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DISINFECTION
by chemicals

basics
Dr.T.V.Rao MD

Dr.T.V.Rao MD

1
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.
Dr.T.V.Rao MD

2
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.
Dr.T.V.Rao MD

3
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)
Dr.T.V.Rao MD

4
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

5
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

Dr.T.V.Rao MD

6
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.
Dr.T.V.Rao MD

7
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

8
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

9
Number of living microbes

Figure 9.1 A plot of microbial death rate

Constant percentage
of the extant population
is killed each minute

90% die
1 min

90% die
1 min

Time (min)
Dr.T.V.Rao MD

10
Chemical Methods
Many matters, substances and
objects cannot be sterilized with
Physical methods
So Need for Disinfectants
Dr.T.V.Rao MD

11
Chemical methods
•
•
•
•
•
•
•
•
•

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

12
Chemical agents
• A variety of chemical agents are used as
antiseptics and disinfectants.
• Factors influencing the potency of a disinfectant:
• Concentration
• Time of action
• pH
• Temperature
• Nature of organism
• Presence of organic matter
Dr.T.V.Rao MD

13
Levels of Disinfection
1. High level
disinfectants
2. Intermediate
level
disinfectants
3. low level
disinfectants
Dr.T.V.Rao MD

14
High level disinfectants:
•
•
•

Glutaraldehyde, Hydrogen peroxide, peracitic acid
and chlorine compounds.
Effectiveness may be equal to that of sterilisation.
Used for:
• Endoscopes
• Cystoscopies
• Surgical instruments with plastic components

Dr.T.V.Rao MD

15
Intermediate level disinfectants
•
•

•

May not effective
against spores
Includes alcohols,
iodophores and
phenols
Used for:
– Laryngoscopes
– Fiber optic
endoscopes
Dr.T.V.Rao MD

16
Low level disinfectants:
– Many organisms can survive on
exposure to these disinfectants.
– Used for items which come in
contact with the patients but they
do not penetrate into tissues.
– Stethoscopes, ECG electrodes etc.
Dr.T.V.Rao MD

17
Alcohols :
– Ethyl alcohol and Isopropyl alcohol are
commonly used.
– Act by denaturing of bacterial proteins.
– No sporicidal and virucidal activity.
– Used as skin antiseptics.
– Isopropyl alcohol is preferred to Ethyl
alcohol as it is better fat solvent, more
bactericidal and less volatile.
Dr.T.V.Rao MD

18
Aldehydes:
2.

a) Formaldehyde
b) Glutaraldehyde

Dr.T.V.Rao MD

19
Formaldehyde:





Bactericidal, sporicidal and virucidal
Used in both aqueous solution and gaseous forms
A 10% aqueous solution is routinely used.
Uses:
1)
2)
3)
4)
5)

preservation of tissues for pathological examination
To sterilize bacterial vaccines
To prepare toxoid from toxin
For killing of bacterial cultures and suspensions
For destroying anthrax spores in hair and wool.
Dr.T.V.Rao MD

20
Glutaraldehyde :
• Effective against bacteria , fungi and viruses.
• Less toxic and irritant to eyes and skin than
formaldehyde
• Used as 2% buffered solution
• Available commercially as CIDEX
• Uses:
1. For sterilisation of cystoscopes, endoscopes and
bronchoscopes
2. For sterilisation of plastic endotracheal tubes, face
masks, rubber anesthetic tubes
Dr.T.V.Rao MD

21
Phenols:
•
•
•
•

Lister,the father of antiseptic surgery, first introduced
the use of phenol ( Carbolic acid) in surgery (1867).
Produced by distillation of coal tar between
temperatures of 170°C and 270°C.
Bactericidal action due to cell membrane damage.
Commonly used Phenol derivatives are
1. cresol
2. chlorhexidine
3. chloroxylenol
4. hexachlorophanes
Dr.T.V.Rao MD

22
Cresols :
•
•
•

Lysol is a solution of
cresols in soap.
Active against a wide
range of organisms.
uses:
1. For sterilisation of
infected glass ware
2. Cleaning of floors
3. Disinfection of excreta

Dr.T.V.Rao MD

23
Chlorhexidine:
–Eg.Savlon – (Chlorhexidine and
Cetrimide)
–More active against Gram positive than
Gram negative
–Good fungicidal activity.
–No action on spores and little activity
against viruses
Dr.T.V.Rao MD

24
Chloroxylenol :
–Eg.dettol
–Less toxic and
less irritant.
–Readily
inactivated by
organic matter
–Inactive against
pseudomonas.
Dr.T.V.Rao MD

25
Hexachlorophanes:
:
• More active against Gram positive than Gram
negative bacteria.
• Bacteriostatic at high dilutions.
• Applied on skin as prophylaxis against
staphylococcal infections.
• Potentially toxic and should be used with care.

