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Notes prepared from Chandrakant kokare

Syllabus:

•Classification of Disinfectants

•Mode of Action of Disinfectants

•Factors influencing the Disinfection and Antiseptics

•Evaluation for bacteriostatic and bactericidal actions


Definition:
Disinfection is the process of destruction or removal of
microorganisms and reducing them to a level not harmful to health.

 Disinfectants generally kills the sensitive vegetative cells but not the
heat resistant endospores. Disinfection is less effective than
Sterilization process

 Usually disinfectants are bacteriocidal. Occasionally it may be


bacteriostatic.
Ideal properties of disinfectants:
 Broad spectrum

 Non toxic and non irritant

 Non corroding and non staining

 Fast acting and odourless

 Economical and easy to use

 Solubility and misibility

 Stabile on long term storage

 Should act as also detergents.


General terminology:
Sterilization: The process of destroying all forms of microbial life. A
sterile object means free of any living microorganism, that is
complete absence or destruction of all microorganisms.
Disinfectant: A Chemical agent that kill the growing forms but not the
resistant spores. These commonly applied in inanimate objects. So
Disinfection is the process of destroying infectious agents.
Antiseptic: These also a disinfectant, chemical agent which prevent
the growth of microorganism. But these are applied in living objects.
Sanitizer: Reduction of mic roorganism to safe level counts as
mentioned by public health requirements. Applied mainly on non
living objects.
Germicide: Disinfectant action but used for all types of germs
(Microbes)
Bactericide: Agent kills the bacteria called bactericide. Same as to
fungicide, virucide, sporicide .
General terminology continues.,
 Bacteriostasis: Agent which prevents the growth of bacteria. Same
mentioned for fungistatic, microbistatic.
Antimicrobial agent: Agent which interferes with the growth and
metabolism of microbes. For mentioning specifice type of
microorganism that is for bacteria it is antibacterial and for fungi it is
antifungal etc. These are used for treatment of infections and so
called chemotherapeutic agents.
Most resistant Prions

Endospores of bacteria

Mycobacteria

Cysts of protozoa

Vegetative protozoa

Gram negative bacteria

Fungi including most spore forms

Viruses without envelope

Gram positive bacteria

Least resistant Viruses with lipid envelope


Classification of Disinfectants
 Based on Consistency
On the basis of consistency or state of matter, they are two types,
1. Liquid (Alcohols and phenols)
2. Gaseous (Formaldehyde vapour)
Based on Spectrum of Activity
On the basis of spectrum, they are three types,
1. High level Disinfectants
2.Intermediate level Disinfectants
3. Low level
Based on Mode of Action
Along with mechanism, they are classified into,
1. Action on plasma membrane
2. Denaturing cellular proteins
3.Oxidation of essential sulfahydral groups of
enzymes (eg., H2O2, halogens)
Classification of Disinfectants continued,
4. Alkylation of amino-, carboxyl- and hydroxyl group
(eg. Formaldehyde)
5. Damaging nucleic acid (eg. Formaldehyde)

The major antimicrobial agents can be grouped as shown below,


1. Acids and alkalies
2. Halogens
3. Heavy metals and their compounds
4. Phenol and Phenolic compounds
5. Alcohols
6. Aldehydes
7. Quarternary ammonium compounds
8. Dyes
9. Detergents and soaps
Acids and alkalis
Strong acid: The germicidal activity of acids is proportional to the
Hydrogen (H+) ions dissociation in solutions. It will irreversibly
denature the proteins of bacteria. It will take very short time to kill
the bacteria. But handling is very difficult. Eg., Most mineral acids
ie., Hydrochloric acid, Sulphuric acid etc
Weak acid: Dissociation of H+ ions is limited. But it can act
differently against microorganism from strong acids. Undissociated
ions directly attack bacterial cell components. Eg., Benzoic acid,
Citric acid etc
Strong alkali: These are completely dissociated and `. These are
very good detergents because they even penetrate the spore
producing bacteria. They are non toxic and have little odour when
diluted. So they are used to clean floors, in food industry etc,
Weak alkali: Have weak dissociation. Eg. Sodium bicarbonate,
Quick lime (Anti infective agent in agri) and Borax used for control
of foot and mouth disease.
Halogens
Chlorine, Iodine, bromine and fluorine in the free state as well as their
compounds strongly act germicidal. Bromine and fluorine are irritants and
are difficult to handle. But Chlorine and Iodine are commonly used for
sterilization.
Mode of Action:
Chlorine used especially in municipal water.
Cl2 + H2O = HCl + HClO (Hypochlorous acid)
HClO = 2HCl + O2
O2 – Oxidizing agent – acts on the cell organelles
Cl2 – acts on the proteins of cell membrane

