Disinfection For PH 2

Disinfection, Preservatives &
Antiseptics
Learning Objectives:
At the end of the course, the student should:
- Understand the basic principles of disinfection

- Describe disinfectant groups and
- Know their MOA
- Factors affecting disinfection
- Dynamics of disinfection
- Evaluation of disinfectant activity
- Define terms associated with disinfection.
References:
W. B. Hugo and A. D. Russell, Pharmaceutical

Microbiology.
Cooper and Gunns Tutorial Pharmacy.
Michael E. Aulton, Aultons Pharmaceutics, The

design and Manufacture of Medicines.
www........
Define the ff terms:
Antiseptic Fungistat
Antisepsis Germicide
Bactericide Preservation
Bacteriostat Preservative
Sporicide
Biocide
Sporostat
Disinfectant Viricide
Disinfection
Fungicide
Disinfection, Antisepses and Preservation
Disinfection
Removing microorganisms, including potentially
pathogenic ones from the surface of
inanimate objects.
Does not necessarily kill all microorganisms but

reduces them to a level acceptable for a
defined purpose.
5
Classes of chemical disinfectants:
High-level disinfectants (sterilants) kill all organisms,

except high levels of bacterial spores.
Intermediate-level disinfectants (tuberculocides) kill all

vegetative bacteria, including mycobacterium
tuberculosis, but may exclude some fungi and viruses
and spores.
Low-level disinfectants (hospital disinfectants) destroy

most vegetative bacteria, fungi and viruses, but not
spores or some resistant microorganisms.
Factors affecting choice of agent for use as

disinfectant
Properties of the chemical agent: such as

concentration, temperature, pH and formulation.
Microbial challenge: The type of microorganisms

present and the degree of contamination
(bioburden).
bioburden = long exposure time
concentrations.
Vegetative bacteria, mycobacterium spp, spores,
fungi and viruses have different susceptibilities.
Factors contnd
Intended application: Active constituent should

not break down the preservative or vice versa.
Disinfection of instruments, the agent must
not corrode, affect clarity, texture or integrity.
Environmental factors: Organic matter (blood,

pus, milk, etc) and ions affect antimicrobial
activity by adsorption or inactivation.
Toxicity of the agent: Potentially toxic agents

like phenolics, formaldehyde and toxic volatile
substances should be stored and used with
care due to their side effects.
Groupings
Use: Iodine
Air Phenols
Surface Alcohols
Air: Aldehydes
Alcohols Hydroxy acids & their
Halogens Esters
Surface active agents
Surface: Dyes
Acids & Alkalis Metals & their salts
Halogens & Chlorhexidine
derivatives
Disinfectant groups
Air disinfectants
Disinfect microorganisms suspended in the air.
Dispersed as vapour at sufficient concentrations in the
air to act.
E.g. dilute bleach and bithionol in theatres and burn
units.
Challenges:
constant interaction between sterilised and
unsterilized air (exchange at doors, windows, etc)
Presence of materials that can absorb and
subsequently remove the disinfectants from the air.
SURFACE DISINFECTANTS
ACIDS AND ALKALIS
Strong Acids:
Dissociate completely, producing H+ that have a very
strong affinity for water.
MOA: Irreversible denaturation of proteins /
withdrawal of water from the bacteria.
Challenge: Safety of handling them.
Weak Acids
Dissociate partially and produce few H+.
Uses: Preservation (e.g. benzoic, acetic, sorbic, etc),
treatment of skin conditions.
Challenge: Rapid decrease in activity above pH 3
Strong Alkalis
Dissociate completely to give OH-, which has a strong
affinity for water.
MOA: ??? An increase in the external
pH renders the cell surface more negatively charged. Biocidal agents that are cationic
in nature thus bind more strongly to the cell surface with a consequent increase in
activity.
Advantages:
Non-toxic when adequately diluted
Leave no residual odour.
Their metal hydroxides are good detergents and
penetrate well. E.g: NaOH and trisodium phosphate.
(Find uses)
Challenge: Corrosive and must be handled with care.
Weak Alkalis
Dissociate partially in aqueous solutions.
Some are salts of a strong alkali and a weak acid.
(e.g. NaHCO3)
MOA: ???
Uses:
- Quicklime - anti-infective agent in Agriculture
- Sodium Borate - foot and mouth disease
In health?????
HALOGENS AND THEIR DERIVATIVES