Dr.T.V.Rao MD

26
Halogens:
4.Halogens:
i. Chlorine
ii. Iodine
 Commonly used
disinfectants
 Bactericidal,
sporicidal and
virucidal
Dr.T.V.Rao MD

27
Chlorine :
 In the form of
 Bleaching powder,
 Sodium hypochlorite
 Chloramine
 Disinfection is due to release of free chlorine.
 Reacts with water to form hypochlorus acid .
 Bactericidal, viricidal, fungicidal and sporicidal.
 Used in water supplies, swimming pools, food
and dairy industries.
Dr.T.V.Rao MD

28
Iodine :
• Used as skin
disinfectant.
• Bactericidal and
moderate
action on
spores.
• Betadine is one
example.
Dr.T.V.Rao MD

29
5.Oxidising agents:
Hydrogen peroxide
• Effective at concentration of 3-6%
• Kills spores at 10- 25%
• Used to disinfect
»Contact lenses
»Surgical prostheses
»Plastic implants

Dr.T.V.Rao MD

30
Salts:
• Salts of heavy metals have toxic effect on
bacteria.
• Salts of copper, silver and mercury are used as
disinfectants.
• Act by coagulation of bacterial proteins.
• Mercuric chloride, once used as disinfectant is
highly toxic.
• Thimersol and mercurochrome are less toxic
• Copper salts are used as fungicides.
Dr.T.V.Rao MD

31
Surface active agents:
• Substances which alter energy relationships at
interfaces , producing a reduction of surface
tension, are known as surface active agents or
surfactants.
– Anionic
– cationic
– nonionic
– amphoteric compounds.
Dr.T.V.Rao MD

32
The anionic compounds:
:

• e.g.:- common soaps,

• Have strong detergent but weak
antimicrobial properties .
• These agents are most active at acidic ph.
• Effective against Gram positive
organisms but are relatively ineffective
against Gram negative species.
Dr.T.V.Rao MD

33
:

Cationic surfactants:

• Quaternary ammonium compounds are
the most important cationic surfactants.
• These compounds are bactericidal for a
wide range of organisms, gram positive
species are more susceptible.
• The common cationic compounds are
acetyl trimethyl ammonium bromide
(cetavalon or Cetrimide) and
benzalkonium chloride.
Dr.T.V.Rao MD

34
Amphoteric compounds:
Known as ‘Tego’ compounds possess
detergent properties of anionic and
antimicrobial activity of cationic
compounds.
• They are active against a wide range of
Gram positive and Gram negative
organisms and some viruses.
Dr.T.V.Rao MD

35
Dyes :
:
• Two groups of dyes have been used extensively as
skin and wound antiseptics
– aniline dyes
– acridine dyes
• Both are bacteriostatic in high dilution but have low
bactericidal action.
• Aniline dyes include crystal violet, brilliant green and
malachite green.
• They are more active against Gram positive bacteria
than Gram negative bacteria.
Dr.T.V.Rao MD
36
Dyes :
• Acridine dyes include acriflavine , euflavine,
proflavine and aminacrine.
• They are more active against Gram positive bacteria
than Gram negative bacteria.
• Gentian violet and acriflavine are two widely used
dyes for skin disinfection especially in Gram positive
bacterial infections.

Dr.T.V.Rao MD

37
Vapour phase disinfectants:
• Formaldehyde
gas
• Ethylene oxide
• Betapropiolactone

Dr.T.V.Rao MD

38
Formaldehyde gas:
• Used for fumigation of operation theatres, wards and
laboratories etc.
• It is generated by adding 150 gm of KMnO4 to 280 ml
of formalin for 1000 cu. Feet of room volume.
• The doors should be sealed and left unopened for 48
hours.
• The gas is toxic and irritant when inhaled.
• After completion of sterilisation the irritant vapors
are nullified by exposure to ammonia vapor.
Dr.T.V.Rao MD

39
Ethylene oxide:
• It is a colorless liquid with a boiling point of 10.7°C.
• Effective against all types of organisms including
viruses and spores.
• It has a potential toxicity to human beings, including
mutagenicity and carcinogenicity .
• It is highly inflammable.
• Used for sterilizing plastic and rubber articles,
respirators, heart lung machines, sutures, dental
equipment's etc.
Dr.T.V.Rao MD

40
Betapropiolactone :
• Condensation product of ketane and
formaldehyde.
• It has a boiling point of 163°C.
• Used in 0.2%.
• Effective against all types of organisms
including viruses.
• More efficient for fumigation than
formaldehyde.
• Used for inactivation of vaccines.
Dr.T.V.Rao MD