Ca(OCl)2 and Na(OCl) – used in diary and eating equipments


Chlorine compounds - used to disinfect the wounds, treat atheletes foot
and other infections
I2- Sporicidal, fungicidal and active against many viruses. As a skin
disinfectant and used for cold sterilization of surgical sutures
I2 + PVP = Iodophores – Non irritating, non staining and odourless. It’s
a oxidizing agent which inactivates essential proteins. I2 compounds used
for disinfection of water, air and sanitization of food utensils.
Heavy metals
Mercury, Silver and Copper – Heavy metals
Mode of Action – combined with cellular proteins and coagulate it.
SH S
Enzyme + HgCl2 Enzyme Hg + 2HCl
SH S

Mercury compounds are, Mercuric chloride, Mercurous chloride,


Mercuric oxide, Ammoniated mercury – these are inorganic
compounds used as bactericidal and bacteriostatic in ointment
preparations and Merthiolate, Metaphen, Mercurochrome – are
organic compounds used as bactericidal, bacteriostatic, fungicidal.
Silver compounds – Silver nitrate – Bactericidal, Antiseptic
Copper compounds – Copper sulphate – Fungicide, prevent algal
growth in lakes and pools
Phenol and its derivatives

Phenol o- Cresol m- Cresol p- Cresol

Chloro cresol Chloroxylenol o- Phenylphenol Hexachlorophane

Mode of Action: Disruption of cells, precipitation of cell protein,


inactivation of enzymes and leakage of amino acids from the cell
 Cresols are more germicidal than phenol when they are emulsified in
soaps and alkalies.
 Lysol is a commercial preparation containing cresol
 Hexylresorcinol used as solution in glycerine and water and used as
mouth gargles.
Alcohols
 These are having rapid bactericidal action against vegetative bacteria when
diluted to 60 to 70% with water
 Ethanol (60-70%) and isopropanol (50-60%) used as skin disinfectants
 Methanol vapour used as fungicide.
 The higher alcohols (Propyl, butyl etc) are more germicidal than ethanol
 Alcohols are used as preservatives in some vaccines. Are used prior to
hypodermic injections and for the disinfection of clinical oral
thermometer. Alcohol conc above 60% are effective against virus.
Mode of Action: Denaturate the proteins. Damage the lipid complexes in the
cell membrane. Also act as dehydrating agents
Eg., Methanol – CH3OH, Ethanol – CH3CH2OH, Isopropanol –
(CH3)2CHOH, n-Butanol – CH3(CH2)3OH, n-Amyl alcohol -
CH3(CH2)4OH, n-Octyl alcohol - CH3(CH2)7OH

2- Phenoxy ethanol 2- Phenylethyl alcohol Chlorobutanol


Aldehydes
 Formaldehyde (HCHO) used as a gaseous sterilization. As formalin
solution BP contains 34 to 38% w/w of CH2O for sterilization of certain
equipments
 Glutaraldehyde CHOCH2CH2CH2CHO have rapid sporicidal and
tuberculocidal action.
It is used as 2% aqueous solution, buffered with sodium bicarbonate to
pH 7.5 to 8.5. It is less toxic and less irritant to skin than formaldehyde.
In medical field, it is used for sterilizing urological, lensed and other
instruments
Quaternary ammonium compounds
 Cationic detergents
 Highly active against G+ve bacteria and some against G-ve bacteria
 Also active against fungi and some pathogenic protozoa
 They are mainly used for control of m.o. on floors, walls, nursing homes
and other public places.
 Also used as skin antiseptics and sanitizing agents in diary, egg and fish
industries
Mode of Action: Disruption of cell walls and membranes. Also inactivate
enzymes and denature proteins

