different types of sterilization

1. Sterilization

2. Sterilization is the process that eliminates, removes, kills, or deactivates all
forms of life and other biological agents such as fungi, bacteria, viruses, spore
forms, unicellular eukaryotic organisms such as plasmodium, etc.
Sterilization can be carried out through heat, chemicals, irradiation, high
pressure, and filtration.
In one sentence, sterilization is the process by which an article, surface, or
medium is freed from all living organisms either in the vegetative or spore state.

3. The various methods of sterilization are:
1. Physical Method
a. Thermal (Heat) methods
b. Radiation method
c. Filtration method
2. Chemical Method
3. Gaseous method

4. Factors affecting sterilization by heat:
• Nature of heat: Moist heat is more effective than dry heat
• Temperature and time: temperature and time are inversely proportional. As
temperature increases the time taken decreases.
• Number of microorganisms: More the number of microorganisms, higher the
temperature or longer the duration required.
• Nature of microorganism: Depends on species and strain of microorganism, sensitivity
to heat may vary. Spores are highly resistant to heat.
• Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure. Certain heat sensitive articles must be sterilized at lower
temperature.
• Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.

5. Physical methods for Sterilization
Sunlight:
The microbicide activity of sunlight is mainly due to the presence of
ultra violet rays in it.
It is responsible for spontaneous sterilization in natural conditions.
In tropical countries, the sunlight is more effective in killing germs
due to combination of ultraviolet rays and heat.
By killing bacteria suspended in water, sunlight provides natural
method of disinfection of water bodies such as tanks and lakes

6. 1. HEAT STERILIZATION:
Heat is considered the most reliable method of sterilization of objects that can withstand
heat.
It is carried out in two ways viz.
Moist and Dry heat. They are the most common sterilizing methods used in hospitals and are
indicated for most materials.
This method of sterilization is applied only to the thermostable products, but it can be used
for moisture-sensitive materials for which dry heat (160-180°C) sterilization, and for
moisture-resistant materials for which moist heat (121-134°C) sterilization is used.

7. Advantages:
•A most common method for controlling microbial growth.
•Very effective in destroying unwanted microbes.
•Low cost.
Disadvantages:
•It can degrade thermally unstable media.
Dry Heat

8. Dry Heat
Dry heat sterilization
requires higher temperatures and longer exposure times than moist heat
sterilization.
It uses high temperatures to kill microorganisms and bacterial spores and is
used on items that cannot get wet and for glassware, oils, powders, metal
instruments, and items wrapped in paper.
There are three types of temperatures are recommended
Temperature Exposure time
150°C 150 minutes
160°C 60 minutes
170°C 30 minutes

9. Thermal death time (TDT):
It is the minimum time required to kill a suspension of an organism at a
predetermined temperature in a specified environment.
Thermal death time is inversely proportional to temperature.
It is increased in presence of an organic substance, proteins, nucleic acid, starch
gelatin, etc.

10. Principle: It causes denaturation of proteins and oxidative damage. There
are several different types of dry heat sterilization such as:
•Flaming: It is used for straight wires, bacterial loops, and spatulas.
Here, the substance is exposed to the flame for just a few minutes. The flame
will burn out the microbes directly.
Incineration – It is an effective method of sterilization in microbe cultures. The
end of the microbe loop is exposed to red hot flame; thus, it kills microorganism.
It is the easiest way to destroy microbes in metals.

11. Hot air oven:
• This method was introduced by Louis Pasteur. Articles to be sterilized are
exposed to high temperature (160°C) for duration of one hour in an
electrically heated oven.
• Since air is poor conductor of heat, even distribution of heat throughout
the chamber is achieved by a fan.
• The heat is transferred to the article by radiation, conduction and
convection.
• The oven should be fitted with a thermostat control, temperature
indicator, meshed shelves and must have adequate insulation.

12. •Glasswares, forceps, scissors, scalpels, etc. are sterilized by
this method but surgical dressings, rubber items, or plastic
materials are not sterilized by this method.

13. Principle of Hot Air Oven
The hot air oven is based on the principle method of dry heat sterilization.
(conduction,convection,radiation)
Since conduction is the basis of dry heat sterilizations, thus the temperature first
reaches the surface of the material to be sterilized, and then it gradually moves
towards the core of the material.
Thus, dry heat sterilization makes sure to sterilize every part of the material. Then,
the whole material gets a uniform supply of heat, and if this heat is employed for a
certain amount of time, then it helps in the sterilization of all different kinds of
microorganisms, such as bacteria, viruses, fungi, and even the resistant
endospores, which escape most of the sterilization procedures.

14. Dry heat sterilizes the material by inducing oxidizing the particles inside it and
damaging their primary component, which results in the ultimate death of the
organism. Usually, the temperature that is set for efficient sterilization is about
an hour.
Since hot air is lighter than cold air, thus increasing the temperature inside the
chamber results in the flow of hot air up to the roof of the chamber while cold air
comes down. Thus, it facilitates the circulation of hot air inside the chamber.

