Chain of Infection, Various physical & chemical methods used for sterilization & disinfection

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2. INFECTION
A pathological condition due to the growth
of microorganisms in host

3. Chain of Infection
Causative
agent
Susceptible
Host
Reservoir
Portal of
Entry
Portal of Exit
Mode of
Transmission

5. Causative agents / Infectious agent:
A microbial organism with the ability to cause disease.
Examples:
Bacteria
Viruses
Fungi
Protozoa
Reservoir / Sources:
A habitat within which microorganisms lives, grows and multiplies.
Examples:
Human beings
Animals
Soil
Water

6. Portal of Exit:
A place of exit providing a way for a microorganism to leave the
reservoir.
Examples:
Upper respiratory tract
Saliva from the oral cavity
Sneezing
Coughing
Urogenital tract
Semen
Vaginal secretions
Infected urine
Gastrointestinal tract
Feces / diarrhoea from the bowel
Vomitus
Skin and mucous membranes
Discharges from infected skin lesions and
infected wounds
Blood
Infected blood

7. Mode Of Transmission:
Method of transfer by which the organism moves or is carried from
one place to another.
Transmission of Infection
Direct
Indirect
Direct contact
Droplet spread
Air-borne
Vehicle-borne
Vector-borne (mechanical or biologic)

8. Direct Transmission:
Direct contact
• Person-to-person contact
• Contact with soil or vegetation harboring infectious organisms.
Droplet spread
• Spray with relatively large, short-range aerosols produced by
sneezing, coughing, or even talking.
• Droplet spread is classified as direct because transmission is by
direct spray over a few feet, before the droplets fall to the ground.
• Eg. Pertussis and meningococcal

9. Indirect Transmission:
Transfer of an infectious agent from a reservoir to a host by suspended
air particles, inanimate objects (vehicles), or animate intermediaries
(vectors).
Airborne transmission:
• Occurs when infectious agents are carried by dust or droplet nuclei
suspended in air.
• Airborne dust → settles on surfaces → become resuspended by air
currents
• Droplet nuclei: less than 5 microns in size.
• Droplets fall to the ground within a few feet
Droplet nuclei may remain suspended in the air for long periods of
time and may be blown over great distances.

10. Vehicles:
• Food
• Water
• Biologic products (blood)
• Fomites: inanimate objects such as handkerchiefs, bedding, or
surgical scalpels
Vectors:
• Mosquitoes
• Fleas and ticks
• Carry an infectious agent through mechanical means or may support
growth or changes in the agent.

11. Portal Of Entry:
An opening allowing the microorganism to enter the host.
Examples:
Upper respiratory tract
inhalation (eg. influenza)
Urogenital tract
sexual contact (eg. Chlamydia)
Gastrointestinal tract
ingestion (eg. gastroenteritis)
Skin and mucous membranes
open wound or punctures
ulcers
Blood
Needlestick injury (eg. hepatitis B)
Tubes placed in body cavities
such as urinary catheters

12. Susceptible Host:
A person who cannot resist a microorganism invading the body,
multiplying, and resulting in infection.
Susceptibility of a host depends on:
• Genetic or constitutional factors
• Specific immunity

13. Important Terminologies
• ASEPSIS:
The state of being free of living pathogenic microorganisms
• STERILIZATION:
Process by which an article, surface or medium is made free of all
living microbes either in vegetative or spore state.
• DISINFECTION:
Process of reducing or eliminating living pathogenic microorganisms
in or on materials, so they are no longer able to cause disease.
The agent used to achieve this state is called Disinfectant.

14. • ANTISEPTICS:
Chemical disinfectants which can be easily applied on skin or mucus
membrane to prevent infection by inhibiting the growth of pathogens
• BACTERICIDAL AGENTS:
Agents which are able to kill bacteria.
• BACTERIOSTATIC AGENTS:
Agents which inhibit the multiplication of bacteria but does not
necessarily kill them.
• DECONTAMINATION:
Process of rendering an article or area free of danger from
contaminants.

15. Various agents used in
Sterilization & Disinfection
Physical agents
Chemical agents

16. PHYSICAL AGENTS
CHEMICAL AGENTS
Sunlight
Alcohols:
Ethanol, isopropyl alcohol
Drying
Aldehydes:
Formaldehyde, Glutaraldehyde
Dry heat :
Hot air oven, Incineration, Flaming
Dyes
Moist heat:
Boiling, Pasteurization, tyndallisation,
Autoclaving
Halogens
Filtration
Phenolic compounds
Radiation
Ionizing, Non Ionizing
Gases
Ethylene oxide, Formaldehyde, beta
propiolactone(BPL).
Ultrasonic vibration
Metallic salts and surface active agents

17. Physical Methods of Sterilisation
Sunlight
• Direct sunlight is a natural method of sterilization of water in
tanks, rivers and lakes.
• Direct sunlight has an active germicidal effect due to the
combined effect of ultraviolet and heat rays.
Drying
• 4/5th of the bacterial weight is due to water.
• Therefore drying in air has deleterious effect on many
bacteria.
• This is unreliable method, spores are unaffected by drying.

