Disinfectant and antiseptic is used for kill the microbes or inhibit the growth of microbes and decreasing their numbers in such a low level that they become unable to impart any harmful effect.

1. Disinfectants & Antiseptic
Mr. S. P. Shinde
Assistant Professor
Pune, Maharashtra

2. Mr. S. P. Shinde 2
Introduction
➢ Disinfection is a process of destruction or elimination of microbes and
decreasing their numbers in such a low level that they become unable to
impart any harmful effect.
➢ The process of disinfection is more effective in killing or removing
vegetative cells than endospores because vegetative cells are less resistant
as compared to endospores (as they are heat-resistant).
➢ The antimicrobial agents that are applied on the surface of non-living or
inanimate objects (e.g. working area, dishes, bench etc.) are termed as
disinfectants.
➢ The term antiseptics is used for those chemical agents that are used on
animate or living bodies (e.g. human body tissue, plant tissue etc.).
➢ Generally disinfectants are bactericidal, though they may sometimes be
bacteriostatic.

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An ideal antiseptic or disinfectant should have the following properties
1) It should have a broad spectrum of activity.
2) It should be stable and active at all pH.
3) It should be active in the presence of organic matter also.
4) It should have long shelf-life.
5) It should have a high penetration power and rapid action.
6) It should be non-toxic, non-allergenic, non-irritative and non-corrosive.
7) It should have a pleasant odour.
8) Its efficacy should not drop on dilution.
9) It should be inexpensive and easily available.
10) It should not leave non-volatile residue or stain.

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Disinfectants are classified on the basis of the following factors:
1) Based on Consistency: On the basis of consistency or state of matter,
disinfectants are of two types:
i) Liquid - e.g. alcohols, phenols
ii) Gaseous- e.g. formaldehyde vapour.
2) Based on Spectrum of Activity: On the basis of spectrum of activity,
disinfectants are of three types:
i) High Level Disinfectants: These disinfectants are used against certain types
of endoscopes, cystoscopes and surgical instruments with plastic components
which cannot withstand sterilisation procedures such as autoclaving.
Examples of high level disinfectants include glutaraldehyde, hydrogen
peroxide, peracetic acid and chlorine compounds.
Classification

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ii) Intermediate Level Disinfectants: These disinfectants may not be effective
against bacterial spores: so are used for instruments where contamination with
spores and other highly resistant organisms is unlikely.
Examples: alcohols, iodophores and phenolic compounds.
iii) Low Level Disinfectants: These disinfectants are used against items which
come in contact with the patients, but they do not penetrate the tissues.
Stethoscopes, electrocardiogram, electrodes etc. are examples of such items.
Examples: quaternary ammonium compounds and phenolic disinfectants.
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3) Based on Mechanism of Action: On the basis of mechanism of action,
disinfectants are classified into:
i. Disinfectants acting on plasma membrane (e.g. alcohol, detergent)
ii. Disinfectants denaturing cellular proteins (e.g. alcohol, phenol)
iii. Disinfectants responsible for oxidation of essential sulphydryl groups of
enzymes (e.g. H2O2, halogens)
iv. Disinfectants responsible for alkylation of amino-, carboxyl- and
hydroxyl group (e.g. formaldehyde)
v. Disinfectants damaging nucleic acid (e.g. formaldehyde)
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Different disinfectants have different mechanism of action. The bacterial
spores, some viruses and bacteria are highly resistant to many disinfectants.
Mode of action and applications of the following disinfectants are discussed
below:
1) Phenols
2) Alcohols
3) Surface active agents
4) Halogens
5) Oxidising agents
6) Dyes
7) Heavy metals
8) Aldehydes
Mode of Action
PASHODHA

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➢ Phenols are derived from coal tar and are used as disinfectants.
➢ It has a corrosive property and may produce harmful effects in sensitive
people.
Mode of Action
➢ Phenols act by disrupting the cell membranes, precipitating the proteins
and by inactivating the microbial enzymes.
➢ They alter the selective permeability of cytoplasmic membrane of
microbial cell, thereby the vital intracellular substances leak out.
➢ Depending on their concentration to be used they may be either
bacteriostatic or bactericidal.
Phenols and Phenolic Compounds

