Disinfectants-A History Over view

The earliest recorded example of chemical disinfection is the use of

copper or silver vessels, instead of pottery ones, to store drinking water
to prevent it becoming foul
This innovation was introduced about 450 BC by the Persians
Both copper and silver have significant antimicrobial activity, although
neither is much used for disinfection purposes today because of their
toxicity
Other ancient disinfectants, used mainly for topical treatment of
wounds were wine, vinegar and honey, while wine and honey now tend
to be used internally, vinegar, or rather its active ingredient, dilute
acetic acid, has been revived as a wound dressing where antibiotic
resistant Pseudomonas bacteria are a problem
 Disinfectants-A History Over view

Mercuric chloride was introduced as a wound dressing in the Middle

Ages by the Arabs, but it was not until the 18th and 19th centuries that
great strides forward in chemical disinfection were made with the
introduction of a range of chemicals such as copper sulphate (1767),
bleaching powder (1798), creosote (literally, 'flesh-saviour' from the
Greek; 1836), iodine (1839), chlorine water (1843) and phenol (1860)

Today, some of these are still used for some disinfection purposes, and
there is a large array of more modern chemicals
 CONTROL OF MICROBIAL GROWTH

Terms Related to destruction of microorganisms

Sterilization – killing of all microbial life
Disinfection – destruction of vegetative (non-spore forming) pathogens
mostly by chemical – disinfectants
Antisepsis – treatment of living tissue to kill pathogens - antiseptic

Antiseptic Disinfectant
 Disinfection is a complex rate process which is dependent upon:
1) physico - chemistry of the disinfectant
2) cellular - chemical nature and physical state of pathogens
3) physical and chemical factors like
a) temperature
b) pH
c) electrolytes
d) interfering substances
Four major pathogen groups in decreasing order of their
resistance to disinfection is:
1) Bacterial Spores or Cysts
2) Protozoan Spores
3) Viruses
4) Vegetative Bacteria.
 CONDITIONS INFLUENCING
MICROBIAL CONTROL

TEMPERATURE
low, inhibits growth
high, promotes disinfectant activity

TYPE OF MICROORGANISM
Gram positive generally more sensitive to
disinfection
Pseudomonas spp. resistant to disinfection
 CONDITIONS INFLUENCING
MICROBIAL CONTROL

PHYSIOLOGICAL STATE OF THE MICROORGANISM

Actively growing organisms are more susceptible to disinfection

ENVIRONMENTT
Organic matter protects against chemical
or physical inactivation
 PHYSICAL METHODS OF DISINFECTION

-Desiccation -Osmotic pressure - Filtration-Freezing –

Radiation - Heat

Desiccation
Very variable effect on microbes, many can survive extended
times (months), some endospore for centuries, others die in
minutes
Great significance for hospital & nursing home microbiology,
bedding, dust, clothing contaminated with mucus, urine, feces
or pus
Osmotic Pressure
High salt or sugar content (hypertonic conditions)
 PHYSICAL METHODS-CONTINUES

• Sterile Filtration
– For heat sensitive solutions/liquids
– Cellulose esters or plastic polymers (pore size from
0.45 – 0.01 µm)
– High Efficiency Particulate Air Filters (HEPA)filters
(pore size 0.3µm)
• Cooling/Freezing
– Refrigeration –(0-7oC) only bacteriostatic
– Slow freezing kills many pathogens and parasites
 PHYSICAL METHODS- CONTINUES

• Radiation
– Ionizing Radiation (<1 nm, high energy) gamma rays, X-rays, high
energy electron beam
• Creation of hydroxyl radicals from water, DNA damage
• Certain foods, pharmaceuticals, surgical supplies, plastic syringes,
etc.
– Non-Ionizing Radiation (>1nm, lower energy) UV light, DNA damage
(260 nm Thymine dimers)
 PHYSICAL METHODS- CONTINUES
UV Disinfection
optimum germicidal λ = 250- 270 nm
Typically Hg vapor (output is 253.7 nm).
Physical method: radiation absorbed by DNA and RNA, death and
inability to replicate
First order process.
Time and distance dependent.
Nature of liquid, competing reactions & turbidity affect.
UV RADIATION
 PHYSICAL METHODS - CONTINUES
HEAT
MOIST HEAT
• Boiling (100o C)
– Kills nonspore-forming bacteria, viruses, mold and fungi
• Pressurized (121o C)
– All organisms (but prions)