18. Heat
• Most reliable method of sterilization
• Dry heat: Oxidative damage & toxic effect of elevated levels of
electrolytes
• Moist heat: Denaturation & coagulation of proteins.
• Time required for sterilization: inversely proportional to temp.
of exposure
Dry Heat:
1. Flaming
2. Incineration
3. Hot air oven

19. FLAMING

Inoculation
loop
or
Wire, the tip of Forceps
and spatulas are held in
a bunsen flame till they
are red hot.

20. INCINERATION
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Combustion at high temp
This is an excellent method of destroying
materials such as contaminated cloths, dressings,
animal carcasses and pathological materials.
Plastics such as PVC & polythene sterilized by
this method.
Polystyrene emits dense black smoke on
incineration.

21. HOT AIR OVEN
• The oven utilizes dry heat to sterilize
articles
• Low penetrating power
• A holding period of 160 C for 1 hr is
desirable.
• At 180 C cotton plugs get charred
• Cutting instruments used in surgery
sterilized at 150 C for 2 hrs
• Oven heated by electricity.
• Fan fitted for even distribution of air.
• There is a thermostat controlling the
temperature.
• Double walled insulation keeps the
heat in and conserves energy.

22. Recommended temp & duration:

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160ºC for at least 45 mins.
170ºC for at least 18 mins.
180ºC for at least 7.5 mins.
190ºC for at least 1.5 mins.
Uses:

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To sterilize Glassware, Forceps, Scissors, Scalpels, Swabs
Pharmaceuticals products like Liquid paraffin, dusting powder, fats
and grease.

23. Sterilization Control:
Clostridium tetani used as microbiological test
Paper strips impregnated with spores: inserted in suitable packs
Sterilized
Removed & inoculated into Thioglycolate broth
Incubated at 37ºC for 5 days

24. Moist Heat:
1. Temp below 100°C:
Pasteurisation
Water bath
Inspissator
2. Temperature at 100°C:
Boiling
Koch/Arnold’s steamer (Steam sterilizer)
Tyndallisation
3. Steam under pressure (above 100°C):
Autoclave

25. TEMPERATURE BELOW 100 C
Pasteurisation:
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Process of killing of pathogens in the milk but does not
sterilize it .
Milk is heated at 63 C for 30 mins. (Holder method) or at
72 C for 15-20 Sec. (Flash process)

Followed by rapid cooling to 13 C

Non-sporing bacteria destroyed

Coxiella burtenii heat resistant & survives Pasteurisation

26. Water Bath:

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To inactivate non sporing bacteria for the preparation of
vaccines - Special vaccine bath at 60 C for one hour is used
Serum or body fluids containing coagulable proteins can be
sterilized by heating for 1 hr at 56 C in a water bath for
several successive days.
Hot water bath

27. Inspissator:
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Sterilizes by heating at 80-85 C for half an hour for 3
successive days
Media such as Lowenstein-Jensen & Loefller’s serum
sterilized in Inspissator.
Non-sporing bacteria killed by moist heat at 60 C for 30 mins
Temp of 80 C for 5-10 mins destroy vegetative forms of
bacteria, yeasts & moulds
Viruses inactivated rapidly at 60 C

28. TEMPERATURE AT 100 C
Boiling:

Kills vegetative forms of bacterial pathogens at 90-100 C.

Process hastened by adding 2% Sodium bicarbonate.

Not recommended for sterilization of surgical instruments

Sporing bacteria exposed for prolonged period.

Hepatitis virus: Can survive up to 30 minutes of boiling.

