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5/9/2017
Preservatives, Antiseptics and Disinfectants
1
PHL424
PRESERVATIVES, ANTISEPTICS,
DISINFECTANTS
Dr. Sarah I Bukhari
Associate Professor of Microbiology
College of Pharmacy
King Saud University
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Preservatives, Antiseptics and Disinfectants
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PRESERVATIVE
• Deterioration of pharmaceutical products may result
from:
• Chemical or physical changes
• Microbial contamination
• Microbial contamination is prevented or minimized by
mainly by adding preservative
• Preservatives added to pharmaceutical or food
products to;
• Prevent microbial spoilage
• keep levels of microorganisms to a low and safe level
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Characters of Ideal
Preservative
1. Effective in low concentration
2. Broad spectrum
3. Non-toxic, non-irritant, non-corrosive and no
4.
5.
6.
7.
8.
offensive odor
Must have adequate stability
Active at room temperature
Compatible with the preparations and its
activity not affected by the ingredients.
Active in presence of large number of
microorganisms and spore
Resist the attack by the microorganisms.
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Which pharmaceutical products need
preservative?
A. Sterile products
• The function of preservative is to maintain sterility
Parenteral injections when dispensed in multi-dose
2. Ophthalmic products
1.
B.
Non-sterile products
• The function of preservative is to destroy or to prevent the
growth of microbes
• In oral and topical use
• Sometimes preservatives combination is required
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Commonly used preservatives
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Esters of parahydroxy benzoic acid (Parabens)
Benzoic acid and sodium benzoate( 0.1-0.2%)
Sorbic acid
Benzalkonium chloride (0.002-0.01%), Cetermide
Chlorhexideacetate（0.02~0.05%）
Alcohol (15-20%)
Chlorobutanol
Phenol (0.1-0.5%)
Cresol (0.1-0.5%)
Phenylmercuric nitrate and acetate ( 0.002-0.01%)
Thiomersal
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Parabens
• Esters of parahydroxy benzoic acid
• Methyl, ethyl, propyl & butyl esters & their sodium salts
• The most widely used group of preservatives
• Most effective at a pH 7~9.
• Concentrations of 0.1~0.2% are normally used
• Suitable for both external and internal use.
• Usually used in combination, such as ethyl:propylester
(1:1) or ethyl:butylester(4:1).
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Benzoic acid and sodium benzoate
Good antifungal and antibacterial preservative used at
a pH of less than 4.
Concentration of 0.03~0.1% is recommended.
Sodium benzoate: 0.1~0.25%.
Only the undissociated fraction or molecular form of a
preservative possesses preservative capacity because the
ionized portion is incapable of penetrating M.O.
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Sorbic acid and its salts
(sorbates)
• Sorbic acid has similar properties to benzoic acid.
• Also only effective in acidic conditions (pH 4.5).
• Concentrations of 0.05~0.3% are used.
• Suitable for oral use.
• In general the salts are preferred over the acid
form because they are more soluble in water, but it
is the acid form that is active
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Preservatives used in injections
• Phenol
• Cresol
• Chlorocresol
• Benzyl alcohol
0.5
0.3
0.1
0.9
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Preservatives used in eye drops
• Chlorobutol
• Chlorhexidine
• Phenylmercuric nitrate
• Benzalkonium chloride
• Phenyl ethyl alcohol
0.5
0.01
0.002
0.01
0.5
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Preservatives used in oral
• Ethyl alcohol
15
• Chloroform
0.2
• Benzoic acid
0.1
• Sodium benzoate
0. 1
• Methyl and propyl hydroxy benzoate
0.1-0.2
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DISINFECTANTS
• Disinfection: Reducing number of pathogenic
organisms to point where they no longer cause
diseases
• May use physical or chemical methods.