Chlorine
Chlorine gas combines with water to form
hypochlorous acid.
MOA: Denaturation of proteins in the cell
membrane
Hypochlorites
Prepared by electrolysis of brine (Large scale)
pH 9, by dilution, with in activity.
Dilute solutions: Personal/Sterilisation in the
home, Less stable (stored carefully after
opening)
Chloramines
Inorganic chloramines are made from chlorine and
ammonia. Monochloramine not very active so dichloro
derivatives (more effective) are made.
NH4+ + HOCI = NH2C1 + H2O + H+
NH2Cl + HOCI = NHCl2 + H2O
The reactions are reversible and the chloramines form
reservoirs of chlorine. Less active than hypochlorous
acid. Superchlorination (addition of excess chlorine -50
ppm) activity.
Uses: Emergency sterilisation of water. Very effective but a
dechlorinating agent, e.g sodium thiosulphate, is added
to make the water palatable.
Organically-combined Chlorine
Chlorine combines with certain nitrogen-
containing chemicals to produce compounds
that provide a depot of chlorine for sustained
antibacterial activity.
Unlike the inorganic chloramines they are stable

in the dry state.
e.g:
Chloramine T and Dichloramine T
Factors Affecting the Action of Chlorine

Compounds
Concentration: Activity with concentration.
pH: activity is highest b/n pH 8-10
Temperature: The T coefficient is about 2 per

10C rise over the range 20-50C. The chemical
stability of chlorine solutions is good within this
range.
Advantages of Chlorine Compounds

Almost odourless at effective concentrations (water
and food sterilisation).
Certain organic forms provide a depot effect.
They are non-irritant to tissues.
Low toxicity of dilute solutions (dairy and feeding
bottles).
They have cleaning and deodorising properties that
are useful in wound treatment.
Cheaper - low concentrations are active (0.05ppm).

Disadvantages of Chlorine Compounds

They do not penetrate organic matter or
bacterial cells, particularly those of fatty
species such as mycobacteria.
Solutions are corrosive to metals, textiles and
biological tissues.
Chlorine combines with organic matter and
this can result in loss of activity.
Chlorine can be tasted at low concentrations
0.04ppm (unpalatable)
IODINE
One of the best bactericides for the skin. Free
iodine is the active constituent of iodine
preparations.
Insoluble in water Preparation???
Most individuals tolerate iodine well.
Iodophores are less toxic.
Iodophores
Iodine combined with complex organic chemicals
e.g polyvinylpyrrolidone (PVP).
Activity of iodophores are equivalent to iodine-
potassium iodide solutions with the same iodine
content.
Advantages
Low toxicity
Well tolerated
Skin stains removable (soap & water).
Low conc. (1% iodine) - Sporicidal.
They are compatible with soaps.

PHENOLS
Phenol (carbolic acid) - Used extensively and serves as
standard for other bactericides in some official tests.
Active against vegetative cells but not spores.
MOA: Protein denaturation
Enters cytoplasmic membrane, disrupts its structure and
functions
OH- form a hydrogen bond with a protein receptor of
enzyme system - the rest of the phenol molecule
constitutes a physical barrier to attachment of the
normal substrate.
Challenge: Causes necrosis of tissues.
Bisphenols and Diphenyls

Derivatives of Phenol.
Halogenation activity against fungi, Gram
ve and gram +ve organisms.
Challenge
Poor solubility of diphenyls
Adv
Low toxicity
Residual antimicrobial activity
Formulations containing phenols

