This document discusses various disinfectants and their properties. It begins by defining disinfection and antisepsis. It then discusses ideal properties of disinfectants and introduces the Spaulding classification system for categorizing disinfectants based on their level of activity. The document goes on to examine specific disinfectants like alcohols, hypochlorites, formaldehyde, glutaraldehyde, ortho-phthalaldehyde and hydrogen peroxide; describing their modes of action, uses, advantages and disadvantages. It concludes by discussing factors that affect the efficacy of disinfection.

1. DR.KAVYA.P

2.  DEFINITION
 SPAULDING CLASSIFICATION SYSTEM
 FACTORS AFFECTING EFFICACY OF DISINFECTION
 EACH DISINFECTANT IN DETAIL
 TESTING OF DISINFECTANTS
 EMERGING PATHOGENS
 HAND WASHING

3. DEFINITION
Process that eliminates many or all
pathogenic microorganisms , except
bacterial spores , on inanimate objects
Antiseptic:
• Germicides applied to living tissue and skin

4. IDEAL DISINFECTANT
• Wide spectrum of activity
• Active in the presence of organic matter
• Effective in acid and alkaline media
• Speedy action
• High penetrating power
• Stable
• Not corrode metals

5. • Nontoxic
• Residual effect on treated surfaces
• Odorless
• Economical
• Soluble
• Stable
• Environment friendly

6. SPAULDING’S CLASSIFICATION

7.  A chemical that kills all microbial
pathogens except large numbers of
bacterial endospores when used according
to labeling
 Glutaraldehyde 2-3.2 %
 Hydrogen peroxide 3-25%
 Chlorine 100-1000ppm free Cl
High level disinfection

8.  A chemical that kills all microbial pathogens
including mycobacteria and non-enveloped viruses
except bacterial endospores
 Isopropyl alcohol 60-95%
 Phenolic compounds 0.4-5% aq
 Iodophores 30-50ppm free iodine
Intermediate level disinfection

9.  A chemical that kills only vegetative bacteria
fungi & lipid enveloped or medium sized viruses
when used according to the labeling
 Quaternary ammonium compounds 0.4-1.6%
Low level disinfectant

10. ANTIMICROBIAL MODES OF ACTION OF
DISINFECTANTS AND ANTISEPTICS
 Denaturation of bacterial proteins by disrupting
hydrogen and disulfide bonds
 —— phenol (high conc.), alcohol, heavy -metal (high conc.),
acids, alkalis, aldehydes)
 Damage to bacterial membrane (lipids and/or
proteins),
causing leakage of intracellular molecules
 —— phenol (low conc.), surfactants, dyes
 Interference of bacterial enzyme and metabolism
 —— oxidants, heavy-metals (low conc.), alkylating agents

11. FACTORS AFFECTING
EFFICACY OF DISINFECTION
Number and location of
microorganisms
Innate resistance of
microorganisms
Concentration and potency of
disinfectants
Prions
Bacterial spores
Coccidia ( Cryptosporidia)
Mycobacteria
Non lipid or small viruses( Polio, Coxsackie)
Fungi ( Aspergillus, Candida)
Vegetative bacteria ( S.aureus, P.aeruginosa)
Lipid or Medium sized Viruses(HIV,HHV,HBV)

12. Physical and chemical factors
Temperature
pH
relative humidity
water hardness
Organic and inorganic matter
Duration of exposure
Biofilms

13. ALCOHOLS
 Ethyl alcohol and Isopropyl
alcohol
 These alcohols are rapidly
bactericidal
 Optimum bactericidal
concentration is in the range of
60% to 90% solutions in water
 They are also tuberculocidal ,
fungicidal, and virucidal
 But do not destroy bacterial
spores

14.  Not recommended for sterilizing medical
and surgical materials, principally because
of their lack of sporicidal action
 Inablility to penetrate protein-rich
materials

15. MODE OF ACTION
 Denaturation of proteins
 Bacteriostatic action-inhibition of
production of metabolites for rapid cell
division

16. USES
 To disinfect oral and rectal thermometers
 Hospital pagers, scissors and stethoscopes
 Disinfect external surfaces of
equipment(stethoscopes, ventilators, manual
ventilation bags),CPR manikins

17. DISADVANTAGES
 Damage the shellac mountings of lensed
instruments
 Damage tonometer tips-cause corneal damage
 Alcohols are flammable, must be stored in cool
well- ventilated area
 Evaporate rapidly-extended exposure time
difficult to achieve unless items are immersed

18. HYPOCHLORITE
 Hypochlorites are the most widely
used of the
chlorine disinfectants
 Available in a liquid form (e.g.,
sodium hypochlorite)
 or a solid form (e.g., calcium
hypochlorite).
 Aqueous solutions of 5.25% to 6.15%
sodium hypochlorite, which usually are
called
“household bleach.”

