Module 17: Control of Microorganism

OBJECTIVES
The present study aims to carry out the following objectives:
 To study the used of moist heat and dry heat in controlling microbes
 To study the importance of filtration in removing microbes
 To study the various forms of radiation for inactivating microorganisms
 To discuss the different types of chemicals used in sterilization.

INTRODUCTION
Prevention, control or elimination of microbes that are potentially harmful to humans is one of the
primary goals of our health care system. The process by which all living cells, viable spores, viruses
and viriods are either destroyed or removed from an object or habitat is termed as sterilization. A
microbial population is not killed instantly when exposed to a lethal agent. Like population growth,
population death is generally exponential or logarithmic i.e. the population will be reduced by the same
fraction at constant interval. When the population has been greatly reduced the rate of killing may slow
due to the survival of more resistant strain of microorganism. There are certain approaches for
controlling microbial growth and the most commonly used are the physical and chemical methods.
USE OF PHYSICAL METHODS IN MICROBIAL CONTROL
In physical methods the most commonly employed physical agents are heat, filtration, and radiation.
Heat: - Heat is the most widely used lethal agent for sterilization. Objects may be sterilized by dry heat
in an oven or by moist heat provided by steam. Of the two methods, sterilization by dry heat requires a
much greater duration and intensity because heat conduction is less rapid in dry than in moist air. In
addition, bacteria can survive in a completely dessicated state and in this state the intrinsic heat
resistance of vegetative bacterial cells is greatly increased, almost to the level characteristic of spores.
Consequently, the death rate is much lower for dry cells than for fully hydrated ones. Dry heat is used
to sterilized glassware or other heat stable solid materials. The objects are exposed to a temperature of
170˚C for 90 minutes in a hot air oven to achieve the dry heat sterilization.

Figure: hot air oven

Moist heat sterilization is usually carried out at temperatures above 100˚C in order to destroy bacterial
endospores and for this it requires the use of saturated steam under pressure. Steam sterilization is
carried out in a device called an autoclave. In autoclave, water is boiled to produce steam which is
released through the jacket and into the autoclave’s chamber. Further, the air initially present in the
chamber is forced out until the chamber is filled with saturated steam and the outlets are closed. Hot,
saturated steam continues to enter until the chamber reaches the temperature of 121˚C and 15 pounds
of pressure. At this temperature saturated steam destroys all vegetative cells and endospores and the
treatment is continued for about 15 minutes to provide a margin of safety.

Figure: Autoclave
Another method of heat sterilization known as pasteurization was developed by Louis Pasteur.
Pasteurization involves heating a particular food such as milk to a specific temperature long enough to
kill the spoilage microorganisms. Many different time and temperature combinations can be used for
pasteurization. The long temperature long time (LTLT) process involves bringing milk to a temperature
of 63˚C for 30 minutes while the high temperature short time (HTST) also known as flash pasteurization
method brings the milk to a temperature of 72˚C for 15 seconds to kill the spoilage microbes.
FILTRATION
An excellent way to reduce the microbial population in the solutions of heat sensitive material is
filtration. There are two types of filters i.e. depth filters and the membrane filters. Depth filters consist
of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels
of small diameter. The solution containing microorganisms is sucked through this layer under vacuum
and microbial cells are removed by physical screening or entrapment and also by adsorption to the
surface of the filter material. Depth filters are made of diatomaceous earth, unglazed porcelain; asbestos
etc. while membrane filters are circular, porous filters with little over 0.1mm thick and pore size of
about 0.2µm in diameter and made of cellulose acetate, cellulose nitrate, polycarbonate or other
synthetic material. The membranes are held in special holders and often preceded by depth filters to
remove larger particles that might clog the membrane filter. The solution is pulled or forced through
the filter with a vacuum and collected in the previously sterilized containers.
 Figure: membrane filter
Another type of filtration is the air filtration and the most common example is the surgical mask and
the cotton plugs on culture vessels that let the air in but keeps the microorganisms out. While laminar
airflow biological safety cabinets employing high efficiency particulate air (HEPA) filter which remove
99.97% of 0.3 µm particles are one of the most effective air filtration systems. This cabinet force air
through the HEPA filter then project a vertical curtain of sterile air across the cabinet opening. They
also used in research labs and industries where a sterile working surface is needed.