Reason? To activity
Use - Household disinfection
Cresol with Soap (Lysol BP)
Cresol BP - Fractional distillation of coal tar.
Lysol effective against vegetative organisms
Bactericidal and detergent properties.
Challenge: Tissue necrosis
Skin irritant (5-10% solutions)
Solution? = Sudol but it is less specific in action.
Black Fluids
Contain the higher boiling fractions of tar acids.
Chlorinated derivatives added to activity.
Non-selective and non-toxic when diluted.
Challenge: Strong odours / Stain linen.
Uses Disinfection of drains.
White Fluids
Emulsions from more refined tar acids
Uses - disinfect linen / wound treatment
Preparations of Chlorinated Phenols

Most are formulations of chloroxylenol in castor
oil soap.
Terpineol is added as carrier of the phenol and
to provide a pleasant odour.
Low toxicity / pleasant odour / persistent
activity
Uses - disinfectant in homes / Obs & Gynae
Soaps containing Phenolic Compounds
Phenols formulated in soaps have little activity.
ALCOHOLS
Non-corrosive, but a fire hazard.
activity in the presence of organic material.
Not sporicidal
Disinfecting ability with in the length of

the carbon chain.
Solubility with in the carbon chain.

Alcohols:
MOA: Denaturation of protein
Acts similar to Phenols.
Uses - disinfectant/antiseptic/preservative
depending on the concentration
Only ethyl and isopropyl are used due to

toxicity and solubility
Alcohols
Most effective when combined with purified
water (aids diffusion through cell membrane)
80% ethanol (C2H5OH) + 5% isopropanol
(C3H9O)
- inactivate lipid-enveloped viruses (HIV,
hepatitis B and C).
70% ethyl alcohol kills most rapidly
It is bactericidal.
Chlorination of Alcohols
e.g. Chlorbutol (chlorbutanol)
Chlorination activity (0.5% is active)
Disadvantages:
Strong camphor-like odour.
Hydrolyses in solution (heat sterilisation & low
pH)
Low solubility in water around room TC
Volatile in steam.
Phenyl and Aryl substituted Alcohols
Phenyl-ethyl alcohol 0.5% - preservative for

eye drops.
Benzyl alcohol 0.9% - preservative for

injections
Chlorine derivatives of benzyl alcohol -

disinfectants, mouthwashes and gargles.
Glycols
Uses?
Glycerol
Antibacterial agent above 50% concentration
lower concentrations some organisms can use it
as an energy source.
ALDEHYDES
Formaldehyde
Formalin B.P. (40% formaldehyde gas in water)
Uses: fumigant for disinfecting rooms
Preservative of pathological specimens.
MOA: Reducing agent on protein and protein-

like materials
Glutaraldehyde:
Students should read on it.
HYDROXY-ACIDS AND THEIR ESTERS

Lactic acid
Uses:Preservative in food (e.g. yoghourt).
Para-hydroxy-benzoic acid
Methyl, ethyl, propyl and butyl esters
Uses: Antifungal preservatives
Challenge: ???
Cationic agents
Bactericidal but not very good detergents
Also known as detergent sanitisers.
The most satisfactory bactericides are the

quaternary ammonium compounds e.g
Cetrimide benzalkonium chloride

and domiphen bromide.
They are very soluble in water.
Uses:
Disinfectants - dairy, food and brewing
Preservative - certain eye drops.
Challenge:
activity in presence of organic matter &
polysorbate 80
Shd not be used as wet dressings due to sensitisation
Haemolytic (Not suitable as preservatives for
injections
Adv:
Strongly adsorbed on to surfaces = Residual activity
DYES
Derivatives of
Acridine, pararosaniline and quinoline are of
interest
Acridines
MOA: DNA intercalation disrupts genetic
code, interferes with protein formation and
DNA replication.
Uses: Disinfectant
Obs & Gynae, gas gangrene
Adv: does not stain

effective at low concentrations
Challenge: Toxic to leucocytes

Retard healing process (wounds)
Pararosaniline (triphenylmethane) dyes
E.gs: Malachite green / Brilliant green

Active against bacteria and fungi
MOA: Change oxidation-reduction potential of

the organism.
Uses:
Paints for application to fungal infections e.g
ringworm.
METALS, THEIR SALTS AND ORGANIC DERIVATIVES