15. Operation:
(i)Articles to be sterilized are first wrapped or enclosed in cardboard, paper,
or aluminum containers.
(ii) Then, the materials are arranged to ensure uninterrupted airflow.
(iii) Oven may be pre-heated for materials with poor heat conductivity.
(iv) The temperature is allowed to fall to 40°C, before the removal of sterilized
material

17. Sterilization process:
• Articles to be sterilized must be perfectly dry before placing them
inside to avoid breakage.
• Articles must be placed at sufficient distance so as to allow free
circulation of air in between.
• Mouths of flasks, test tubes and both ends of pipettes must be plugged
with cotton wool.
• Articles such as petri dishes and pipettes may be arranged inside metal
canisters and then placed.
• Individual glass articles must be wrapped in Kraft paper or aluminum
foils

18. Sterilization cycle:
• This takes into consideration the time taken for the articles to reach the
sterilizing temperature, maintenance of the sterilizing temperature for a
defined period (holding time) and the time taken for the articles to cool down.
• Different temperature-time relations for holding time are
• 60 minutes at 160°C,
• 40 minutes at 170°C and
• 20 minutes at 180°C.
• Increasing temperature by 10 degrees shortens the sterilizing time by 50%
• The hot air oven must not be opened until the temperature inside has fallen
below 60°C to prevent breakage of glassware

19. Sterilization control:
• Three methods exist to check the efficacy of sterilization process, namely physical,
chemical and biological.
• Physical: Temperature chart recorder and thermocouple.
• Chemical: Browne’s tube No.3 (green spot, color changes from red to green)
• Biological: 106 spores of Bacillus subtilis varniger or Clostridium tetanion paper strips
are placed inside envelopes and then placed inside the hot air oven.
• Upon completion of sterilization cycle, the strips are removed and inoculated into
thioglycollate broth or cooked meat medium and incubated at 37°C for 3-5 days.
• Proper sterilization should kill the spores and there should not be any growth

20. • Advantages:
• It is an effective method of sterilization of heat stable articles.
• The articles remain dry after sterilization.
• This is the only method of sterilizing oils and powders.
Disadvantages:
• Since air is poor conductor of heat, hot air has poor penetration.
• Cotton wool and paper may get slightly charred.
• Glasses may become smoky.
• Takes longer time compared to autoclave

21. Applications of Dry Heat Sterilization:
• It has huge applications in dry glassware, sterilizing N95 masks,
general instruments, and packaging items in the microbiology
laboratory.
• It also has applications in the pharmaceutical, chemical, food,
textile, and beverage industries.
• Used in curing, drying, baking, and annealing because it can
eliminate moisture from material.
• Used for measurement of mixed liquor suspended solids (MLSS)
• It can also be used to store material at a constant temperature.

22. Moist Heat Sterilization
Moist heat sterilization involves the use of steam in the range of 121-
134˚C.
Steam under pressure is used to generate high temperature needed
for sterilization.
Saturated steam acts as an effective sterilizing agent.

23. Principle of Moist Heat sterilization
Moist heat destroys microorganisms by the irreversible denaturation of enzymes
and structural proteins. The temperature at which denaturation occurs varies
inversely with the amount of water present. Sterilization in saturated steam thus
requires precise control of time, temperature, and pressure.
Pressure serves as a means to obtain the high temperatures necessary to
quickly kill microorganisms. Specific temperatures must be obtained to ensure
microbicidal activity.
Minimum sterilization time should be measured from the moment when all the
materials to be sterilized have reached the required temperature throughout.

24. Autoclave
• Autoclaves use pressurized steam to destroy microorganisms, and are
the most dependable systems available for the decontamination of
laboratory waste and the sterilization of laboratory glassware, media,
and reagents.
• For efficient heat transfer, steam must flush the air out of the
autoclave chamber. Generally the conditions employed are
Temperature upto121-134˚C for 15-20 min under 15lbspressure,based
on type of material used.
• Sterilization can be effectively achieved at a temperature above 100°C
using an autoclave.

25. • Stages of operation
• Air removal and steam admission
• Heating up and exposure
• Drying and cooling

26. • Water boils at 100°C at atmospheric pressure, but if pressure is raised, the
temperature at which the water boils also increases.
• In an autoclave the water is boiled in a closed chamber. As the pressure rises, the
boiling point of water also raises.
• At a pressure of 15 lbs inside the autoclave, the temperature is said to be 121°C.
• Exposure of articles to this temperature for 15 minutes sterilizes them.
• To destroy the infective agents associated with spongiform
encephalopathies(prions), higher temperatures or longer times are used; 135°C
or 121°C for at least one hour are recommended.

27. Monitoring of steam sterilization process
Like other sterilization systems, the steam cycle is monitored by mechanical,
chemical, and biological indicators. Steam sterilizers usually are monitored
using a printout (or graphically) by measuring temperature, the time at the
temperature, and pressure.
Chemical indicators are affixed to the outside and incorporated into the pack
to monitor the temperature or time and temperature. Autoclave indicator tapes
are commercially available and a change in color of the tape suggests proper
sterilization.
Temperature-monitoring probes should be inserted into representative
containers, with additional probes placed in the load at the potentially
coolest and least accessible parts of the loaded chamber. The conditions
should be within ±2 °C and ±10 kPa (±0.1 atm) of the required values. Each
cycle should be recorded on a time-temperature chart or by other suitable
means.