Cetrimide Cetyl pyridinium


chloride
Dyes
 Basic dyes are more effective bactericidal than acidic dyes.
 Acridine and Triphenylmethane dyes are used as antimicrobial agents
 Acridine dyes are, acriflavine, proflavine, aminacrine and enflavine. They are
effective against G+ve bacteria. Used for treatment of burns, opthalmic
application and bladder irrigation
Mode of Action: Impair the DNA complex and destroy the reproduction
 Triphenylmethane or aniline dyes are brilliant green, malachite green and crystal
violet. These dyes are added to selective culture media to inhibit the growth of
G+ve bacteria and helps in isolaton of G-ve bacteria from materials
contaminated with G+ve bacteria. Crystal violet also has fungicidal action

Aminacrine hcl
Crystal Violet- X-(CH3)2N, Y-(CH3)2N, Z-(CH3)2.Cl
Brilliant green- X-(C2H5)2N, Y-No group, Z-(CH3)2.Cl
Acridine
Malachite green- X-(CH3)2N, Y-No group, Z-(CH3)2
Detergents
 They are used as surface active agents, wetting agents and emulsifiers.
 They are classified into four main groups such as anionic, cationic,
nonionic and amphoteric.
 Cationic surface active agents are the most antibacterial agents eg.
Cetrimide, benzalkonium chloride.
 Anionic surfactants are Soaps, Sodium lauryl sulfate.
 Soaps prepared from saturated fatty acids are more effective against
Gram negative bacteria
 While prepared from unsaturated fatty acids are effective against G+ve
bacteria and Neisseria group
 Non ionic detergents are not ionized and also it donot posses
antimicrobial activity
 Amphoteric compounds have properties of both anionic and cationic
detergents
Factors affecting Disinfectant action
The rate and extent of antimicrobial action of chemical disinfectants are influenced
by a number of factors,
1. Concentration of disinfectant

2. Temperature

3. Time of contact

4. pH of the environment

5. Surface tension

6. Formulation of the disinfectant

7. Chemical structure of disinfectant

8. Type and number of microorganisms present

9. Interfering substances in the environment

10. Potentiation, synergism and antagonism of disinfectants


(1) Concentration of disinfectant
 Rate of killing of microorganism is directly proportional to concentration
of the disinfectant.
 The effectiveness is generally related to the concentration exponentially
not linearly
 The dilution coeffecient can be calculated from the following equation,

 Where n is the concentration exponent or dilution coeffecient for the


disinfectant, t1 is the death time with disinfectant concentration C1 and
t2 is the death time with disinfectant concentration C2.
 Concentration exponent or dilution coeffecient is an important
characteristic of each disinfectant and is useful in determining the effect
of dilution on the disinfectant.
(2) Temperature
 Rate of disinfection increases with the temperature.
 The effect of temperature on bactericidal activity may be means of
temperature coeffecient. The temperature coeffecient per degree rise in
temperature is denoted by Ɵ. Where 10ºC rise in temperature is
expressed by Ɵ 10 or Q10 values.
Ɵ 10 or Q10 = Time required to kill at Tº
Time required to kill at (T+10)º
Ɵ (T2-T1) = t1/t2
Where T2 and T1 are two temperatures differing by 10ºC; t2 and t1 are
the corresponding lethal times.
(3) Time of contact
 Suffecient time of contact is must to exert the disinfectant action
 Principles of first order kinetics is implied for the time of contact,
K = 1/t log N0/Nt
Where, t= time for the viable count to fall from N0 to Nt
N0= initial number of microorganisms
Nt= final number of microorganisms
 The survivor / time curve may not be constant and its shape is influenced
by number of factors mainly the concentration of the disinfectant.
(4) pH of the environment

 A change of pH can affect the rate of growth of micro organism due to


change of potency and binding capacity of disinfectant.

pH of 6-8 is optimal for growth of many bacteria

Rate of growth decreases on either side of this range.

Phenolic and acidic compounds have greatest activity in acidic conditions

Acridine dyes and quartenary ammonium compounds have more activity


in alkaline conditions.