• Disinfectant: Chemical or Physical agent applied
to inanimate objects to destroy microbes that
causing disease but not necessary resistant spore
forming
• Disinfectants are used on inanimate objects
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IDEAL DISINFECTANT
• Effective at room temperature
• Not activated by organic matter
• Compatible with soaps and detergents
• Noncorrosive, nonirritant, non-staining nontoxic
• Inexpensive
• Capable of killing all pathogenic organisms
• Require limited time of exposure
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ANTISEPTIC
• Agent applied to living tissue animate (skin and
mucous membranes) to inhibit or kill microbes
• Must be capable of preventing sepsis
• Non-toxic, non-irritating, non-statining
• Usually less efficient than disinfectants
• Examples: 70% ethanol, Povidone Iodine
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CHEMICAL CLASSES OF
DISINFECTANTS/ANTISEPTICS
1. Phenol and phenolics
2. Surface active agents
3. Biguanides
4. Heavy metals
5. Alcohols
6. Halogens
7. Aldehydes
8. Oxidizing agents
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CLASSIFICATION ACCORDING TO THE
ACTIVITY
1. High level disinfectant
• Called chemical sterilants
• Destroy vegetative bacteria, mycobacteria, fungi and enveloped
(lipid) and nonenveloped (non lipid) viruses
• Not necessarily bacterial spores
2. Intermediate level disinfectiant
• Kill vegetative bacteria, mycobacteria, most fungi, most viruses
• Have little or no sporicidal activity
2.
Low level disinfectants
• Kill most vegetative bacteria, enveloped viruses, some fungi
• Do not kill mycobacteria or bacterial spores
•
Typically used to clean environmental surfaces
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CLASSIFICATION OF ITEMS
1. Non Critical items:
• Those that either come in contact with only intact skin but not mucous
membranes or do not directly contact the patient
• Involves cleaning and/or low level disinfection
2. Semi Critical items:
• Devices that come in contact with non-intact skin or mucous membranes
but ordinarily do not penetrate them
• Involves careful cleaning followed preferably by high-Level-disinfection
3. Critical items:
• Instruments & devices that enter sterile tissues
• High risk of infection if the item is contaminated
• Involves particular cleaning followed by sterilization
Disinfectants
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Low-level
Phenols,
Biguanides, Quats
Intermediatelevel
Alcohols,
Halogens
High-level
Aldehydes,
H2O2, Paracetic acid
-QUAT=quaternary ammonium compounds
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Microbicial activity
Bacteria
G+ G- TB
Viruses
Spore Lipophilic Hydrophilic
Phenols
HS HS
R
R
S
R
Quats
HS HS
R
R
S
R
Biguanides
HS MS
R
R
V
R
Alcohols
HS HS
S
R
S
V
Iodine/Hypochlorite HS HS
S
S
S
R/S
Aldehydes
S
S
S
MS
HS HS
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TYPES OF
DISINFECTANTS/ANTISEPTICS
Low-Level Disinfectants
1.
Phenolics
2.
quaternary ammonium compounds
3.
Cationic Biguanides
4.
Mercurials
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Types of Disinfectants/Antiseptics
1. Phenol, Phenolics and bisphenols
• Low to Intermediate-level disinfectants
•
Denature proteins & disrupt cell membranes
A. Phenol: oldest surgical antiseptic (Not used)
• Now, neither used as disinfectant or antiseptic
• Corrosive effect on tissues & carcinogenic
• Disagreeable odor and possible side effects
• Now, just used for evaluating disinfectants
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Types of Disinfectants/Antiseptics
B.
•
•
•
•
Phenolics: derivatives of phenol to diminish
side effects
Detergents are added to clean & remove organic
material and to increase antimicrobial activity
Stable and persist for long periods
Used to disinfect hard surfaces in hospitals and lab
Good for disinfecting things like pus, saliva, and
feces
• Cresols are phenolics that are derived from coal tar
• Cresol(O-phenylphenol): main ingredient in Lysol
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Types of Disinfectants/Antiseptics
C. Bisphenols: have 2 phenolic groups
Hexachlorophene
• Particularly good against gram-positive strep &
staph
• Can cause neurological damage
ii. Triclosan
• Used in soaps, toothpaste
• Broad spectrum, especially against G+ & fungi
i.
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Phenol and phenolics
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Types of Disinfectants/Antiseptics
2. SURFace ACTive AgeNT (SURFACTANTS)
• Reduce surface tension
• Types of surfactants: anionic, cationic, nonionic,
amphoteric
a. Anionic (Soaps and detergents)
• Soaps have hydrophilic and hydrophobic ends
•Good degerming agents but not antimicrobial
• Detergents are positively charged organic
surfactants
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Quaternary ammonium compounds
Benzathonium
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Types of Disinfectants/Antiseptics
b. Cationic surfactants: quaternary ammonium
compounds (Quats)
• Derivatives of ammonium chloride
• Substitutions → compounds with variable degree of
antimicrobial activity
• Benzalkonium
chloride,
benzathonium
cetylpyridinium chloride, Cetrimonium bromide
•
chloride,
Inactivation
of
energy-producing
enzymes,
denaturation of proteins & disruption of cell membrane
• Used in sanitation of non-critical surfaces
• Instruments that contact skin (Blood pressure cuffs)
• Used in mouth rinses and sore throat remedies.