19.  Bactericidal, virucidal, fungicidal,
mycobactericidal, sporicidal
 Do not leave toxic residues
 Unaffected by water hardness
 Inexpensive
 Fast acting
 Remove dried or fixed organisms and
biofilms from surfaces.
 Have a low incidence of serious toxicity

20. MODE OF ACTION OF FREE CHLORINE
 Oxidation of sulfhydryl enzymes and amino acids
 Ring chlorination of amino acids
 Loss of intracellular contents
 Decreased uptake of nutrients
 Inhibition of protein synthesis
 Decreased oxygen uptake
 Depressed DNA synthesis

21. DISADVANTAGES
 Produce ocular irritation , oropharyngeal ,
esophageal and gastric burns
 Corrosive to metals in high concentrations
 Inactivation by organic matter
 Discoloring or bleaching of fabrics
 Release of toxic chlorine gas when mixed with
ammonia or acid

22.  Concept of electrolyzing saline
 The basic materials of saline and electricity are
cheap and the end product (i.e.,water) is not damaging to
the environment
 The main products of this water are
hypochlorous acid and chlorine
 The antimicrobial activity of super oxidized water
is strongly affected by the concentration
of the active ingredient (available free chlorine)
Super oxidized water

23.  Freshly generated super oxidized water is
rapidly effective(<2 minutes) in achieving a
5log10 reduction of pathogenic
microorganisms
 i.e. M. tuberculosis, Mycobacterium chelonae,
poliovirus
Human Immunodeficiency Virus [HIV], MRSA, E. coli
Candida albicans
Enterococcus faecalis, P. aeruginosa

24. USES OF HYPOCHLORITE
 1:10-1:100 dilution of 5.25%-6.15% sodium hypochlorite,
an EPA registered tuberculocidal disinfectant for
decontaminating blood spills
 Inorganic chlorine solution used for disinfecting tonometer
heads
 Spot disinfection of countertops and floors
 Irrigating agent in endodontic treatment
 Disinfectant for manikins,laundry,dental appliances,
hydrotherapy tanks, water distribution system in hemodialysis
centers and hemodialysis machines

25.  Chlorine is the preferred disinfectant in water
treatment
 Hyper chlorination of a Legionella-contaminated
hospital water system resulted in a dramatic
decrease in the isolation of Legionella
pneumophila from water outlets and a cessation
of health care–associated legionnaires’ disease in
the affected unit
 Chloramine T and hypochlorites have been used to
disinfect hydrotherapy equipment

26. FORMALDEHYDE
 Liquid and gaseous states
 Water based solution
formalin (37%
formaldehyde by weight)
 Aqueous solution is a
bactericide,tuberculocide
fungicide,virucide and
sporicide

27. MODE OF ACTION
 Alkylating the amino and sulfhydryl groups of
proteins and ring nitrogen atoms of purine bases

28. USES
 High level disinfectant
 To prepare viral vaccines(poliovirus and
influenza)
 Embalming agent and to preserve anatomic
specimens
 Formaldehyde-alcohol is a chemical
sterilant
 Destroying anthrax spores on hair and
wool

29. DISADVANTAGES
 Irritating fumes and its pungent odour
 Suspected human carcinogen linked to
nasal cancer and lung cancer

30. INCIDUR SOLUTION
 Formaldehyde free disinfectant
 Active ingredients are glutaraldehyde
and glyoxal
 Non carcinogenic , non toxic , non irritant
non corrosive and ecofriendly
 Very economical (costs less than Rs.10
for a theatre size of 1000 cubic feet

31.  Low concentration ,1% dilution enough ,no eye
irritation
 Takes only an hour to disinfect-helpful in
emergencies and between two patients

32. USAGE
•10 ml of Incidur in 1 ltr of portable water for high
risk area and 5 ml of Incidur in 1 ltr of portable
water for low risk area
•Use mop or applicator for mopping and aerial
fumigation

33. Benefits of Incidur solution
 Formaldehyde free complex formulation
 Short reaction time enables daily disinfections of
Operation Theatres
 Long residual effect & sustained-release capacity
 Disinfects and cleans surfaces in a single Cycle
 Low Application Concentration ( 1% in high risk areas &
0.5% in non-critical areas)
 Fresh, pleasing fragrance

34. INCIDUR SPRAY
•Extremely short reaction time
•Ready-to-use, instant disinfectant spray
•Formaldehyde free, non-corrosive and eco-
friendly
•Good material compatibility
•Longer residual effect

35. USAGE
•Spray undiluted 40 ml Incidur Spray to
1 square meter. of surface area from a
distance of 1ft. and let it dry.

36. Areas of Application
•High risk areas: OT, ICCU, ICU, NICU
•General areas: wards, clinics, laboratories,
toilets in hospitals and sterile and production
areas in pharmaceutical industries

37.  Active ingredients are :PROPANOL, ETHANOL,
GLUTARAL AND BENZALKONIUM CHLORIDE
 Reacts in very short time of 10 -15 minutes
 Highly effective disinfectant for water resistance
surfaces & equipments like screens, monitors,
trolleys, electronic devices and control panels

38.  High-level disinfectant and chemical sterilant
 Only when the solution is “activated” (alkaline)
by use of alkalinizing agents to a pH of 7.5 to 8.5
does the solution become sporicidal
 Once “activated,” these solutions have a shelf life
of minimally 14 days because of the
polymerization of the glutaraldehyde molecules
at alkaline pH levels
Glutaraldehyde

39.  The use of glutaraldehyde -based solutions in
health care facilities is common because:-
 They have excellent biocidal properties
 They are active in the presence of organic
matter
 They do not corrode endoscopic equipment,
thermometers, rubber, or plastic equipment

40. MODE OF ACTION
 Alkylation of sulfhydryl , hydroxyl ,
carboxyl, and amino groups of
microorganisms , which alters RNA , DNA ,
and protein synthesis