28. Biological Indicators
The effectiveness of steam sterilization is monitored with a biological
indicator using an envelope containing spores of Geobacillus
stearothermophilus (formerly Bacillus stearothermophilus; e.g. ATCC 7953
or CIP 52.81) for which the
D-value (i.e. 90% reduction of the microbial population) is 1.5-2.5 minutes at
121 °C, using about 106 spores per indicator (this is based on a worst-case
scenario that an item may contain a population of 106 spores having same
resistance as that of Bacillus stearothermophilus).
After sterilization is over the strip is removed and inoculated into tryptone
soy broth and incubated at 56°C for 5 days. No growth of Geobacillus
stearothermophilus indicates proper sterilization.

29. Advantages of Steam Sterilization Method
1.Nontoxic to patient, staff, environment
2.Cycle easy to control and monitor
3.Rapidly microbicidal
4.Least affected by organic/inorganic soils among sterilization
processes listed
5.Rapid cycle time
6.Penetrates medical packing, device lumens
Disadvantages of Steam Sterilization Method
1.Deleterious for heat-sensitive instruments
2.Microsurgical instruments damaged by repeated exposure
3.May leave instruments wet, causing them to rust

30. Construction and Operation of Autoclave:
• A simple autoclave has vertical or horizontal cylindrical body with a
heating element, a perforated try to keep the articles, a lid that can
be fastened by screw clamps, a pressure gauge, a safety valve and a
discharge tap.
• The articles to be sterilized must not be tightly packed.
• The screw caps and cotton plugs must be loosely fitted.
• The lid is closed but the discharge tap is kept open and the water is
heated.

31. • As the water starts boiling, the steam drives air out of the discharge tap.
• When all the air is displaced and steam start appearing through the discharge tap,
the tap is closed.
• The pressure inside is allowed to rise up to 15 lbs per square inch.
• At this pressure the articles are held for 15 minutes, after which the heating is
stopped and the autoclave is allowed to cool.
• Once the pressure gauge shows the pressure equal to atmospheric pressure, the
discharge tap is opened to let the air in.
• The lid is then opened and articles removed.
• Articles sterilized: Culture media, dressings, certain equipment, linen etc

32. • Precautions
• Articles should not be tightly packed; the autoclave must not be overloaded.
• Air discharge must be complete and there should not be any residual air trapped
inside
• Caps of bottles and flasks should not be tight.
• Articles must be wrapped in paper to prevent drenching, bottles must not be
overfilled.
.

33. Disadvantages of Moist Heat:
Non-stainless steel metal items corrode in moist heat.
It may damage rubber and plastic items.
This method is not suitable for oils, fats, ointments, oily injections, etc.
Applications of Moist Heat Sterilization:
This method is a most essential biocidal agent. It is used for surgical dressings,
sheets, surgical and diagnostic equipment, containers, closures, aqueous injections,
ophthalmic preparations, etc.

34. Moist heat Dry heat
1. It has water and steam. 1. It does not require water and steam.
2. It is based on protein denaturation and
coagulation.
2. It is based on protein denaturation.
3. Latent heat is liberated when steam
condenses on a cooler surface.
3. It is based on oxidative damage.
4. Spores are killed by exposure to heat at
121°C for 10-30 minutes.
4. Spores are killed by exposure of temperature
at 160°C for 1 hour.
5. This process is under pressure. 5. This process is on direct flame.
6. This process takes less time. 6. This process takes more time.
7. It is mainly dependent on boiling and
autoclaving.
7. It is mainly dependent on flame and
incineration.

35. • Fractional sterilization/tyndallization
• Sometimes, culture media contain substances that are destroyed at 120°C
and for such media autoclaving is not possible.
• An alternative to moist heat is the so-called Fractional sterilization or
Tyndallization. In this method, the medium is exposed to steam at 100°C in
a steamer for 30 min on three consecutive days. After each exposure, the
flasks are incubated at 30°-37°C.
• The basis of this procedure is that most common bacteria are killed at
100°C, except bacterial endospores, certain fungal spores and some yeast.
• During the intervening incubations these spores germinate and become
vulnerable during the next exposure.

36. Boiling water
• Vegetative cells will be destroyed
• But some bacterial spores can withstand
• Disinfection rather than sterilization
• Cannot be used as a sterilization technique in lab

37. Pasteurization
• Pasteurization (or pasteurization) is the process by which heat is
applied to food and beverages to kill pathogens and extend shelf life.
Typically, the heat is below the boiling point of water (100 °C or
212 °F).
• While pasteurization kills or inactivates many microorganisms, it is
not a form of sterilization, because bacterial spores are not destroyed.
Pasteurization extends shelf life via heat inactivation of enzymes that
spoil food.

38. • Pasteurization is the process of applying low heat to kill pathogens
and inactivate spoilage enzymes.
• It does not kill bacterial spores, so pasteurization does not truly
sterilize products.
• Pasteurization is named for Louis Pasteur, who developed a method
to kill microbes in 1864. However, the process has been in use since
at least 1117 AD.