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Types of Disinfectants/Antiseptics
3.
Cationic Biguanides
• Chlorhexidine: most popular antiseptic of this group
•
Disrupts the cytoplasmic membrane
• Relatively ineffective against other G-ve, fungi, most viruses
• Antiseptic solution, mouth wash, skin cream
• Savlon is combination of chlorhexidine & cetermide
• Chlorhexidine is deactivated by forming insoluble salts
with anionic compounds
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Chlorhexidine
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Types of Disinfectants/Antiseptics
4. Heavy Metals
• Heavy-metal ions denature proteins
• Combine with sulfhydryl (—SH) groups
• Silver, mercury, copper, and zinc are used as
germicidals
• Low-level bacteriostatic and fungistatic agents
• Copper controls algal growth
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Silver
• 1% silver nitrate to prevent ophthalmia neonatorum
• Silver impregnated wound dressings are protective
against antibiotic-resistant bacteria
• Silver sulfadiazine - topical cream for burns
• Surfacine - water-insoluble silver iodide in a polymer
carrier, persistent for at least 13 days, can be used on
animate and inanimate objects
• Silver nanoparticle infused commericial products plastic food storage containers keep food fresher,
minimizes odors in athletic clothing.
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Mercury
• Primarily bacteriostatic but broad range of activity
i. Mercuric chloride was used to treat syphilis
ii. Other mercurials: Mercurochrome and Merthiolate
 Mercurochrome (merbromin®) compound containing
mercury & bromine
Topical antiseptic used for minor cuts and scrapes
Its use has been old-fashioned
Replaced by other agents (Betadine®)
Still used in developing countries (Low price)
 Merthiolate
(thimerosal®) compound containing
mercury & sodium
Thimerosal used to preserve vaccines
Tincture Topical antiseptic solution (NOT USED NOW)
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Mercurochrome (merbrominR)
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Merthiolate (thimerosal®)
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Copper and Zinc
• Copper
• Copper sulfate is used to control green algae
growth in ponds, pools, reservoirs and fish tanks
• Copper compounds are sometimes used in paint
to prevent mildew
• Zinc
• Zinc chloride is used in some mouthwashes
• Zinc pyrithione is fungistatic and bacteriostatic
• 1-2% used in antidandruff shampoos
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TYPES OF
DISINFECTANTS/ANTISEPTICS
Intermediate-Level Disinfectants
5. Alcohols
6. Halogen
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Types of Disinfectants/Antiseptics
5. Alcohols
• Intermediate-level disinfectants/antiseptic
• Bactericidal (G + & G-), Tuberculocidal, Virucidal
(Lipophilic & some hydrophilic viruses), Fungicidal
• Non-sporicidal
• Denature proteins & disrupt cytoplasmic membrane
• Aqueous ethanol (60-95%) & isopropanol (62-65%)
• 70% is the best concentration
• Absolute alcohols are not active WHY?
• Because denaturation requires water
• Alcohol evaporate quickly & leave no residue behind
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Types of Disinfectants/Antiseptics
5. Alcohols
• Isopropanol is slightly active than ethanol for
E.coli and S. aureus
• More effective than soap
• Not good for wound disinfection because
proteins coagulate and form a protective coat
around bacteria
• In tinctures, they enhance the effectiveness of
other antimicrobial chemicals
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Types of Disinfectants/Antiseptics
6. Halogens and halogens containing compounds
• Some halogens (iodine and chlorine) are used
alone or as components of inorganic or organic
solutions
• Intermediate-level disinfectant/antiseptic
• Denatures proteins by breaking disulfide bonds
• Combine with certain amino acids to inactivate
enzymes and other cellular proteins
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Types of Disinfectants/Antiseptics
6. Halogens and halogens containing compounds
• Iodine kills rapidly than chlorine
• Iodine formulations
i. Iodine tincture (2% I2 & 2.5% KI in alcohol)
ii. Iodophors (povidone/ polyvinylpyrrolidone -iodines,
Betadine®) are preparation containing free iodine &
povidone (inert carrier) that maintain iodine conc by
complexing with free iodine
• Povidone is surface active – improves wetting actions
• Iodophors are less irritating and less likely to produce
hypersensitivity than iodine tincture
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Types of Disinfectants/Antiseptics
• Chlorine
• The germicidal action is based on the formation of
•
•
•
•
hypochlorous acid when chlorine is added to water
Good oxidizing agent
Chlorine is used as a disinfectant in gaseous form (Cl2 ) or in
the form of a compound, such as calcium hypochlorite
(Semmelweis's lime water), sodium hypochlorite (NaOCl,
Clorox), sodium dichloroisocyanurate, and chloramines.
