PHL424 PRESERVATIVES, ANTISEPTICS, DISINFECTANTS Dr. Sarah I Bukhari Associate Professor of Microbiology College of Pharmacy King Saud University 12/16/2015Preservatives,

PHL424PRESERVATIVES, ANTISEPTICS,

DISINFECTANTS

Dr. Sarah I BukhariAssociate Professor of

MicrobiologyCollege of PharmacyKing Saud University

04/21/23 Preservatives, Antiseptics and Disinfectants 1

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


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.


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


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


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).


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.


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


Preservatives used in injections

•Phenol 0.5•Cresol 0.3•Chlorocresol 0.1•Benzyl alcohol 0.9


Preservatives used in eye drops

•Chlorobutol 0.5•Chlorhexidine 0.01•Phenylmercuric nitrate 0.002•Benzalkonium chloride 0.01•Phenyl ethyl alcohol 0.5


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



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


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


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


CHEMICAL CLASSES OF DISINFECTANTS/ANTISEPTICS

1. Phenol and phenolics2. Surface active agents3. Biguanides4. Heavy metals5. Alcohols6. Halogens7. Aldehydes 8. Oxidizing agents


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


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



-QUAT=quaternary ammonium compounds

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


TYPES OF DISINFECTANTS/ANTISE

PTICS

Low-Level Disinfectants1.Phenolics2.quaternary ammonium compounds3.Cationic Biguanides4.Mercurials


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


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


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


Phenol and phenolics



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


Quaternary ammonium compounds


Benzathonium

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.



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


Chlorhexidine04/21/23 Preservatives, Antiseptics and Disinfectants 30


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


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.


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)


Mercurochrome (merbrominR)


Merthiolate (thimerosal®)


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



TYPES OF DISINFECTANTS/ANTISE

PTICS

Intermediate-Level Disinfectants5.Alcohols6.Halogen


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



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




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



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



• 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



TYPES OF DISINFECTANTS/ANTISEPTICS

High-Level Disinfectants7.Aldehydes8.Oxidizing agents


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


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


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


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



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


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)


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)


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.


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.


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


RW test


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




• 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.



• 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 .


Determination of Chick-Martin Coefficient



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.


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


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.



• 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.


• 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.


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.

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.


ASSESSMENT OF A NEW ANTIBIOTICS

• Determination of the antimicrobial

spectrum

• Determination of MIC

• Determination of MBC

• Determination of antibiotic synergism and

antagonism

• Microbiological assay


Documents

PHL424 PRESERVATIVES, ANTISEPTICS, DISINFECTANTS Dr. Sarah I Bukhari Associate Professor of Microbiology College of Pharmacy King Saud University 12/16/2015Preservatives,