BiochemD-112

Lecture 1 and 2

UNIT IV

Control of Microorganisms: Physical methods

of Sterilization - Moist heat, dry heat
 Control of Microorganisms

When environmental conditions become unfavorable and

nutrient supply are limiting, the microbial growth starts to
decline. In this decline phase, or death phase, the
number of live cells decreases at a logarithmic rate.
During the decline phase, many cells undergo
involution—that is, they assume a variety of unusual
shapes, which makes them difficult to identify.
In cultures of spore-forming organisms, more spores
than vegetative (metabolically active) cells survive.
In order to control the microbial growth, the effects of
microorganisms can often be controlled by simply
limiting or inhibiting growth.

Antimicrobial agents can be chemical, physical, mechanical, and biological. A biocide means all
antimicrobial agents that can be used to control microorganisms.
Physical methods of microbial growth control are used extensively in industry, medicine, and home.
 Microbial Growth Control: Terminology

A biocide means all or any antimicrobial agents that can be used to control microorganisms.
Sterilization (Latin sterilis, unable to produce offspring or barren), is the process by which all living cells,
spores, and acellular entities (e.g., viruses, viroids, and prions) are either destroyed or removed from an
object or habitat. A sterile object is totally free of viable microorganisms, spores, and other infectious agents.
A sterilizing agent is called a sterilant.
Disinfection is the killing, inhibition, or removal of microorganisms that may cause disease; disinfection is the
substantial reduction of the total microbial population and the destruction of potential pathogens.
Disinfection, may not however eliminate all microorganisms. A disinfecting agent is called a disinfectant.
Sanitization is closely related to disinfection. In sanitization, the microbial population is reduced to levels that
are considered safe by public health standards. The inanimate object is usually cleaned as well as partially
disinfected. A sanitizing agent is called a sanitizer.
Antisepsis (Greek anti, against, and sepsis, putrefaction) is the destruction or inhibition of microorganisms on
living tissue; it is the prevention of infection or sepsis. Antiseptics are chemical agents applied to tissue to
prevent infection by killing or inhibiting pathogen growth; they also reduce the total microbial population.
A disinfectant or antiseptic can be particularly effective against a specific group, in which case it may be called
a bactericide, fungicide, or viricide.
Chemotherapy is the use of chemical agents to kill or inhibit the growth of microorganisms within host tissue.
 Terminology Related to the Control of Microbial Growth

Some treatments only inhibit the growth and multiplication of bacteria; their names have the suffix -stat or -stasis,
meaning to stop or to steady, as in bacteriostasis. Growth resumes when the agent is removed.
Sepsis, from the Greek for decay or putrid, indicates bacterial contamination, Aseptic means that an object or area is
free of pathogens. Asepsis is the absence of significant contamination.
 Impact of Biocide Exposure
The growth of microorganisms can be controlled by physical,
chemical, mechanical, or biological means.
The exposure of microorganisms to increasing biocide
concentrations decreases the number of viable organisms.
Figure shows three possible population reduction curves
resulting from three different biocides.
The shape of the curve reflects various conditions that
influence biocide effectiveness.
Note that in each case, the eventual decline in viable
microorganisms can occur as a staged interval of viability from
antisepsis to sterilization.

A microbial cell is often defined as dead if it does not grow when inoculated into culture medium that would
normally support its growth. In like manner, an inactive virus cannot infect a suitable host.
This definition has flaws, however, some microbes are temporarily unable to reproduce referred to as viable but
nonculturable (VBNC).
It is worth noting here that resistance to antimicrobial biocides has been increasing nearly as rapidly as resistance
to antibiotics.
 Kinetics of Biocide Action
A microbial population is not killed instantly when exposed to a lethal agent.
Population death is generally exponential (logarithmic); that is, the population is
reduced by the same fraction at constant intervals.
If the logarithm of the population number remaining is plotted against the time
of exposure of the microorganism to the agent, a straight-line plot will result.
When the population has been greatly reduced, the rate of killing may slow due
to the survival of a more resistant strain of the microorganism.
A biocide’s killing efficiency is measured through decimal reduction time (D) or
D value.
D value is the time required to kill 90% of the microorganisms or spores in a
sample under specified conditions.
In a semilogarithmic plot of the population remaining versus the time of heating, the D value is the time required for the
line to drop by one log cycle or tenfold.
In other words, for each minute the treatment is applied, 90% of the remaining population is killed.
The lower the D-value, the faster the biocide kills microbes. A lower D-value indicates that the biocide is highly
effective and requires less time to achieve microbial inactivation.
Factors like temperature, biocide concentration, and microbial type influence the D-value and the overall efficiency of the
biocidal process.
 Factors Affecting Microbial Control

Several factors influence the effectiveness of antimicrobial treatments:

