Physical and Chemical Methods of Sterilization

Mary Ann V.
Alcon, RMT
▪ Sterilization- the process of killing or removing all microbial forms, including
spores.
▪ Disinfection- the process by which most forms on inanimate objects killed
without necessarily destroying saprophytes and bacterial endospores.
▪ Antiseptics- use of chemical agents on living tissue to prevent the spread of
microorganisms either by inhibiting their growth or destroying them.
▪ Bactericidal or germicidal agent- agent, physical or chemical, that kills bacteria.
▪ Bacteriostatic agent- agent, physical or chemical, capable of inhibiting the
growth of bacteria without necessarily killing them.
▪ Sporicidal, fungicidal, viricidal- agents capable of destroying spores, fimgi, and
viruses, repectively.
▪Heating- is the most common physical method of
sterilization. The rate of killing expressed in thermal
death time.
▪Mechanism of action of heating include:

1. Formation of single –strand breaks in the bacterial DNA.
2. Coagulation and denaturation protein.
3. Accumulation of toxic levels of electrolytes.
4. Alteration pf cell membrane structure.
1. Nature of the heat- moist heat has greater killing action than dry heat.
2. Temperature and time- as temperature increases, the time taken to sterilize decreases.
3. Number of microorganisms- the more organism there has the higher the temperature and the
longer the duration of the process require to destroy all of them.
4. Nature of microorganism- spore forming microorganism are more difficult to destroy than non-
forming ones.
5. Type of material- the temperature required to sterilize materials depends on the sensitivity of
the material to heat.
6. Presence of organic material- the presence of organic material such as fats, proteins, and sugars
may necessitate higher temperatures.
1. Moist Heat
1. Temperature below 100 degree Celsius.
1. Pasteurization
2. Vaccine bath
3. Serum bath
4. Inspissation
2. Temperature 100 degree Celsius.
1. Boiling
2. Fractional sterilization (Tyndallization)
3. Temperature above 100 degree Celsius.
1. Autoclave
2. Dry Heat
1. Red flame
2. Open flame
3. Incineration
4. Hot air oven
5. Infrared rays
▪DESSICATION
▪FREEZING
▪FILTATION
▪RADIATION
▪ ULTRA VIOLET LIGHT/ NON IONIZING RADIATION
▪ IONIZING RADIATION
▪ ELECTRON BEAMS
▪ ELECTROMAGNETIC RAYS
▪SONIC AND ULTRASONIC VIBRATION

▪OSMOTIC PRESSURE
▪ Pasteurization
▪ This method of destroying disease- producing organisms in
the milk and milk products as well as other beverages.
These are several variations of this method based on the
temperature utilized.
▪ Conventional method where the milk is heated at 60-65 deg. Cel.
Followed by the rapid cooling.
▪ Flash method involves heating at 72 deg. Cel. For 15 seconds
followed by quick cooling to 13 deg Cel.
▪ Ultra high temperature (UHT) method- heating is done at 140
deg. Cel. For a period of 15 sec’s and 149 deg Cel/ for 0.5 sec’s
▪ It is used to destroy
contaminating bacteria in vaccine
preparation.
▪ This method is heated in a water
bath at 60 deg. Cel for one hour.
▪ This procedure is not sporicidal.
▪ Only vegetative forms are destroy
▪It used to inactivate bacteria
contaminating serum
preparations and is done by
heating at 56 deg. Cel for
several days.
▪Similar to vaccine bath, only
vegetative forms are destroy
since higher temperature will
cause coagulation of proteins
present in the serum.
▪This technique is used to solidify
and disinfect egg containing and
serum containing media.
▪The culture medium is placed in
the slopes at 80 – 85 deg. Cel for
30 mins. for 3 days.
▪The basic method is that:
▪ 1st day: Vegetative forms will die.
▪ 2nd day: spores will germinate.
▪Boiling
▪It involves utilizing water at boiling temperature 100
℃.
▪Its is not sporicidal and will destroy only the vegetative
forms.
▪The killing action can be enhanced by addition of 2%
sodium carbonate.
▪Metals and glassware's can be disinfected for 10-2-0
mins. using this method without opening the lid of the
boiler.
▪ AKA intermittent sterilization
▪ Involve exposing the material to be sterilized to live steam at 100 ℃ for 30-90
minutes for 3 consecutive days.
▪ Example: TCBS, Selenite Broth
Note: Only vegetative form of bacteria are destroyed in this method.