Figure: Laminar air flow

RADIATION
Radiation in various forms from high-energy radiation to sunlight can be used to kill microbes or inhibit
their growth. Generally there are two types of radiation used in sterilization i.e. ionizing and non-
ionizing radiation. Ionizing radiation is an excellent sterilizing agent and can penetrate deep into the
objects. They include the X- rays and the gamma rays and are strong enough to pass through the cell
where it alters the molecular structures and damages the cell components. They introduces break in the
double stranded DNA which causes mutation to occur and ultimately when the mutation accumulates
it lead to the death of the cell. While the most common form of non-ionizing radiation is the UV
radiation. They effectively reduce the microbial population where direct exposure takes place. UV rays
exert its mutagenic effect by exciting electrons in molecules. This excitation of electrons in the DNA
molecules results in the formation of extra bonds between adjacent pyrimidines specifically the thymine
in the DNA. The bonding of the two thymines is called the thymine dimers. These dimers change the
shape of the DNA in the cell and caused problems during replication and ultimately kill the cell.
 Figure: Effects of UV radiation on the microbial DNA

USED OF CHEMICAL AGENTS IN MICROBIAL CONTROL

Chemical agents reduces the microbial population but generally not intended to achieve sterilization.
Chemicals are more often employed in disinfection and antisepsis. Here, disinfection is the elimination
of microbes from the inanimate objects or surfaces while the removal of microbes from the living tissues
are termed as antisepsis. An ideal disinfectant or antiseptic must be effective against a wide variety of
infectious agent at high dilutions and in the presence of organic matter. Although the chemical must be
toxic for infectious agent it should not be toxic to people or corrosive for common materials. It should
also be stable on storage, soluble in water and lipids for penetration into microorganisms and have a
low surface tension so that it can enter cracks in surfaces. A large number of chemicals are in common
use which includes –
Phenol and phenol derivatives: - Phenol was the first widely used chemical as disinfectant and
antiseptic. Phenol and phenolics such as cresols, xylenols and orthophenylphenol are used as
disinfectants in laboratories and hospitals. Phenolics damages the cell membrane and denature the
proteins of the microorganisms. They are also effective in the presence of organic material and remain
active on surfaces long after application.
Alcohol: - Alcohol is also one of the most effective and widely used antiseptic and disinfectant. They
are bactericidal and fungicidal but are not effective on bacterial spores. Ethanol and isopropanol are
among the most popular alcohol germicides and are usually used in the concentration of 70-80%. The
mode of action of alcohol is that it solubilizes the lipid bilayer of the cell wall and the cell membranes
and creates pores which further enters into the cell cytoplasm and denature cellular proteins and
ultimately killing the bacteria. As alcohols evaporate quickly they leave no residues and are useful in
degerming the skin before injections. Further, for disinfecting thermometers and small instruments a 10
to 15 minutes soaking in alcohol is sufficient.
Halogens: - Among the halogens the iodine and chlorine are used as an antimicrobial agent. Iodine is
used as the tincture of iodine containing a 2% solution of iodine and sodium iodide in water – ethanol
solution. They are commonly used as skin antiseptic but it may damage the skin and the stain is left
which results in iodine allergies. A combination of iodine and organic molecules called iodophore is
developed which are less irritating than iodine and do not stain. Iodophore are water soluble, stable and
non- staining and release iodine slowly to minimize skin burns and irritation. Iodine are bactericidal,
fungicidal virucidal and even sporicidal. Chlorine is widely used to disinfect municipal water supplies,
swimming pools and they are also employed in dairy and food industries. They are used as sodium
hypochlorite and calcium hypochlorite. The mode of action of chlorine is that it carry out oxidation of
cellular materials and destruction of vegetative bacteria and fungi but not spores.
Heavy metals: - Heavy metals such as mercury, silver, arsenic, zinc and copper are used as
antimicrobial agents. Heavy metals and their compounds inactivate cellular proteins and enzymes by
combining with them. When present in higher concentration heavy metal salts coagulates and
precipitates cellular proteins and kills the microorganisms.
Aldehydes: - Among the aldehydes formaldehydes and glutareldehyde are commonly used
antimicrobials and are highly reactive molecules that combine with cellular proteins and inactivate
them. They are sporicidal also. Formaldehyde before used is usually dissolved in water. They are stable
in higher concentration and at higher temperature because in room temperature they are polymerized to
form para-formaldehyde. Another aldehyde i.e. glutareldehyde is used in 2% buffered solution as a
disinfectant. It is less irritating than formaldehyde and is used to disinfect hospitals and laboratory
equipments. Glutareldehyde usually kills the vegetative cells within 10 minutes of exposure but requires
about 12 hours to destroy the spores.
Gaseous agent: - Ethylene oxide and the β-propiolactone are the commonly used gaseous sterilizing
agents. Heat sensitive items are sterilized by the used of ethylene oxide. They are both microbicidal and
sporicidal. It is one of the most effective sterilizing agent because it can rapidly penetrate packing
material and plastic wraps. Extensive aeration of the sterilized material is necessary to remove residual
ethylene oxide as it is very toxic. While β-propiolactone is occasionally used as sterilizing gas. They
kill microbes more rapidly than the ethylene oxide but cannot penetrate materials well and is
carcinogenic and hence they are not used extensively as ethylene oxide.
CONCLUSION
Although many microorganisms are beneficial and necessary for human well-beings, microbial
activities may have undesirable consequences such as food spoilage and diseases. Therefore, it is very
much essential to kill the microbes or inhibit their growth to minimize their destructive effects. So,
when working with microbes, operations that might generate infectious aerosols should be carried out
in biological safety cabinets, bench tops and incubators should be disinfected regularly and autoclaves
must be maintained and operated properly to ensure adequate sterilization.

