Physical and chemical control of microorganisms

1. PHYSICALAND CHEMICAL
CONTROL OF
MICROORGANISMS
&
METHODS OF STERILIZATION
By
Dr. Harinatha Reddy
Dpt of Biotechnology
8th class

2. • Sterilization is the process by which all living cells, viable spores,
viruses, and viroids are either destroyed or removed from an
object.
• A sterile object is totally free of viable microorganisms, spores, and
other infectious agents.
• Joseph Lister ("father of modern surgery) a British surgeon used
carbolic acid (phenol) as disinfectant or to sterilise surgical
instruments and to clean wounds.
• Louis Pasteur is the father of sterilization techniques (Ex:
Autoclave, Pasteurization)

3. • Any Substances or Chemical agent that kill organisms often have
the suffix “cide” (Ex: Bactericide, Fungicide and Algicide).
• Other chemicals do not kill, but they do prevent growth. Their
names end with “static” (Ex: Bacteriostatic and Fungistatic).
• Two types of methods for sterilization:
• 1.Physical methods
• 2.Chemical methods

4. Physical Methods:
• Heat
• Filters
• Radiation
1.HEAT:
Thermal death point (TDP):
The lowest temperature at which all microorganisms in a microbial suspension
can be killed in 10 minutes.
Thermal death time (TDT):
This is the shortest time needed to kill all microorganisms in a microbial
suspension at a specific temperature and under defined conditions.
Decimal reduction time (D) or D value :
The decimal reduction time is the time required to kill 90% of the
microorganisms or spores in a sample at a specified temperature.

5. Decimal reduction time (D) or D value :
• The decimal reduction time is the time required to kill 90% of
the microorganisms or spores in a sample at a specified
temperature.
• The Z-value is the number of degree of temperature change
necessary to change the D value by a factor of 10.
• For example:
• if D value at 121 °C is 15 min.
• Then D Value at 131 °C is 1.5 min. (Here Z Value is 10 °C )

6. Dry Heating:
• 1.Flaming: is widely used for sterilization of needles and
inoculation loops.
• Small objects like inoculating loops and needles that are not
easily injured by heat are sterilized by thrusting them into the
flame.
• Hot air sterilization: Commonly used for sterilization of
Glassware's (Test tubes, Petri dishes, flasks, Pipettes), powders
and oils.
• Instrument used for Hot air sterilization is Hot Air Oven.

7. Hot Air Oven:
• The oven consists of big chamber, the walls of the chamber is
made up of steel or aluminium.
• A fan is set at the bottom of the chamber, it forces steam of hot
dry air in to the chamber, resulting rise in chamber temperature.
• Temperature at 160 ºC sterilizes the glassware in a period of
two hours (60 min).

8. Moist heating:
• Moist heat sterilization must be carried out at temperatures above
100°C in order to destroy bacterial endospores, and this requires the
use of steam under pressure.
• Steam sterilization is carried out with an autoclave a device
somewhat like a fancy pressure cooker.
• The development of the autoclave by Louis Pasteur and his
associate Chamberland in 1884.
• It is used for sterilization of medias, plastic, rubber materials and
glassware.
• But effective method for glassware sterilization is HOT AIR
OVEN.

9. • In autoclave the water is boiled to produce steam, which is released
into the autoclave’s chamber.
• Hot steam continues to enter until the chamber reaches the desired
temperature and pressure, usually 121°C and 15 pounds of pressure.
• At this temperature saturated steam destroys all vegetative cells and
endospores within 12 to 15 minutes.
• Moist heat is thought to kill so effectively by degrading nucleic acids
and by denaturing enzymes and other essential proteins. It also may
disrupt cell membranes.

10. Filters:
• Filtration is an excellent way to reduce the microbial population in
heat-sensitive solutions contaminating microorganisms, the filter
simply removes them.
• There are two types of filters: 1.Depth filters, 2.Membrane filters.
• Depth filters: consist of fibrous or granular materials.
• The solution containing microorganisms is sucked through this
layer under vacuum, and microbial cells are removed by physical
screening or entrapment.

11. Membrane filters
• Membrane filters have replaced depth filters for many purposes.
These filters are porous membranes, over 0.1 mm thick, made of
cellulose acetate, cellulose nitrate, polycarbonate and fluoride, or
other synthetic materials.
• Although a wide variety of pore sizes are available, membranes with
pores about 0.2 µm in diameter are used to remove most vegetative
cells, but not viruses.
•

12. • Air also can be sterilized by filtration:
• Two common examples are:
• Surgical masks and cotton plugs: on culture vessels that let air in but
keep microorganisms out.
• HEPA: Laminar flow biological safety cabinets contains high-efficiency
particulate air (HEPA) filters, which remove 99.97% of 0.3 µm particles,
are one of the most important air filtration systems.
• Laminar flow biological safety cabinets force air through HEPA filters.
• A person uses these cabinets when working with dangerous agents such as
Mycobacterium tuberculosis, tumor viruses, and recombinant DNA.
• They are also employed in research labs and industries, when a sterile
working surface is needed for conducting assays, preparing media,
examining tissue cultures.