Amphoteric surface active agents (Tego compounds) have optimum


activities at widely differing pH values, depending on the number of
nitrogen groups
(5) Surface tension:
 The contact between aqueous solutions of disinfectants and structure of
microorganism is facilitated by surfactant properties.

 This helps in adsorption of disinfectant on the surface of the cells as well


as helps in wetting and spreading properties in solutions

 A combination of soap with crude phenol (Carbolic acid) has excellent


disinfectant properties

 Presence of soap reduces the extinction time of bacteria since it increases


the contact time of disinfectant with bacteria by lowering the surface
tension of the solution.
(6) Formulation of the disinfectant:
 Formulation may be important for the effectiveness

Chlorhexidine and quartenary ammonium compounds have greater


activity when mixed with 70% alcohol than in aqueous solution.

Iodine is insoluble in water and so dissolved in alcohol or pot. Iodide


solution or with solutions of surfactants.

Presence of surfactants in iodine solution will moderate the staining and


corrosive properties and increase the stability of preparation

For convenient and economic purpose, the formulation of disinfectant


always be concentrated and it is diluted with water at the time of use.
(7) Chemical structure of the disinfectant:
 Chemical structure may change the disinfectant activity.
Substitution of alkyl chain in para position to phenolic OH group
increases the activity but greater than 6 carbon chains addition will
decreases water solubility and obviously disinfectant.
Halogenation increases the activity of phenol but nitration increases
activity and systematic toxicity also.
(8) Type and number of microorganism present:
 Disinfectant efficiency depends on the nature and number of
contaminating m.o. and presence or absence of spores.
 Most vegetative bacteria except Acid fast bacilli are easily killed by most
chemical disinfectants.
 Bacterial spores are difficult to destroy but aldehydes are sporicidal.
 Aldehydes with Halogens and Beta propiolactones are virucidal.
Acid fast bacilli are resistant to many disinfectants but are susceptible to
iodine, formaldehyde, alcohol and phenolic compounds.
(9) Interfering substances in the environment:
 Material such as blood, body fluids, pus, milk, food residues or colloidal
proteins may reduce the effectiveness of disinfectant.
 This is due to adsorption or shielding of micro organism or interaction
with disinfectants.
Presence of oils and fats reduce the activity of phenolics.
(10) Potentiation, synergism and antagonism of disinfectants:
 Potentiation – enhanced antimicrobial activity by addition of any
excipients. Eg., Polysorbate 80 addition with disinfectants
 Synergism – enhanced antimicrobial activity by addition of two
antimicrobial or disinfectants Eg., Different esters of p-hydroxybenzoate
are available, when combined together exhibit synergistic activity
Antagonism – Decreased antimicrobial activity by addition of any
excipients Eg., Sodium thiosulphate, lubrol, W+lecithin etc.
Factors affecting choice of Antimicrobial agent
Selection depends upon
1. Properties of chemical agents
2. Environment
3. Types of microorganism
4. Intended application
5. Toxicity of agents
6. Cultural state

 Properties of Chemical agents: The rate and extent of killing m.o. is


influenced by the concentration, solubility, pH ie., properties of
chemical agent
 Environment: The substance from the infected environment like
blood, pus, colloidal proteins, milk, dried organic deposit will interfere
and reduce the effectiveness of the antimicrobial agent.
 Types and count of microorganism: Antimicrobial activity is
influenced by types and count of m.o. (bio burden). Long exposure and
higher concentration is needed to kill the bio burden. Antimicrobial
agents donot have equal action against all m.o. It can kill easily the
vegetative cell of bacteria but not the spores, mycobacterium.
 Intended application: Sometimes the intended application of
antimicrobial agents ie., preservatives, antiseptic, disinfectant will give
adverse effects eg., agent which is used for kill the m.o. also corrode
the equipments, may change the texture of polymers
 Toxicity of agent: COSHH – Control of Substances Hazardous to
Health regulations specify precaution while handling toxic agents eg.,
Phenols, formaldehyde and glutaraldehyde. Toxic volatile substances
are kept in covered containers to reduce irritation. Alcohols, chlorine
compounds and phenolics mainly affect the eyes and skin. Aldehyde
affect the respiratory system.
 Cultural state: M.O. are more sensitive at the log phase of growth,
because during replication of nucleic acid and protein synthesis they
may easily interfered by chemical agents.
EVALUATION OF ANTIMICROBIAL AGENTS AND
DISINFECTANTS (Bactericidal and Bacteriostatic activity)

