To give knowledge about this concept.

1. STERILIZATION
&
DISINFECTION
Ms. Harshada. R. Bafna
Asst. Professor

2. • Control of microbes is possible either by inhibiting the growth of
microorganism or by destructing or killing the microbes.
• The agent which inhibits microbial growth is known by name with
suffix - stasis.
• The agents which destruct/kill the microorganism are known by
name with suffix- cide.
• Sterilization : Destruction or separation of all forms of microbial life
including endospore.
• Disinfection : Destruction of pathogenic vegetative forms.
• Antisepsis : When process of destruction of pathogenic microbe is
associated with living tissue it is called as antisepsis.
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3. • Sanitization : Lowering the microbial count to a safe public health
level to lower the risk of disease transmission is called as sanitization.
• Pasteurization : Brief heat treatment to reduce spoilage in food
products e.g. milk or wine.
• Preservation : It means delay of spoilage by inhibiting microbial
growth.
• Thermal death point (TDP) : The lowest temperature at which all
microorganisms in the broth get killed in 10 minutes.
• Thermal death time (TDT) : The minimum time period to kill all
microbes at given temperature.
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4. • Decimal reduction time (DRT or D value) : The time in minutes
needed to destruct 90% of population at given time.
• Inactivation Factor (IF) : Inactivation factor (IF) is also sometimes
used to describe the total microbial inactivation by a sterility process.
IF is defined as follows :
IF = 10 t/D
t = Exposure time
D = D-value
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5. Heat is used by two ways — Moist heat and Dry heat.
A. Moist heat
• Moist heat occurs in the form of hot water, boiling water or steam.
• Practically temperature of moist heat ranges from 60°C to 135°C.
Mode of action : Moist heat is responsible for disruption cell
components. It coagulates and denatures proteins. Proteins are
denatured more rapidly at lower temperature if moisture is present.
Other cell components including - cell membrane, ribosome, DNA and
RNA also get denatured by moist heat.
The five ways in which moist heat is employed to control microbes are
1. Steam under pressure (Autoclaving).
2. Intermittent sterilization / Tyndalisation.
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6. 3. Boiling water.
4. Pasteurization.
5. Canning.
• In the pharmaceutical industry, moist heat sterilization is used for :
Aqueous injections.
Surgical dressings.
Diagnostic and surgical equipments.
Containers and closures.
Ophthalmic preparations.
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7. Steam under pressure
• Steam under pressure can be efficiently utilized for sterilization by
using special apparatus called autoclave.
• It is a pressure cooker like apparatus.
• The word autoclave means "self closing", as the lid is closed by
excess pressure within the vessel.
Construction :
• Autoclave is an upright aluminum or stainless steel cylindrical vessel
of various capacity (5 Lit. to 100 Lit.).
• The parameters within autoclave are indicated solely by pressure
gauge, as thermometer would likely be damaged.
• Inside the autoclave the heater is equipped at bottom level.
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9. • It is electrically heated.
• Autoclave is also equipped with exhaust safety valve to release
pressure and a valve to maintain pressure at a particular level. (Fig.)
Applications :
• To sterilize aqueous solutions e.g. bacteriological media like nutrient
broth, nutrient agar, etc.
• To sterilize surgical dressings, plastic and rubber fabrics.
• Occasionally to sterilize glasswares, like petriplate, pipettes, flasks,
etc.
• To sterilize the metal instruments, immediate drying is required to
protect the instruments against corrosion.
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10. Disadvantages :
• During autoclaving, the pH of aqueous solutions changes due to
water loss.
• Oils do not get sterilized in autoclave as they are hydrophobic in
nature and do not allow the steam to penetrate through it.
• The most common conditions used for sterilization using an
autoclave are as follows :
Temperature Holding time
(min.)
Pressure (PSI – pounds
per square inch)
121 15 15
126 10 20
134 03 30
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11. Fractional sterilization / Tyndalisation
• Some materials cannot withstand the high temperatures of
autoclaving.
• If no other method is available, alternative method of sterilization is
intermittent sterilization (Tyndalisation).
• John Tyndall developed it.
• This technique requires a chamber to hold the materials and a
reservoir for boiling water.
• Items are exposed at 100°C for 30 min.
• This temperature inactivates all vegetative cells but does not
inactivate spores.
