ANTISEPTICS AND DISINFECTANTS They have specific use and their selectivity is very low.
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PHL424PRESERVATIVES, ANTISEPTICS,
DISINFECTANTS
Dr. Sarah I BukhariAssociate Professor of
MicrobiologyCollege of PharmacyKing Saud University
04/21/23 Preservatives, Antiseptics and Disinfectants 1
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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 concentration2. Broad spectrum3. Non-toxic, non-irritant, non-corrosive and
no offensive odor4. Must have adequate stability5. Active at room temperature6. Compatible with the preparations and its
activity not affected by the ingredients.7. Active in presence of large number of
microorganisms and spore8. 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 sterility1. Parenteral injections when dispensed in multi-dose 2. Ophthalmic products
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. Esters of parahydroxy benzoic acid (Parabens)2. Benzoic acid and sodium benzoate( 0.1-0.2%) 3. Sorbic acid 4. Benzalkonium chloride (0.002-0.01%), Cetermide 5. Chlorhexideacetate ( 0.02~0.05% ) 6. Alcohol (15-20%) 7. Chlorobutanol 8. Phenol (0.1-0.5%) 9. Cresol (0.1-0.5%) 10.Phenylmercuric nitrate and acetate ( 0.002-
0.01%)11.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 benzoateGood 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 0.5•Cresol 0.3•Chlorocresol 0.1•Benzyl alcohol 0.9
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Preservatives used in eye drops
•Chlorobutol 0.5•Chlorhexidine 0.01•Phenylmercuric nitrate 0.002•Benzalkonium chloride 0.01•Phenyl ethyl alcohol 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 phenolics2. Surface active agents3. Biguanides4. Heavy metals5. Alcohols6. Halogens7. 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 spores2. Intermediate level disinfectiant• Kill vegetative bacteria, mycobacteria, most fungi, most
viruses• Have little or no sporicidal activity2. 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
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04/21/23 Preservatives, Antiseptics and Disinfectants 19
-QUAT=quaternary ammonium compounds
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Microbicial activity
Bacteria VirusesG+
G- TB 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 HS HS
S S S MS
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TYPES OF DISINFECTANTS/ANTISE
PTICS
Low-Level Disinfectants1.Phenolics2.quaternary ammonium compounds3.Cationic Biguanides4.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/AntisepticsB. 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/AntisepticsC. Bisphenols: have 2 phenolic groups
i.Hexachlorophene• Particularly good against gram-positive
strep & staph• Can cause neurological damage
ii.Triclosan• Used in soaps, toothpaste• Broad spectrum, especially against G+ &
fungi
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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, amphoterica.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
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Benzathonium
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Types of Disinfectants/Antisepticsb. Cationic surfactants: quaternary
ammonium compounds (Quats)•Derivatives of ammonium chloride•Substitutions → compounds with variable degree of antimicrobial activity
• Benzalkonium chloride, benzathonium chloride, cetylpyridinium chloride, Cetrimonium bromide
•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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Chlorhexidine04/21/23 Preservatives, Antiseptics and Disinfectants 30
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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 activityi.Mercuric chloride was used to treat syphilisii.Other mercurials: Mercurochrome and Merthiolate Mercurochrome (merbromin®) compound containing mercury & bromineTopical 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 & sodiumThimerosal used to preserve vaccinesTincture 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/ANTISE
PTICS
Intermediate-Level Disinfectants5.Alcohols6.Halogen
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Types of Disinfectants/Antiseptics5. 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 formulationsi. 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 Disinfectants7.Aldehydes8.Oxidizing agents
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Types of Disinfectants/Antiseptics7. 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
8. 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
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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 tests2. 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
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The Chick-Martin (CM) test
1. It is sometimes difficult to compare other
disinfectant with phenol as standard.2. In this test it was used only one M.O.
as test strain.3. The results is numerical thus difficult
to interpret.4. The M.O. was transferred with loop.5. 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 testThe 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 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 non-ionic 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
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