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1. DISINFECTION
By: Dr. Manish Tiwari
Department Of
Microbiology
Smc Medical College
Lucknow

2. Disinfection
Many matters, substances and objects which is cannot be
sterilized by Physical methods, So Need for Disinfectants.
It refers to a process that destruction or removal most if not
all pathogenic organisms but not bacterial spores.

3. Use of disinfectant
Disinfectan
t
kitchen
Dairy
industry
Food
industry
Brewery
industry
bathroom Biological
lab
floor
hand wash Pharmaceutic
al industry
hospital

4. Properties of disinfectant
 Should be non corrosive, inexpensive and effective
 It should be broad spectrum and should not cause deterioration
of the object to which it is applied
 It should not be harmful to human and animals
 It should be have a pleasant odour
 I must be soluble in water and safe for regular use
 It should not cause any kind pollution when disposed on it
environment.

5. Chemical methods
1. Alcohols
2. Aldehydes
3. Phenols
4. Halogens
5. Oxidizing agents
6. Salts
7. Surface active agents
8. Dyes
9. Vapour phase disints

6. Chemical agents:
• A variety of chemical agents are used as
antiseptics and disinfectants.
• Factors influencing the potency of a disinfectant:
1. Concentration
2. Time of action
3. pH
4. Temperature
5. Nature of organism
6. Presence of organic matter

7. Spaulding classification
1. High level
disinfectants
2. Intermediate level
disinfectants
3. low level
disinfectants

8. 1. High level disinfectants
 It is chemical that kill the all microbial pathogen except
large no. of spore.
• 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

9. 2.Low level disinfection:
 It chemical substance , kills the only vegetative
form of bacteria , fungi and lipid virus.
 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.

10. 3.Intermediate level disinfectants
 It chemical substance , kills the all microbial pathogens
including MTB and NEV except spore.
 Includes alcohols, iodophores and phenols
 Used for:
– Laryngoscopes
– Fiber optic endoscopes

11. 1. 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.
Concentration 60-90%

12. 2. Aldehydes:
a) Formaldehyde
b) Glutaraldehyde

13. A. Formaldehyde
 Bactericidal, sporicidal and virucidal
 Used in both aqueous solution and gaseous forms
 A 10% aqueous solution is routinely used.
 Uses:
1. Preservation of anatomical specimen.
2. Fumigation of closed area such as operation theaters & lab.
3. Preparation of toxoid from toxin.
4. Sterilize the bacterial vaccine.

14. B. Glutaraldehyde :
• Effective against MTB, fungi and viruses.
• Less toxic and irritant to eyes and skin than
formaldehyde
• Used as 2% concentration.
• commercially available as "CIDEX"
• Available in inactive forms; has to activated by
alkalinization before use.
• Uses:
1. For sterilisation of cystoscopes, endoscopes and
bronchoscopes
2. For sterilisation of plastic endotracheal tubes, face
masks, rubber anesthetic tubes

15. 3. 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

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

17. Chlorhexidine:
 Eg. Savlon – (Chlorhexidine an Cetrimide)
 –More active against Gram positive than Gram negative
 –Good fungicidal activity.
 –No action on spores and little activity against viruses

18. Chloroxylenol :
 It is active ingredients of
DETTOL.
 Eg.dettol
 Less toxic and less irritant.
 Readily inactivated by organic
matter
 Less active against pseudomonas.

19. Hexachlorophanes:
• More active against Gram positive than Gram negative
bacteria.
• It can causes the brain damage, hence its uses as antiseptics
is restricted.
• Bacteriostatic at high dilutions.
• Applied on skin as prophylaxis against
staphylococcal infections.
• Potentially toxic and should be used with care.

20. 4. Halogens:
i. Chlorine
ii. Iodine
 Commonly used disinfectants
 Bactericidal, sporicidal and virucidal

21. 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.

22. Iodine :
• it an aqueous and
alcoholic solution,
that are widely used
as skin disinfectant.
• Bactericidal and moderate
action on spores.
• Betadine & wescodyne is
example.

23. 5.Oxidising agents:
Hydrogen peroxide
It is strong oxidizer agent
Effective at concentration of 3-6%
Kills spores at 10%
Used to disinfect
 Contact lenses
 Surgical prostheses
 Plastic implants

24. 6.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.
Silver nitrate (1%) eye drops were used for infants to prevent to
ophthalmia neonatorum.
Silver sulfadiazine is used on burns surfaces.

25. 7. 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.

26. 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 bacteria .

27. 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 effective , but
not effective for MTB or spores.
 The common cationic compounds are acetyl trimethyl
ammonium bromide (cetavalon or Cetrimide)

28. 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.
 Its uses as a antiseptics in dental practice .

29. 8.Dyes :
 Two groups of dyes have been used extensively as skin and
wound antiseptics:
1. aniline dyes
2. 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.

30. 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.

31. 9 .Vapour phase disinfectants:
1. Formaldehyde gas
2. Ethylene oxide
3. Betapropiolactone
4. Hydrogen peroxide fogging.