• The surviving spores will germinate to vegetative cells, during
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12. incubation at appropriate temperature for 24 hrs and the material is
again subjected to steam (100°C for 30 min.).
• This cycle is repeated for three days.
• Steam Arnold is an apparatus used for Tyndalisation.
Application :
• Tyndalisation is used mostly for sterilization of heat sensitive culture
media such as those containing sera, egg and also for the control of
microbes in the food.
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13. Boiling water :
• It is not actually sterilization but is widely used for control of
microorganisms; 100°C will not kill all resistant cells.
• When material is exposed to boiling water for 30 min., all non-
spores forming pathogens will be killed; including resistant species
like tubercle bacillus and staphylococci.
Application :
• At home, boiling water is used to sanitize and disinfect materials for
babies.
• Boiling is also useful in cleaning the utensils, bedding and clothing.
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14. Pasteurization :
• Louis Pasteur developed this technique.
• A technique in which heat is applied to liquids to kill agents of
infection and spoilage; while at the same time retaining flavour and
food value.
• Pasteurization can be done by one of the following methods :
1. Batch Method / LTH : (Low temperature holding method) Heating
of liquid at 62.8°C (145°F) for 30 min.
2. Flash Method / HTST : (High temperature short time). Heating of
liquid at 71.7°C, (161°F) for 15 seconds. This method is used for
things like eggs.
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15. Ultra high temperature method (UHT)-
• Holding liquid at temperature 134°C (273.2°F) for 1- 2 seconds.
• This method is used for milk and milk products.
• For pasteurization, typically temperature is adjusted in order to
destruct Mycobacterium tuberculosis and Coxiella burnetti,
infectious agent of tuberculosis and Q-fever respectively.
Applications :
• Pasteurization is most often employed to prevent the transmission of
milk borne diseases from infected cows.
• The primary target of pasteurization is the control of non-spore
forming pathogens.
• To kill unwanted contaminants in beer and wine.
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16. Limitation :
• It does not destructs thermoduric organisms and their spore.
Canning :
• The sensitivity of bacterial endospore is affected by pH of the
material being sterilized.
• Low acid containing foods such as meat, fish, vegetables and eggs
require extensive heating, so they are canned at temperature between
121°C and 151°C.
• Acidic food such as tomatoes, pineapples, berries and certain fruits
having pH 3 to 4.5 can be canned by boiling for 15-30 min.
• Acid environment makes endospore much more sensitive to heat.
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17. Dry Heat
• The dry heat air, heated by a flame or electric heating coil, is used for
sterilization purpose.
• Practically the temperature of dry heat ranges from 160°C to several
thousand degrees Celsius.
• Microbes are generally more resistant to dry heat than moist heat.
• This is because of less penetration and diffusion of dry heat
compared to moist heat.
Mode of action :
• Dry heat is responsible for denaturation of proteins.
• It inactivates membranes and DNA.
• Other cell structures like ribosome and RNA are also inactivated.
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18. • It oxidizes the cells and dehydrates the cell.
• Extremely high temperature reduce cells to ash.
• The two ways that dry heat is employed-to sterilize microbes are :
Incineration
Hot air oven
1. Incineration :
Incineration means the direct flaming/burning. The flame of Bunsen
burner reaches 187°C at its hottest point. Direct exposure to such
intense heat ignites and reduces the microbes to ash.
Applications :
• To sterilize inoculating loops.
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19. • For complete destruction and disposal of infectious materials such
as syringes, needles, culture materials, dressings, bandages, wastes
and pathological samples.
Disadvantage :
• Method is limited to use for metal and heat resistant glassware.
• It contributes to air pollution.
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20. 2. Hot air oven :
• Hot air oven is equipment for the dry heat sterilization by hot air
under atmospheric pressure.
• It is having chamber, usually a closed electric coils, which radiates
heat within enclosed compartment.
• Hot air is circulated within compartment making contacts with the
items and transferring the heat. (Fig.)
• Here sterilization is accomplished by exposure of items to high
temperature.
• The most common conditions used for sterilization using dry heat
are as follows:
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21. 2/28/2024 HRB 21

22. Temperature Holding time (min)
1600C 120
170C 60
180C 30
Applications :
• To sterilize needles, porcelain and metal equipments.
• To sterilize oils and fats, including oily injections.
• Various powders can be sterilized by this method e.g. talc.