41.  2% or higher aqueous solutions of glutaraldehyde,
buffered to a pH of 7.5 to 8.5
with sodium bicarbonate, were effective in
killing vegetative bacteria in less than 2
minutes
 M. tuberculosis, fungi and viruses in less than 10
minutes
 Spores of Bacillus and Clostridium
species in 3 hours
MICROBICIDAL ACTIVITY

42. Microorganisms with relative resistance to
glutaraldehyde
 M. chelonae, Mycobacterium avium-intracellulare,
Mycobacterium xenopi
 Methylobacterium mesophilicum
 Trichosporon organisms,
 Fungal ascospores (e.g., Microascus cinereus, Chaetomium
globosum),
 Cryptosporidium organisms

43.  During use, glutaraldehyde commonly becomes
diluted
Studies have revealed a glutaraldehyde
concentration decline after a few days of use in
an automatic endoscope washer
 This occurs because instruments are not
thoroughly dried

44.  Chemical test strips or liquid chemical monitors for
determining an effective concentration is present despite
repeated use and dilution
 Frequency of testing based on how frequently the
solutions are used (used daily, test daily; used weekly,
test before use; used 30 times a day, test each 10th use)
 The concentration should be considered
unacceptable or unsafe when the test result
indicates a dilution below the product’s MIC
(generally to 1.0% to 1.5% )
 This result is shown by the indicator’s not changing
colour.

45. USES
 High level disinfectant for endoscopes,spirometry
tubing,dialyzers,transducers,anesthesia and
respiratory therapy equipment
 Noncorrosive to metal and does not damage lensed
instruments, rubber or plastics

46. DISADVANTAGES
 Too toxic and expensive-should not be used for cleaning
noncritical surfaces
 Colitis from residual disinfecting solution in endoscope
solution channels(endoscope rinsing)
 Keratopathy and corneal decompensation caused by
ophthalmic instruments inadequately rinsed after soaking in
2% glutaraldehyde
 Acute or chronic exposure-skin irritation or
dermatitis, mucous membrane irritation, pulmonary symptoms
 Pungent and irritating odor

47. ORTHO-PHTALALDEHYDE
 High-level disinfectant
contains 0.55% 1,2
benzenedicarboxaldehyde
 clear, pale-blue liquid with a
pH of 7.5.

48.  Interact with amino acids ,proteins and microorganisms
 Kill spores by blocking the spore germination process
 Excellent microbicidal activity with superior
mycobactericidal activity
 Active against glutaraldehyde-resistant
mycobacteria and Bacillus atrophaeus spores

49. ADVANTAGES OVER GLUTARALDEHYDE
 Excellent stability over a wide pH range(pH 3-9)
 Do not irritate the eyes and nasal passages
 Does not require exposure monitoring
 Barely perceptible odour
 Requires no activation
 Like glutaraldehyde, has excellent compatibility
with materials

50. DISADVANTAGES
 Stains proteins gray including unprotected skin
Must be handled with caution
 Personal protective equipment should be worn
 Equipment must be thoroughly rinsed to prevent discoloration
of skin and mucous membrane
 Reports of anaphylaxis after cystoscopy
 Contraindicated in the reprocessing of all
urologic instrumentation for patients with a
history of bladder cancer

51. Hydrogen Peroxide
 Bactericidal,virucidal,fungicidal,
sporicidal
 Produce destructive free radicals
that attack membrane lipids,DNA
and other essential cell
components

52. USES
 3% hydrogen peroxide is a stable and effective
disinfectant
 3% to 6% for the disinfection of soft contact lenses,
tonometer biprisms, ventilators,
fabrics and endoscopes
 Hydrogen peroxide is effective in spot disinfecting fabrics
in patients’ rooms
 Has been instilled into urinary drainage bags in an
attempt to eliminate the bag as a source of bladder
bacteriuria and environmental contamination

53.  No activation required
 May enhance removal of organic matter and organisms
 No disposal issues
 No odour or irritation issues
 Does not coagulate blood or fix tissues to surfaces
 Inactivates Cryptosporidium organisms
Advantages-hydrogen peroxide

54. DISADVANTAGES
 Epidemic of pseudo membrane- like enteritis and colitis in a
gastrointestinal endoscopy unit associated with inadequate
rinsing of 3% hydrogen peroxide from the endoscope
 Compatibility testing with 7.5% hydrogen peroxide found both
cosmetic changes(discoloration of black anodized metal finishes)
and functional changes with tested endoscopes
 Dilution of the hydrogen peroxide must be monitored by
regularly testing the minimum effective concentration(7.5-6%)

55. PERACETIC ACID
 Lacks harmful decomposition
products(i.e. acetic acid water
oxygen hydrogen peroxide)
 Enhances removal of organic
material and leaves no residue
 Rapid action against all
microorganisms
 Remains effective in presence of
organic matter and is sporicidal
even at low temperatures

56. MODE OF ACTION
 Denatures proteins disrupts the cell wall
permeability ,oxidizes sulfhydryl and sulfur bonds
in proteins enzymes and other metabolites

57. USES
 An automated machine using per acetic acid to
chemically sterilize medical(e.g., endoscopes
arthroscopes) surgical and dental instruments
 The sterilant, 35% per acetic acid is diluted to 0.2%
with filtered water at a temperature of 50°C
 Excellent microbial killing and no clinical failures
leading to infection