Used to disinfect drinking water and swimming pools
2 drops Clorox/L (4 if cloudy) and let sit 30 minutes
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TYPES OF
DISINFECTANTS/ANTISEPTICS
High-Level Disinfectants
7.
8.
Aldehydes
Oxidizing agents
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Types of Disinfectants/Antiseptics
7. ALDEHYDES
• Compounds containing terminal –CHO groups
• Alkylating
•
•
•
•
agents- denature proteins and
inactivate nucleic acids
Chemical disinfectants and sterilant
High level disinfection
Glutaraldehyde, formaldehyde
See under chemical sterilization
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Glutaraldhyde
• 2% aqueous solution
• Bactericidal
• Resist spores between 10-30
min – Sporicidal within 10 h
• Used to sterilize and disinfect
medical instruments such as
endoscopes,
respiratory
therapy, for kidney dialysis
machines, endoscopes
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Ortho-phthalaldehyde (OPA)
• OPA is a chemical sterilant
• Similar to gluteraldehyde in antimicrobial activity
• OPA has advantages compared to gluteraldehyde
• Has stability over a wide pH range (pH 3-9)
• Not irritant to the eyes and nasal passages
• Does not require exposure monitoring
• Requires no activation
• Has excellent material compatibility like gluteraldehyde
• Potential disadvantage of OPA
• It stains proteins (Skin) gray
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Formaldhyde
• High level disinfection (1-10%)
• Limited use
• Used
in
unusual
conditions
requiring
decontaimination of large rooms
• Used to sterilize and disinfect medical instruments
such as endoscopes
• Bactericidal
• Resist spores between 10-30 min – Sporicidal 10
h
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Types of Disinfectants/Antiseptics
Oxidizing Agents
Peroxides, ozone, and peracetic acid
Kill by oxidation of microbial enzymes
High-level disinfectants and antiseptics
H2O2 (10-25%) can disinfect and sterilize surfaces
• Not used for wounds because of catalase activity
• Ozone treatment of drinking water
• The combination of peracetic acid and H2O2 has been
used for disinfecting hemodialyzers
• 0.23% peracetic acid plus 7.35% H2O2
8.
•
•
•
•
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Peracetic Acid (40%)
• Very rapid action against all microorganisms
• Effective in the presence of organic matter
• Sporicidal even at low temperatures
• No harmful decomposition products
• Leaves no residue
• Corrode copper, brass, bronze, plain steel, and galvanized
iron but these effects can be reduced by additives and pH
modifications
• Unstable particularly when diluted (1%)
• Used in automated machines to sterilize medical, surgical
& dental instruments (e.g., endoscopes)
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Evaluation of Biocidal action
(disinfectants)
• It classified into suspension tests & counting methods
• Principle:
 Contact and removal methods:
i. Contact : M.O & AMA (reaction mixture),
ii. Remove samples from the reaction medium into:
iii. Recovery medium in the absence of the AMA to check
end point which may be :
1. Sterility: absence of living M.O (Extinction time method)
2. Change in viable count of survivors (Counting method)
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Extinction time methods
• These are essentially tests for sterility upon
bacterial suspensions performed after treatment
with AMA for a prescribed time and under
controlled conditions
• They may be subdivided into
1. Phenol coefficient-type tests
2. Tests carried out at use-dilutions & test overall
performance without reference to phenol.
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Phenol coefficient tests
• Also called suspension tests.