 The number of microbes: The more microbes there are to begin with, the longer it takes to
eliminate the entire population.
 Environmental influences: Most disinfectants work somewhat better in warm solutions. Heat is
also measurably more effective under acidic conditions.
 Time of exposure: Chemical antimicrobials often require extended exposure to affect more-
resistant microbes or endospores.
 Microbial characteristics: Microbes in surface biofilms, when they are encased in the mucoid
matrix, are difficult for biocides to reach effectively.
 The presence of organic matter often inhibits the action of chemical antimicrobials. In hospitals,
the presence of organic matter in blood, vomitus, or feces influences the selection of disinfectants.
 Temperature: Because their activity is due to temperature-dependent chemical reactions,
disinfectants work somewhat better under warm conditions.
 The nature of the suspending medium is also a factor in heat treatment. Fats and proteins are
especially protective, and a medium rich in these substances protects microbes, which will then
have a higher survival rate.
 Mechanism of Action of Biocides

Biocides can act on multiple structural and functional components of bacteria, including cell walls, cell
membranes, proteins, and nucleic acids. Understanding the mechanisms of action of biocides can
help in the development of new biocides and biocidal systems.
Weak interaction in biomolecules are influenced by agents like heat, pH or chemicals to interfere in
their normal functions and disruption of metabolic processes of microbes.
Reactive biocides: These biocides can attack multiple targets in a cell, depending on their
reactivity. For example, hypohalous acids are powerful oxidants that react with most organic
molecules, while isothiazolones are less reactive electrophiles that only react with strong
nucleophiles.
Membrane active biocides: Quaternary ammonium compounds (QACs) are membrane lytic biocides
that are often used as disinfectants in healthcare and food preparation.
Physicochemical interactions: Biocides can interact with microbial target structures through
physicochemical means.
Biological molecule reactions: Biocides can react with biological molecules in specific ways.
Metabolic or energetic processes: Biocides can disrupt selected metabolic or energetic processes.
 Mode of Action of Various Biocides

Biocide Mode of Action Target Organisms

Alcohols Membrane disruption, Bacteria, fungi, some
protein denaturation viruses
Quaternary Ammonium Membrane disruption Bacteria, fungi
Compounds (QACs)

Oxidizing Agents Oxidation of proteins, Bacteria, viruses, spores

lipids, DNA
Halogens (Chlorine, Protein and enzyme Bacteria, viruses, fungi,
Iodine) inactivation, ROS spores
generation
Aldehydes Cross-linking of proteins Bacteria, viruses, spores
(Formaldehyde, and nucleic acids
Glutaraldehyde)
Heavy Metals (Silver, Enzyme inhibition Bacteria
Mercury)
Methods of Microbial Growth Control
 Physical Methods of Sterilization: HEAT

The physical method of sterilization includes steam, dry heat,

boiling water, radiation and filtration.
Heating is the most common method used for killing microbes,
including the most resistant forms, such as endospores.
Heat kills microbes by damaging their structural components and
disrupting their essential life processes. Heat can kill microbes in
several ways, including:
Denaturing proteins: Heat can denature proteins and nucleic
acids, which can impair metabolic processes in membranes.
Coagulation: Dry heat can cause proteins to coagulate.
Oxidation: Dry heat can cause oxidation of cellular molecules.
Desiccation: Dry heat can dry out bacteria.
The effectiveness of heat as a sterilant is quantified by the decimal
reduction time (D).
It is also possible to determine the temperature change at a given D value that decreases the
microbial population by one log cycle (90%). This temperature change is referred to as the Z value
and is predicted from a semilogarithmic plot of D values versus temperature.
 Moist Heat
The four ways that moist heat is employed to control
microbes are;
Approximate Conditions for Moist Heat Killing
 Steam under pressure,
 Nonpressurized steam,
 Boiling water, and
 Pasteurization.
A moist heat sterilization method is that which uses
pressurized steam to kill microbes on or within a
product.
Moist heat destroys cells and viruses by degrading
nucleic acids and denaturing enzymes and other
essential proteins. It also disrupts cell membranes.
Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores.
However, the temperature is not enough to eliminate bacterial spores. Therefore, boiling does not sterilize
but only disinfects.
Many heat-sensitive substances, such as milk, are treated with controlled heating at temperatures well
below boiling (55 to 60°C), a process known as pasteurization in honor of its developer, Louis Pasteur.
Pasteurization does not sterilize a beverage, but it does kill any pathogens present and drastically
slows spoilage by reducing the level of nonpathogenic spoilage microorganisms.
 Autoclaving
To destroy bacterial endospores, moist heat sterilization must be
carried out at temperatures above 100°C, and this requires the use
of saturated steam under pressure.
Steam sterilization is carried out with an autoclave a device
Invented by Charles Chamberland in 1884.
Water is boiled to produce steam, which is released into the
autoclave’s chamber.
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
desired temperature and pressure, usually 121°C and 15 pounds
per sq. in. (psi) of pressure. Bacterial spores will be killed only if
they are kept at 121°C for 10 to 12 minutes.
Autoclaving must be carried out properly so that the saturated steam can destroy all vegetative cells and
spores within the processed materials. If all air has not been flushed out of the chamber, it will not reach
121°C, even though it may reach a pressure of 15 psi.
Sometimes indicator tape or paper that changes color upon sufficient heating or a culture tube
containing spores of Geobacillus stearothermophilus is autoclaved with a load of material.
 Autoclaving Cntd…
Autoclaving cycles should be set as per the
weight, volume and state of the material to be
sterilized.
The temperature of the object rises and falls
more slowly than the temperature of the
autoclave. The temperature of the object must
reach the target temperature and be held for 10–15
min to ensure sterility, regardless of the
temperature and time recorded in the autoclave.