Zinc, Gold, silver, Al, and copper, have antimicrobial
activity (oligodynamic) effect.
Mercury - Bacteriostatic
Challenge: High toxicity
MOA: Combines with proteins, polypeptides and
some enzymes which prevents their utilisation by
the organism.
Uses: Preservatives (injections / eye drops / creams
Antiseptic - Topical applications
Treatment of fungal infections of the skin.
Salts of Zn, Cu, and Al Astringents / antiseptics.
CHLORHEXIDINE
Bactericide from guanidine compounds. It is
used as its salts.
Soluble in water, ethyl alcohol and propylene
glycol.
Non-toxic
Active against pathogenic fungi
Not sporicidal / virucidal
Uses:
Antiseptic: Before surgery / cleaning wounds
Preservative - eye drops (acetate)
DISINFECTANT GROUPS - recap

Phenols
Alcohols
Aldehydes
Biguanides
Surface active agent
Halogens
Heavy metals
Dyes
Acids and Esters
Hydrogen peroxide: Hydrogen peroxide
Peracetic Acid: Peracetic acid.
Diamidines: Propamidine, Dibromopropamidine.
Quinoline derivatives: Dequalinium, Laurolinium.
MODES OF ACTION
Different disinfectants have different MOA
All disinfectants act by harming microorganisms
in one way or another.
The modes of action include:
Protein denaturation
Membrane disruption
Nucleic acid damage
Inhibition of metabolism
PROPERTIES OF AN IDEAL DISINFECTANT

Fast acting even in the presence of organic
substances
Effective against all types of infectious agents.
Offer complete and full sterilisation without
harming other forms of life.
Inexpensive and non-corrosive.
Non-toxic to humans or animals.
Break down quickly into harmless components.
PROPERTIES OF AN IDEAL DISINFECTANT cont.

Easily penetrate the material being
disinfected without damaging or discolouring.
Should be stable; even when exposed to light,
heat and other environmental factors.
Should be easy to prepare, obtain and use.
Should not have an unpleasant odour.
NOTE: There is no IDEAL disinfectant!!!
DYNAMICS OF DISINFECTION
Introduction:
Changes in the population of viable bacteria in
an environment determined by a viable count,
and a plot of this count against time gives a
dynamic picture of pattern of change.
death/mortality curve which usually follows the

principles of first order kinetics
The rate or velocity constant K of disinfection
can be measured by
K = 1/t log (B/b)
T = time for the viable count to fall from B to b

B = initial number of organisms
b = final number of organisms
If the rate of kill is fast, the plot would be a straight
line. If the concentration was lower, resulting in a
slower rate of kill, an s-shaped (sigmoid) graph would
be obtained (B)
What is happening in C? 49
FACTORS AFFECTING DISINFECTANT ACTION
Factors connected with the Organism
Species and strain nature, resistance, gram +/-
Morphological state spores, slime layers,

capsules, water-repellent layers
Cultural State (metabolic state)

lag/log/decline phase
Previous history growth in thiol containing

media leads to the inactivation of Hg2+
Factors connected with the Reaction Medium
Nature of the medium aqueous soln are more

active than organic or oily solvent
Inoculum size Any suggestions???
Concentration of the bactericide

The concentration coefficient is expressed as
= logt2-logt1
logc1-logc2
Where t1 and t2 are the times taken to produce a given

reduction in survivors with antibacterial concentrations
c1 and c2 respectively
In SIMPLER terms
Concentration coefficient is expressed as
Cn x t = K
Where
C=[]
n = [ ] exponent or dilution coefficient
t = death time
K =???
Questions?
If 5% w/v concentration of a bactericide of
dilution coefficient of 3 takes 25 minutes to
effect killing, what will be the time of kill when
it is diluted 8 times.
A sanitizer with dilution coefficient of 0.5