• Qualitative tests: Activity of disinfectant is compared
with that of phenol against bacterial suspension
1. The Rideal-Walker (RW) test
 A set of 5 dilutions of the substance to be tested
 A set of 5 dilutions of phenol
 Both sets are inoculated with Salmonella typhi
 At
intervals of 2.5, 5, 7.5 & 10 min,
disinfectant/bacterial mixture is 'tested for sterility by
subculturing a loopful into nutrient broth, which is
then incubated for 48-72 hours at 370C.
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The Rideal-Walker (RW) test
•
Presence or absence of growth is recorded. The test
itself is conducted at 17- l8oC (room temp)
•
The test is a special example of what is known as a
phenol coefficient test
•
The RW coefficient is calculated from the as
following formula:
•
•
RW coefficient = Dilution of unknown which kills in 7.5 but not in 5 min.
Dilution of phenol which kills in 7.5 but not in 5 min
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RW test
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The Chick-Martin (CM) test
• Modification of Rideal-Walker method
• Done mimicking naturally: in presence of organic
matter
• 3% dried human faeces should be added
• Garrod use dried yeast as a substitute and the test was
made the subject of a British Standard (BS 808:1986).
• Contact period is 30 min at 30oC
• Both Salmonella typhi and S. aureus are used
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The Chick-Martin (CM) test
• Subcultures are made in duplicate into nutrient broth
• Incubate for 48 h at 37oC and the presence or absence of
growth noted
• The concentration of phenol which prevents growth in
both is determined and the mean value calculated; a
similar value is obtained for the unknown.
• The coefficient is calculated by dividing the value found
for phenol by the value found for the unknown.
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The Chick-Martin (CM) test
•
If in a pair of tubes, growth is obtained in one but
not
the
other,
the
concentration
value
corresponding to that pair is used.
•
A typical experimental result and the method of
calculating the coefficient is shown in the
following table .
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Determination of Chick-Martin
Coefficient
62
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The Chick-Martin (CM) test
1.
2.
3.
4.
5.
It is sometimes difficult to compare other
disinfectant with phenol as standard.
In this test it was used only one M.O. as test
strain.
The results is numerical thus difficult to
interpret.
The M.O. was transferred with loop.
The contact time is short.
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The AOAC test
• In the USA a modification of the Rideal-Walker test
have been made by United States Association of
Official Analytical Chemist (AOAC).
• Compare the activity of the unknown with phenol
but permit the choice of the medium and utilizes 3
organisms; S. typhi, Ps. aeruginosa and S. aureus
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Capacity use-dilution test
The Kelsey-Sykes (KS) test
• Its triplicate challenge test, designed to determine conc
of disinfectant that will be effective in clean and dirty
conditions
• Bacterial inoculum was added to the disinfectant in 3
successive lots at 0,1 and 5 minutes.
• This is the principle of capacity test where the capacity
or lack of capacity of the disinfectant to destroy
successive additions of a bacterial culture is tested
• It’s performed in separate repeats using: S. aureus, E.
coli, Ps. aeruginosa & Proteus vulgaris.
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• It’s
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more realistic choice than employed test
organism in RW & CM tests.
• The organisms are grown on a synthetic medium
and survival is tested in a broth containing the nonionic surface active agent (Tween 80).
• The disinfectant reaction is at 20oC and recovery of
organisms at 32oC.
• Calibrated and dropping pipettes rather than loops
are used for inoculation and other liquid
manipulations, and disinfectants diluted at
approximately the dilutions recommended for use
are made in hard water.
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• The test outlined above is carried out under
clean and dirty conditions (compare RW,
clean, and CM, dirty), the latter being
simulated by dried yeast as in the CM test.
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 In summary, therefore, the KS suspension test
differs from the RW and CM tests in:
1. It is a capacity test
2. It reports the data as a pass or fail and not
as a
coefficient
3. It uses a range of microorganisms.
4. It combines an individual feature of the RW test in
that it can report on disinfectant activity under both
clean and dirty conditions.
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The Criticisms of KS test
• The test was suitable for white and clear,
soluble disinfectants providing due care was
taken on interpreting the pass concentration.
• Further modification of the test is necessary
before it can be applied to other disinfectants.
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ASSESSMENT OF A NEW ANTIBIOTICS
• Determination of the antimicrobial spectrum
• Determination of MIC
• Determination of MBC
• Determination of antibiotic synergism and
antagonism
• Microbiological assay