

Heat :
Principle:
Dry heat kills the organism by

denaturation of the bacterial proteins, oxidative damage toxic effect of elevated levels of electrolytes.

12/2/2012 Dr.T.V.Rao MD 26

Heat :
Dry heat: 1.Red heat 2.Flaming 3.Incineration 4.Hot air oven
12/2/2012 Dr.T.V.Rao MD 27

Dry-Heat Sterilization
Involves heating at atmospheric pressure and often use a fan to obtain uniform temperature by circulation. Heat at 180 for half hour , 170 for 1 hr., or 160 C for 2 hrs. Times are the periods during which object is maintained at the respective temp.
12/2/2012 Dr.T.V.Rao MD 28

Dry heat:
Dry heat:
1. Red heat: Materials are held in the flame of a bunsen burner till they become red hot. Inoculating wires or loops Tips of forceps Needles

12/2/2012

Dr.T.V.Rao MD

29

Dry heat:
Dry heat:
2. Flaming: Materials are
passed through the flame of a bunsen burner without allowing them to become red hot. Glass slides scalpels Mouths of culture tubes
12/2/2012 Dr.T.V.Rao MD 30

Incineration:
Materials are reduced to ashes by burning. Instrument used was incinerator. Soiled dressings Animal carcasses Bedding Pathological material

12/2/2012

Dr.T.V.Rao MD

31

Dry-Heat Sterilization
Disadvantages

Disadvantages:
Less reliable than autoclaving Large temp difference may arise within device. sharp instruments get dulled Many materials do not tolerate dry heat
12/2/2012 Dr.T.V.Rao MD 32

Hot air oven:

Most widely used method Electrically heated and fitted with a fan to even distribution of air in the chamber. Fitted with a thermostat that maintains the chamber air at a chosen temperature. Temperature and time: 160 C for 2 hours. 170 C for 1 hour 180 C for 30 minutes.
12/2/2012 Dr.T.V.Rao MD 33

Uses of Hot Air Oven

Sterilisation of
1.Glassware like glass syringes, petri dishes, pipettes and test tubes. 2.Surgical instruments like scalpels, scissors, forceps etc. 3.Chemicals like liquid paraffin, fats etc.
12/2/2012 Dr.T.V.Rao MD 34

 Precautions :
1. Should not be overloaded 2. Arranged in a manner which allows free circulation of air 3. Material to be sterilized should be perfectly dry. 4. Test tubes, flasks etc. should be fitted with cotton plugs. 5. petridishes and pipetts should be wrapped in paper. 6. Rubber materials and inflammable materials should not be kept inside. 7. The oven must be allowed to cool for two hours before opening, since glass ware may crack by 12/2/2012 35 sudden cooling. Dr.T.V.Rao MD

Sterilisation controls :
Sterilisation controls

1.Spores of Bacillus subtilis subsp. niger

2. Thermocouples

3.Brownes tube
12/2/2012 Dr.T.V.Rao MD 36

Sterilizing below100C 1. temperature below 100

Pasteurization of milk

1.Inspissation 2.Vaccine bath

12/2/2012

Dr.T.V.Rao MD

37

Principle of Pasteurization

12/2/2012

Dr.T.V.Rao MD

38

A temperature at 100C
II. A temperature at 100C
1. Boiling
2. Tyndallisation

3. Steam sterilisation
12/2/2012 Dr.T.V.Rao MD 39

Boiling :
1 Boiling for 10 30 minutes may kill most of vegetative forms but spores with stand boiling. 2. Tyndallisation : Steam at 100C for 20 minutes on three successive days Used for egg , serum and sugar containing media. 3. Steam sterilizer : Steam at 100C for 90 minutes. Used for media which are decomposed at high temperature.
12/2/2012 Dr.T.V.Rao MD 40

Temperatures above 100C

III. A temperature above 100C
Autoclave : -Steam above 100C has a better killing power than dry heat. -Bacteria are more susceptible to moist heat.
12/2/2012 Dr.T.V.Rao MD 41

Components of autoclave:
Components of 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 washer. Lid bears a discharge tap for air and steam, a pressure gauge and a safety valve.

