Physical and Chemical Control of Microorganism

SECOND SEMESTER 2023-24
The way microorganisms die

Physical controls on microorganisms
BITS Pilani, Pilani Campus

The Control of Microbial Growth
• Sepsis refers to microbial contamination.
• Asepsis is the absence of significant contamination.
• Aseptic surgery techniques prevent microbial contamination of wounds.
• Sterilization: (Latin sterilis, unable to produce offspring)-Removal or destruction

of all microbial life
• Sterilant: Sterilizing agent
• Commercial sterilization (for food): Absolute sterility is not required. Killing

Clostridium botulinum endospores. Some thermophilic bacteria may survive: food
spoilage if kept at high temp.

Disinfection: Removal of vegetative pathogens
Antisepsis: Removal of pathogens from living tissue
Degerming: Removal of microbes from a limited area
Sanitization: Lower microbial counts on eating utensils
Biocide/Germicide: Kills microbes
Bacteriostasis: Inhibit bacterial growth

Effectiveness of Antimicrobial Treatment
Depends on:
 Number of microbes
 Environment (organic matter, temperature, biofilms)
 Time of exposure
 Microbial characteristics

Actions of Microbial Control Agents
 Alternation of membrane permeability

 Damage to proteins
 Damage to nucleic acids

Methods of Microbial Control

Filtration
 Filters are porous membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose
nitrate, polycarbonate, polyvinylidene 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, from liquids
 These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils,
antibiotics, and other heat-sensitive solutions.
 Air also can be filtered to remove microorganisms.
 Two common examples are N95 disposable masks (exclude 95% of particles that are larger
than 0.3 μm) used in hospitals and labs, and high-efficiency particulate air (HEPA) filters
(made from fiberglass) that let air move freely but restrict microorganisms.
 HEPA filters remove 99.97% of particles 0.3 μm or larger by both physical retention and
electrostatic interactions.
Microbial Control Agents
Heat
 Thermal death point (TDP): The lowest temperature at which all cells in a culture
are killed in 10 min.
 Thermal death time (TDT): Time to kill all cells in a culture at a particular
temperature.
 Decimal reduction time (DRT): Time in minutes in which 90% of bacteria will be
killed at a given temperature
 Moist heat denatures proteins
 Free-flowing steam has the same temp as boiling water – may not be very
effective.
 Autoclave: Steam under pressure: preferred method for sterilization

Temperature
 Low temperature inhibits microbial growth

• Refrigeration
• Slow freezing is more harmful to bacteria
• Deep freezing
 High pressure can inactivate bacteria by altering protein

 Desiccation prevents metabolism, but can remain viable
 Hypertonic solutions preserve material by removing water

Heat
 Many objects are best sterilized by dry heat.
 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.

 Microbial death results from the oxidation of cell constituents and protein
denaturation.
 Dry air heat is less effective than moist heat.

 The endospores 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 slow and not suitable for heat-sensitive materials such as
plastic and rubber.
Pasteurization
 Pasteurization reduces spoilage organisms and pathogens

 Depends on viscosity and fat content.
 Phosphatase is done test to determine sterility.
Methods
 Batch pasteurization 63°C (145.4°F) for 30 min

 High temperature, short time: 72°C for 15 sec
 Ultra high temperature: 140°C (284°F) for ~3 sec
 Thermoduric organisms survive

Moist heat killing

Heat
Pressure (psi) in excess of Temperature (°C)

atmospheric pressure
atmospheric pressure+0 100

Heat
 Moist heat destroys cells and viruses by degrading nucleic acids, denaturing proteins, and
disrupting cell membranes.
 Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and
eukaryotic spores.
 Unfortunately, the temperature of boiling water (100°C at sea level) is not sufficient to
destroy bacterial endospores. Therefore boiling can be used for disinfecting drinking water
and objects not harmed by water, but boiling does not sterilize.
 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 somewhat like a pressure
cooker.
 The development of the autoclave by Charles Chamberland in 1879 played a significant role
in the so-called “golden age of microbiology” from 1850–1915.
 Usually 121°C and 15 pounds per square inch (psi) of pressure.
 biological indicator is often included in the autoclave Geobacillus stearothermophilus and
bacillus stearothermophilus .
Steam Sterilization
 Steam must contact the item’s surface or in an aqueous medium.
 Under these conditions, steam at a pressure of about 15 psi (121°C) will kill all
organisms and their endospores in about 15 min.
 Heat need extra time to reach the core of solid material.
 Aluminum foil – impervious to steam. Paper should be used.
 Trapped air needs to be removed by steam
 Some materials cannot withstand the high temperature of the autoclave, and
endospore 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 between exposures.
 The incubations permit remaining endospores to germinate into heat-sensitive
vegetative cells that are destroyed upon subsequent steam exposures
Ionizing and Non-Ionizing Radiation