Flaming/Hot air sterilization, 170o C, 2 hours

Heat – destruction of proteins
Autoclave – 121oC 1 atm pressure for 15 min will kill all
organisms ad their endospores
Pasteurization – milk, yogurt, beer, etc. brief heating kills most
spoiling microbes without altering taste (milk 72oC 30 s)
UHT – ultra high temperature treatment (coffee creamer, etc. need
no refrigeration (140 oC ca. 2 s)
 PHYSICAL METHODS - CONTINUES

DRY HEAT
Sterilizing Oven

This Class 100 Sterilization Oven is Used

for the depyrogenation of glass,
teflon and other heat resistant materials
 MECHANISM OF ACTION OF CHEMICAL
AGENTTS

• Reactions that affect proteins (DNA)

• Reactions that affect cell membranes
• Reactions that affect other cell components

Alteration or denaturation of the protein structure occurs when

the hydrogen and disulfide bonds are disrupted, resulting in
alteration of the functional shape.

This prevents the normal function of the protein to occur.

Permanent denaturation will kill an organism while temporary

denaturation only weakens and not kill the organism.

Reactions which denature proteins include the following:

 MECHANISM OF ACTION OF CHEMICAL
AGENTTS
Hydrolysis breaks down a molecule by the addition of water
with acids or strong alkalis.
Oxidation is the addition of a oxygen atom to or the removal of a
hydrogen atom from a molecule. Oxidizing agents are
electron acceptors, examples are hydrogen peroxide or potassium
permanganate by oxidizing the disulfide linkages or sulfhydryl
groups. Sometimes compounds containing halogens (chlorine,
fluorine, bromine and iodine) which can also act as oxidizing agents
Heavy metals (silver and mercury) attach to sulfhydryl groups.
Alkylating agents contain a methyl group (CH3) and donate these
groups to proteins, such as formaldehyde will.
Halogens (chlorine, bromine, iodine) can replace the hydrogen
atom in the carboxyl (COOH), sulfhydryl (SH), amino (NH2) and
alcohol (OH) groups.
 MECHANISM OF ACTION OF CHEMICAL
AGENTTS
Reactions can affect the cell membrane of the microorganisms
because not only do the cell membranes contain protein but also
lipids which can be dissolved and thus disrupt the cell membranes.

Surfactants are substances which reduce the surface tension. These

substances include alcohols, detergents and quaternary ammonium
compounds.

Phenolics are one type of alcohol which denatures proteins but also
dissolves lipids.

Wetting agents and detergent solutions will not kill microorganisms

but help to dissolve lipids so that other agents can get at them.
 MECHANISM OF ACTION OF CHEMICAL
AGENTTS

There are other components such as nucleic acids and energy

capturing systems of microorganisms which can be attacked by
chemicals.

Alkylating agents can replace a hydrogen, amino or alcohol group

in nucleic acids of the microorganisms.

Lactic acid and propionic acid are end products of fermentation

and thus prevent energy capture in certain bacteria.
 TYPES OF CHEMICAL DISINFECTANTS

• Heavy metals
• Alcohols
• Phenol and phenolics
• Quaternary ammonium
compounds
• Soaps
• Surfactants
• Chlorhexidine
• Organic acids
• Halogens
• Aldehydes
• Gaseous chemosterilisers
• Oxidising agents
 HEAVY METALS

• Silver
• Mercury
• Copper
• Zinc

OLIGODYNAMIC ACTION

“small force”
effective in very low concentrations

• The ability of trace amounts of heavy metals to disinfect

• Heavy metals denature proteins by combining with -SH groups
• Disrupts microbial metabolism
 HEAVY METALS

Silver
• Silver (Ag) is an antimicrobial agent
• Ag impregnated dressings for burned victims
• Ag is also incorporated into indwelling catheters

Copper
•CuSO4 used to destroy green algae in swimming pools and fish tanks
Zinc
Cu + Zn treated shingles are available to create anti-fungal roofs
ZnCl2 is a common ingredient in mouthwashes
 HEAVY METALS

MERCURY

• Mercuric chloride
– Toxic and corrosive
– Mildew in paints

COPPER

• Copper sulfate
• Algicide
– reservoirs, swimming pools, fish tanks
• Mildew in paints
 ALCOHOL
Ethanol
Isopropanol
• Not strong disinfectants
• Kill bacteria, fungi and enveloped viruses
• Do not kill endospores, or naked viruses

MODE OF ACTION

• Dissolves lipids Disadvantages

• Denatures proteins not effective against spores
– Needs water inactivated by organic matter
– More effective when
diluted (usually 70%)
 ALCOHOL