takes 3 minutes to effect killing. If it takes 18
minutes to kill when it is diluted 5times, what
is the initial concentration?
Factors connected with the Organism and the
Reaction Medium
1. Temperature: ToC efficacy of most
disinfectants.
The temperature coefficient, , is expressed as
(T2-T1) = k2
k1
k1 and k2 = death rate constants (reaction rate)
at T1 and T2 respectively.
This relationship holds true for temperatures
between 15oC and 45oC.
An arithmetic in temperature = geometric
in antimicrobial action of disinfectant.
As temp , Extinction time ( time at which no

living cell can be detected in sample taken)
56
Factors connected with the Organism and the Reaction Medium
2. pH:
a. Rate of growth of the microorganisms
optimum pH for bacterial growth = 6 to 8.
b. Degree of ionization of antimicrobial substance

phenol, salicylic acid, benzoic acid, = active when
unionized, acridine compounds active when ionized
c. adsorption of the antimicrobial substance at the cell

surface.
GV paint, brilliant green and cationic surfactants have
increasing antimicrobial activity with increasing pH
3. Reaction between bactericide and constituents of the
reaction medium:
a. Chemical: eg for thiol containing compounds and Hg2+
b. Physico-chemical: for biphasic preps, adsorption or

partition can occur
c. Biochemical: specific metabolic pathways

egs???
4. Oxidation Reduction potential:

Affects agents that act by oxidation or reduction. Egs???
OCl-, KMNO4
5. Surface effects
Surfactants the surface tension and penetration of
disinfectants into bacteria
Anionic surfactant in phenol solution the surface

tension of the phenol, extinction time
Alcohol formulations penetration power, activity
Some solubilizing agents or emulsifying agents affect the

activity of phenol (/ )
Other Factors
Time of contact: % survivor vrs time follows
the principles of 1st order kinetics
K =1/t log (B/b)
60
Chemical structure of the disinfectant:
Eg. For phenols;
Alkyl chains 6C in length activity, alkyl groups >6C
water solubility and activity
Straight alkyl chains activity than branched chains
Halogenation activity
In both alkyl and halogen substitution, activity is

obtained with the alkyl group in the ortho position to
the OH group, and the halogen in the para position to
the phenolic group.
Nitration (NO3) antibacterial activity, systemic

toxicity.
61
Organic matter:
Protects microbes. HOW?? Any examples???
Age of the disinfectant: Freshly prepared disinfectants

are more potent than older ones
Surface microtopography:
Synergism and antagonism of disinfectants:
Water hardness:
Relative humidity:
Application method:
Compatibility: some surfaces are incompatible with
certain types of disinfectants
62
EVALUATION OF DISINFECTANTS
EVALUATION OF DISINFECTANTS
Phenol coefficient:
The disinfectant to be tested is compared with
phenol on a standard microorganism, usually
Salmonella typhi or Staphylococcus aureus.
Disinfectant must belong to the phenol group.
Rideal-Walker test:
Chick-Martin test:
Filter paper method

Use-dilution test
Kelsey-Sykes test: (Quarternary Ammonium compounds)
ANTIMICROBIAL
PRESERVATIVES
FUNCTION:
Prevent growth of unwanted microorganisms in
a product without introducing unwanted
effects.
FACTORS THAT AFFECT THEIR CHOICE

The types of microorganisms likely to be
present and the effects they may have on the
product:
The nature of medicament:

Factors that affect choice cont..
The nature of adjuncts:
The properties of the preservatives:
The nature of the container:
The climate and microbial flora of the country

to which the product is being exported:
Some Preservatives used in official Preparations
Benzoic acid / solution 0.1
Salicylic acid 4.0
Sorbic acid 0.2
Sodium benzoate 0.1 0.2
Benzyl alcohol 0.9
Chlorbutol 0.5
Ethyl alcohol 15.0
Glycerol 50.0
Cresol 0.3 0.5
Phenol 0.5
Chloroform 0.02
Sucrose 6.5
Tablets See Lecture notes
Mixtures/Creams
FACTORS THAT AFFECT PRESERVATION