58.  Peraceticacid removes surface contaminants
especially proteins on endoscopic tubing
 Rigid endoscopes are placed within a lidded
container
 The sterilant fills the lumens either by
immersion in the circulating sterilant or by
use of channel connectors to direct the flow
into the lumen or lumens

59.  Potential incompatibility with materials
 Used only for immersible instruments
 Biologic indicator may not be suitable for routine
monitoring
 Only a small number of instruments can be processed in a
cycle
 More expensive than high-level disinfection
 Serious eye and skin damage (concentrated solution)
 Point-of-use system, no sterile storage
Disadvantages

60. Per acetic acid and Hydrogen Peroxide
 Two chemical sterilants are available that contain per
acetic acid plus hydrogen peroxide
 0.08% per acetic acid plus 1% hydrogen peroxide
 0.23% per acetic acid plus 7.35% hydrogen peroxide
 Combination inactivated all microorganisms except
bacterial spores within 20 minutes
 0.08% per acetic acid plus 1% hydrogen peroxide
effectively inactivated glutaraldehyde-resistant
mycobacteria

61. Indicators
 The manufacturers suggest the use of biologic
monitors (G. stearothermophilus spore strips)
both at the time of installation and routinely to
ensure effectiveness of the process
 A chemical monitoring strip that detects that
the active ingredient is greater than 1500ppm is
available for routine use as an additional process
control

62. USES
 Combination of per acetic
acid and hydrogen peroxide
has been used for
disinfecting hemodialyzers

63. DISADVANTAGES
 Concerns about compatibility with materials
(lead, brass, copper, zinc), both cosmetic and
functional
 Limited clinical experience
 Potential for eye and skin damage

65. IODOPHORES
 Iodine solutions or
tinctures have been
used by health
professionals as
antiseptics in skin or
tissue

66.  Iodophores have been used as both
antiseptics and disinfectants
 Iodophore is a combination of iodine and
a solubilizing agent or carrier
resulting complex provides a sustained-
release reservoir of iodine and releases small
amounts of free iodine in aqueous solution
 Best known and most widely used iodophor
is povidone-iodine,compound of
polyvinylpyrrolidone with iodine

67.  Iodophores retain the germicidal efficacy of
iodine
 Nonstaining and relatively free of toxicity and
irritancy
 “Free” iodine contribute to the bactericidal
activity of Iodophores
 Dilutions of Iodophores have more rapid
bactericidal action than does a full strength
povidone-iodine solution
 So Iodophores must be diluted according to the
manufacturers’ directions to achieve
antimicrobial activity

68.  Bactericidal, mycobactericidal
virucidal require prolonged
contact times to kill certain
fungi and bacterial spores
 Commercial Iodophores are not
sporicidal ,but they are
tuberculocidal
fungicidal,virucidal,and
bactericidal at their
recommended use-dilution

69. USES
 For disinfecting blood culture
bottles and medical equipment
such as hydrotherapy tanks
thermometers and endoscopes
 Iodine or iodine based
antiseptics should not be used
on silicone catheters because
they can adversely affect the
silicone tubing

70. PHENOLICS
 Phenol was used as a
germicide by
Sir Joseph Lister in
his pioneering work on
antiseptic surgery

71.  Two phenol derivatives commonly found as
constituents of hospital disinfectants are
 ortho-phenyl phenol
 orthobenzyl-para-chlorophenol
 Bactericidal, fungicidal, virucidal, and
tuberculocidal
 For disinfection of non critical items

72. MODE OF ACTION
 High concentration-gross
protoplasmic poison
 Low concentration-
bacterial death by
inactivation of essential
enzyme systems and
leakage of essential
metabolites from cell
wall

73. USES
 Disinfection of bedside tables,bedrails,laboratory
surfaces and noncritical medical devices
 To preclean or decontaminate critical and semi critical
devices before terminal sterilization or high level
disinfection
 Phenolics and other disinfectants should not be used to
clean infant bassinets and incubators

74.  Effective against bacteria especially gram positive bacteria
and enveloped viruses
 Not effective against non enveloped viruses and spores
 Maintain their activity in presence of organic material
 Generally solids with low melting point or oily liquids at room
temperature
 Widely used as antiseptics and disinfectants
 Residual disinfectant may cause tissue irritation even when
thoroughly rinsed

75. Chlorophenols and Chloroxyphenols
 Less toxic and irritant
 Less active and more readily inactivated by organic
matter
 Both are relatively inactive against Pseudomonas
 Various combinations used in the control of pyogenic
cocci in surgical and neonatal units in hospitals

76. Chlorhexidine(Hibitane)
 Non toxic skin antiseptic
 Most active against Gram-
positive organisms
 Fairly effective against
Gram-negative organisms

77. Hexachlorophene
 White crystalline powder
phenolic odor
 Topical anti infective
antibacterial
 Used in soaps and
toothpaste
 Potentially toxic

78. Triclosan
 Polychlorophenoxy phenol
 Antibacterial , antifungal
 Used in shampoos,
toothpastes
deodorants, mouthwashes

79. Chloroxylenol
 (4-chloro-3,5-dimethylphenol)
 To control bacteria , algae and
fungi in adhesives emulsions
paints wash tanks
 Used in antibacterial soaps and
household antiseptics
 Not significantly toxic to humans
 Mild skin irritant ,may trigger
allergic reactions