39. • Methods Used in Milk Pasteurization
• High-Temperature Short Time (HTST) Pasteurization
• This type of pasteurization is also known as flash pasteurization.
• Flash pasteurization involves heating milk to 71.7°C/161°F for 15 seconds to
kill Coxiella burnetii, which is the most heat-resistant pathogen in raw milk.
• Since it is technically impossible to bring the milk to that exact temperature, it is
always safe to work with a range of temperatures. To be safe, you can heat the
milk to between 72°C to 74°C for 15 to 20 seconds.
• This will ensure that the milk is heated uniformly to the required temperature.
• This method is most suitable in continuous pasteurization systems.
• Flash pasteurized milk will keep for between 16 and 21 days. For commercial
reasons, some manufacturers intentionally reduce the number of days to push
the products out of the shelves.

40. Low-Temperature Long Time (LTLT) pasteurization
•Here, the temperatures used for pasteurization are reduced to 62.8°C
and held for 30 minutes.
•The prolonged holding period alters the structure of the milk proteins
making them better suited for making yogurt.
•This method is best for batch pasteurization where the milk is held in a
jacketed vat for effective pasteurization.
•There are many designs of batch pasteurizers in the market that are
suitable for both domestic and commercial use.

41. Ultra-High Temperature (UHT) Pasteurization
•This is a completely closed pasteurization method. The product is never
exposed even for a fraction of a second during the entire process.
•It involves heating milk or cream to between 138°C to 150°C for one to
two seconds, then chilling it immediately and aseptically packaging it in a
hermetic (air-tight) container for storage.
•Despite the risk of Millard browning, UHT pasteurization remains the most
popular milk preservation method for safe and stable milk.

42. Significance of Pasteurization
Proper pasteurization is necessary for the following reasons:
•The chief objective of milk pasteurization is to destroy pathogenic bacteria
that could have a public health concern. By destroying these
microorganisms, the product becomes safe for public consumption.
•Secondly, pasteurization eliminates destructive bacteria and enzymes that
could cause spoilage of the product. This leads to the prolonged shelf life of
the milk.
•There is a need to ensure that the product can keep for longer periods
without expensive storage equipment. Pasteurization will eliminate spoilage
bacteria and enzymes and extend the shelf life of the product.

43. Mechanical Methods of Sterilization
Filtration:
This method does not destroy but removes the microorganisms. It is
used for both the clarifications and sterilization of liquids and gases as
it is capable of preventing the passage of both viable and non-viable
particles.
Mechanical Method Of Sterilization to the solution to be sterilized is
pass through depth filter or screen filter which includes
Particulate filter
Microbial filter
Final filter

44. The major mechanisms of filtration are sieving, adsorption, and
trapping within the matrix of the filter material.
Based on the size of the pores of the membrane filter, various types
of microbes are trapped. Like, 0.01 µM is used to trap the smallest
viruses, 0.025 µM is used to trap larger viruses, 0.45 µM is used to
trap the largest bacteria, 1.2 µM is used to trap protozoa and
smallest unicellular algae, etc.

45. Some observations are essential for successful sterilization by
filtration:
1. The whole apparatus must be sterile.
2. An aseptic technique should be followed to minimize the risk of
contamination.
3. The assembly of the filtration unit should be so adjusted that there is
minimal exposure of filtrate to the atmosphere.
4. The filter selected must be fine enough to obstruct the passage of all
bacteria.

46. Four steps for sterilization by filtration:
1. Filtration of the solution through one of the bacteria-proof filters.
2. Aseptic distribution of the filtered solution into the previously sterilized final
containers.
3. Aseptic closure of the containers.
4. Performing the sterility test.

47. 1. Ceramic filters:
These are also known as filter candles, made of porcelain or kieselguhr,
and are available in a range of pore sizes. Kieselguhr filters are usually
softer than the porcelain type.
The candle is placed in the solution to be sterilized and its opening is
attached to the vacuum system. When the vacuum has applied the
pressure inside the candle is decreases.
Due to the difference in pressure between the outside and inside the
candle, the candle moves into the candle. The filtrate is collected in a
sterile container. The main disadvantage of ceramic filters is their tendency
to absorb materials from aqueous solutions.

48. Seitz filter:
It consists of two parts. The lower part holds a perforated disc and the
upper part is a compressed asbestos sheet.
Two parts are joined together with the help of nuts.
There is a valve on the upper part through which a vacuum is applied. Due
to the fibrous nature of asbestos pads, they may shed fibers into the filtrate
and also absorb drugs from the solution.
Hence, a few ml of filtrate should always be rejected and sintered glass
disc may also be fixed in the filtration unit immediately after the Seitz filter.

49. 3. Sintered glass filters:
• They are made from borosilicate glass. The glass is finely powdered
• The particles of the required sizes are separated and then packed into disc
molds. These discs are fused to funnels of suitable shape and size.
• Sintered glass filters are available in different pore sizes and are numbered
accordingly. For bacteria-proof filtration number 5 or 3 is used. The filtration is
carried out under reduced pressure.
• Sintered glass filters do not absorb the medicaments from the solution. These
filters are made from borosilicate glass, so a change of pH of the solution not
occurred.