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Applications
1) Phenol functions as bactericidal, fungicidal and mycobactericidal, but is
ineffective against spores and viruses.
2) Cresol (lysol) is a soap solution of phenol-like compounds, It is useful in
disinfecting the inanimate objects like floors, walls, table surfaces and
contaminated hospital items (like rectal thermometers, excreta etc.).
3) Chlorhexidine in isopropanol solution is used for skin disinfection and its
aqueous solution is used for wound irrigation. It is also used as an antiseptic
hand wash.
4) Chlorhexidine gluconate solution (20%) is used as a general skin
disinfectant and also as a preoperative hand and skin disinfectant.
Chlorhexidine gluconate solution with quaternary ammonium compounds
show stronger and broader antimicrobial effects (e.g. Savlon).
5) Chloroxylenols (Dettol) are topically suitable due to less irritant property.
These compounds are more effective against gram-positive bacteria than
against gram-negative bacteria.

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Alcohols show broad spectrum antimicrobial activity.
Ethyl alcohol, methyl alcohol and isopropyl alcohol are most frequently used.
Mode of Action
➢ Alcohols act by dehydrating the cells, by disrupting bacterial cell
membranes and by coagulating the protein content of microorganisms.
➢ The ethyl alcohol or ethanol solutions of concentration between 70% and
90% are very effective against the vegetative forms of microorganisms.
➢ But ethyl alcohol does not completely sterilize an object since it does not
affect bacterial endospores; for example, it is reported that the endospores
of Bacillus anthracis (causative agent of anthrax) can survive in alcohol
for 20 years.
Alcohols

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Applications
1) Antiseptic Action: A solution of ethyl alcohol (70%) and isopropyl alcohol
(90%) work as a skin antiseptic. This solution is used as a disinfectant for
clinical oral thermometers and other surgical instruments.
2) Cleaning Agent: Wipes of alcohol are used to clean skin before taking
blood samples.
3) Isopropyl Alcohol: It is preferred over ethanol for disinfecting surfaces.
4) Methyl Alcohol: It kills fungal spores, therefore is best suited for
disinfecting inoculation hoods.

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➢ Soaps or detergents are the examples of surface active agents.
➢ Detergents carrying negatively charged long chain hydrocarbons are
anionic detergents (e.g. soaps and bile salts)
Mode of Action
➢ Surface active agents carry long chain of fat-soluble hydrocarbons and
water-soluble charged ions.
➢ Under the influence of this dual nature of fat and water solubility, they
concentrate on the membrane surfaces and disrupt the membrane, thus
resulting in the leakage of cell constituents.
➢ Quaternary ammonium compounds act by the denaturation of cell
proteins, interference with metabolic processes, and damage to
cytoplasmic membrane.
Surface Active Agents

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Applications
1) They inhibit the growth of vegetative cells, Mycobacteria and
enveloped viruses.
2) Solutions of 1-2% of dilution are widely used as disinfectants for
domestic and hospital purposes.
3) The quaternary ammonium compounds are germicidal in nature with
low toxicity, high water solubility, high stability in solution and with
less corrosive properties; hence preferred as useful antiseptics,
disinfectants and sanitising agents.
4) They are used for disinfecting floors, walls and other surfaces in
hospitals, nursing homes and other public places.
5) They are also used for sanitising food and beverage utensils in
restaurants, surfaces and equipment in food-processing industry.

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➢ Halogens being strong oxidising agents are highly reactive and destructive
towards vital cellular components of microbes.
➢ Members of the halogen family (especially iodine, chlorine and bromine)
are used as key components in various antimicrobial chemicals.
➢ Iodine is the oldest microbicidal agent with highest efficacy.
Mode of Action
➢ Halogens act as oxidising agents and damage the essential sulfydryl
groups of enzymes by oxidation reaction.
➢ Iodine is a strong oxidising agent and can destroy essential microbial
metabolic compounds by oxidation.
Halogens

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Applications
1) Iodine acts as a germicidal agent, highly effective against all types of
bacteria.
2) Iodophores diluted with 50% alcohol are used for preparing hand washes.
3) 10% Povidone iodine in undiluted form is used as a pre- and post-operative
skin disinfectant.
4) Sometimes iodine vapour is used for disinfecting air.
5) Liquefied chlorine gas is preferred for disinfecting drinking water and
swimming pool water. It is also used for treating effluents from sewage
treatment plants.
6) Products having calcium hypochlorite are used to sanitise utensils in
restaurants and equipment in dairy plants.
7) 1% Sodium hypochlorite solution is used for personal hygiene and for
affordable household disinfection.
8) Higher concentrations (around 5-12%) of sodium hypochlorite are used as
household bleaches, disinfectants and as sanitisers in dairy and food-
processing plants.