– 70% ethanol, or isopropanol (“rubbing alcohol)

– Mode of action: Protein denaturation & coagulation
therefore membrane disruption
– Advantage of evaporation after disinfection
– Poor effect on wounds! Superficial coagulation of
proteins → secludes deeper layers of microbes
– Alcohols kill vegetative forms of bacteria (including TB)
and fungi, but have no action on spores or viruses
– Their effect depend on concentration and type of alcohol
– The following three solutions have similar effect:
Ethanol 70% , isopropanol 60% and n-propanol 40%
70-80% alcohol inactivates HIV and Hepatitis B in 2-10
minutes.
 PHENOL

Hard surface disinfection

Phenol

Phenol no longer used as a disinfectant because of its

toxicity to tissues
Many non-pathogens attack organic matter, producing
chemicals which may be highly odorous, corrosive or
staining

Disadvantages
effectiveness reduced by presence of organic
matter
some toxicity
 PHENOLICS

Mode of action: Damage lipid cell membranes

Quite effective in the presence of organic materials
(pus, saliva, feces)
E.g. Lysol® (O-phenylphenol)
Very good surface disinfectants
PHENOL DERIVATIVES
– Less of an irritant, and more effective than phenol
• Mode of action
– Damages plasma membrane
– Enzyme inactivation
– Protein denaturation
 QUATERNARY AMMONIUM COMPOUNDS

•cleaning non-critical environmental surfaces

• Products used for this purpose generally contain low-level
disinfectants such as quaternary ammonium compounds (quats) or
phenolics
•effective against easier to kill vegetative bacteria
minimum of 10 minutes of contact time
 QUATS continues

•Quats have negative effects on humans

•are considered to be hazardous by OSHA criteria
•Some products registered as disinfectant cleaners may claim to be
non-hazardous because they haven’t been tested yet
•They should not be considered free of toxic health effects
 SOAPS/SURFACTANTS

•mechanical removal of microbes through scrubbing

•soap breaks oily film on skin (emulsification); little value as an
antiseptic
•detergents-more effective against gram positive than gram negative
-- nonionic detergents have no germicidal activity
-- anionic (acid) sanitizes, food and dairy industry
-- cationic (positive)-antiseptic for skin, instruments, utensils
 SURFACTANTS

Surface active agents (surfactants).

4 types, cationic, anionic, non-ionic & amphoteric agents
the anionic & non-ionic compounds have only very weak antimicrobial
activity
They are active against bacteria and enveloped viruses (such as feline
herpesvirus) but not non-enveloped viruses (such as feline calicivirus)
 SURFACTANTS CONTS

•main use is in wound disinfection and as preservatives in

pharmaceutical preparations, eg. eye drops.
•amphoteric compounds (ie. possess both anionic and cationic
characters) are the most active disinfectants of the 4 types of
surfactants, and are less affected by organic matter

•Surfactants are non-ionic, usually alcohol ethoxylates which are more

efficient at penetrating skin than many other surfactants
•surfactants enhance the toxicity of other toxic chemicals and concerns
have been raised about low-level exposure to toxic chemicals
How surfactants work
 CHLORHEXIDINE

• A non-phenolic compound
• Skin and mucous membranes, low toxicity
• Surgery
– Scrubbing and skin preparation

Effect:

•Chlorhexidine is efficient against both gram+ and gram- bacteria

(Pseudomonas, Proteus and Providencia might be resistant).
•No effect is seen on TB, spores and viruses.
•The bactericidal effect is enhanced by alcohol
 ORGANIC ACIDS

Organic Acids - various organic acids and their salts are common
antimicrobials in foods
- preservatives to control mold growth
- sorbic acid (Ca, Na, K) used in cheeses, baked goods, soft drinks, fruit
juices, jams, jellies
- benzoates (sodium benzoate, methy-p-hydroxybenzoate
[methylyparaben]) fruit juices, jam, jellies, soft drinks, salad dressings,
margarine, and many pharmaceutical products
- boric acid used in eye washes
- calcium propionate-prevents mold growth in bread
 HALOGENS

• Iodine
• Chlorine

• Halogens
– Iodine I2
– Halogenation of microbial proteins
– Iodine tincture (solution in diluted alcohols)
– Iodophores – organic molecules slowly releasing I2, less staining than straight I2
preparations (e.g. Betadine®, Isodine®)
– Extremely effective, skin disinfection, wound treatment (I2 tablets water treatment)
 IODINE (I2)

• Wide spectrum
– Antiseptic
• Bacteria, viruses, many endospores and fungi
• Combines with tyrosine and oxidizes -SH groups
– Alters proteins

CHLORINE Cl2 (gas)

– Formation of ClO- in water (“bleach”)

– Strong oxidizing activity destroys microbial enzymes
– Drinking water, swimming pools, sewage treatment
 IODINE & ITS COMPOUNDS (cont.)