50. 4. Sintered metal filters: They are the metallic counterpart of sintered
glass filters. These are usually made from stainless steel. They have the
advantage of having greater mechanical strength.
5. Membrane filters: These are made of cellulose acetate or cellulose
nitrate. These are fixed in metallic holders similar to those used with
asbestos pads.
The pore size in the membranes lies in the range of 100-150 µ. They are
also called millipores filters. They are suitable for sterilizing aqueous and
oily solutions but are not suitable for organic solvents like alcohol, ketones,
esters, or chloroform.

51. 6. Air filter: (HEPA filter): It is high-efficiency particulate air or originally called
High-Efficiency Particulate Absorber (HEPA). It is used to describe filters that
can trap 99.97 percent of particles that are 0.3 microns (Fig). Air particles are
circulated through the HEPA filter in four directions viz.
1.Interception:- Larger particles such as dust and dirt, etc. whose size is more
than 0.3 microns will come in contact with the glass fibers and then get
trapped.
2.Inertial Impaction:- When air goes through the filter it can easily divert its
direction based on the path of flow. On the other hand, particles tend to move
in the same path due to the phenomena known as the inertia of motion. So,
due to this glass fibers will trap the particles.

52. 3. Diffusion:-
Particles whose size is less than or equal to 0.1 microns (e.g. viruses) follow
the Brownian motion. In other words, there are molecules in the air.
When the size and mass of the particles are very small, the collision of
particles with air molecules will cause the particles to move in a random
direction.
Since the particles of a size less than 0.1 microns move in a zigzag direction,
they will be easily trapped by the filter.

53. Advantages:
•The method is suitable for the sterilization of thermolabile medicaments,
such as blood products, insulin, and enzymes.
•All types of bacteria i.e., living as well as dead, are removed from the
preparation.
•Both clarification and sterilization are done side by side.
•It is an excellent method for the rapid supply of a small volume of a
parenteral solution in an emergency.

55. Applications:
This filtration method is useful for sterilization of parenteral solutions
containing thermolabile medicaments without any decomposition e.g. insulin,
blood serum, and other products containing protein matters, heat-sensitive
injections, biological products, etc.

56. Cold Sterilization Definition – It is a process in which sterilization is carried
out at low temperatures with the help of chemicals, filters, radiation and all
other means excluding high temperatures. It is done for products that
contain heat-sensitive ingredients and yet require sterilization

57. SONIC AND ULTRASONIC VIBRATIONS:
• Sound waves of frequency >20,000 cycle/second kills bacteria and some
viruses on exposing for one hour.
• Microwaves are not particularly antimicrobial in themselves, rather the
killing effect of microwaves are largely due to the heat that they
generate.
• High frequency sound waves disrupt cells.
• They are used to clean and disinfect instruments as well as to reduce
microbial load.
• This method is not reliable since many viruses and phages are not
affected by these waves

58. Radiation Sterilization
• Many types of radiation are used for sterilization like
electromagnetic radiation (e.g. gamma rays and UV light),
particulate radiation (e.g. accelerated electrons).The major target for
these radiation is microbial DNA.
• Radiation sterilization with high energy gamma rays or accelerated
electrons has proven to be a useful method for the industrial
sterilization of heat sensitive products.

59. • Two types of radiation are used, ionizing and non-ionizing.
• Non-ionizing rays are low energy rays with poor penetrative power
while ionizing rays are high-energy rays with good penetrative
power. Since radiation does not generate heat, it is termed "cold
sterilization".
• In some parts of Europe, fruits and vegetables are irradiated to
increase their shelf life up to 500 percent.

60. Ionizing radiations
Ionizing radiation is an excellent agent for sterilization/disinfection, it
kills organisms without increasing the temperature; so aptly called
cold sterilization. It destroys bacterial endospore and vegetative
cells, both eukaryotic and prokaryotic; but not always effective
against viruses.
Mechanism of Sterilization by Ionizing Radiations
When ionizing radiation collides with particles, they produce electrons (e−)
and other reactive molecules such as hydroxyl radicals (•OH), and hydride
radicals (H•). Each of these reactive molecules is capable of degrading and
altering biopolymers such as DNA and protein. Breakage of DNA and
degradation of enzymes lead to the death of the irradiated cells

61. Several sources of ionizing radiation are available, including X-ray
machines, cathode ray tubes (electron-beam radiation), and radioactive
nuclides (sources of gamma/x-rays).
X-Rays
X-rays are lethal to microorganisms and higher forms of life but are rarely used in
sterilization because their production is expensive and efficient utilization is
difficult (since radiations are given off in all directions from the point of origin).
Gamma Rays: Gamma rays are attractive for use in commercial sterilization of
materials of considerable thickness or volume, eg., packaged food or medical
devices.
Cathode Rays (Electron-Beam Radiation)
Cathode rays or electron-beams can sterilize materials at room temperature with
brief exposure. They have limited penetrating power and are used for the
sterilization of surgical supplies, drugs, and other materials.