Disadvantages :
• Not suitable for aqueous solutions, plastic, paper and cloths.
• It takes longer time compared to autoclaving.
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23. Radiation
• Radiation is defined as any form of energy emitted from atomic
activities that travels at high velocity through matter or space.
• The energy content of the radiation is inversely proportional to
wavelength.
• Hence, shorter the wavelength greater is the energy and thus more
the killing power.
• High-energy radiation includes UV light, X-ray and gamma-ray.
• These rays can kill living cells including microorganisms.
• Radiation is grouped into two types - Ionizing radiation and Non-
ionizing radiation.
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24. 1. Ionizing radiation :
• The radiation, which is responsible for ionization of atoms/
molecules.
• The radiation with short wavelength and high energy when strike,
eject the electrons from atoms, this forms ions.
Mode of action :
• Ionizing radiation disrupts the atomic structure of molecule by
dislodging/ejecting the electron and thus results in an electrical
imbalance and formation of ions.
• One of the most sensitive targets for ionizing radiation is the DNA
molecule.
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26. • Ionizing radiations induce mutation which generally do not get
repaired.
• The radiation dosage is measured in rads (radiation absorbed dose).
• Depending on application, exposure ranges from 0.5 to 5 megarads.
• Although all ionizing radiation can penetrate solids & liquids but the
gamma rays have the highest penetrating power, whereas X-rays
have intermediate & cathode rays have least penetrating power.
(Fig.)
Applications :
• Foods like, meat can be sterilized.
• Sterilizing medical products including drugs, vaccines, syringes,
surgical gloves and tissues like bone, cartilage, skin & heart valves.
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27. Limitation :
• Radiation irradiated foods are altered in flavour.
• Main risk is to the machine operator from exposure of radiation.
2. Non-ionizing radiation (UV-rays)
• Ultraviolet rays have wavelength ranges from approximately 100
nm - 400 nm.
• It is most lethal from 240 - 480 nm (Peak 260 nm).
• Mercury vapor lamp is commonly used source of UV rays.
• UV radiation passes readily through air, slightly through liquid and
poorly through solids.
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28. Mode of action :
• UV radiation possesses moderate energy and do not ionize the
atoms.
• It acts on DNA molecule by forming dimer between adjacent
pyrimidines. (Thymine-thymine dimer).
• These dimers are responsible for induction of mutation.
• As thymine binds to thymine instead of adenine of complementary
strands it results in poor transcription and bad proteins.
• The harmful effect of UV rays is not so detrimental compared to
ionizing radiation; many times effect can be repaired (Fig.).
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29. 2/28/2024 HRB 29

30. Applications :
• It is used to control air-bone contaminants in hospital room,
operation theatre, food preparation areas, etc.
• It is also used for purification of liquid including milk, fruit, juice,
wine, beer, etc.
Disadvantage:
• UV rays can damage eyes and are known to cause sun burns and
skin cancers in humans.
Microwaves :
• Microwaves as such do not kill microbes directly.
• But they can destroy microbes because of the heat they generate and
hence are considered to act in a "indirect" way.
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31. Chemical Sterilization
The chemicals used for sterilization are called as Sterilent. These
agents possess following characteristics :
1. They affect proteins, DNA or cytoplasmic membranes.
2. Most of these agents have some kind of toxicity to humans. Hence,
risk of harm needs to be weighed against potential benefits.
3. These agents may also cause detrimental effects on environment.
Ethylene oxide :
• Ethylene oxide is the gas that is used on a large scale for industrial
and medical sterilization.
• At room temperature it is a colorless gas with a characteristic
ethereal odour.
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32. • It can be liquefied easily and the liquid boils at 10.8°C.
• Mode of action : Ethylene oxide is responsible for the alkylation
of nucleic acid and proteins.
Factors affecting sterilization :
• Sterilization efficiency depends on the humidity.
• A relative humidity of 25-33% is found to be optimal.
• Sterilization can be achieved at room temperature but a long
exposure time is necessary; sterilization time can be decreased with
rise in temperature.
• The concentration of ethylene oxide for sterilization range from 250
to 1000 mg/litre.
• The ethylene oxide has the ability to penetrate paper, fabrics, etc.
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33. Advantages of ethylene oxide:
• It is suitable for thermoliable substances because sterilization can be
carried out at room temperature.