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Oxidising agents oxidise the cell membrane of microorganisms, resulting in
the loss of cellular structure and cell-lysis or death of microbes. The key
elements in oxidising agents are chlorine and oxygen. Hydrogen peroxide
(H2O2) is a chief oxidising agent which in 3-6% concentration is effective
against almost all organisms.
Mode of Action
Hydrogen peroxide releases nascent oxygen, therefore is used as an
antimicrobial agent. It also gives hydroxyl-free radical which denatures
proteins and DNA molecules.
Applications
1) At 6% concentration, it is used for decontaminating the instruments and
equipment.
2) At 3% concentration, it is used for disinfecting the skin and deodorising
wounds and ulcers.
3) Strong solutions of hydrogen peroxide act as sporicidal.
Oxidising Agents

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Dyes show better antibacterial property for gram-positive bacteria than gram-
negative bacteria and are more bacteriostatic in nature.
Mode of Action
Both aniline and acridine dyes have low bactericidal action and are
bacteriostatic in high dilution. Aniline dyes interrupt the synthesis of
peptidoglycan (a component of bacterial cell wall) and acridine dyes interrupt
the synthesis of cellular nucleic acids and proteins of bacteria.
Applications
1) They have better antibacterial action for gram-positive bacteria.
2) They are topically applied as antiseptics for treating mild burns.
3) Gentian violet and acriflavine are applied as paint over the skin to treat
bacterial infections
4) Melachite green is incorporated in medium for inhibiting the growth of all
bacteria, except Mycobacterium tuberculosis.
Dyes

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The term ‘heavy metal’ is used for denoting mercury, lead, zinc, silver and copper.
In the early 20th century, mercuric chloride (HgCl2) had been widely used as a
general disinfectant; but later, it was placed with other less toxic and corrosive
agents.
Mode of Action
Heavy metals bind to the cellular proteins and inactivate them. For example
mercuric chloride combines with sulphydryl (--SH) groups that inactivate
enzymes.
Applications
1) Silver sulphadiazine is applied topically for preventing colonisation and
infection of burned tissues.
2) Copper salts are used as fungicides. It also acts as a fungicide and is a
constituent of Bordeaux mixture used as a garden spray to protect plants
against fungal infections.
3) Zinc compounds are fungicidals and are used in formulation of ointments and
powders used for treating athlete's foot.
Heavy Metals

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➢ Formaldehyde works as a bactericidal, sporicidal and virucidal agent.
➢ It is used in aqueous as well as gaseous form.
➢ For general purposes a 10% aqueous formalin solution is used.
➢ Glutaraldehyde is highly effective against bacteria (e.g. M. tuberculosis),
fungi and viruses (like HIV).
➢ Its toxicity and irritancy to skin and eyes is comparatively less than
formaldehyde. Generally, it is used as a 2% buffered solution
Mode of Action
➢ Aldehydes cause alkylation of amino-, carboxyl- or hydroxyl groups and
damage nucleic acids.
➢ They are effective against all microorganisms and spores.
Aldehydes

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Applications
1) A 40% formaldehyde solution (formalin) is used to disinfect surface. It is
used to fumigate rooms, chambers, operation theatres, biological safety
cabinets, wards, sick rooms etc.
2) It is used as a preservative for histological examination of tissues.
3) Fumigation process is performed by boiling formalin, heating
paraformaldehyde or treating formalin with potassium permanganate.
4) It is also used for sterilizing bedding, furniture and books.
5) 2% Gluteraldehyde solution is used for sterilizing thermometers,
cystoscopes, bronchoscopes, centrifuges, anesthetic equipment etc.
6) It is also used for sterilizing plastic endotracheal tube, face masks,
corrugated rubber anesthetic tubes and metal instruments.
7) 2% Formaldehyde solution is used at 40°C for 20 minutes for disinfecting
wool; and 0.25% formaldehyde solution is used at 60°C for 6 hours for
disinfecting animal hair and bristles.