Tyndallization:
Some materials cannot withstand the high temperature of the autoclave, and spore contamination precludes
the use of other methods to sterilize them. For these materials, a process of intermittent sterilization, also
known as tyndallization (for John Tyndall (1820–1893) is used. The process also uses steam (30–60 minutes)
to destroy vegetative bacteria. However, steam exposure is repeated for a total of three times with 23- to 24-
hour incubations (3 days) between steam exposures. The incubations permit remaining spores to germinate
into heat-sensitive vegetative cells that are then destroyed upon subsequent steam exposures.
 Dry Heat

Many objects are best sterilized by dry heat. Dry heat sterilization works by conduction, where the
heat is absorbed by the object's exterior surface and then conducted to the next layer.
The temperatures and times employed in dry heat vary according to the particular method, but in
general, they are greater than with moist heat.
Incineration in a flame or electric heating coil is the most rigorous of all heat treatments. The flame
of a Bunsen burner reaches 1,870oC at its hottest point, and furnaces/incinerators operate at
temperatures of 800oC to 6,500oC. Direct exposure to such intense heat ignites and reduces
microbes and other substances to ashes and gas. The microbial death therefore, results from the
oxidation of cell constituents and denaturation of proteins.
Some items are sterilized by incineration. For instance, inoculating loops, which are used
routinely in the laboratory, can be sterilized in a small, bench-top incinerator. Other items are
sterilized in an oven at 160 to 170°C for 2 to 3 hours.
The hot-air oven provides another means of dry-heat sterilization. The dry oven is usually electric
(occasionally gas) and has coils that radiate heat within an enclosed compartment. Heated,
circulated air transfers its heat to the materials in the oven.
 Advantages and Disadvantages of Dry Heat Sterilization

Dry heat sterilization can kill or deactivate bacteria, viruses, fungi, spore forms, and prions.
Dry heat sterilization is used for materials that are damaged by moist heat or can't tolerate it, such as
powders, oils, anhydrous fats, and some metals.
It does not corrode glassware and metal instruments as moist heat does, and it can be used to
sterilize powders, oils, and similar items.
Dry heat sterilization is nontoxic, noncorrosive, and has low operating costs.
It is very effective in permanent disposal of waste material from laboratories; burning reduces
bulky packages and garbage to ashes.
Despite these advantages, dry heat sterilization is slow and not suitable.
Dry air heat is less effective than moist heat. The spores of Clostridium botulinum, the cause of
botulism, are killed in 5 minutes at 121°C by moist heat but only after 2 hours at 160°C by dry heat.
Dry heat sterilization is time-consuming, and high temperatures aren't suitable for most materials.
It also doesn't work well on some items, like those that resist moist heating.
Dry heat is not a good choice for liquids, rubber, plastics etc. which are routine used in
microbiology laboratories.
 Cold Treatment

As a general rule, chilling, freezing, and desiccation should not be construed as methods of
disinfection or sterilization because their antimicrobial effects are erratic and uncertain.
It must be emphasized that cold merely retards the activities of most microbes. Although it is
true that some sensitive microbes are killed by cold temperatures, most are not adversely
affected by gradual cooling, long-term refrigeration, or deep-freezing.
The principal benefit of cold treatment is to slow growth of cultures and microbes in food and
other perishable materials during processing and storage.
In fact, freezing temperatures, ranging from -70°C to -135°C, provide an environment that can
preserve cultures of bacteria, viruses, and fungi for long periods. Pathogens able to survive
several months in the refrigerator are Staphylococcus aureus; Clostridium species
(sporeformers); Listeria; and several types of yeasts, molds, and viruses.
It is interesting to note that a combination of freezing and drying—lyophilization—is a common
method of preserving microorganisms and other cells in a viable state for many years. Pure
cultures are frozen instantaneously and exposed to a vacuum that rapidly removes the water. This
method avoids the formation of ice crystals that would damage the cells. Although not all cells
survive this process, enough of them do to permit future reconstitution of that culture.
Thermal death time
Another way to characterize the heat sensitivity of an organism is to measure its thermal death time, the
time it takes to kill all cells at a given temperature. To determine the thermal death time, samples of a
cell suspension are heated for different times, mixed with culture medium, and incubated. If all the cells
have been killed, no growth is observed in the incubated samples.
Commercial sterilization.
One would think that canned food in the supermarket is completely sterile. In reality, the heat treatment
required to ensure absolute sterility would unnecessarily degrade the quality of the food. Instead, food is
subjected only to enough heat to destroy the endospores of Clostridium botulinum, which can produce a
deadly toxin. This limited heat treatment is termed commercial sterilization.