Sterilisation Physical agent

Question 1

Define Sterilization and Disinfection

Answer 1

Sterilization is defined as the process by which an article, surface or medium is freed of all living microorganisms (Sporing and non sporing bacteria, viruses, fungi and protozoa) either in the vegetative or spore state.

Disinfection means the destruction or removal of all pathogenic organisms, or organisms capable of giving rise to infection

Question 2

What are the uses of sterilization and Disinfection?

Answer 2

in microbiology for preventing contamination by extraneous organisms,
in surgery for maintaining asepsis,
in food and drug manufacture for ensuring safety from contaminating organisms.

Question 3

Explain the terms a. Antisepsis b. Antiseptics c. Bacteriostatic agents d. Decontamination

Answer 3

antisepsis is used to indicate the prevention of infection, usually by inhibiting the growth of bacteria in wounds or tissues.

Antiseptics are chemical disinfectants, which can be safely applied to skin or mucous membrane and are used to prevent infection by inhibiting the growth of bacteria.

Bacteriostatic agents only prevent the multiplication of bacteria, which may however remain alive.

Decontamination refers to the process of rendering an article or area free of danger from contaminants, including microbial, chemical, radioactive and other hazards.

Question 4

What are the Physical agents of sterilisation and disinfection?

Answer 4

1.Sunlight
2. Drying
3. Dry heat: flaming, incineration, and hot air.
4. Moist heat: pasteurization, boiling, steam under normal pressure, and steam under pressure.
5. Filtration: candles, asbestos pads, membranes.
6. Radiation.
7. Ultrasonic and sonic vibrations.

Question 5

Name the Chemicals agents used in sterilisation and disinfection

Answer 5

Ethylene oxide, Formaldehyde, Betapropiolactone , Alcohols, Aldehydes, Dyes, Halogens, Phenols, Surfaceactive agents, Metallic salts etc.

Question 6

What is Wet sterilization?

Answer 6

Sterilisation using steam or water at high temperature

Question 7

What is Dry sterilisation?

Answer 7

sterilisation using hot air

Question 8

Define thermal death time

Answer 8

minimum time required to kill a suspension of organisms at a predetermined temperature in a specified environment.

Question 9

Explain various dry heat sterilisation methods

Answer 9

Flaming:
Inoculating loop or wire, the tip of forceps and searing spatulas are held in a Bunsen flame till they become red hot.Inoculation loops carrying infective material may be dipped in a disinfectant before flaming to prevent spattering.

Incineration
excellent method for safely destroying materials such as contaminated cloth, animal carcasses, and pathological materials that can be burnt.

Hot air oven
electrical devices
developed by Louis Pasteur.
uses dry heat to sterilize articles
most widely used method
is fitted with a fan to ensure even distribution of air and elimination of air pockets.
heated by electricity, with heating elements in the wall of the chamber.
The material to be sterilised :glass wares, forceps, scissors, scalpels, all-glass syringes, swabs , pharmaceutical products such as liquid paraffin, dusting powder, fats and grease in small containers.

Question 10

11. Explain various moist heat sterilisation methods

Answer 10

Temperatures below 1000
C:
Pasteurization of milk: originally employed by Louis Pasteur.employed in food and dairy (milk and milk products) industry.There are two methods:
Holder method: The temperature employed is 630
C for 30 minutes
❖ Flash process: The temperature
employed is 720
C for 15-20 seconds
❖ Both methods are followed by cooling
quickly to 100
C or lower
❖ Destroy all nonsporing pathogens
❖ Coxiella burnetti may survive the holder method

Vaccine bath: Vaccines of nonsporing
bacteria are heat inactivated in special
vaccine bath at 60 o C for 1 hour

Serum bath: Serum or body fluids can be
sterilized by heating for one hour at 560
C
in a water bath for several days

Inspissation: Media such as LJ are
sterilized at 80-85 0
C for half an hour on
three successive days in an inspissator
• On the first day, the vegetative bacteria
would die and those spores that
germinate by next day are then killed the following day.