• Skin irritant
• Tincture
– I2 + alcohol
• Iodophor (Betadine and Isodine)
– I2 + organic molecule
– Less irritant
– Pseudomonas resistant
 CHLORINE (Cl2)

• 1846 Semmelweiss
– Puerperal fever
• Since 1908 main water disinfectant in the USA

REACTION OF CHLORINE WITH WATER

Cl2 + H2O  H+ + HOCl

HOCl H+ + OCl-

 HOCl, OCl- and pH

HOCl %

OCl %
-
4 pH 10
 Cl2 disinfection and pH

Disinfection %

4 pH 10
 HYPOCHLORUS ACID VS. HYPOCHLORITE

• Hypochlorus acid has no charge (neutral) HOCl

– Moves freely through membrane

• Hypochlorite ion negatively charged (HCl-)

• Cannot enter cell freely

MODE OF ACTION

• Strong oxidizing agent

• Interferes with enzyme activity
 CHLORINE LIMITATIONS IN WATER DISINFECTION

• Generates toxic trihalomethanes

– Chloroform related chemicals

CHLORAMINES

Cl2 and ammonia

Long lasting, but slow
Effective with organic matter
 ALDEHYDES

• Formaldehyde and glutaraldehyde

• Strong antimicrobials
• Sporicidal
– Inactivate proteins/Nucleic acids
• Used for vaccine preparation
Aldehyde group

Disadvantages
toxic
requires extensive rinsing
allergic reactions
 OXIDISING AGENTS

• Gaseous chemosterilizers (ethylene oxide—

effective all forms of life incl. endospores)
• Oxidizing agents
Oxidation: Types of Oxidants

•Oxygen (O2)
•Chlorine (Cl2, HOCl or OCl-)
•Chloramines (NH2Cl or NHCl2)
•Ozone (O3)
•Chlorine Dioxide (ClO2)
•Permanganate (MnO4-)
•Others: Peroxide, Bromine, UV light
 GASEOUS CHEMOSTERILIZERS
Ethylene oxide
Advantages
- Denatures proteins
- Sterilizes (4 to 18 hours)
- Disposable plasticware

Disadvantages
Toxic,
carcinogenic
Corrosive,
explosive
 Hydrogen peroxide

Mode of Action
oxidizes organic compounds
inactivates many cellular components
Antiseptic

Disadvantages
Breaks down quickly
Strong solutions can burn skin

Advantages
O2 released when it breaks down
effective against anaerobic bacteria
 Ozone(O3)

Ozone
Ozone (O3) is a powerful oxidizing agent and is used as a disinfectant
in water treatment.
O3 is a highly reactive gas formed by electrical discharges in the
presence of O2.

O3 is energy intensive because a large

O3 + H2O →HO3+ + OH- amount of energy is usually required
HO3+ + OH- →2HO2 to split the stable oxygen covalent
O3 + HO2 → HO + 2O2 bond to form ozone.
O3 readily reverts to elemental
HO + HO2 → H2O + O2
oxygen during oxidation-reduction
reactions.
 Chlorine Dioxide: more virucidal

Chlorine Dioxide
Although ClO2 has not been widely used in the past, there is
recent interest in its use because it does not produce
significant amounts of THMs as by-products from
reactions with organic compounds. Chlorine chemistry is
complex. In an acidic solution, reduction to chloride
predominates.
• generated by reacting chlorine and sodium chlorite:
– Cl2 + 2NaClO2 → 2ClO2 + 2NaCl
• pH 6-8: 2ClO2 + 2OH2 →ClO2- + ClO3-
• chorite chlorate
• effective as HOCl and more than OCl- or chloramines
• disrupts protein synthesis
• doesn’t form toxic chloramines, or THMs
 Chlorine Dioxide

• ClO2 has 1.4 times the oxidation power as Cl2

• At neutral pH values typically found in natural waters,
ClO2 has only about 70% of the oxidizing capacity as Cl2.

ClO2 produces inorganic byproducts such as chlorite (ClO2-)

and chlorate (ClO3-) for which health effects are not well
understood.
 Discussion ???

• Some water quality managers are proposing to disinfect

water before it discharges to the coastal ocean. Is this a
good idea?