62. Uses of Ionizing Radiations
The major method in use for radiation sterilization is gamma irradiation.
Gamma radiation is used in the sterilization of;
•Disposables such as plastic syringes, infusion sets, catgut sutures, catheters,
gloves, and adhesive dressings before use.
•Bone, tissue grafts, antibiotics, and hormones.
•Irradiation of food (permitted in some countries).

63. Advantages of Ionizing Radiations
1.High penetrating power: products can be processed in their fully sealed,
final packaging thus limiting the risk of contamination following sterilization.
2.Rapidity of action: saves and efforts.
3.Temperature is not raised: compatible with temperature-sensitive materials,
such as pharmaceuticals and biological samples.
4.Flexibility: can sterilize products of any phase (gaseous, liquid, or solid
materials), density, size, or thickness.
Disadvantages
•Capital costs are high and specialized facilities are often needed e.g. for
gamma irradiation.
•Use of gamma radiation requires handling and disposal of radioactive material.
•Not compatible with all materials and can cause breakdown of the packaging
material and/or product. For example, Common plastics such as polyvinyl
chloride (PVC), acetal, and polytetrafluoroethylene (PTFE) are sensitive to
gamma radiation.

64. Sterilization/disinfection control: The efficacy of ionizing radiation is tested
using Bacillus pumilus.
Non-ionizing Radiation:
Non-ionizing radiations are lethal but do not penetrate glass, dirt, films,
or water; hence their use is restricted to disinfection of clean surfaces in
operation theaters, laminar flow hoods and water treatment. The
recommended dose is 250-300 nm wavelength, given for 30 minutes.
Examples of non-ionizing radiation include infrared and ultraviolet
radiation.

65. Infra-Red Radiation
Infra-red rays are low energy type electromagnetic rays, having
wavelengths longer than those of visible light. They kill microorganisms by
oxidation of molecules as a result of heat generated. Infra-red rays are
used for the rapid mass sterilization of syringes and catheters.
Ultraviolet Light (UV) Sterilization
Sunlight is partly composed of UV light but most shorter wavelengths of
light are filtered out by the ozone layer. There are three types of UV
radiation; UVA, UVB, and UVC, classified according to their wavelength.
Short-wavelength UVC is the most damaging type of UV radiation.
Mechanisms of UV Sterilization
Many cellular materials, including nucleic acids, absorb ultraviolet light. It
causes the bonding of two adjacent pyrimidines, i.e., the formation of
pyrimidine dimer, resulting in the inhibition of DNA replication. This leads
to mutation and death of exposed organisms

66. Uses of UV Sterilization
UV lights are useful for disinfecting surfaces, air, and water that do not absorb
the UV rays. Certain types of UV lights can kill the flu (influenza) virus.
Ultraviolet radiation is used for disinfecting enclosed areas such as bacterial
laboratory, nurseries, inoculation hood, laminar flow, and operation theaters.
For example, laboratory biological cabinets all come equipped with a
“germicidal” UV light to decontaminate the surface after use.
Disadvantages
Damages skin and eyes: Conventional UV light can penetrate and damage skin and
also cause cataracts.
Does not penetrate paper, glass, and cloth.

67. What is Chemical Sterilization?
•Chemical Sterilization is the process of removal of microorganisms by the
use of chemical bactericidal agents.
•Even if physical methods of sterilization are more appropriate for effective
sterilization, it is not always appropriate to use for heat-sensitive materials
like plastics, fiber optics, and biological specimens.
•Under such conditions, chemical either in liquid or gaseous state can be
used for sterilization. However, it is crucial to ensure that the materials
undergoing sterilization are compatible with the chemical being used.
•Besides, it is important to adopt safety rules in the workplace safety during
the use of chemical agents.
•The chemical method of sterilization can be categorized as liquid and
gaseous sterilization.

69. Ideal Properties of Chemical Sterilization:
•A wide spectrum of activity.
•Active in presence of organic matter.
•Effective in acid as well as alkaline media.
•High penetration power.
•Stable.
•Speedy action.
•Compatible with other antiseptics and disinfectants.

70. 1. Gaseous Sterilization
•Gaseous sterilization involves the process of exposing equipment or
devices to different gases in a closed heated or pressurized chamber.
•Gaseous sterilization is a more effective technique as gases can pass
through a tiny orifice and provide more effective results.
•Besides, gases are commonly used along with heat treatment which
also facilitates the functioning of the gases.
•However, there is an issue of release of some toxic gases during the
process which needs to be removed regularly from the system.
•The mechanism of action is different for different types of gases.

71. Gas sterilization involves exposing equipment to chemical gases in an enclosed
heated or pressurized chamber.
Liquid sterilizing agents are impractical or ineffective for sterilizing items.
Gaseous agents are more effective sterilants because they can permeate small
openings and crevices easily.
Gas chemicals also sterilize faster than liquids because they usually are combined
with high heat. The gas residue is also easier to remove from sterilized articles
but requires more expensive equipment.