Temperature at 100 0
C:
Boiling:
▪ Vegetative bacteria are killed immediately
at 90-1000
C
▪ Material should be immersed in the water
and boiled for 10-30 minutes
Boiling is not recommended for the
sterilization of instruments used for
surgical procedures
▪ Autoclaving at high pressure can destroy
spores and ensure sterilization

Steam at atmospheric pressure (1000
C):

Free steam:
❖ Used to sterilize culture media, which
may decompose if subjected to higher
temperatures
❖ A single exposure of ninety minutes
usually ensures sterilization
❖ Ex: Koch or Arnold steamer

Tyndallization: (Intermittent heating)
• For media containing sugars or gelatin an
exposure of 1000
C for 20 minutes on three
successive days is used
• Principle is that the first exposure kills all
vegetative bacteria
• Spores, since they are in a favorable
medium, will germinate
• Will be killed on the subsequent occasions
• May fail with spores of certain anaerobes
and the thermophiles

Moist heat above 100 0 C (Steam under
pressure):
Autoclave:
A strong, pressurized,Steam heated vessel
for Sterilization or cooking
Principle:
• Water boils when its vapour pressure
(pressure exerted by a vapour on the
surface of the liquid) equals that of
surrounding atmosphere
• Hence when pressure inside a closed
vessel increases, the temperature at
which water boils also increases
• Saturated steam thus produced has
penetrative power
• When steam comes into contact with a
cooler surface it condenses to water
Gives up its latent heat (The quantity of
heat absorbed or released by a substance
undergoing a change of state) to that
surface

• Process continues till the temperature of
that surface is raised to that of steam
• This result in the denaturation and
coagulation of protein inside the
microorganism
• Usual holding time is 121 0C for 15
minutes at 15 PSI
Simple laboratory Autoclave:
• Consists of a vertical or horizontal
cylinder of gunmetal or stainless steel, in
a supporting sheet iron case
• Lid or door is fastened by crew clamps
• Made airtight by an asbestos washer
Autoclave has on its lid or upper side:
• A discharge tap for air and steam
• A pressure gauge
• A safety valve
• Heating is by
gas or electricity

Question 11

What are the factors which affect the sterilisation? How it affect sterilisation?

Answer 11

Nature of heat: Moist heat is more
effective than dry heat.
• Temperature and time: temperature and
time are inversely proportional
• 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: Protein,
sugars, oils and fats increase the time
required for sterilisation
• The presence of disinfectants and high
acid or alkaline pH speed up the bacterial
killing

Question 12

What is Pasteurization?

Answer 12

This process was originally employed by Louis Pasteur. Currently this procedure is employed in food and dairy (milk and milk products) industry. There are two methods.
Holder method: The temperature
employed is 630
C for 30 minutes
❖ Flash process: The temperature
employed is 720
C for 15-20 seconds
❖ Both methods are followed by cooling
quickly to 100
C or lower
❖ Destroy all nonsporing pathogens
❖ Coxiella burnetti may survive the holder
method

Question 13

What is Vaccine bath?

Answer 13

Vaccines of nonsporing
bacteria are heat inactivated in special
vaccine bath at 60 o C for 1 hour

Question 14

What is Serum bath?

Answer 14

Serum bath: Serum or body fluids can be
sterilized by heating for one hour at 560
C
in a water bath for several days

Question 15

What is Inspissation?

Answer 15

Media such as LJ are
sterilized at 80-85 0
C for half an hour on
three successive days in an inspissator
• On the first day, the vegetative bacteria
would die and those spores that
germinate by next day are then killed the
following day.

Question 16

What is Tyndallization?

Answer 16

For media containing sugars or gelatin an
exposure of 1000
C for 20 minutes on three
successive days is used
• Principle is that the first exposure kills all
vegetative bacteria
• Spores, since they are in a favorable
medium, will germinate
• Will be killed on the subsequent occasions
• May fail with spores of certain anaerobes
and the thermophiles

Question 17

What is Flaming and its uses?

Answer 17

Inoculating loop or wire
✔ Tip of forceps
✔ Searing spatulas are held in a Bunsen
flame till they become red hot
✔ Inoculation loops carrying infective
material may be dipped in a disinfectant
before flaming

Question 18

What is Incineration ?

Answer 18

Excellent method for safely destroying
materials such as:
ø Contaminated cloth
ø Animal carcasses
ø Pathological materials
ø Polystyrene materials emit clouds of
dense black smoke
ø Hence should be autoclaved in
appropriate containers

Question 19

What is boiling? What is its disadvantage?