Endospores: Can survive up to 20 hours or more of boiling

29. Steam sterilizer:

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Steam is generated using a steamer (Koch/ Arnold)
Consists of a Tin cabinet
Has a conical lid to enable the drainage of condensed steam
Perforated tray above water: ensures materials are surrounded by
steam.
For routine sterilization exposure of 90 mins is used

30. Tyndallisation:

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For media containing sugar and gelatin exposure of
100 C for 20 min for 3 successive days is used
Process is also termed as Intermittent Sterilization
Principle:
- 1st exposure kills vegetative bacteria
- In intervals spores germinate into vegetative cells
which are killed on subsequent heating

31. STEAM UNDER PRESSURE
Autoclave
Principle:

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Steam under pressure (above 100 C)
has better killing power as compared to
dry heat: better penetration power
Bacteria more susceptible to moist
heat: bacterial protein coagulates
rapidly
Steam coming in contact with cooler
surfaces condenses to water &
liberates latent heat to that surface

32. Components:
 Autoclave consists of a vertical or a horizontal
cylinder. (Gunmetal or stainless steel in supporting
iron case)
 Lid: fastened by screw clamps
 The lid is provided with:
Discharge tap: for air & steam
Pressure gauge: to measure the pressure
 A safety valve is present to permit the escape of
steam from the chamber

33. Procedure:
 Sufficient water added
 Articles to be sterilised are placed on tray & autoclave is
heated
 Lid screwed tight with discharge tap open
 When no more air comes out, discharge tap is closed
 Sterilisation can be carried out under following conditions:
121 C for 15 mins.
126 C for 10 mins.
134 C for 3 mins.
 Heater turned off & autoclave allowed to cool
 Discharge tap opened to allow the entry of air in autoclave

34. Sterilization control:
1. Spores:
 Spores of Bacillus stearothermophilus used as test organism
 Spores get killed on exposure of 12 mins at 121 C
 Spores impregnated on paper strips: packed in envelopes →
inserted in different parts of autoclave → Sterilization →
inoculated into suitable medium → incubated
2. Thermocouples:
Records temp directly by potentiometer
3. Chemical indicators

35. Filtration
Filtration helps to remove bacteria from heat labile liquids such as
serum and solutions of sugar, Antibiotics.
Uses:
• To sterilize serum and solutions of sugar, Antibiotics for preparation
of culture media
• To obtain bacteria-free filtrates of clinical samples for virus isolation
• Separation of Toxins & bacteriophages from bacteria
• Filter discs to retain bacterial cells from liquid
Types:
• Candle filters
• Asbestos filters
• Sintered glass filters
• Membrane filters

36. CANDLE FILTERS


Widely used for purification of
water for Industrial & drinking
purpose
Two types
1. Unglazed ceramic filter:
Eg. Chamberland filter & Doulton
filter
2. Diatomaceous earth filters:
Eg. Berkefeld filter & Mandler
filter

37. ASBESTOS FILTER

Disposable, single-use discs

High adsorbing capacity

Carcinogenic

Eg: Seitz filter, Sterimat filter

38. SINTERED GLASS FILTER

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Prepared by heat fusing powdered
glass particles of graded size
Low absorptive property & can be
Cleaned easily
brittle, expensive.

39. MEMBRANE FILTERS

Made of cellulose esters or other
polymers
Uses:
 Water purification & analysis
 Sterilization & sterility testing
 Preparation of solutions for
parenteral use

40. Radiation
Two types:
1. Non-ionising: IR, UV
2. Ionising: Gamma, high energy electrons
Non- Ionising radiation:
• Electromagnetic rays with longer wavelength
• Absorbed as heat
• Infrared rays: Can be considered as hot air sterilisation
• IR: rapid mass sterilisation of prepacked Syringes and catheters
• UV: disinfecting enclosed areas like operation theatres, laboratories

41. IONISING RADIATIONS:

X- rays, gamma rays & cosmic rays.

Lethal to DNA & other vital constituents

High penetrative power

No appreciable increase in the temperature – Cold sterilisation

Sterilise plastics Syringes, swabs, catheters, animal feed, grease,
fabrics, metal foils
Ultrasonic & Sonic Vibrations


Bactericidal
Microorganisms vary in their sensitivity, hence no practical value
in sterilisation and disinfection

42. Chemical Methods
Chemicals can be used as Antiseptics & Disinfectants
Potency of disinfectants depends on:

Concentration of substance

Time of action

pH of medium

Temperature

Nature of organism

Presence of extraneous material

43. Characteristics of Ideal Antiseptic or Disinfectant:

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Wide spectrum of activity (effective against all microbes)
Active in presence of organic matter
Effective in acid or alkaline media
Rapid action
High penetrating power
Stable
Should not lead to corrosion of metals
Should not cause local irritation
No interference with healing
Should not be toxic if absorbed into circulation
Inexpensive & easily available & easy to use

44. Chemical agents act in one of the following ways:

Protein coagulation

Disruption of the cell membrane


Removal of free Sulphydryl groups (required for enzyme
action)
Substrate competition: compound resembling essential
substrate.