80. Savlon
 Combination of cetrimide and
chlorhexidine
 Best for disinfection of plastic appliances
 1 in 20 aqueous solution for 20 mts
 Savlon in spirit is more effective. [ 1:6 ]
80

81. SURFACE ACTIVE AGENTS
 Cationic
 Anionic
 Non-ionic
 Amphoteric

82. Cationic compounds
 Bactericidal ,fungicidal
 Active against Gram- positive organisms and to a lesser
extent on Gram-negative organisms
 No action on spores, tubercle bacilli and most viruses
 Act on phosphate groups of cell membrane, membrane
loses its semi permeability and the cell proteins are
denatured

83.  Quarternary ammonium compounds as well as 70%
isopropyl alcohol, phenolic and a chlorine
containing wipe effectively remove or inactivate
contaminants from computer keyboards within 5
sec
 Environmental sanitation of noncritical surfaces
such as floors, furniture and walls
 EPA registered ones for disinfecting medical
equipment that contacts intact skin(BP cuffs)

84. Octenidine dihydrochloride
 Cationic
surfactant

85.  Common compounds are
 acetyl trimethyl ammonium bromide(Cetavlon or
Cetrimide)
 Benzalkonium chloride
 Most active at alkaline pH
 Organic matter reduces their action
 Anionic surface active agents render them inactive

86. Anionic compounds
 Common soap have moderate action
 Soaps from saturated FA-more effective against
Gram-negative bacilli
 Soaps from unsaturated FA-greater action against
Gram-positive organisms

87. Amphoteric or Ampholytic compounds
 Tego compounds
 Active against wide range of Gram-positive
and Gram-negative organisms and some
viruses

88. DYES
 Aniline dyes
 Acridine dyes
 Used as skin and wound antiseptics
 Both are bacteriostatic in high dilution
 Low bactericidal activity

89. Aniline dyes
 Brilliant green , malachite green and crystal violet
 More active against Gram-positive organisms than Gram-
negative organisms
 Not tuberculocidal (use of malachite green in LJ medium)
 Nonirritant , nontoxic
 Inhibited by organic material
 Used in lab as selective agents in culture media

90. Acridine dyes
 Proflavine , acriflavine , euflavine and aminacrine
 More active against Gram-positive than Gram-
negative organisms
 Very little affected by presence of organic matter
 Impair the DNA complexes of the organisms ,kill
or destroy the reproductive capacity of the cell

91. Betapropiolactone (BPL)
 Condensation product of ketane and
formaldehyde
 For fumigation
 Has carcinogenic activity

92. TESTING OF DISINFECTANTS

93.  There are several methods of testing disinfectants
 Carrier test
 Suspension test
 Capacity test
 Practical test
 Field test or in-use test

94. 
Disinfection process validation is defined as
"establishing documented evidence that a disinfection
process will consistently remove or inactivate known or
possible pathogens from inanimate objects."

95.  ROBERT KOCH 1881
 GEPPERT 1890
EARLIER TESTS FOR DISINFECTANTS

96. CARRIER TESTS
 Oldest tests
 Use-dilution test by AOAC
 PROCEDURE:
 the carrier such as a silk or catgut thread or a
penicylinder (a little stick) is contaminated by
submersion in a liquid culture of the test organism
 The carrier is then dried and is brought in contact
with the disinfectant for a given exposure time
 After the exposure, it is cultured in a nutrient broth;
no growth indicates activity of the disinfectant
tested whereas growth indicates a failing activity

97.  By multiplying the number of test concentrations
of the disinfectant and the contact times, a
potentially active concentration-time
relationships of the disinfectant is obtained
 Limitations:
 the number of bacteria dried on a carrier is hard to
standardize
 the survival of the bacteria on the carrier during drying
is not constant

98. AOAC Use-dilution test
 Carrier based test
 The organisms used are Salmonella cholerasuis, S. aureus
and P. aeruginosa
 Carriers (stainless steel cylinders) are meticulously
cleaned, sterilized by autoclaving in a solution of
asparagine cooled and inoculated with a test organism by
immersing in one of the culture suspensions
 The cylinders are drained on filter paper, dried at 370C
for 40 minutes, exposed to the use-dilution of the
disinfectant for 10 minutes and cultured to assess the
survival of the bacteria

99. A single test involves the evaluation of 60 inoculated
carriers (one organism) against one product sample. In
addition to the 60 carriers, 6 carriers are required to
estimate carrier bacterial load and 6 more are included
as extras
 Use-dilution test is performed to
confirm the efficiency of disinfectant dilution derived
from phenol coefficient test

100. Suspension tests
 Qualitative tests
 Tests for determining phenol coefficient (Rideal
and Walker)
 Quantitative tests

101.  In these tests, a sample of the bacterial culture is
suspended into the disinfectant solution and after
exposure it is verified by subculture whether this
inoculum is killed or not
 Suspension tests are preferred to carrier tests as
the bacteria are uniformly exposed to the
disinfectant

102. In quantitative methods, the number of surviving
organisms is counted and compared to the original
inoculum size
By subtracting the logarithm of the former from the
logarithm of the latter the decimal log reduction or
microbicidal effect (ME) is obtained
An ME of 1 equals to a killing of 90% of the initial number
of bacteria, an ME of 2 means 99% killed
A generally accepted requirement is an ME that equals
or is greater than 5: at least 99.999% of the germs are
killed