UV
• Ultraviolet (UV) radiation around 260 nm is quite lethal.
• It causes thymine-thymine dimerization of DNA, preventing replication and transcription
• Commercial UV units are available for water treatment (Penetration?)
Thymine Dimers in the Same DNA Strand Are Formed by Ultraviolet Radiation

Ionizing Radiation
 Ionizing radiation is an excellent sterilizing agent that penetrates deep into objects.
 Ionizing radiation has sufficient energy to dislodge electrons from atoms or molecules,
producing
 Chemically reactive free radicals, damage DNA, destroy bacterial endospores, not always
effective against viruses
 Ionizing radiation (X-rays, gamma rays, electron beams) – damages many molecules:
gamma-ray can penetrate deeply but need more time; high energy electron beams can not
penetrate deep but need short exposure.
 Principle effect: dislodge e- from atoms - production of free radicals and other highly
reactive molecules
 ionization of water – formation of hydroxyl radicals.
 Used for sterilization of heat-sensitive materials: pharmaceuticals and disposable medical

and dental supplies: syringes, gloves, suturing materials, etc.

Ionizing radiation
 Gamma radiation (from a cobalt 60 source) and

 Beta radiation (accelerated electrons from high-voltage electricity)
 Used in the cold sterilization of antibiotics, hormones, sutures, and plastic
disposable supplies such as syringes
 Used to sterilize and “pasteurize” meat and other foods.
 Irradiation can eliminate the threat of such pathogens
 E. coli O157:H7 (life-threatening intestinal disease)
 Staphylococcus aureus, (skin and blood infections, and readily colonizes medical
devices)
 Campylobacter jejuni, which contaminates poultry, causing intestinal disease when
undercooked meat is eaten.
 Both the FDA and WHO approved irradiated food and declared it safe for human
consumption.
 Currently irradiation is used to treat meat, fruits, vegetables, and spices
SECOND SEMESTER 2023-24
Chemical controls on Microorganisms

Chemical antimicrobial Agents
 The potency, or effectiveness, of a chemical anti microbial agent is affected by

time, temperature, pH, and concentration
 The death rate of organisms is affected by the length of time the organisms are
exposed to the antimicrobial agent
 The death rate of organisms subjected to a chemical agent is accelerated by

increasing the temperature
 Increasing temperature by 10°C roughly doubles the rate of chemical reactions

and thereby increases the potency of the chemical agent
 Acidic or alkaline pH can increase or decrease the agent’s potency

 antiseptic and disinfectant
THE PHENOL COEFFICIENT
 Phenol (carbolic acid)- As a disinfectant 1867

 Standard disinfectant to which other disinfectants are compared under the same
conditions (called the phenol coefficient)
2. THE FILTER PAPER METHOD
3. THE USE-DILUTION TEST

Disinfectant Selection
An ideal disinfectant should:
1. Be fast acting even in the presence of organic substances, such as those in body
fluids.
2. Be effective against all types of infectious agents, without destroying tissues or

acting as a poison if ingested.
3. Easily penetrate material to be disinfected without damaging or discoloring the

material.
4. Be easy to prepare and stable even when exposed to light, heat, or other
environmental factors.
5. Be inexpensive and easy to obtain and use.
6. Not have an unpleasant odor.

Mechanism of action
Denaturation of proteins
 Much of a cell is made of protein, and all its enzymes are proteins. The alteration of
protein structure is called Denaturation
 In denaturation, hydrogen and disulfide bonds are disrupted, and the functional
shape of the protein molecule is destroyed
Bactericidal (permanently alteration),

Bacteriostatic (temporarily alteration)

Mechanism of action
 Boric acid, and strong alkalis destroy protein by hydrolyzing it.
 Oxidizing agents (electron acceptors), such as hydrogen peroxide and potassium
permanganate, oxidize disulfide linkages (—S—S—) or sulfhydryl groups (—SH).
 Agents that contain halogens—the elements chlorine, fluorine, bromine and

iodine—also sometimes act as oxidizing agents
 Halogens can be substituted for hydrogen in carboxyl (—COOH), sulfhydryl, amino

(—NH2), and alcohol (—OH) groups.
 Heavy metals, such as mercury and silver, attach to sulfhydryl groups

 Alkylating agents, which contain methyl (—CH3) or similar groups, donate these
groups to proteins
 All these reactions can kill microorganisms.