▪Autoclave
▪ is the most efficient method of
sterilization because it can destroy
all microbial forms.
▪ 15 pounds per square inch (psi), the
temperature inside the vessel will
reach 121 ℃ (15 to 20 minutes)
▪ This method sterilized the following:
▪ Surgical bandages
▪ Culture media.
▪ Applicator sticks
▪ Tubes
A. Red Flame
-It is used to sterilize articles like bacteriological wire loops, straight wires, tips of the forceps, and searing spatulas.
It is limited only to articles that can be heated to redness in flame.
B. Open Flame
-Make use of the Bunsen burner or alcohol lamp.
-It is aimed at burning the organism into ashes, and used to sterilize such articles as mouths of test tubes, scalpel,
glass slide and cover slip.
C. Incineration
-This method is aimed at burning the organism into ashes.
D. Hot Air Oven

-Articles to be sterilized are placed in the oven with the temperature of 160 ℃ for one hour.
E. Infrared Rays
- This method of sterilized are places in a conveyor belt and passed through a tunnel that is heated by infrared
radiations.
- The temperature to which the materials are subjected to 180 ℃ for 7.5 minutes.
- It can used to sterilized metallic equipment and glassware.
▪ This method is based on the principle of depriving the microorganism of moisture.
▪ It is used mainly for food preservation, such as in the preparation of dried fish and
fruits.
▪ It may destroy vegetative form.
▪ Endospores are resistant to drying.
▪ It is not a reliable method for sterilization because most pathogenic organism are
resistant to low temperature.
▪ Its main use in the laboratory is for the preservation of microorganism in a process
called lyophilization or freeze drying where the organism is rapidly frozen.
▪ This is a form of mechanical sieving that does not kill microorganism but merely
separate them from the fluid.
▪ Cellulose ester filter with a pore size of 0.22 mm- 0.45 mm.
▪ ULTRAVIOLET LIGHT/ NON IONIZING RADIATION
▪ The effective wavelength is in the range of 200nm- 280nm as the most effective.
▪ UVL acts by inducing formation of thymine- thymine dimers resulting in lethal
mutations.
▪ This used a disinfectant in hospital wards, operating room laboratories.
▪ IONIZING RADIATION
▪ ELECTRON BEAMS
▪ Particulate in nature.
▪ It is used to sterilize syringes, gloves, dressing packs, foods, and medicine.
▪ ELECTROMAGNETIC RAYS
▪ This requires longer exposure time. The high energy radiation produced
cause damage to the microorganism nucleic acid.
▪ This used to sterilize commercially sterilize disposable petri dish, plastic
syringes, vitamins, antibiotics, hormones, fabrics, and glasswares.
▪ Exposure to soundwaves at a frequency of approximately
20,000 cycles/ second for one hour can kill some bacteria
and viruses.
▪ High frequency soundwaves acts by disrupting cells. They
are used to disinfect and clean instruments and to reduce
microbial loads.
▪ This is a method based on the principles of osmosis, so that
when the concentration of the fluid surrounding the
organism is altered, this will cause the bacterial cell to
collapse.
▪ This is used for preservation of fruits in syrup and meats in
brine.
FACTORS THAT AFFECT THE EFFICACY OF CHEMICAL AGENT:
1. CONCENTATION AND POTENCY OF

CHEMICAL AGENT
2. DURATION OF EXPOSURE
3. TEMPERATURE
4. NATURE OF SURROUNDING MEDIUM
5. MATURE OF THE ORGANISM
6. NUMBER OF ORGANISM/ SIZE OF INOCULUM
CHARACTERISTICS OF GOOD CHEMICAL AGENT ARE THE FOLLOWING
1. Its should be broad spectrum, 1. It should be a non toxic, non allergic,
able to destroy a wide variety of non irritative and non corrosive.
microorganism 2. It should be soluble in water and easy
to apply.
2. It should be fast acting, able to 3. It should leave a residual antimicrobial
destroy microbes within a short film on the treated surface.
period of time. 4. It should have a high penetrating
3. It should be active in the power.
presence of organic matter. 5. It should not be expensive and must be
4. It should be active in any pH. easily available.
6. It should be safe under storage and
5. It should be stable. shipping for reasonable period of time.
7. It should not have a bad ordor.
CLASSIFICATION OF DISINFECTANT BASED ON THE FOLLOWING:
1. CONSISTENSY (LIQUID OR GASEOUS)

2. SPECTRUM OF ACTIVITY
3. MECHANISM OF ACTION
1. SURFACE AVTIVE AGENT
1. CATIONIC AGENTS
2. ANIONIC AGENTS
2. PHENOLIC COMPUNDS
1. PHENOLS
2. CRESOLS
3. CHLORHEXIDINE
4. CHLOROXYLENOLS
5. HEXACHLOROPHENE
6. TRICLOSAN
3. ALCOHOLS
1. EHTYL ALCOHOL
2. ISOPROPHYL ALCCOHOL
3. BENZYL ALCOHOL
4. METHYL ALCOHOL

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Mary Ann V.

▪Mechanism of action of heating include:

▪SONIC AND ULTRASONIC VIBRATION

Note: Only vegetative form of bacteria are destroyed in this method.

D. Hot Air Oven

1. CONCENTATION AND POTENCY OF

1. CONSISTENSY (LIQUID OR GASEOUS)

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