103. RIDEAL WALKER
 Phenol is diluted from 1:95 to 1:115 and the test disinfectant is
diluted from 1:400 to 1:800
.
 Their bactericidal activity is determined against Salmonella typhi
suspension
 Subcultures are performed from both the test and phenol at intervals
of 2.5, 5, 7.5 and 10 minutes
 The plates are incubated for 48-72 hours at 37°C
 That dilution of disinfectant which disinfects the suspension in a given
time is divided by that dilution of phenol which disinfects the
suspension in same time gives its phenol coefficient

104. Chick Martin Test
 Determines the phenol coefficient of the test disinfectant

106.  Including disinfectant inactivators in recovery medium
 The recovery medium Letheen broth contains the
inactivator Lecithin and Polysorbate 80
 In separate tests, the bacterial suspensions are added to
standard dilutions of pure phenol and several dilutions of
the test disinfectant
 Contact time of 5,10,15 min
 Highest dil of disinfectant that kills at 10 min & not 5 min
PHENOL COEFFICIENT TEST BY AOAC

107.  This test was designed to demonstrate log reduction
values over time for a disinfectant against selected
bacteria, fungi, and/or mold
 The most common organisms tested include: Bacillus
subtilis, Bacillus atrophaeus, Bacillus thuringiensis,
Staphylococcus aureus, Salmonella cholerasuis,
Pseudomonas aeruginosa, Aspergillus niger, and
Trichophyton mentagrophytes
Disinfectant kill time test

108.  A tube of disinfectant is placed into a water bath for
temperature control and allowed to equilibrate
 Once the tube has reached temperature, it is inoculated
to achieve a concentration of
approximately 106 CFU/mL

109.  At selected time points (generally five points are used
including zero) aliquots are removed and placed into a
neutralizer blank
 Dilutions of the neutralizer are made and selected
dilutions plated onto agar
 Colonies are enumerated and log reductions are
calculated

110.  ABILITY TO RETAIN ACTIVITY IN PRESENCE OF INCREASED
LOAD
 The disinfectant is challenged repeatedly by successive
additions of bacterial suspension until its capacity to kill
has been exhausted
 Capacity tests simulate the practical situations of
housekeeping and instrument disinfection
 The best known capacity test is the Kelsey-Sykes test
(Kelsey and Sykes, 1969)
CAPACITY TESTS

111.  TRIPLE CHALLENGE TEST
 The duration of test-30 minutes
 The concentration of the disinfectant is reduced by half
by the addition of organic matter (autoclaved yeast cells),
which builds up to a final concentration of 0.5%
 Depending on the type of disinfectant, a single test
organism is selected from S. aureus, P.aeruginosa,
P.vulgaris and E. coli
KELSEY SYKES TEST

112.  The method can be carried out under 'clean' or 'dirty’
conditions
 The dilutions of the disinfectant are made in hard water for
clean conditions and in yeast suspension for dirty
conditions
 Test organism alone or with yeast is added at 0, 10 and 20
minutes interval
 The contact time of disinfectant and test organism is 8 min

113.  The three sets of five replicate cultures corresponding to
each challenge are incubated at 32oC for 48 hours
 Growth is assessed by turbidity
 The disinfectant is evaluated on its ability to kill
microorganisms or lack of it and the result is reported as
a pass or a fail and not as a coefficient
 Sets that contain two or more negative cultures are
recorded as a negative result

115.  Specimen result of a test
Conc Inoculum 1 2 3 Result
 1.0 2 x 109 +++++ +++++ +++++ Fail
 1.5 2 x 109 - - - -+ - -+++ +++++ Pass
 2.0 2 x 109 - - - - - - - - - - - - - -+ Pass

116.  The capacity test of Kelsey and Sykes gives a good
guideline for the dilution of the preparation to be
used
 Disadvantage of this test is the fact that it is
complicated

117.  Surface tests assess the effectiveness of the selected
sanitizer against surface-adhered microorganisms
 The test surface (a small tile, a microscopic slide, a piece
of PVC, a stainless steel disc, etc.) is contaminated with a
standardized inoculum of the test bacteria and dried
 A definite volume of the disinfectant solution is
distributed over the carrier
 After the given exposure time the number of survivors is
determined by impression on a contact plate or by a
rinsing technique, in which the carrier is rinsed in a
diluent
Surface disinfection tests

118.  The number of bacteria is determined in the rinsing fluid
 In order to determine the spontaneous dying rate of the
organisms caused by drying on the carrier, a control series
is included in which the disinfectant is substituted by
distilled water
 From the comparison of the survivors in this control series
with the test series, the reduction is determined
quantitatively
 Surface tests can reflect in-use conditions like contact
time , temperatures ,use dilutions and surface properties

119.  A 24 hour culture in nutrient broth culture is prepared
 A volume of microbial culture (usually 0.010mL to 0.020
mL) is placed onto the center of each of a number of
sterile test surfaces
 This inoculum can be spread over the sterile test surface
in a circular pattern to achieve a thin, uniform coverage
with the test microorganism
 The inoculated test surfaces are treated with the
disinfectant, each for a different length of time
Surface Time kill Test

120.  Immediately after the treatment times have elapsed, the
test surfaces are placed into a solution that neutralizes
the disinfecting action of the product
 Microorganisms surviving treatment are cultured and
enumerated
 Results of the time kill study are tabulated and reported
 Charting microbial concentrations on the test surfaces as
a function of treatment time with the disinfectant or
sanitizer