41
• Programme Created by Dr.T.V.Rao MD
for Medical and Paramedical Students in
the Developing World
• Email.com
• doctortvrao@gmail.com

Dr.T.V.Rao MD

42

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Disinfection by Chemicals basics

  • 2. 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. Dr.T.V.Rao MD 2
  • 3. 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. Dr.T.V.Rao MD 3
  • 4. 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) Dr.T.V.Rao MD 4
  • 5. 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 5
  • 6. 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 Dr.T.V.Rao MD 6
  • 7. 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. Dr.T.V.Rao MD 7
  • 8. 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 8
  • 9. 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 9
  • 10. Number of living microbes Figure 9.1 A plot of microbial death rate Constant percentage of the extant population is killed each minute 90% die 1 min 90% die 1 min Time (min) Dr.T.V.Rao MD 10
  • 11. Chemical Methods Many matters, substances and objects cannot be sterilized with Physical methods So Need for Disinfectants Dr.T.V.Rao MD 11
  • 13. Chemical agents • A variety of chemical agents are used as antiseptics and disinfectants. • Factors influencing the potency of a disinfectant: • Concentration • Time of action • pH • Temperature • Nature of organism • Presence of organic matter Dr.T.V.Rao MD 13
  • 14. Levels of Disinfection 1. High level disinfectants 2. Intermediate level disinfectants 3. low level disinfectants Dr.T.V.Rao MD 14
  • 15. High level disinfectants: • • • Glutaraldehyde, Hydrogen peroxide, peracitic acid and chlorine compounds. Effectiveness may be equal to that of sterilisation. Used for: • Endoscopes • Cystoscopies • Surgical instruments with plastic components Dr.T.V.Rao MD 15
  • 16. Intermediate level disinfectants • • • May not effective against spores Includes alcohols, iodophores and phenols Used for: – Laryngoscopes – Fiber optic endoscopes Dr.T.V.Rao MD 16
  • 17. Low level disinfectants: – Many organisms can survive on exposure to these disinfectants. – Used for items which come in contact with the patients but they do not penetrate into tissues. – Stethoscopes, ECG electrodes etc. Dr.T.V.Rao MD 17
  • 18. Alcohols : – Ethyl alcohol and Isopropyl alcohol are commonly used. – Act by denaturing of bacterial proteins. – No sporicidal and virucidal activity. – Used as skin antiseptics. – Isopropyl alcohol is preferred to Ethyl alcohol as it is better fat solvent, more bactericidal and less volatile. Dr.T.V.Rao MD 18
  • 20. Formaldehyde:     Bactericidal, sporicidal and virucidal Used in both aqueous solution and gaseous forms A 10% aqueous solution is routinely used. Uses: 1) 2) 3) 4) 5) preservation of tissues for pathological examination To sterilize bacterial vaccines To prepare toxoid from toxin For killing of bacterial cultures and suspensions For destroying anthrax spores in hair and wool. Dr.T.V.Rao MD 20
  • 21. Glutaraldehyde : • Effective against bacteria , fungi and viruses. • Less toxic and irritant to eyes and skin than formaldehyde • Used as 2% buffered solution • Available commercially as CIDEX • Uses: 1. For sterilisation of cystoscopes, endoscopes and bronchoscopes 2. For sterilisation of plastic endotracheal tubes, face masks, rubber anesthetic tubes Dr.T.V.Rao MD 21
  • 22. Phenols: • • • • Lister,the father of antiseptic surgery, first introduced the use of phenol ( Carbolic acid) in surgery (1867). Produced by distillation of coal tar between temperatures of 170°C and 270°C. Bactericidal action due to cell membrane damage. Commonly used Phenol derivatives are 1. cresol 2. chlorhexidine 3. chloroxylenol 4. hexachlorophanes Dr.T.V.Rao MD 22
  • 23. Cresols : • • • Lysol is a solution of cresols in soap. Active against a wide range of organisms. uses: 1. For sterilisation of infected glass ware 2. Cleaning of floors 3. Disinfection of excreta Dr.T.V.Rao MD 23
  • 24. Chlorhexidine: –Eg.Savlon – (Chlorhexidine and Cetrimide) –More active against Gram positive than Gram negative –Good fungicidal activity. –No action on spores and little activity against viruses Dr.T.V.Rao MD 24
  • 25. Chloroxylenol : –Eg.dettol –Less toxic and less irritant. –Readily inactivated by organic matter –Inactive against pseudomonas. Dr.T.V.Rao MD 25
  • 26. Hexachlorophanes: : • More active against Gram positive than Gram negative bacteria. • Bacteriostatic at high dilutions. • Applied on skin as prophylaxis against staphylococcal infections. • Potentially toxic and should be used with care. Dr.T.V.Rao MD 26