• It does not damage moisture sensitive substances and equipments
because only a low humidification is needed.
• Pre-packed articles can be sterilized because of the great penetrating
power of ethylene oxide.
• Although ethylene oxide is a highly reactive compound;
comparatively few materials are damaged by the process of
sterilization.
Disadvantages of ethylene oxide sterilization:
• Long exposure is necessary. So, it is unsuitable in emergencies.
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34. • It has a toxic effect on human beings.
• It is carcinogenic.
• It is expensive.
Formaldehyde :
• Formaldehyde (HCHO) is a gas.
• At room temperature it polymerizes forming a solid substance.
• The important polymer is paraformaldehyde, a colorless substance
that rapidly yields formaldehyde upon heating.
• Formaldehyde is also marketed in aqueous solution as formalin,
which contains 37 to 40% formaldehyde.
• Glutarldehyde is another aldehyde used for sterilization.
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35. Mode of action :
• Formaldehyde is an extremely reactive chemical, it readily combines
with vital organic nitrogen compounds such as proteins and nucleic
acids.
• It is likely that interaction of formaldehyde with these cellular
substances accounts for its antimicrobial action.
• It is also effective against killing spores.
Applications :
• Formaldehyde in solution is useful for sterilization of certain
instruments.
• Formaldehyde in gaseous form can be used for disinfection and
sterilization of enclosed areas.
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36. • Formalin and paraformaldehyde are two main sources of
formaldehyde when it is used for gaseous sterilization.
• Vaporization of formaldehyde from either of these source into an
enclosed area for an adequate time will cause sterilization.
• Vegetative cells are killed more quickly than spores.
• Humidity and temperature have a pronounced effect on the
microcidal action of formaldehyde.
• Sterilisation is possible at room temperature and the relative
humidity between 60 to 80 percent.
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37. Filtration
• To sterilize the fluids (liquid or gas), they are passed through various
bacteriological filters.
• This is a mechanical way of sterilization and is generally used for heat
sensitive fluids.
• Important filters are discussed below :
1. Seitz filter :
• It is an asbestos filter.
• The disk of pressed asbestos is tightly clamped between two smooth
metal rings by screw.
• The liquid to be filtered is poured into a funnel like attachment and
drawn through the asbestos disk by vacuum.
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38. • After completion of filtration the disc can be replaced.
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39. 2. Membrane filter :
• Membrane filters are prepared from a variety of polymeric material
like polycarbonate and polyesters.
• As the pores of membrane filter are uniform these are superior
compare to others.
• They also possess a faster rate of filtration.
• Membrane filters are not only used for sterilization, but also used
for separation of microorganisms and for collecting microbial
sample e.g. viruses can be separated from sample.
• Membranes with pore sizes of 0.2 to 0.4 μm are used to remove
bacteria.
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40. Advantages of membrane filters:
1. All microorganisms are separated by process of sieving.
2. Membranes have a high and uniform porosity permitting a rapid
rate of filtration.
3. Membranes are disposable. Hence, there is no cross contamination
between filtered products.
4. Adsorption is very less.
Disadvantages of membrane filters are as follows :
1. Prefilter is used before the membrane filter to avoid clogging and
breaking.
2. They have less chemical resistance to certain organic solvents such
as chloroform, ketones and esters.
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41. 2/28/2024 HRB 41

42. 3. Sintered glass filters (fritted glass filter/morton filters) :
• Borosilicate glass is finely powdered in a ball mill and packed into
disc moulds and heated until suitable adhesion takes place between
the granules.
• The sintered discs are finally fused into funnels of suitable size and
shape (Fig. 16.6).
• Sintered glass filters are available in several different porosities but
for filtration sterilization a number or grade 5 or 5 on 3 must be
used.
• They have a low adsorptive property and can be cleaned easily.
• They are brittle and expensive and have a small area of filtration
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43. 2/28/2024 HRB 43

44. 4. HEPA filter : (High efficiency particulate air)
• These filters are widely used for getting sterile air.
• HEPA filters are made up of cellulose acetate and are available in
various pore sizes.
• A generally available size of HEPA filter pores is 0.3 μ.
• It provides 99.9% efficiency.
• The HEPA filters are generally equipped with laminar airflow,
which is very important instrument for providing aseptic condition.