1. Tube dilution and agar plate method

2. Filter paper and cup plate method

3. Ditch – plate method

4. Phenol coeffecient method

5. Kelsey Skyes method


1. Tube dilution and agar plate method:
 The anti-microbial agent + nutrient broth/ agar medium + test
m.o. Incubated at 30-35 ºC for 2 to 3 days
 Turbidity or colonies are observed and the results are observed
2. Filter paper, Cup-plate and Cylinder plate method:
 The agar is melted and cooled. Inoculated with test m.o. and
poured in sterile petri-plate.
 Make holes about 9 mm in dia made by a sterile cork borer.
 Different antimicrobial agent is directly placed in the holes by
using filter paper disc and cylinder respectively.
 The zone of inhibition is observed after incubation at 30-35 ºC
for 2 to 3 days.The diameter of zone of inhibition gives an
indication of action of different antimicrobial agent


3. Ditch –plate method:
 A solution of antimicrobial substance is carefully run into the
ditch which is prepared in an agar plate.
 A loopful of eact test m.o. is then streaked outwards from the
ditch on the agar surface.
 M.O. resistant to the antimicrobial grow right upto the ditch
whereas sensitive m.o. shows a zone of inhibition adjacent to
the ditch or centre of plate.
 Width of inhibition indicates the Antimicrobial action
4. Phenol coefficient method:
 Various disinfectants are tested for its microbicidal property and
they are compared with phenol activity as reference.
 The same quantities of micro organisms are added to the
different dilutions of phenol and disinfectants
 In UK, Sal. typhi and in USA, Sal.typhi, Staph. Aureus and
Pseud. aeruginosa are used as test micro organisms
 The pheno coeffecient test includes,
1. Rideal – Walker test (RW test)
2. Chick – Martin test (CM test)
3. USFDA test
4. The US Association of Official Agricultural Chemists test
(AOAC test)
 These tests is only suitable for testing water soluble
disinfectants and exerts the microbicidal action as phenol
 In Rideal – Walker test , Rideal – Walker broth (Meat extract, Peptone,
Sodium chloride and Water) is used and Salmonella typhi as sensitive micro
organism.
 Different dilutions of test disinfectants and phenol are prepared and 5ml of
each dilution is inoculated with 0.5ml of 24 hrs broth culture of organisms.
 All tubes are placed in water bath (20 ºC). Subcultures of each reaction
mixture are taken and transferred to 5 ml sterile broth after 2.5, 5, 7.5 and 10
min
 The broth tubes are incubated at 37 ºC for 48 to 72 hours and examined for
the presence or absence of growth.
 The Rideal – Walker coeffecient of the test disinfectant is then calculated as
follows,
 If a phenol coeffecient or ridal Walkar coeffecient is 1,
then the test disinfectant has the same effectiveness and
if it is less than 1 it means less effective and if it is more
than 1 it means more effective than phenol.
Advantages of Phenol Coeffecient test:
1. Inexpensive and can be performed quickly
2. Have reproducible results
3. They are helpful to eliminate to useless products
Disadvantages of Phenol Coeffecient test:
1. Have only limited information (only one M.O. – S.typhi)
2. Activity of bactericides done at only one concentration with
fixed death time and reaction temperatures
3. In presence of organic matter, the test has no indication of
activity of disinfectants
4. No information about toxicity
5. Sampling errors are large
5. Kelsey – Sykes method (1969):
 In this method, several test bacteria are used. They are, Staph.
Aureus, Proteus vulgaris, E. coli and Pseud. aeruginosa.
 The final conc. of bacterial cells is about 109/ml. Basic procedure is
outlined in below table.
 The samples taken at 8, 18 and 28 minutes are then incubated at 30
to 32 °C and the number of tubes showing growth or the number of
colonies from surface plate culture is recorded.
 Result:
 No growth occurs in 2 or more of 5 tubes of 18 min samples ie.,
subcultures taken after second incremental addition of bacteria or
 There are not more than 5 colonies from the 5 drops on the agar
plate.