72. 2. Liquid Sterilization
•Liquid sterilization is the process of sterilization which involves the submerging
of equipment in the liquid sterilant to kill all viable microorganisms and their
spores.
•Although liquid sterilization is not as effective as gaseous sterilization, it is
appropriate in conditions where a low level of contamination is present.
•Different liquid chemicals used for liquid sterilization includes the following:

73. Mode of Action of Chemical Disinfectants:
•It acts by protein coagulation.
•Disruption of cell membrane resulting in exposure, damage, or loss of the
contents.
•Removal of free sulfhydryl group essential for the functioning of enzymes.
•Substrate competition: A compound resembling the essential substrate of
the enzyme diverts the enzymes necessary for the metabolism of the cell
and causes cell death.

74. Alcohol:
Principle: It is based on denaturing coagulating proteins and dissolving membrane
lipid of microorganisms. propyl, butyl, amyl alcohols
Advantages:
It is inexpensive and non-toxic.
Widely available.
Rapidly effective.
Active against bacteria, viruses, mycobacterium.
Disadvantages:
Not effective against bacterial spores.
Not effective with organic materials.

75. Aldehydes:
Formaldehyde, glutaraldehyde
Formaldehyde is also a common aldehyde which is a gas in high concentration but at
room temperature,
it polymerizes and forms a solid substance. Formaldehyde solution as well as in
gaseous form is used for sterilization
and disinfection of enclosed areas respectively where vegetative cells are killed
more quickly than spores.
Formalin and paraformaldehyde are two important sources of formaldehyde when it
is used for gaseous disinfection.
Formalin is the aqueous solution of 40% formaldehyde. Formalin is extensively used
for the
preservation of specimens, inactive viruses, and bacteria in vaccines.

76. Principle:
Aldehyde combines with important proteins and nucleic acids of the bacterial
cells. These interactions of an aldehyde with these cellular substances produce
antimicrobial action. Formaldehyde inactivates microorganisms by alkylating
the amino acids and sulfhydryl groups of proteins and ring nitrogen atoms of
purine bases.
Advantage:
•They have good activity against spores, viruses, and fungi.
Disadvantages:
•They are toxic.
•They need a long exposure time for the action, a minimum of 3 hours.
•Freshness and pH are critical.
•They are irritant and carcinogenic.
•Glutaraldehyde is moderately active against TB.

77. Phenolics and Phenolic Agents:
Phenols are used as a standard chemical to determine the
antimicrobial activity of other similar chemical compounds by the
Phenol coefficient method.
This method is used for evaluating the effectiveness of disinfectants.
Especially Staphylococcus aureus or Salmonella typhi are used for
testing.

78. hexylresorcinol is a phenolic derivative that is marketed in a solution
of glycerin and water, acts as a strong surface tension reductant as
well as high bactericidal activity. Pure crystalline phenol is colorless,
its 5% aqueous solution is used as a disinfectant of sputum, urine,
feces, etc.
Quaternary ammonium compounds e.g. cetrimide are used in
combination with other agents and acts as good detergent
properties. Some other examples of phenolic compounds are amyl
phenol, benzyl-4-chlorophenol, etc.

79. Principle:
They are having various modes of action. They act by destroying
plasma membranes and denature proteins. They also affect plasma
membrane, inactivate enzymes, and denature proteins. Some
phenolics are mild enough for use as antiseptics.
Advantages:
•They are stable, persist for long times after application, and remain
active in the presence of organic compounds.
•They are active against a wide range of organisms.
•They have good antimicrobial activity and are rapid bactericidal.
•They are more active in acid pH.
•They remain active on surfaces long after application.

80. Disadvantages:
•They are having caustic effects on the skin.
•They are having systemic toxicity.
•They are not effective against spores.
•They are not effective at low temperatures.
•They are incompatible with non-ionic and cation surfactants.
•They have a disagreeable odor

81. Halogens, mainly, iodine and chlorine are used as antimicrobial
agents. Iodine is used as an antiseptic against all microbes, fungi, and
viruses. It inhibits protein synthesis and oxidizes the sulphydryl group
of amino acids.
These agents can irreversibly oxidize and inactivate essential metabolic
compounds like proteins with sulphhydryl groups. It also has halogenations
action of tyrosine unit of enzymes and other cellular proteins require
tyrosine for activity.
Advantages of Iodine compounds:
Iodine compounds are effective against gram-positive bacteria.
They produce residual activity.
They retain microbial action in the presence of organic debris.
They are available in solutions, sprays, and in-gel preparations.

82. Disadvantages of iodine compounds:
•Iodine compounds when used alone are major irritants.
•They are weak against mycobacteria, fungi, viruses.
•They are absorbed into the skin and can be toxic.
•They can cause 1st and 2nd degree burns.
•Chlorinated compounds show antimicrobial activity due to the formation of
hypochlorous acid in water. This hypochlorous acid is further decomposed into
nascent oxygen which shows a strong oxidation reaction.

83. Heavy Metals:
Most heavy metals have a detrimental effect on microorganisms.
Compounds such as mercury, silver, copper are more effective against
microorganisms. Mercury compounds such as mercuric chloride, mercurous
chloride, mercuric oxide are in dilution form act as bactericidal.
Heavy metals and their compounds have antimicrobial activity due to the
combination with cellular proteins and inactivating their function. Example:
mercuric chloride inhibits the action of the enzyme by acting on the sulfhydryl
group of enzymes and form inactive enzymes. A high concentration of heavy metal
salt coagulates cytoplasmic proteins of microorganisms that cause the death of the
cells.