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Factors Influencing Disinfection
1) Concentration of Disinfectant: Concentration of the disinfectant directly
influences the rate of killing microorganisms. The curve obtained between
the efficiency and concentration of disinfectant is generally exponential,
rather than linear.
2) Temperature: Normally, on increasing the temperature the rate of
disinfection increases. Temperature coefficient (Q10) is used for
quantitative representation of temperature effect on bacterial activity.
3) Time of Exposure: Each disinfectant takes sufficient time of contact to
produce its action and to explain this first-order kinetics. The velocity or
rate constant (K) is the measurement of the efficiency of the disinfectant.
4) pH of Environment: The rate of growth of inoculum and the potency and
ability of the disinfectant to combine with the cell surface during the
disinfection process depends on the pH changes.

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5) Surface Tension: The surface properties influence the contact between the
aqueous solutions of disinfectants and it help in adsorption on the surfaces of
cell, and also in spreading and wetting properties of the solution.
6) Formulation of the Disinfectant: Formulation of a disinfectant is an
important factor as it influences the effective use of disinfectant. For
example, quaternary ammonium compounds and chlorhexidine are efficient
in 70% alcohol than in aqueous solution.
7) Chemical Structure of Disinfectant: Any change in the molecular structure of
the chemical compound may alter the activity and efficiency of the
disinfectant. For example, para-substitution of an alkyl chain up to 6 carbons
in length increases antimicrobial activity, but greater than 6-carbons in
length decreases water solubility and disinfectant activity.
8) Type and Number of Microorganisms Present: Number and nature of the
contaminating microorganisms and bacterial spores directly influence the
efficiency of disinfection, as most of the disinfecting agents are ineffective
on bacterial spores and viruses.
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9) Interfering Substances in the Environment: Presence of many compounds
(e.g. body fluid, food residues, blood, milk, pus or colloidal proteins) even
in small amount may decrease the effectiveness of many disinfecting
agents.
10) Synergism, Antagonism and Potentiation of Disinfectants: Synergism or
synergistic effects are often shown by two antimicrobial agents which
gives an increased activity. Antagonism or antagonistic effects result in
decreased antimicrobial activity and is used in the elimination of
antimicrobial properties of materials e.g. sodium thiosulphate, lubrol W+
lecithin, etc. Potentiation of a disinfectant leads to enhanced antimicrobial
activity e.g. polysorbate 80
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For the evaluation of efficiency, potency and reactivity of disinfectants
following techniques are used:
1) Tube dilution and agar plate method
2) Cup-plate, filter paper and cylinder plate method
3) Ditch-plate or giant colony method
4) Kelsey-Sykes test
5) In-use dilution test
6) Phenol coefficient test - Rideal-Walker test
7) Chick-Martin test
Evaluation of Disinfection

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1) Tube Dilution and Agar Plate Method
The chemical disinfecting agent is introduced into the agar medium or nutrient
broth medium. After that the medium are inoculated with the test
microorganisms. The test tube containing medium and microorganism is
incubated at 30-35°C for 2-3 days and then the results are observed in the form
of turbidity or colonies. These turbid colonies are recorded and the activity of
given disinfectant is compared.

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➢ In this method, heating is performed for melting the agar and cooled up to
45°C. The agar medium is inoculated with the test microorganisms and
poured into a sterile petri dish.
➢ In the cup-plate method when the inoculated agar has been solidified, holes
of about 9mm in diameter are made by sterile cork borer and the
antimicrobial agents are directly placed in these holes.
➢ In the filter paper and cylinder plate method the antimicrobial agents are
applied on the surface of solidified inoculated agar medium, by using filter
paper disc and cylinder respectively.
➢ On incubation at the temperature of 30-35°C for 2-3 days, a zone of
inhibition is observed.
➢ The diameter of this zone gives an indication of the relative activities of
different antimicrobial substances against the test microbes.
2) Cup-Plate, Filter Paper and Cylinder Plate Method

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Cup Plate Method

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3) Ditch-Plate or Giant Colony Method
A ditch is prepared in agar plate and the solution of antimicrobial substances is
carefully poured into the ditch. On the agar surface, a loopful of each test
microorganism is streaked outwards from the ditch. Microbes resistant to the
antimicrobial agent grow on right of the ditch; whereas susceptible
microorganisms show a zone of inhibition adjacent to the ditch or centre of
plate. The width of the zone of inhibition indicates the relative activity of
antimicrobial substance.