Mechanism of action
REACTIONS THAT AFFECT MEMBRANES

 Membranes also contain lipids and thus can be disrupted by substances that
dissolve lipids.
 Surfactants reduce surface tension, just as soaps and detergents break up grease
particles in dishwater
 Detergent solutions, also called wetting agents, are often used with other chemical
agents to help the agent penetrate fatty substances.
 Alcohols, detergents, and quaternary ammonium compounds (quats, Zephiran),

such as benzalkonium chloride, can dissolve lipids.
 Phenols, which are alcohols, dissolve lipids and also denature proteins.
Soaps and detergents
 Soaps and detergents remove microbes, oily substances, and dirt.

 Mechanical scrubbing greatly enhances their action.
 vigorous hand washing is one of the easiest and cheapest means of preventing the
spread of disease
 Soaps contain alkali and sodium and can kill many species of Streptococcus,
Micrococcus, and Neisseria and destroy influenza viruses.
 Detergents can be cationic if they are positively charged (sanitize food utensils)
and anionic (for laundering clothes) if they are negatively charged.
 Anionic detergents are less effective than cationic detergents

Alcohols
 Alcohols are among the most widely used disinfectants, antiseptics, and
sanitizers.
 They are bactericidal and fungicidal but not sporicidal; some enveloped viruses
are also destroyed.
 The two most popular alcohol germicides are ethanol and isopropanol,
 usually used in about 60 to 80% concentration.
 They act by denaturing proteins and possibly by dissolving membrane lipids.

Phenolics
 Phenolics denature proteins and disrupt cell membranes

 Phenolics are tuberculocidal, effective in the presence of organic material, and
remain active on surfaces long after application

Halogens
 Iodine and chlorine are important antimicrobial agents
 Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and
iodinating proteins (first skin antiseptics to come into use)
 At higher concentrations, it may even kill some endospores.

 Iodine often is applied as tincture of iodine, 2% or more iodine in a water-ethanol
solution of potassium iodide
1. USP Tincture of Iodine: 2% free iodine, (0.08 M, I2), which provides about 1 mg of
free iodine per 50μL drop
2. USP Strong Iodine Tincture-7% tincture solution

3. Lugol's iodine(aqueous iodine/strong iodine solution)-Soln of potassium iodide with
iodine in water, at 2% or 5%, without dilution-irritating and destructive to mucosa
Halogens
 Although it is an effective antiseptic, the skin may be damaged, a stain remains,

and iodine allergies can result.
 Often seafood allergies are triggered by iodine in the seafood.

 Iodine can be complexed with an organic carrier to form an iodophor.
 Iodophors are water-soluble, stable, and nonstaining, and release iodine slowly to
minimize skin irritation.
 Povidone-iodine (PVP-I)-A chemical complex of Polyvinylpyrrolidone (PVP),

hydrogen iodide, and elemental iodine.
 The recommended strength solution contains 10% Povidone with 1% total

titratable iodine.
Halogens
 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 (Cl2), sodium hypochlorite (bleach, NaOCl), or

calcium hypochlorite [Ca(OCl)2], all of which yield hypochlorous acid (HOCl)
 Oxidation of cellular materials and destruction of vegetative bacteria and fungi

 Two important waterborne eukaryotic pathogens (protozoa), Cryptosporidium
(cryptosporidiosis) and Giardia (giardiasis) are not killed by chlorine
 household bleach (diluted to 10% in water) can be used to disinfect surfaces

contaminated by human body fluids and can become more effective by the addition of
vinegar
Halogens
 However, chlorine itself is easily inactivated by the presence of organic

materials. That is why a substance such as copper sulfate is used to control
algal growth in water to be purified with chlorine.
 Chloramine, a combination of chlorine and ammonia, is less effective than

other chlorine compounds at killing microbes, but superior at eliminating taste
and odor problems, (Root canal therapy, water treatment procedures)

HEAVY METALS
 Selenium, mercury, copper, and silver. Even tiny quantities are very effective in
inhibiting bacterial growth
 Silver nitrate was once widely used to prevent gonococcal infection in newborn
infants.
 Phenylmercuric nitrate and mercuric naphthenate inhibit both bacteria and fungi and
are used as laboratory disinfectants
 Selenium sulfide kills fungi, including spores (commonly used to treat fungal skin
infections, dandruff etc.)
 Copper sulfate is used to control algal growth (a problem in maintaining water quality
in heating and air-conditioning systems and outdoor swimming pools.
 Few pennies, for a clear aquarium

ALKYLATING AGENTS
 Alkylating agents disrupt the structure of both proteins and nucleic acids (should not
be used in situations where they might affect human cells)
 Formaldehyde, glutaraldehyde, and β-propiolactone are used in aqueous solutions.