45. ALCOHOLS
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Ethanol /Isopropyl alcohol are frequently used
As skin antiseptics: denature proteins
No action on spores
Concentration recommended 60-90% in water
Isopropyl alcohol preferred: better fat solvent, more bactericidal,
less volatile
Uses:
 Disinfection of clinical thermometer.
 Methanol: effective against fungal spores → Used for treating
cabinets & incubators

46. ALDEHYDES

Formaldehyde & Glutaraldehyde are frequently used
Formaldehyde:
 Bactericidal, sporicidal & has a lethal effect on viruses
 Used to preserve anatomical specimens
 Destroying Anthrax spores in hair and wool
 10% Formalin+0.5% Sodium tetraborate is used to sterilise metal
instruments
 Formaldehyde gas: irritant & toxic when inhaled
used for sterilizing instruments & heat-sensitive catheters,
fumigating wards, laboratories

47. Glutaraldehyde:
 Action similar to formaldehyde
 Effective against Tubercle bacilli, fungi & viruses
 Less irritant & toxic to eyes & skin
 Used to treat corrugated rubber anesthetic tubes, Face masks,
Plastic endotracheal tubes, Metal instruments and polythene tubing

48. DYES
Bacteriostatic in nature
Aniline dyes:
 Are Brilliant green, Malachite green & Crystal violet
 Active against Gram positive bacteria
 No activity against tubercle bacilli. Hence, malachite green used in
LJ medium
 Non-toxic & non-irritant to tissue but inhibited by organic material
like pus
Acridine dyes:
 Impair the DNA complexes in bacterial cell
 Effective against Gram positive
 Important dyes are Proflavine, Acriflavine, Euflavine, Aminacrine

49. HALOGENS
Iodine:

Iodine in aqueous and alcoholic solution has been used widely
as a skin disinfectant

Actively bactericidal with moderate action against spores

Active against Tubercle bacilli & viruses
Chlorine:

Chlorine and its compounds have been used as disinfectants in
water supplies, swimming pools, food & dairy industries

Used commonly as hypochlorites (ClO)

Wide spectrum of action against viruses

50. PHENOLS

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Obtained by distillation of coal tar at 170-270 C
Damage cell membrane releasing cell contents
Phenolic derivatives have been widely used as disinfectants for
various purposes in hospitals
Lysol, cresol: active against wide range, not inactivated by organic
matter, but toxic to humans
Hexachlorophene: toxic, should be handled with care
Chlorhexidine: non-toxic antiseptic, most active against Gram +ve
Uses:
 Various combinations are used in the control of pyogenic cocci in
surgical & neonatal units in hospitals.
 Aqueous solutions are used in treatment of wounds

51. GASES
Ethylene Oxide:
 Colourless, Highly penetrating gas with a sweet ethereal smell.
 Effective against all types of microorganisms including viruses and
spores
 Inflammable & explosive at high concentration
 Alkylate carboxyl, hydroxyl & amino group in protein and also
react with RNA & DNA
 Toxic, mutagenic & carcinogenic
Uses:
 Specially used for sterilising heart-lung machines, respirators,
sutures, dental equipments, books and clothing.
 Also used to sterilise Glass, metal and paper surfaces, plastics, soil,
some foods and tobacco.

52. Formaldehyde gas:


Widely employed for fumigation of operation theatres and other
rooms
When formaldehyde vapour is generated, doors should be sealed &
left unopened for 48 hrs
Beta Propiolactone (BPL):

Low penetrating power but more efficient than formaldehyde for
fumigation

Used in fumigation

For sterilisation 0.2% BPL is used

Has a rapid biocidal activity, but carcinogenic

Very effective against viruses

53. SURFACE ACTIVE AGENTS & METALLIC SALTS
Surface active agents:
 Substances which reduce the surface tension
 Used as wetting agents, detergents & emulsifiers
 Classified as: Anionic, Cationic, Nonionic & Amphoteric
 Cationic compounds act on PO4 group in cell membrane
 Cations are widely used in the form of quaternary ammonium
compounds.
 Markedly bactericidal, active against Gram positive organisms.
 No action on spores, tubercle bacilli, viruses
 Anionic compounds (soaps): moderate action
 Soaps from saturated fatty acids: against Gram –ve
 Soaps from unsaturated fatty acids: against Gram +ve

54. Metallic Salts:

Salts of heavy metals: greater action

The salts of silver, copper and mercury are used as disinfectants.

Act by coagulating proteins

Organic compounds: marked bacteriostatic, weak bactericidal and
limited fungicidal activity

Mercuric chloride: highly toxic

Copper salts: fungicides

55. TESTING OF DISINFECTANATS
Rideal-Walker test:

Activity of disinfectant to be tested is compared to the activity of
phenol of varying concentration
Chick-Martin Test:

Disinfectant is tested in the presence of organic matter

56. Thank You