121. Disinfection of computer key boards
 Disinfectants included: quaternary ammonium
compounds, 70% isopropyl alcohol, phenolic, chlorine
(80ppm)
 All tested products were effective (>95%) in removing
and/or inactivating the test pathogens (MRSA,
P.aeruginosa) No functional/cosmetic damage after 300
wipes
 At present, recommend that keyboards be disinfected
daily (for 5 sec) and when visibly soiled

122.  Time required for the death of 90 % of
organisms at a particular temperature
D value

123.  The z-value of an organism is the temperature, in
degrees Fahrenheit or Celsius, that is
required for the thermal destruction curve to
move one log cycle
 It is the reciprocal of the slope resulting from
the plot of the logarithm of the D-value versus
the temperature at which the D-value was
obtained
 It may be simplified as the temperature required
for one log reduction in the D-value
Z value

124.  While the D-value gives the time needed at a certain
temperature to kill an organism, the z-value relates the
resistance of an organism to differing temperatures
 The z-value calculates a thermal process of equivalency, if
given one D-value and the z-value

125.  Example: if it takes an increase of 10°F to move the curve one log,
then our z-value is 10.
 Given a D-value of 4.5 minutes at 150°F, the D-value can be
calculated for 160°F by reducing the time by 1 log.
 The new D-value for 160°F given the z-value is 0.45 minutes.
 This means that each 10°F increase in temperature will reduce our D-
value by 1 log.
 Conversely, a 10°F decrease in temperature will increase our D-value
by 1 log.
 So, the D-value for a temperature of 140°F would be 45 minutes.

126.  D-value refers to decimal reduction time and is the time
required at a certain temperature to kill 90% of the
organisms being studied.
 Thus after a colony is reduced by 1 D, only 10% of the
original organisms remain.
 The population number has been reduced by one decimal
place in the counting scheme.
 Generally, each lot of a sterilization-resistant organism is
given a unique D-value.

127.  When referring to D values it is proper to give the
temperature as a subscript to the D
 For example, a hypothetical organism is reduced by 90%
after exposure to temperatures of 300 degrees Fahrenheit
for 20 minutes, thus the D-value would be written as
D300F = 20 minutes
 D-value determination is often carried out to measure a
disinfectant's efficiency to reduce the number of
microbes, present in a given environment

128.  Reactions that have small Z values are highly
temperature dependent, whereas those with large Z
values require larger changes in temperature to
reduce the time. A Z value of 10°C is typical for a
spore forming bacterium
 Z (°C) D121 (min)
bacteria 5-10 1-5
enzymes 30-40 1-5
vitamins 20-25 150-200
pigments 40-70 15-50

129.  A working example of how to use D and Z values in
pasteurization calculations:
 Pooled raw milk at the processing plant has bacterial
population of 4x105/mL
 It is to be processed at 79°C for 21 seconds
 The average D value at 65°C for the mixed population is 7
min. The Z value is 7°C

130.  Cryptosporidium parvum
 Helicobacter pylori
 E. coli O157:H7
 Rotavirus
 Human papilloma virus
 Noro virus
 Severe acute respiratory syndrome (SARS) Coronavirus
 Avian influenza virus
 Creutzfeldt-Jacob disease (CJD),
 Multidrug-resistant bacteria such as VRE and MRSA
EMERGING PATHOGENS

131.  C. parvum is not completely inactivated by most
disinfectants used in health care
 The only chemical disinfectants and sterilants able to
inactivate greater than 3 log10 of C. parvum organisms-
6% and 7.5% hydrogen peroxide
 Sterilization methods—including steam, ETO and
hydrogen peroxide gas plasma fully inactivate C. parvum
C.parvum

133.  Current cleaning and disinfection practices appear
satisfactory to prevent health care– associated
transmission
 For example, endoscopes are unlikely to represent an
important vehicle for the transmission of C. parvum
because according to the results of bacterial studies,
mechanical cleaning removes approximately 104
organisms, and drying rapidly results in loss of
C. parvum viability

134.  Chlorine at approximately 1 ppm has been found
of eliminating approximately 4 log10 of E. coli O157:H7
organisms within 1 minute in a suspension test
 Electrolyzed oxidizing water at 23°C was
effective in 10 minutes in producing a 5 log10
decrease in E. coli O157:H7 organisms
 The following disinfectants eliminated more than
5 log10 of E. coli O157:H7 within 30 seconds:
 Quaternary ammonium compound, Phenolic,
Hypochlorite (1:10 dilution of 5.25% bleach) and
Ethanol
E.coli

136.  Ethanol (80%) and glutaraldehyde (0.5%) killed all strains within 15seconds
 Chlorhexidine gluconate ,benzalkonium chloride
povidone-iodine (0.1%), and sodium hypochlorite
(150ppm) killed all strains within 30 seconds
 In the presence of organic matter both ethanol and
glutaraldehyde retained similar bactericidal activity
 ** Disinfection of experimentally contaminated
endoscopes with 2% glutaraldehyde or with the
peracetic acid system has been demonstrated to be
effective in eliminating H. pylori
H. pylori