Moisture: Powder and tablets, zinc chloride,
digitalis and ergot must be preserved in air
tight containers with dehydrating agents.
Light: Direct sun light could destroy active
components / change colour. Store in amber
or dark coloured bottles and at dark places.
Temperature: Biological products, antibiotics,
vaccines and insulin must be kept in
refrigerators (2 - 15C) to prevent
decomposition.
Air:
O2, CO2 and moisture may cause deterioration of
Caffeine, codeine sulphate, Castor oil,
cinnamon and ferrous sulphate.
Age:
Many tinctures and fluid extracts deteriorate
with age in spite of best preservations.
Biological drugs and antibiotics also lose their
potency with time. There is the need for
constant watch on the expiry dates of these
products.
Preparation procedures:
Drugs collection Drying
Washing Cleaning
Cutting Grinding
Boiling Frying
Chopping Pounding
Distilling, etc. should be carried out
properly.
Storage of raw materials and equipment
Store house: Should be constructed with good

material and well ventilated.
Each container should be labelled, with name

and quantity of the drug, date of storage,
place of collection, etc.
Stored drugs should be dried regularly by

exposing them to the sun.
Strong smelling drugs, such as Asafoetida,

Camphor, etc. should be kept isolated in
tightly closed bottles, so that the odours may
not contaminate other drugs.
PRESERVATIVE EFFICACY TESTS

Challenge tests These are tests applied to the
formulated product in its final container to
determine whether it is adequately protected
against microbial spoilage.
Students should read the details of these tests.

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Disinfection, Preservatives &

- Understand the basic principles of disinfection

W. B. Hugo and A. D. Russell, Pharmaceutical

Cooper and Gunns Tutorial Pharmacy.

Michael E. Aulton, Aultons Pharmaceutics, The

Does not necessarily kill all microorganisms but

High-level disinfectants (sterilants) kill all organisms,

Intermediate-level disinfectants (tuberculocides) kill all

Low-level disinfectants (hospital disinfectants) destroy

Factors affecting choice of agent for use as

Properties of the chemical agent: such as

Microbial challenge: The type of microorganisms

Intended application: Active constituent should

Environmental factors: Organic matter (blood,

Toxicity of the agent: Potentially toxic agents

HALOGENS AND THEIR DERIVATIVES

Unlike the inorganic chloramines they are stable

Factors Affecting the Action of Chlorine

Concentration: Activity with concentration.

pH: activity is highest b/n pH 8-10

Temperature: The T coefficient is about 2 per

Advantages of Chlorine Compounds

Cheaper - low concentrations are active (0.05ppm).

Disadvantages of Chlorine Compounds

Skin stains removable (soap & water).

Low conc. (1% iodine) - Sporicidal.

They are compatible with soaps.

Bisphenols and Diphenyls

Formulations containing phenols

Preparations of Chlorinated Phenols

activity in the presence of organic material.

Disinfecting ability with in the length of

Solubility with in the carbon chain.

Only ethyl and isopropyl are used due to

Phenyl and Aryl substituted Alcohols

Phenyl-ethyl alcohol 0.5% - preservative for

Benzyl alcohol 0.9% - preservative for

Chlorine derivatives of benzyl alcohol -

MOA: Reducing agent on protein and protein-

HYDROXY-ACIDS AND THEIR ESTERS

The most satisfactory bactericides are the

Cetrimide benzalkonium chloride

Adv: does not stain

Challenge: Toxic to leucocytes

Pararosaniline (triphenylmethane) dyes

E.gs: Malachite green / Brilliant green

MOA: Change oxidation-reduction potential of

METALS, THEIR SALTS AND ORGANIC DERIVATIVES

DISINFECTANT GROUPS - recap

PROPERTIES OF AN IDEAL DISINFECTANT

PROPERTIES OF AN IDEAL DISINFECTANT cont.

death/mortality curve which usually follows the

K = 1/t log (B/b)

T = time for the viable count to fall from B to b

Morphological state spores, slime layers,

Cultural State (metabolic state)

Previous history growth in thiol containing

Nature of the medium aqueous soln are more

Inoculum size Any suggestions???

Concentration of the bactericide

Where t1 and t2 are the times taken to produce a given

A sanitizer with dilution coefficient of 0.5

As temp , Extinction time ( time at which no