GLOSSARY
Disinfection: The process of killing microbes and resistant spores from an inanimate objects
Antisepsis: The process of preventing the growth of microbes in the living tissue
Sporicidal: Capacity to kill spores
Bactericidal: Capacity to kill or inactivate bacteria
Mutation: A sudden and heritable change in the DNA of the organism
Endospores: A structure produces by bacteria which are dormant, metabolically inactive and cannot
reproduce
Dessication: State of extreme dryness
DNA replication: A process by which the DNA makes the copy of itself during cell division

Antimicrobial: Agents that kills or inhibit microbes

Virucidal: Capacity to destroy or inactivate viruses

FAQS (frequently asked questions)

1. What is sterilization?

Answer: The process by which all living cells, viable spores, viruses and viriods are either destroyed or
removed from an object or habitat is termed as sterilization.
2. State the working principle of an autoclave?

Answer: Moist heat sterilization is usually carried out in autoclave. It uses saturated steam under
pressure for sterilization. In autoclave, water is boiled to produce steam which is released through the
jacket and into the autoclave’s chamber. Further, the air initially present in the chamber is forced out
until the chamber is filled with saturated steam and the outlets are closed. Hot, saturated steam continues
to enter until the chamber reaches the temperature of 121˚C and 15 pounds of pressure. At this
temperature saturated steam destroys all vegetative cells and endospores and the treatment is continued
for about 15 minutes to provide a margin of safety.
3. What are LTLT and HTLT pasteurization?

Answer: LTLT is long temperature long time and HTST id high temperature long time pasteurization.
4. Write a note on depth filters?

Answer: Depth filters are used to sterilize heat sensitive solutions. Depth filters consist of fibrous or
granular materials that have been bonded into a thick layer filled with twisting channels of small
diameter. The solution containing microorganisms is sucked through this layer under vacuum and
microbial cells are removed by physical screening or entrapment and also by adsorption to the surface
of the filter material. Depth filters are made of diatomaceous earth, unglazed porcelain; asbestos etc.
5. Explain the mode of action of UV radiation in controlling microbes?

Answer: UV radiation effectively reduce the microbial population where direct exposure takes place.
UV rays exert its mutagenic effect by exciting electrons in molecules. This excitation of electrons in
the DNA molecules results in the formation of extra bonds between adjacent pyrimidines specifically
the thymine in the DNA. The bonding of the two thymines is called the thymine dimers. These dimers
change the shape of the DNA in the cell and caused problems during replication and ultimately kill the
cell.
6. Differentiate between disinfection and antisepsis?

Answer: disinfection is the elimination of microbes from the inanimate objects or surfaces while the
removal of microbes from the living tissues are termed as antisepsis.
7. Name some phenol and phenolic compounds that are used in sterilization.

Answer: Cresols, xylenols and orthophenylphenol

8. Write a note on the use of alcohol in controlling microbes?
Answer: Alcohol is also one of the most effective and widely used antiseptic and disinfectant. They are
bactericidal and fungicidal but are not effective on bacterial spores. Ethanol and isopropanol are among
the most popular alcohol germicides and are usually used in the concentration of 70-80%. The mode of
action of alcohol is that it solubilizes the lipid bilayer of the cell wall and the cell membranes and creates
pores which further enters into the cell cytoplasm and denature cellular proteins and ultimately killing
the bacteria. As alcohols evaporate quickly they leave no residues and are useful in degerming the skin
before injections.
9. What are iodophore?

Answer: Iodophore is a combination of iodine and organic molecules and are water soluble, stable and
non- staining.
10. Write the mode of action of phenolics on microbial cells?

Answer: Phenolics damages the cell membrane and denature the proteins of the microorganisms.

REFERENCES
Prescott, L.M., Harley, J.P. and Klein, D.A. 1999. Microbiology. 4th Edition, McGraw-Hill.
Pelczar, M.J., Chan, E.C.S. and Krieg, N.R. 1998. Microbiology. TATA McGRAW-HILL.
Stanier, R.Y., Ingrahm, J.L. and Painter, P.R. 2003. General Microbiology. MACMILLAN Press Ltd.
Tortora, G.J., Funke, B.R. and Case, C.L. 2013. Microbiology: An Introduction. Pearson publication.

LINKS
http://www.clayton.edu/portals/438/Microsoft%20Word%20-%20UV%20lab.pdf
https://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/sterilize/radiation.html
https://bio.libretexts.org/Demos%2C_Techniques%2C_and_Experiments/Microbiology_Labs_II/Lab
_19%3A_Use_of_Chemical_Agents_to_Control_of_Microorganisms