138.  Disinfectants with demonstrated efficacy (>3 log10
reduction in virus) against rotavirus within 1 minute
 95% ethanol
 70% isopropanol
 some phenolics
 2% glutaraldehyde
 0.35% per acetic acid
 some quaternary ammonium compounds
Rota virus

140.  Effect of chlorine on the H5N1 subtype of the
avian influenza virus was evaluated
 Free chlorine concentrations typically used in
drinking water treatment (0.52 to 1.08ppm)
were sufficient to inactivate the virus by more
than 3 log10 with an exposure time of 1 minute
Avian influenza

142.  Improper disinfection of environmental surfaces
contaminated by the faeces or vomitus of infected
patients is believed to play a role in the spread of
Noro viruses
 Chlorine, glutaraldehyde, iodine-based products,
accelerated hydrogen peroxide effective
 Quaternary ammonium compound, detergent, and
ethanol were less effective
Noro virus

144.  Sodium hypochlorite (at a free chlorine
concentration of 1000ppm and 5000ppm)
 70% ethyl alcohol
 Povidone-iodine(1% iodine) effective with a
contact period of 1 minute
 SARS coronavirus is stable in faeces and urine at
room temperature for at least 1 to 2 days
SARS

146. CJD: Disinfection and Sterilization
 Critical/Semi critical-devices contaminated with high-
risk tissue from high risk patients requires special prion
reprocessing
„ NaOH and steam sterilization (e.g., 1N NaOH 1h,
1210C 30 m)
 „134oC for 18m (prevacuum)
 „132oC for 60m (gravity)
 No low temperature sterilization technology effective

147.  Immerse in a solution of 40g NaOH in 1L of water for
1hour
Remove and rinse in water, then transfer to an open
pan put in autoclave [121°C gravity displacement or
134°C porous or prevacuum sterilizer] 1hour
(However, the combination of sodium hydroxide and
steam sterilization may be deleterious to surgical
instruments , sterilizers, as well as harmful to sterilizer
operators)
Prions

148.  Place in autoclave at 134°C for 18 minutes in a
sterilizer
 Place in autoclave at 132°C (270°F) for 1 hour in a
gravity displacement sterilizer
 Copper plus per acetic acid
 Vaporised hydrogen peroxide (Sterrad NX)

150.  Prion-contaminated medical device impossible or
difficult to clean should be discarded
 For disinfection and sterilization of contaminated
medical devices instruments should be kept wet or
damp until they are decontaminated
 They should be decontaminated as soon as
possible after use
 Dried films of tissue are more resistant to prion
inactivation by steam sterilization than are tissues
that were kept moist

151. Human papilloma virus
 Studies with the bovine
papilloma virus have
shown a 99.9%
inactivation with
0.3% povidone-iodine
solution

152.  Current disinfection and sterilization
practices are appropriate for the
management of equipment in a health care facility
after exposure to a bioterrorist agent
 For example, sodium hypochlorite may be
used for surface disinfection
Bio terrorist agents

153.  In instances in which the health care facility
is the site of a bioterrorist attack,
environmental decontamination may require
special decontamination procedures (e.g.,
chlorine dioxide gas for anthrax spores)

154.  Where spillage has occurred involving CJD or body
fluids
10000p.p.m. hypochlorite for not < 30 min
 Where there is overt contamination with blood or
viral contamination 10000p.p.m. hypochlorite
Disinfection of surfaces & spillages

155.  Contamination with other body fluids & infectious
agents
 10000p.p.m. hypochlorite or
 2 % phenolic soln

156.  Liquid spillages must be immediately covered
with a disposable cloth or absorbent paper soaked
in disinfectant & left for 10 min
 If very large spillage a germicidal powder such as
Presept granules
 Granules with >100000 p.p.m.Cl
 Not to be used on acidic fluids

158.  Washed with appropriate disinfectant after each day’s use
 Fumigation with formaldehyde if any maintenance work
requires to be done as cleaning of grids or change of
filters
Disinfection of Safety Cabinets

159. PROCEDURE:
 Place 25 ml formaldehyde soln (40%) in an electrically heated
dish inside cabinet or in an approved vaporizer built into the
cabinet
 Replace the front closure of cabinet if necessary seal with
adhesive tape
 Vaporize formaldehyde
 Leave cabinet closed overnight
Fumigation

160.  Next day switch on extract fan to remove vapor
 Expel it through HEPA filter into exhaust duct
 After a few moments ,open front closure about 1 cm to
increase airflow
 Once vapours extracted, remove front closure
 Check rate of air flows before using

161.  Disinfection by formaldehyde vapors un certain
 Clean &disinfect individually all accessible surfaces
Disinfection of rooms

162.  Wash with soap & water
 Use foot operated ,elbow or wrist taps
 Dried under hot air or with disposable paper
towels
 This removes most transient surface contaminants
& resident skin commensal bacteria
 Some may remain
Disinfection of skin

163.  If there is likely contamination with pathogens:
 Use surgical scrubs-chlorhexidine or iodine
detergent
 20-30 times effective than soap & water wash
 Additional precaution-
Hand rub with alchoholic solution of chlorhexidine

165.  DENSITY OF RESIDENT
FLORA ON SKIN -102- 10 3
CFU/cm2
PATHOGENS FOUND ON HANDS
OF Health care worker
 S.AUREUS > 18 %
 KLEBSIELLA
 ACINETOBACTER spp
 ENTEROBACTER spp
 CANDIDA spp