  • 27. Halogens: 4.Halogens: i. Chlorine ii. Iodine  Commonly used disinfectants  Bactericidal, sporicidal and virucidal Dr.T.V.Rao MD 27
  • 28. Chlorine :  In the form of  Bleaching powder,  Sodium hypochlorite  Chloramine  Disinfection is due to release of free chlorine.  Reacts with water to form hypochlorus acid .  Bactericidal, viricidal, fungicidal and sporicidal.  Used in water supplies, swimming pools, food and dairy industries. Dr.T.V.Rao MD 28
  • 29. Iodine : • Used as skin disinfectant. • Bactericidal and moderate action on spores. • Betadine is one example. Dr.T.V.Rao MD 29
  • 30. 5.Oxidising agents: Hydrogen peroxide • Effective at concentration of 3-6% • Kills spores at 10- 25% • Used to disinfect »Contact lenses »Surgical prostheses »Plastic implants Dr.T.V.Rao MD 30
  • 31. Salts: • Salts of heavy metals have toxic effect on bacteria. • Salts of copper, silver and mercury are used as disinfectants. • Act by coagulation of bacterial proteins. • Mercuric chloride, once used as disinfectant is highly toxic. • Thimersol and mercurochrome are less toxic • Copper salts are used as fungicides. Dr.T.V.Rao MD 31
  • 32. Surface active agents: • Substances which alter energy relationships at interfaces , producing a reduction of surface tension, are known as surface active agents or surfactants. – Anionic – cationic – nonionic – amphoteric compounds. Dr.T.V.Rao MD 32
  • 33. The anionic compounds: : • e.g.:- common soaps, • Have strong detergent but weak antimicrobial properties . • These agents are most active at acidic ph. • Effective against Gram positive organisms but are relatively ineffective against Gram negative species. Dr.T.V.Rao MD 33
  • 34. : Cationic surfactants: • Quaternary ammonium compounds are the most important cationic surfactants. • These compounds are bactericidal for a wide range of organisms, gram positive species are more susceptible. • The common cationic compounds are acetyl trimethyl ammonium bromide (cetavalon or Cetrimide) and benzalkonium chloride. Dr.T.V.Rao MD 34
  • 35. Amphoteric compounds: Known as ‘Tego’ compounds possess detergent properties of anionic and antimicrobial activity of cationic compounds. • They are active against a wide range of Gram positive and Gram negative organisms and some viruses. Dr.T.V.Rao MD 35
  • 36. Dyes : : • Two groups of dyes have been used extensively as skin and wound antiseptics – aniline dyes – acridine dyes • Both are bacteriostatic in high dilution but have low bactericidal action. • Aniline dyes include crystal violet, brilliant green and malachite green. • They are more active against Gram positive bacteria than Gram negative bacteria. Dr.T.V.Rao MD 36
  • 37. Dyes : • Acridine dyes include acriflavine , euflavine, proflavine and aminacrine. • They are more active against Gram positive bacteria than Gram negative bacteria. • Gentian violet and acriflavine are two widely used dyes for skin disinfection especially in Gram positive bacterial infections. Dr.T.V.Rao MD 37
  • 38. Vapour phase disinfectants: • Formaldehyde gas • Ethylene oxide • Betapropiolactone Dr.T.V.Rao MD 38
  • 39. Formaldehyde gas: • Used for fumigation of operation theatres, wards and laboratories etc. • It is generated by adding 150 gm of KMnO4 to 280 ml of formalin for 1000 cu. Feet of room volume. • The doors should be sealed and left unopened for 48 hours. • The gas is toxic and irritant when inhaled. • After completion of sterilisation the irritant vapors are nullified by exposure to ammonia vapor. Dr.T.V.Rao MD 39
  • 40. Ethylene oxide: • It is a colorless liquid with a boiling point of 10.7°C. • Effective against all types of organisms including viruses and spores. • It has a potential toxicity to human beings, including mutagenicity and carcinogenicity . • It is highly inflammable. • Used for sterilizing plastic and rubber articles, respirators, heart lung machines, sutures, dental equipment's etc. Dr.T.V.Rao MD 40
  • 41. Betapropiolactone : • Condensation product of ketane and formaldehyde. • It has a boiling point of 163°C. • Used in 0.2%. • Effective against all types of organisms including viruses. • More efficient for fumigation than formaldehyde. • Used for inactivation of vaccines. Dr.T.V.Rao MD 41
  • 42. • Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing World • Email.com • doctortvrao@gmail.com Dr.T.V.Rao MD 42