13. Radiation:
• The radiation destroys microorganisms by damaging the DNA.
• The ultraviolet and ionizing radiation is used for sterilization.
• Ultraviolet (UV) radiation: around 260 nm less penetration power,
Because of this disadvantage, UV radiation is used as a sterilizing
agent only in safety cabinets, operation theatres and in LAF.
• Uv rays destroy the microorganisms by using pyrimidine dimers.

14. Ionizing radiation:
• Ionizing radiation is an excellent sterilizing agent and penetrates deep
into objects.
• It will destroy bacterial endospores and vegetative cells, both
procaryotic and eucaryotic; however, ionizing radiation is not always as
effective against viruses.
• Gamma rays and x-rays destroy the microorganisms by breaking the
phosphodiester bond.
• Gamma radiation from a cobalt 60 source is used in the cold
sterilization of antibiotics, hormones, and food and drugs.
• Irradiation can eliminate the threat of such pathogens as E. coli,
Streptococcus, Staphylococcus aureus and Salmonella.

15. Chemical methods:
• Many different chemicals are available for use as disinfectants, and
each has its own advantages and disadvantages.
• Ideally the disinfectant must be effective against a wide variety of
infectious agents (gram-positive and gram-negative bacteria, acid-
fast bacteria, bacterial endospores, fungi, and viruses) at high
dilutions.
• A wide varieties of disinfectants are available

16. Phenolics:
• Phenol was the first widely used antiseptic and disinfectant. In
1867 Joseph Lister employed it to reduce the risk of infection
during operations.
• Today phenol and phenol derivatives such as cresols and
orthophenylphenol are used as disinfectants in laboratories and
hospitals.
• Phenolics act by denaturing proteins and disrupting cell
membranes.

17. Alcohols:
• Alcohols are among the most widely used disinfectants and antiseptics.
They are bactericidal and fungicidal but not sporicidal; some lipid-
containing viruses are also destroyed.
• They dissolve membrane lipids and denature proteins and inactivate
enzymes.
• The two most popular alcohols used in sterilization are ethanol (ethyl
alcohol) and isopropyl alcohol.
• Ethanol is effective in 70% concentration.
• Alcohols kills all vegetative cells of bacteria but not endospores.
• Bacillus anthracis have survived for more than 20 years in ethanol.
• Ethanol used to reduce skin microbial flora and disinfection of clinical oral
thermometers.

18. Halogens:
• Fluorine, chlorine, bromine, and iodine are halogens they form salt like
compounds with sodium and other metals.
• The halogens iodine and chlorine are important antimicrobial agents.
• Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and cell proteins.
• At higher concentrations, it may even kill some spores.
• Iodine often has been applied as tincture of iodine (Inorganic), 2% or
more iodine in a aqueous solution of potassium iodide.
• More recently iodine has been complexed with an organic carrier to
form an iodophor (Organic).
• Iodophors are water soluble, stable, and nonstaining, and release iodine
slowly to minimize skin burns.
•

19. Chlorine:
• Chlorine is the usual disinfectant for municipal water supplies
and swimming pools and is also employed in the dairy and food
industries.
• It may be applied as chlorine gas, sodium hypochlorite, or
calcium hypochlorite.
• All of which yield hypochlorous acid (HClO) and then atomic
oxygen.
• The result is oxidation of cellular materials and destruction of
vegetative bacteria and fungi, although not spores.

20. Aldehydes:
• The commonly used aldehydes, formaldehyde and
glutaraldehyde, are highly reactive molecules that combine with
nucleic acids and proteins and inactivate them.
• They are sporicidal and can be used as chemical sterilants.
• Formaldehyde (40%) is usually dissolved in water or alcohol
before use.
• A 2% glutaraldehyde is an effective disinfectant. It is less
irritating than formaldehyde and is used to disinfect hospital and
laboratory equipment.

21. Heavy Metals:
• For many years the heavy metals such as mercury, silver, arsenic,
zinc, and copper were used as germicides.
• A 1% solution of silver nitrate is often added to the eyes of infants
to prevent ophthalmic gonorrhea.
• Copper sulfate is an effective algicide in lakes and swimming
pools.
• 1:1000 dilution of Mercury used as bactericidal and fungicidal.
• Heavy metals combine with proteins, often with their sulfhydryl
groups, and inactivate them. They may also precipitate cell
proteins.

22. Sterilizing Gases:
• Many heat-sensitive items such as disposable plastic petri dishes
and syringes, heart-lung machine components, are now sterilized
with ethylene oxide gas.
• Ethylene oxide (EtO): is both microbicidal and sporicidal and
kills by combining with cell proteins.
• It is a particularly effective sterilizing agent because it rapidly
penetrates packing materials, even plastic wraps.
• Betapropiolactone (BPL): is occasionally employed as a
sterilizing gas.
• It has been used to sterilize vaccines and sera.
• It also destroys microorganisms more readily than ethylene oxide
but does not penetrate materials well and may be carcinogenic.

23. THANK U……