Control of microbial growth
Different methods to control microbial
growth
Different methods to remove microbial
contaminations
• Sterilization is the process by which all living cells, spores, and
acellular entities are destroyed.
• Disinfection is the killing, inhibition, or removal of
microorganisms that may cause disease. It does not kill the
spores.
• In sanitization, the microbial population is reduced to levels that
are considered safe by public health standards.
• Antisepsis 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.
• Chemotherapy is the use of chemical agents to kill or inhibit the
growth of microorganisms within host tissue.
Pattern of microbial death
• A microbial population is not killed instantly when exposed
to a lethal agent. Population death is generally exponential.
• One measure of an agent’s killing efficiency is the decimal
reduction time (D) or D value.
• The decimal reduction time is the time required to kill 90%
of the microorganisms or endospores in a sample under
specified conditions.
• 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.
Pattern of microbial death under heat
Filtration
• Depth filter
• Membrane filter
Filtration
• Depth filters consist of fibrous or granular materials that
have been bonded into a thick layer filled with narrow,
twisting channels.
• Membrane 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.
N95 and HEPA filters
• Air also can be filtered to remove microorganisms.
• Two common examples are N95 disposable masks
used in hospitals and labs, and high-efficiency
particulate air (HEPA) filters that let air move freely
but restrict microorganisms.
• N95 masks exclude 95% of particles that are larger
than 0.3 μm. HEPA filters (a type of depth filter made
from fiberglass) remove 99.97% of particles 0.3 μm
or larger by both physical retention and electrostatic
interactions.
Heat
• Moist heat (autoclave, pasturization,
tyndallization)
• Dry heat (Incineration, Oven)
Autoclaving
• 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.
• Bacterial endospores will be killed only if they are kept at 121°C
for 10 to 12 minutes.
• When a large volume of liquid must be sterilized, an extended
sterilization time is needed because it takes longer for the center
of the liquid to reach 121°C.
• A biological indicator is often included in the autoclave. This
indicator commonly consists of a culture tube containing a
sterile ampule of medium and a paper strip covered with
endospores of Geobacillus stearothermophilus.
• Sometimes indicator tape or paper that changes color upon
heating is autoclaved with a batch of material. These indicate
heating has occurred and are convenient.
Pasteurization
• Many heat-sensitive substances, such as milk, are
treated with controlled heating at temperatures well
below boiling, a process known as pasteurization.
• Pasteur discovered that a brief heating at 55°C to 60°C
would destroy these microorganisms and preserve
wine for long periods.
• Pasteurization does not sterilize, but it does kill
pathogens and drastically slows spoilage by reducing
the level of nonpathogenic spoilage microorganisms.
Tyndallization
• 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.
• 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.
Radiation
• UV ray (260 nm)
• Gamma Ray (Cobalt 60 isotope)
Chemical agents
• Phenolics
• Alcohol
• Halogens
• Heavy metals (silver, arsenic, zinc, and copper)
• Aldehydes (formaldehyde and glutaraldehyde)
• Detergents (benzalkonium chloride and
cetylpyridinium chloride)
• Sterilizing gas (Ethylene oxide, Chlorine dioxide,
Vaporized hydrogen peroxide)
Phenolics
• Phenol and phenol derivatives (phenolics) are
used as disinfectants in laboratories and
hospitals.
• Phenolics denature proteins and disrupt cell
membranes. They have some important
advantages as disinfectants: Phenolics are
tuberculocidal, effective in the presence of
organic material, and remain active on
surfaces long after application
Alcohols
• Alcohols are among the most widely used
disinfectants, antiseptics, and sanitizers.
• 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. A 10- to 15-
minute soaking is sufficient to disinfect.
Halogens (Iodine)
• The halogens iodine and chlorine are important
antimicrobial agents.
• Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and iodinating proteins. 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.
• 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.
Halogens (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 (Cl2), sodium
hypochlorite (bleach, NaOCl), or calcium
hypochlorite [Ca(OCl)2], all of which yield
hypochlorous acid (HOCl).