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4) Kelsey-Sykes Test
➢ This test is the measurement of the capacity of a disinfectant to retain its
activity, when it is used repeatedly in different microbiological operations.
This test is also known as capacity test.
➢ In this test, standard microbes (e.g. E. coli, Ps. aeruginosa and
Staphylococcus aureus) are added to the disinfectant in three continuous
lots at time difference of 0, 10 and 20 minutes.
➢ These three lots are kept in contact with disinfectant for 8 minutes and
samples are transferred at the difference of 8, 18 and 28 minutes
respectively to a recovery medium.
➢ Thus efficiency of disinfectant is determined by its ability to kill bacteria
and not by comparing with phenol.
➢ This test is performed both in dirty and clean conditions; therefore it also
measures the effectiveness of a disinfectant in presence of an organic
matter.

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Kelsey-Sykes Test

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5) In-Use Dilution Test
This test is used for examining the quantity of viable organisms
1) Staphylococcus aureus,
2) Salmonella choleraesuis
3) Pseudomonas aeruginosa.
➢ Standardised cultures of the test organisms are grown in liquid media.
➢ This media is standardised and metal carrier rings are dipped into it and
removed, and dried at 37°C for a short time.
➢ After that, the dried cultures are placed into a disinfectant solution, made at
a concentration specified by the manufactures.
➢ The culture is left for 10 minutes at 20°C.
➢ In the next step, the carrier rings are transferred to a culture medium that
allows the growth of any surviving organisms.
➢ Number of organisms in the resulting culture is calculated showing the
effectiveness of the disinfectants.

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➢ In phenol coefficient or Rideal-Walker test, a suspension with similar
quantities of organisms is used in the estimation of efficiency and action of
different concentration of phenol and the disinfectant to be tested.
➢ A solution of the test disinfectant that sterilises the suspension in a given
time is divided by the corresponding dilutions of phenol; this gives the
phenol coefficient.
➢ However, phenol coefficient is not the measurement of the practical
functioning of the test disinfectant in the presence of organic matters.
➢ By Rideal-Walker test (that uses Rideal-Walker broth and Salmonella typhi)
the phenol coefficient of test disinfectant may be calculated.
6) Phenol Coefficient Test or Rideal-Walker Test

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➢ By using the testing disinfectant and phenol, different dilutions are
prepared and 5ml of each dilution are inoculated with 0.5ml of the 24
hours broth culture of the organisms.
➢ All incubated tubes (disinfectant + organisms and phenol + organisms) are
kept at the temperature of 17.5°C in a water bath.
➢ Sub-cultures of each reaction mixture is prepared and transferred to 5ml
sterile broth, at time intervals of 2.5, 5, 7.5 and 10 minutes.
➢ The broth tubes are incubated at the temperature of 37°C for 48-72 hours,
and examined for the absence or presence of the growth of
microorganisms.
➢ The Rideal-Walker coefficient of the test disinfectant is calculated,
according to the data obtained.
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Conclusion
1) If the Rideal-Walker or phenol coefficient for a given test disinfectant is 1,
the disinfectant has the same effectiveness as phenol.
2) If phenol coefficient of test disinfectant is 20, the disinfectant is 20 times
more active than phenol.
3) If phenol coefficient of test disinfectant is less than 1, it is less effective,
and if more than 1, it is more effective as compared to phenol.

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➢ Chick and Martin suggested that the disinfectants are essential to act in the
presence of organic matter, so they recommended the use of dried human
faeces in the test system.
➢ As a substitute to human faeces, Garrod suggested to use dried yeast.
➢ In this test, Salmonella typhi (test organism) is inoculated into solutions
having graded concentrations of test substance (or phenol in case of
standard) along with dried yeast.
➢ The components are left for 30 minutes at 20°C to interact and lastly
duplicate sub- cultures are made into nutrient broth. These sub-culture
tubes are incubated for 48 hours at 37°C.
➢ Observations are made regarding the presence or absence of growth of
microorganisms.
➢ The phenol concentration that prevents growth in both systems is
determined and the mean value is calculated. The same value would be
obtained for the unknown.
➢ For calculating the coefficient, the value obtained for phenol is divided by
the value found for the unknown.
7) Chick-Martin Test

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