84. Advantages:
•They are powerful biocides.
•They form a complex with proteins
of microorganisms and converts
inactive form of microorganisms.
Disadvantage:
•They are highly toxic to the animals.

85. Surface Active Agents:
Soaps and detergents are mainly used as a surfactant. It depends on
the alkali content in the soap how they show their action.
They act as germicidal for pneumococci, streptococci, gonococci etc.
Detergents are ionized in water and are made up of fats. They quickly
dissolve in cold and hard water
Quaternary ammonium compounds are important cationic detergents
that act as germicidal as well as bactericidal, mainly against Gram-
positive bacteria. They also show fungicidal activity and destroy the
pathogenic protozoa.
Benzalkonium chloride is used as a disinfectant. Diaparene chloride
acts as a bacteriostatic against Brevibacterium ammoniagenes.

86. Principle:
They denature proteins, interfere with glycolysis, and damage the membrane
of the microorganisms. They also damage the cell cytoplasmic membrane
and alter the cell structure.
Advantages:
•They are effective against vegetative bacteria.
•They are widely available.
•They are less expensive.
•They are non-irritant.
Disadvantages:
They are ineffective against spores.
They are not effective against non-enveloped viruses.
They may become contaminated.
They are easily inactivated by the presence of anionic detergents, soaps, and hard
water.

87. Dyes:
There are two types of dyes exhibiting antimicrobial activity viz.
triphenylmethane and acridine. Triphenylmethane dyes such as brilliant
green, crystal violet, malachite green inhibit gram-positive bacteria and
fungi. Malachite green is used to inhibit Staphylococcus aureus.
Acridine dyes are the compounds that are derived from acridine such
as acriflavine, tryptoflavine, proflavine, etc. They inhibit bacteria
especially Staphylococci and gonococci. Gonococci especially are
inhibited by tryptoflavine. Acriflavine, proflavine are used to inhibit the
growth of Gram-negative bacteria.
Principle: They have their inhibitory effect by interfering with cellular
oxidation processes.

88. Advantages:
•They are bacteriostatic in high dilution.
•They are more active against Gram-positive bacteria.
Disadvantages:
•They are less active against Gram-negative bacteria.
•They have low bactericidal activity.

89. Gaseous Agents For Sterilization
Some important medical devices are required for sterilization but not
possible through heat sterilization due to damage to the materials. In such
cases, gaseous sterilization is required. Like Plastic syringes, blood
transfusion apparatus, plastic pipettes, Petri dishes, etc. The main gaseous
agents are used for sterilization are ethylene oxide, formaldehyde, beta-
propiolactone, etc. They act on the principle of denaturing proteins and
DNA by cross-linking functional groups. But this method has some
disadvantages.

90. Disadvantages:
•Gas can be hazardous to people.
•They are often highly explosive.
•Some time gases are extremely poisonous.
•They are potentially carcinogenic.
Factors affecting gaseous sterilization:
The efficiency of the sterilization method is influenced by the concentration
of ethylene oxide.
The humidity of the sterilizing atmosphere.
The temperature of sterilization.
Time of exposure.
Physical nature and permeability of the load.
Atmospheric preconditioning of the load before sterilization

91. Formaldehydes:
It is also a group of alkylating agents. It inactivates
microorganisms by alkylating the amino acid and sulfhydryl
groups of proteins and ring nitrogen atoms of purine bases. It is a
gaseous disinfectant and biocide.
It is a strong, broad-spectrum disinfectant and biocide that can
kill bacteria, viruses, fungi, and endospores. It is very irritating to
live tissues and also carcinogenic, therefore it is not used as an
antiseptic.

92. Ethylene Oxides:
It is a type of alkylating agent that is used for gaseous
sterilization. It is highly penetrating and can sterilize items within
plastic bags such as catheters, disposable items in laboratories
and clinical settings.
•It is gaseous above 10.8°C.
•Ethylene oxide has high antimicrobial activity, it kills even
endospores.
•It is used for the sterilization of heat-sensitive materials such as
spices, oils, plastics, etc.
•Ethylene oxide is used in formulation with CO2 as Freon (CClFe

93. β-Propiolactone:
It binds to DNA, thereby inactivating it. It is a clear liquid with a strong odor
and can kill endospores. It has been used in either liquid form or as a
vapor for the sterilization of medical instruments and tissue grafts, and it is
a common component of vaccines, used to maintain their sterility. It is also
used for the sterilization of nutrient broth, as well as blood plasma, milk,
and water.
•It is gas above 15.5°C.
•Penetration power of β-propiolactone is less than ethylene oxide but it is
more active in killing microorganisms.
•Due to its carcinogenic effects, it is not commonly used.

94. Applications of Gaseous Sterilization:
•This method is used for sterilizing thermolabile substances like
hormones, proteins, various heat-sensitive drugs, etc.