12/2/2012

Dr.T.V.Rao MD

42

Figure 9.6 Autoclave-overview

12/2/2012

Dr.T.V.Rao MD

43

Autoclave: Closed Chamber with High Temperature and Pressure

12/2/2012

Dr.T.V.Rao MD

45

Sterilisation conditions
Sterilisation conditions: Temperature 121 C Chamber pressure -15 lb per square inch. Holding time 15 minutes Others : 126C for 10 minutes 133C for 3 minutes
12/2/2012 Dr.T.V.Rao MD

46

Sterilization instrument Packing

Often instruments are packed for sterilization to be stored and handled without being contaminated. Packing depend on the intended shelf life after sterilization. The available packing options are:
Textile has shelf life of 1 month Paper has shelf life of 1 6 months Nylon, glass, and metal have shelf life of 1 year if 12/2/2012 Dr.T.V.Rao MD tightly closed

47

Uses of Autoclaves:
Uses :
1. Useful for materials which can not withstand high temp. 2. To sterilize culture media, rubber material, gowns, dressings, gloves etc.
12/2/2012 Dr.T.V.Rao MD 48

Sterilisation controls:
Sterilisation controls: 1. Thermocouples 2. Bacterial sporesBacillus stearothermophilus

3. Brownes tube 4. Autoclave tapes

12/2/2012

Dr.T.V.Rao MD

49

Sterility Controls
Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Nutrient medium containing pH color indicator Endospore strip After autoclaving, flexible vial is squeezed to break ampule and release medium onto spore strip. Incubation Yellow medium means spores are viable; autoclaved objects are not sterile.

Red medium means spores were killed; autoclaved objects are sterile.

12/2/2012

Dr.T.V.Rao MD

50

Filtration:
. Filtration:

12/2/2012

Useful for substances which get damaged by heat. To sterilize sera, sugars and antibiotic solutions. To obtain bacteria free filtrates of clinical samples. Purification of water.
Dr.T.V.Rao MD

51

 This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 m will effectively remove bacteria. If viruses must also be removed, a much smaller pore size around 20 nm is needed. Prions are not removed by filtration. The filtration equipment and the filters themselves may be purchased as presterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes.

FILTRATION STERILIZATION

To ensure sterility, the filtration system must be tested to ensure that the membranes have not been punctured prior to or during use. To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air (HEPA filtration) or in a laminar flow cabinet or "flowbox", a device which produces a laminar stream of HEPA filtered air. HEPA filters are critical in the prevention of the spread of airborne bacterial and viral organisms and, therefore, infection. Typically, medical-use HEPA filtration systems also incorporate high-energy ultraviolet light units to kill off the live bacteria and viruses trapped by the filter media.

Several Types of Filters

Types of filters: 1. Candle filters 2. Asbestos disc filters 3. Sintered glass filters 4. Membrane filters 5. Air filters 6. Syringe filters

12/2/2012

Dr.T.V.Rao MD

53

Filtration
Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents.
12/2/2012 Dr.T.V.Rao MD 54

The filtering Depends on Pore Size

The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 m (filtration membranes are most popular for this purpose).

12/2/2012

Dr.T.V.Rao MD

55

Candle filters
12/2/2012 Dr.T.V.Rao MD 56

The roles of HEPA filters in biological flow Safety glass Exhaust HEPA viewscreen filter safety cabinets
Blower Supply HEPA filter Light

High-velocity air barrier

12/2/2012

Dr.T.V.Rao MD

57

Radiations :
Radiations :

Ionizing radiations Non Ionizing radiations

12/2/2012 Dr.T.V.Rao MD 58

Ionising radiations:
Ionizing radiations:
1. X rays 2. Gamma rays 3. Cosmic rays Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation)
12/2/2012 Dr.T.V.Rao MD 59

Physical Methods of Microbial Control

Radiation
Nonionizing radiation
Wavelengths greater than 1 nm Excites electrons, causing them to make new covalent bonds
Affects 3-D structure of proteins and nucleic acids

UV light causes pyrimidine dimers in DNA UV light does not penetrate well Suitable for disinfecting air, transparent fluids, and surfaces of objects
12/2/2012
2012 Pearson Education Inc.

Dr.T.V.Rao MD

60

Forms of Radiation

12/2/2012

Dr.T.V.Rao MD

61

Physical Methods of Microbial Control:

Radiation: Three types of radiation kill microbes:
Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesnt penetrate paper, glass, and cloth.
12/2/2012 Dr.T.V.Rao MD 62

Non-Ionising radiation:
1. Infra red rays 2. Ultraviolet (UV) rays Infra red is used for rapid mass sterilisation of syringes and catheters. Ultraviolet radiation is used for disinfecting enclosed areas such as bacterial laboratory, inoculation hood, laminar flow and operation theatres.
12/2/2012 Dr.T.V.Rao MD 63

Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students

Email doctortvrao@gmail.com

12/2/2012

Dr.T.V.Rao MD

64