Ethylene oxide is used in gaseous form.
 Formaldehyde inactivates viruses and toxins without destroying their antigenic

properties.
 Glutaraldehyde kills all kinds of microorganisms, including spores, and sterilizes

 equipment exposed to it for 10 hours.
 Betapropiolactone destroys hepatitis viruses, as well as most other microbes, but
penetrates materials poorly (used to inactivate viruses in vaccines.

ALKYLATING AGENTS
 Gaseous ethylene oxide has extraordinary penetrating power.

 Used at a concentration of 500 ml/l at 50°C for 4 hours, it sterilizes rubber goods,
mattresses, plastics, and other materials destroyed by higher temperatures.
 Also, NASA has used ethylene oxide to sterilize space probes that might otherwise
carry Earth microbes to other planets.
 All articles sterilized with ethylene oxide must be well-ventilated for 8 to 12 hours
with sterile air to remove all traces of this toxic gas, which can cause burns if it
reaches living tissues and is also highly explosive
 Both the toxicity and flammability of ethylene oxide can be reduced by using it in
gas that contains 90% carbon dioxide.

Physical and Chemical Control of Microorganism

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Physical and Chemical Control of Microorganism

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SECOND SEMESTER 2023-24

The way microorganisms die

BITS Pilani, Pilani Campus

• Sepsis refers to microbial contamination.

• Asepsis is the absence of significant contamination.

• Aseptic surgery techniques prevent microbial contamination of wounds.

• Sterilization: (Latin sterilis, unable to produce offspring)-Removal or destruction

• Sterilant: Sterilizing agent

• Commercial sterilization (for food): Absolute sterility is not required. Killing

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

 Alternation of membrane permeability

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

 Air also can be filtered to remove microorganisms.

BITS Pilani, Pilani Campus

 Low temperature inhibits microbial growth

 High pressure can inactivate bacteria by altering protein

BITS Pilani, Pilani Campus

 Other items are sterilized in an oven at 160° to 170°C for 2 to 3 hours.

 Dry air heat is less effective than moist heat.

 Pasteurization reduces spoilage organisms and pathogens

 Batch pasteurization 63°C (145.4°F) for 30 min

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

Pressure (psi) in excess of Temperature (°C)

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

 ionization of water – formation of hydroxyl radicals.

 Used for sterilization of heat-sensitive materials: pharmaceuticals and disposable medical

BITS Pilani, Pilani Campus

 Gamma radiation (from a cobalt 60 source) and

Chemical controls on Microorganisms

BITS Pilani, Pilani Campus

 The potency, or effectiveness, of a chemical anti microbial agent is affected by

 The death rate of organisms subjected to a chemical agent is accelerated by

 Increasing temperature by 10°C roughly doubles the rate of chemical reactions

 Acidic or alkaline pH can increase or decrease the agent’s potency

 Phenol (carbolic acid)- As a disinfectant 1867

2. THE FILTER PAPER METHOD

3. THE USE-DILUTION TEST

BITS Pilani, Pilani Campus

2. Be effective against all types of infectious agents, without destroying tissues or

3. Easily penetrate material to be disinfected without damaging or discoloring the

5. Be inexpensive and easy to obtain and use.

6. Not have an unpleasant odor.

Bactericidal (permanently alteration),

BITS Pilani, Pilani Campus

 Agents that contain halogens—the elements chlorine, fluorine, bromine and

 Halogens can be substituted for hydrogen in carboxyl (—COOH), sulfhydryl, amino

 Heavy metals, such as mercury and silver, attach to sulfhydryl groups

 All these reactions can kill microorganisms.

REACTIONS THAT AFFECT MEMBRANES

 Alcohols, detergents, and quaternary ammonium compounds (quats, Zephiran),

 Soaps and detergents remove microbes, oily substances, and dirt.

 Anionic detergents are less effective than cationic detergents

BITS Pilani, Pilani Campus

BITS Pilani, Pilani Campus

 Phenolics denature proteins and disrupt cell membranes

BITS Pilani, Pilani Campus

 At higher concentrations, it may even kill some endospores.