Answer 19

Vegetative bacteria are killed immediately
at 90-1000
C
▪ Material should be immersed in the water
and boiled for 10-30 minutesBoiling is not recommended for the
sterilization of instruments used for
surgical procedures
▪ Autoclaving at high pressure can destroy
spores and ensure sterilization

Question 20

What is free steam sterilisation? Give one example

Answer 20

Used to sterilize culture media, which
may decompose if subjected to higher
temperatures
❖ A single exposure of ninety minutes
usually ensures sterilization
❖ Ex: Koch or Arnold steamer

Question 21

Write a note on hot air oven

Answer 21

It is an electrical devices used in
sterilization
• Developed by Louis Pasteur
• The oven uses dry heat to sterilize articles
• Most widely used method of sterilization
by dry heat
• Oven is fitted with a fan to ensure even
distribution of air and elimination of air
pockets
• The oven is usually heated by electricity,
with heating elements in the wall of the
chamberThe material to be sterilised under this
method include
• Glass wares
• Forceps
• Scissors
• Scalpels
• All-glass syringes
• Swabs
• Pharmaceutical products such as
• Liquid paraffin
• Dusting powder
• Fats and greaseA holding period of 1600
C for one hour is
employedHow to operate Working of hot air oven:
• Materials to be sterilised is kept inside the
oven
• Then adjust temperature control to the
temperature of 1600
C
• Close the doors and switch on the
machineThe total processing time of a hot air oven
consists of three periods:
• The heating up period: which is the time it
takes the materials to heat up to the
required temperature.Apx. 1hr
• Holding periods: which is the time at
which sterilisation is taking place at a
known temperature e.g. 1600
C for one
hour or 1500
C for 2 hours
• The cooling down time: The temperature
should be allowed to cool down by itself
and this may takes about 2 hours.

Question 22

What are the advantages and disadvantages of hot air oven?

Answer 22

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

Question 23

What is an autoclave? Explain the Structure, working and principle of Autoclaves

Answer 23

A strong, pressurized,Steam heated vessel
for Sterilization or cookingPrinciple:
• Water boils when its vapour pressure
(pressure exerted by a vapour on the
surface of the liquid) equals that of
surrounding atmosphere
• Hence when pressure inside a closed
vessel increases, the temperature at
which water boils also increases
• Saturated steam thus produced has
penetrative power
• When steam comes into contact with a
cooler surface it condenses to waterGives up its latent heat (The quantity of
heat absorbed or released by a substance
undergoing a change of state) to that
surface

• Process continues till the temperature of
that surface is raised to that of steam
• This result in the denaturation and
coagulation of protein inside the
microorganism
• Usual holding time is 121 0C for 15
minutes at 15 PSISimple laboratory Autoclave:
• Consists of a vertical or horizontal
cylinder of gunmetal or stainless steel, in
a supporting sheet iron case
• Lid or door is fastened by crew clamps
• Made airtight by an asbestos washerAutoclave has on its lid or upper side:
• A discharge tap for air and steam
• A pressure gauge
• A safety valve
• Heating is by
gas or electricityProcedure:
❖ Sufficient water is put in the cylinder
❖ Material to be sterilized is placed on the
tray
❖ Autoclave is heated
❖ Lid is screwed tight
❖ Discharge tap is kept opened for some
time to remove air from the autoclaveSteam air mixture is allowed to escape
freely till all the air inside has been
displaced
❖ Then the discharge tap is closed
❖ Safety valve is adjusted to the required
pressure (15 PSI)
❖ Steam pressure rises inside
❖ When it reaches the desired set level, the
safety valve opensThe excess steam escapes to maintain the
required pressure
❖ From this point, the holding period is
calculated
❖ When the holding period is over, the heater
is turned off
❖ Autoclave allowed to cool till the pressure
gauge indicates that the pressure inside is
equal to the atmospheric pressure
❖ The discharge tap is opened slowly and air
is let into the autoclave

Question 24

What are the precaution we have to take while operating an autoclave

Answer 24

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 (Wet thorougly).
• Bottles must not be overfilled.If the tap is opened when the pressure inside
If the tap is opened when the pressure inside
is high:
Liquid media will tend to boil violently Spill from their containers
If opened after the pressure inside has fallen below atmospheric pressure:
Excessive amount of water would have evaporated and lost from the media

Question 25

What are the advantages and disadvantages of autoclaves?

Answer 25

Advantage: Very effective way of sterilization, quicker than hot air oven •Disadvantages: Drenching and wetting or articles may occur, trapped air may reduce the efficacy, takes long time to cool

Question 26

What are the uses of Autoclaves

Answer 26

To sterilize: •Media •Instruments •Dressing laboratory articles •Pharmaceutical products •Infectious materials etc

Question 27

What are the common reasons for failure to Sterilize by autoclaving?