• The laminar airflow is widely used to carry out sterility testing, drug
packing, and ampoule fillings.
• Laminar airflow is equipped with UV light.
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45. Mechanism :
• The pores of filters are adjusted in such a way that it does not allow
the bacteria and other microorganisms.
• The pore size is varies from 8 to 0.02 μ.
• Many times this phenomenon does not happen due to the choking
(blocking) of filters. Hence, continuous replacement is necessary.
• To enhance the rate of filtration vacuum is created in lower flask.
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46. Applications :
1. Filtration is used to sterilize liquids that cannot withstand heat. e.g.
serum, other blood products, vaccines, drugs, intravenous fluids,
enzymes and culture media.
2. To sterilize milk and other heat labile beverages, to reduces
microbial load of water.
3. It is an efficient means of removing air-borne contamination that
are common source of infection and spoilage.
4. Particulate filters are used to sterilize respiratory and anesthetic
gases and to provide steady flow of sterile air in sterile room.
Disadvantage :
• It cannot remove viruses and toxins.
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47. 5. Filter candles (ceramic/Berkefield filter) :
• These are manufactured in different grades of porosity and have been
used widely for purification of water for industrial drinking
purposes.
• They are made of either porous porcelain or Kiesulghur.
• They are usually encountered as cylindrical candles with
comparatively thick walls (Fig. 16.7).
• These depth filters with cellular walls and are available in various
sizes.
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48. 2/28/2024 HRB 48

49. DISINFECTION
• Disinfection means the killing or removing of organisms capable of
causing infection.
• Disinfection generally kills the sensitive vegetative cells but not
heat-resistant endospore.
• Disinfection is usually accomplished by chemicals; called as
disinfectant. The term is often used synonymously with antiseptic.
• However disinfectants are applicable to inanimate objects like floors,
dishes, laundry and bedding.
• The terms sanitizer and sanitization are often used with this
meaning, while antiseptics are applicable on living tissues.
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50. Characteristics of an Ideal Disinfectant
1. Antimicrobial activity: A good disinfectant should kill large variety
of microorganisms even at low concentration.
2. Solubility: To be effective, chemical must be soluble in water and
commonly used other solvents.
3. Stability: Chemical should not change its germicidal power even after
long period of storage.
4. Non-toxicity: The compounds must be non-toxic to human beings and
other animals.
5. Homogeneity: The preparation of disinfectant must be homogenous.
6. Toxicity at room temperature and body temperature: The
chemical should be effective at room and body temperatures against
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51. microorganisms and it should not need specific temperature for its
antimicrobial activity.
7. Capacity to penetrate: More the penetration power more effective
is the disinfectant.
8. Non-corroding and non-staining: It should not damage or stain
the object on which it is applied.
9. Deodorizing ability: It must have a deodorizing ability.
10. Detergent ability: It must possess a detergent like ability.
11. Availability: The compound must be readily available in large
quantities at a reasonable price.
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52. Disinfectants
1. Phenol and phenolic compounds :
• Joseph Lister (1867) first used phenol to reduce the infection of
surgical wounds.
• He initiated the practice of applying a solution of phenol (carbolic
acid) to surgical apparatus,
• Phenol related compounds are called phenolics and are also used as
effective disinfectants. e.g. cresol butyl phenols, lysol, etc.
Mode of action :
• Exposure of microbial cell to phenolic compound produces a variety
of effects depending upon the concentration.
• Researchers have described phenolics which can damage
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53. cytoplasmic membrane, precipitate cellular proteins and
inactivate enzymes.
• Phenolic compounds are generally active in the presence of organic
contaminants.
Spectrum of action : Bactericidal, fungicidal, virucidal but not
sporicidal.
Applications :
1. It is used as disinfectant in many commercial antiseptic and
disinfectant preparations.
2. Aqueous solutions of phenols (2-5%) are used to disinfect materials
like sputum, urine, feces, etc.
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54. 3. The derivatives of phenol are also used to disinfect surgical
instruments e.g. cresol.
4. Hexachlorophene is available as an antiseptic.
Disadvantages :
1. Most phenolic are too toxic to use as antiseptic and are relatively
insoluble.
2. Some phenolic have objectionable odour.
3. Phenol produces toxic effect and irritation.
4. Phenol has carcinogenic effect.
5. Hexachlorophene can cause neurological damage.
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55. 2. Alcohols :
• Ethyl alcohol in concentration 50-95 % is effective against
microorganisms.