32. 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.

33. Ethylene oxide (ETO):
 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.

34. Betapropiolactone(BPL) :
 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.
 It was earlier used for fumigation but is no longer used as
it has carcinogenic activity.
 Used for inactivation of vaccines.

35. Plasma sterilization
 Plasma sterilization is a recently introduced sterilization
method; increasingly used nowadays.
 Plasma is known as the fourth state of matter and consists of
ions, electrons or neutral particles.
 Principle: plasma refers to a gaseous state consisting of ions,
photon and free electrons and neutral uncharged particles.
These active agents present in the plasma such as photons of
ultravoilet rays and radicals ( e.g. O & H) are capable of killing
microorganisms and spores efficiently.

36. Conti….
Uses :
 it is used for sterilization of materials and device that cannot
tolerate high temperature and humidity of autoclave, such as
some plastics, electrical devices and corrosin- susceptible
metals alloys.
 Sterilization control: efficacy of plasma sterilization is tested
by using Geobacillus Stearothermophilus.

37. Testing
1. Kochs method
2. Minimum Inhibition Concentration
3. Rideal Walker Method
4. Chick Martin test & Garrod’s test
5. Capacity use dilution test (Kelsey-Sykes test)
6. In-use test
7. Stability test

38. BASIC PRINCIPLE
 All these tests use the same principle in evaluating the efficacy
of the test disinfectant:
 After the indicator bacterium have been exposed to contact
with the disinfectant, their viability is tested by sub-culturing
them on media prepared without disinfectant
 The disinfectant carried over to the sub-cultures should
therefore neutralized either by diluting them to sub- inhibitory
conc. Or by adding a substance that inactivates it.
 1/10 dilution for phenols, aldehydes
 0.5% Na.thiosulphate for chlorine comp’s, iodophores
 1% Na. bisulphate for HCHO and glutaraldehude

39. 1. KOCHS METHOD
 Spores of Bacillus anthraces were dried on silk thread and were
subjected to action of disinfectants.
 Later, it was washed and transferred to solid medium.
2. MIC
 Measures the lowest concentration of the disinfectant that
inhibits the growth of known strain of organism.
Disadvantages:
i. No. of organisms considered-too low
ii. Time of exposure- too long
iii. No sufficient organic matter

40. 3. RIDEAL WALKER METHOD
 PHENOL-COEFFICIENT TEST.
 Bactericidal potency is assessed by measuring the rate of
killing a selected range of bacteria under specified conditions.
 B.cidal power of a particular disinfectant as compared with
that of pure PHENOL.
 Minimum time of 10min.
 Usually employed cultures are S.typhi& St.aureus.
 Phenol coefficient is considered to be 1.

41. 4. CHICK-MARTIN TEST& GARROD’S TEST
 Modification of Rideal Walker Method
 Is done conditions mimicking naturality i.e., in the presence of
organic matter.
 3% dried human feces is added
 Contact time is increased to 30min
 Both S.typhi & St.aureus are used for testing efficacy

42. GARROD’S TEST
 Modification of Chick-Martin Test.
Contains yeast instead of feces.
5g of dry yeast is mixed with 100ml DW.
48ml suspension is added to 2ml broth of S.typhi

43. 5. IN-USE TEST/ TEST OF MAURER
 Intended to estimate the number of living organism in a vessel of
disinfectant in actual use.
 The disinfectant that is already in use is diluted 1/10 by mixing 1 ml
of the disinfectant with 9 ml of sterile nutrient broth.
 Ten drops of the diluted disinfectant (each 0.02 ml) is placed on two
nutrient agar plates
 One plate is incubated at 37oC for 3 days while the other is held at
room temperature for 7 days
 The number of drops that yielded growth is counted after incubation
 If there growth in more than five drops on either plate, it represents
failure of disinfectant.

44. Method
 9ml of quarter strength Ringer soln
 1ml of test disinfectant, 9ml isotonic soln that inactivates
 it Prepare a suspension
 Transfer 10 small drops onto separate areas of the surface of
each of two dried agar plates Incubate at 37 C-48hr
 Incubate at room temp
 Examine the plates Score the growth from each drop Growth
from >5drops = failure

45. 6. CAPACITY USE DILUTION TEST /KELSEY-
SYKES TEST
 Kelsey-Sykes test is a triple challenge test, designed to
determine concentrations of disinfectant that will be effective
in clean and dirty conditions.

46. 7. STABILITY TESTS
 It measures the stability & long-term effectiveness of the
diluted disinfectant in clean and dirty medium.
 Used to supplement the information obtained from capacity use
dilution tests.
 There is no single reliable test to determine the efficacy of a
disinfectant. This is due to the fact that many parameters play a
role in influencing the action of disinfectant

47. Write short notes on:
a) COLD STERILISATION
b) INSPISSATION
c) METHODS OF TESTING DISINFECTANTS
d) USE OF ANTISEPTICS