Heavy metals
• heavy metal ions such as those of mercury,
silver, arsenic, zinc, and copper were used as
germicides.
• Silver sulfadiazine is used on burns. Copper
sulfate is an effective algicide in lakes and
swimming pools.
• Heavy metals combine with proteins, often
with their sulfhydryl groups, and inactivate
them. They may also precipitate cell proteins.
Quaternary Ammonium Compounds
• Detergents that have broad spectrum antimicrobial activity and
are effective disinfectants used for decontamination purposes.
• They are amphipathic, having both polar hydrophilic and
nonpolar hydrophobic components. The hydrophilic portion of a
quaternary ammonium compound is a positively charged
quaternary nitrogen; thus quaternary ammonium compounds
are cationic detergents.
• Their antimicrobial activity is the result of their ability to disrupt
microbial membranes; they may also denature proteins.
• Cationic detergents such as benzalkonium chloride and
cetylpyridinium chloride kill most bacteria.
Aldehydes
• Both of the commonly used aldehydes,
formaldehyde and glutaraldehyde, are highly
reactive molecules that inactivate nucleic
acids and proteins, probably by cross-linking
and alkylating molecules.
• They are sporocidal and can be used as
chemical sterilizing agents.
Sterilizing gases (EtO)
• Many heat-sensitive items such as plastic Petri dishes, heart-lung
machine components, sutures, and catheters are sterilized with
ethylene oxide gas.
• Ethylene oxide (EtO) is both microbicidal and sporocidal. It is a
strong alkylating agent that kills by reacting with DNA and
proteins to block replication and enzymatic activity.
• It is a particularly effective sterilizing agent because it rapidly
penetrates packing materials, even plastic wraps.
• Because pure EtO is explosive, it is usually supplied in a 10 to 20%
concentration mixed with CO2.
• Because it is so toxic to humans, extensive aeration of the
sterilized materials is necessary to remove residual EtO.
Chlorine dioxide (ClO2)
• ClO2 is typically aerosolized in a humidified environment, 1
mg/liter of air in 60% relative humidity, for at least 4 hours.
• At this concentration, ClO2 provides a greater than 6 log
reduction of endospores and vegetative bacteria.
• It has been used to sterilize hospital operating and patient
rooms.
• ClO2 fumigation is used in the food industry to sanitize fruits
and vegetables of contaminating yeasts and molds.
• It reacts readily with amino acids cysteine, tryptophan, and
tyrosine, denaturing proteins; with free fatty acids, lysing
membranes; and with nucleic acids, inhibiting replication.
Vaporized hydrogen peroxide
(VHP)
• It is produced from a solution of hydrogen peroxide in water
that is passed over a vaporizer to achieve a vapor concentration
between 140 and 1,400 parts per million (ppm), depending on
the agent to be destroyed.
• VHP is then introduced as a sterilizing vapor into the enclosure
for some time, depending on the size of the enclosure and the
materials within.
• Hydrogen peroxide and its oxy-radical by-products are toxic (75
ppm are dangerous to human health) and kill a wide variety of
microorganisms.
• During the course of the decontamination process, VHP breaks
down to water and oxygen, both of which are harmless.
Factors affecting the functions of anti-
microbial agents
• Population size
• Population composition
• Concentration of the agent applied
• Contact time
• Temperature
• Local environment
Phenol coefficient
• A series of dilutions of phenol and the disinfectant being
tested are prepared. Standard amounts of Salmonella
enterica serovar Typhi and Staphylococcus aureus are
added to each dilution;
• The dilutions are then placed in a 20° or 37°C water bath.
At 5-minute intervals, samples are withdrawn from each
dilution and used to inoculate growth medium, which is
incubated and examined for growth.
• The highest dilution (i.e., the lowest concentration) that
kills the bacteria after a 10-minute exposure but not after
5 minutes is used to calculate the phenol coefficient.