• For practical application 70 % concentration of alcohol is generally
used.
Mode of action :
• Alcohol causes coagulation of proteins and lipids. It also
dehydrates the cell.
Spectrum of action :
• Acts mainly on bacterial vegetative cell and fungi but it is not
sporicidal.
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56. Applications :
1. Alcohol is effective in reducing the microbial flora of skin.
2. Ethanol (70 - 95 %) acts as germicide in clinical laboratory.
3. Physicians use denatured alcohol (spirit) to degerm the skin.
Disadvantages :
1. Alcohol is volatile, irritating and is inactivated by organic matter.
2. Does not inactivate polio and hepatitis-B viruses.
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57. 3. Heavy metals :
• Most of the heavy metals either alone or in certain compounds exert
a detrimental effect on microorganisms.
• Silver and mercury are popularly used as disinfectants.
Mode of action :
• Heavy metals and their compounds act as antimicrobial by combining
with cellular protein and inactivating them enzymes containing
sulfide group.
• High concentration of salt of heavy metals like Hg, Cu
and Ag coagulate cytoplasmic proteins resulting in the damage of
cell components.
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58. • The heavy metals are effective antimicrobial in trace concentration;
this effect is called as oligodynamic Power.
Spectrum of action: Bactericidal, fungicidal, virucidal but not
sporicidal.
Applications:
1. Mercury: It is mostly used in ointments as antiseptic.
2. Silver: The eyes of newborns are treated with a few drop of 1%
silver nitrate solution to prevent ophthalmic and gonococci infections
of eyes.
3. Copper: Copper sulphate is most effective against algae, it is used in
swimming pools and open water reservoir.
Drawback: Metal solutions are toxic and may cause allergies.
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59. 4. Halogen:
• The name halogen is from Greek roots (HAL - means salt; GEN
means to produce).
• All halogens, are component of salts.
• Chlorine, iodine, bromine and fluorine are halogens.
• The chlorine and iodine are widely used as disinfectant.
Spectrum of action : Chlorine -Bactericidal, fungicidal, virucidal
but not sporicidal. Iodine - Bactericidal, fungicidal, virucidal and
sporicidal.
Mode of Action :
Chlorine — Cl2 + H20HC1 + TIOC1 (Hypochlorous acid)
HOC1 HC1 + [0](Nascent oxygen)
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60. The nascent oxygen is strong oxidizing agent and is responsible for
protein denaturation. Iodine — Interferes with interamolecular bonding
in proteins.
Applications :
1. Chlorine is used for disinfection of municipal drinking water and
swimming pools.
2. Household bleach is common and cheap source of chlorine and
contains 5.25% sodium hypochlorite (NaOC1).
3. But high concentration of chlorine can cause irritation of eyes, skin
and mucous membranes.
4. Iodine is used for cleaning the wound.
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61. 5. Dyes :
• Certain dyes can be used as antimicrobial agents such as aniline and
acridine
Mode of action : Dyes are effective probably by binding to nucleic
acid, thus are responsible for mutation in DNA.
Spectrum of action : Mostly inhibitory for Gram-positive bacteria
and fungi.
Application : For selective growth of microbes, dyes are used as
media component.
6. Detergents :
• Surface tension reducers employed primarily for cleaning surfaces,
are called as detergents.
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62. • The different forms of cationic (quaternary ammonium compound)
detergents are popularly used as disinfectant.
Mode of action :
• A variety of damaging effects of detergent on microorganisms have
been observed.
• These include, denaturation of proteins, interference with
glycolysis and the membrane damage.
• These are also responsible for reducing the surface tension due to
this there is disruption of the cell.
Spectrum of action : Bactericidal, fungicidal but not sporicidal.
Applications :
• They are used to sanities food and beverage utensils in restaurants
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63. and homes.
• They are used as disinfectants in cosmetic preparation.
• Detergents are widely used for control of microorganism on floors,
walls and other surfaces in hospitals, nursing homes and other
public places.
7. Hydrogen Peroxide :Hydrogen peroxide is a oxidizing agent.
Spectrum of action : Mainly effective against anaerobic
bacteria, fungicidal but is not virucidal.
Application : Generally used for cleaning wounds.
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