Answer 27

Failure to achieve the correct temperature •Failure to hold the process for the correct time •Improper evacuation of air •Tight load (Over load)

Question 28

What is meant by sterilisation control? Explain the methods used for autoclaves and Hot air oven

Answer 28

Sterilization control: Biological method: ❖ Paper strips impregnated with 106 spores of a non-toxigenic strain of Clostridium tetani are placed in envelopes and inserted into suitable packs ❖ After sterilization, the strips are removed and inoculated into thioglycollate or cooked meat media ❖ Incubated for sterility test under strict anaerobic conditions for five days at 37° C
Chemical method: ❑Browne’s tube (green spot) is available for dry heat ❑Convenient for routine use ❑After proper sterilization a green colour is produced (after 60 minutes at 160° C)
Thermocouples may also be used periodically

Sterilization control: Testing the efficacy of an autoclave: Physical methods: a. By thermocouples b. By pressure gauge
Chemical Methods: •By chemical indicator strips which change colour from red to green after proper sterilization
Biological Methods: ❖By using strips impregnated with spores of bacillus sterothermophilus ❖Spore strips are introduced into the autoclave ❖After sterilization they are tested for sterility

Question 29

What is filtration? Write a short notes on membrane filtration

Answer 29

Filtration helps to remove microorganisms from heat labile liquids
Membrane filters: •Made of cellulose esters or other polymers Used for: 1. For water purification and analysis 2.Sterilization and sterility testing 3.For the preparation of solutions for parenteral use 4.For sterilization of serum, toxins, antitoxins and antibiotic solutions

Question 30

Explain sterilisation by Radiation

Answer 30

Two types of radiation are used for sterilization 1. Non ionizing radiation : low energy rays with poor penetrative power UV Rays from sun 2. Ionising radiation: ionizing rays are high-energy rays with good penetrative powerNon ionizing radiation: • Ex: Ultraviolet rays. • Here electromagnetic rays with wavelengths longer than those of visible light are used. • These are absorbed as heat. • Hence Infrared radiation can be considered as a form of hot air sterilization. • UV rays are generated using a high-pressure mercury vapor lamp. • UV rays inhibit DNA replication. Microorganisms that are exposed to the effective UV radiation are inactivated within seconds •UV rays don’t kill spores •UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. •Disadvantages of using uv rays include low penetrative power, limited life of the UV bulb, some bacteria have DNA repair enzymes that can overcome damage caused by UV rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes

Ionizing radiation: •Ionizing rays are of two types, particulate and electromagnetic rays •Electron beams are particulate in nature while gamma rays are electromagnetic in nature •High speed electrons beams are produced by a linear accelerator from a heated cathode •Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticalsSterilization is accomplished in few seconds • Unlike electromagnetic rays, the instruments can be switched off • Disadvantage includes poor penetrative power and requirement of sophisticated equipment • Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain radioactive isotopes • They have more penetrative power than electron beam but require longer time of exposureThese high-energy radiations damage the nucleic acid of the microorganism •Kills all bacteria, fungi, viruses and spores •Since this radiation does not generate heat, it is termed “cold sterilization” •It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics •Disadvantages include; unlike electron beams, they can’t be switched off, glasswares tend to become brownish, loss of tensile strength in fabric •Gamma irradiation impairs the flavour of certain foods
Ultrasonic And Sonic Vibration: •Bactericidal but the results have been variable •No practical value in sterilization and disinfection

Question 31

What is Non ionizing radiation and how it is useful in sterilisation and disinfection?

Answer 31

Non ionizing radiation: • Ex: Ultraviolet rays. • Here electromagnetic rays with wavelengths longer than those of visible light are used. These are absorbed as heat. UV rays are generated using a high-pressure mercury vapor lamp. • UV rays inhibit DNA replication. Microorganisms that are exposed to the effective UV radiation are inactivated within seconds •UV rays don’t kill spores •UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc.

Question 32

What Ionizing radiation and how it is useful in sterilisation and disinfection?

Answer 32

Ionizing rays are of two types, particulate and electromagnetic rays •Electron beams are particulate in nature while gamma rays are electromagnetic in nature •High speed electrons beams are produced by a linear accelerator from a heated cathode •Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticalsSterilization is accomplished in few seconds • Unlike electromagnetic rays, the instruments can be switched offElectromagnetic rays such as gamma rays emanate from nuclear disintegration of certain radioactive isotopes • They have more penetrative power than electron beam but require longer time of exposureThese high-energy radiations damage the nucleic acid of the microorganism •Kills all bacteria, fungi, viruses and spores •Since this radiation does not generate heat, it is termed “cold sterilization” •It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics

Question 33

What is cold sterilisation?

Answer 33

this radiation does not generate heat,