Microbial Control

Physical Methods
Chemical Methods
Lecture-13
Faculty: Kazi Fahmida Rahman
Topics
• Targets for Antimicrobial Agents
• Terminologies
• Microbial Death
• Conditions that Effect Antimicrobial
Activity
• Mechanical Removal Methods Rely on
Barriers
• Physical Methods
• Chemical Methods
 If you can starve or poison, or inhibit or
prevent growth or replication, you can
control microorganisms.

Microbial Growth and Replication

Pathways: Targets for Control

1. Alteration of permeability
2. Damage DNA
3. Damage protein
❖Alteration of permeability:
This membrane regulates the passage of nutrients & wastes in & out of cell.
Damage to the membrane lipids/protein causes cellular contents to leak into the
surrounding.

❖Damage DNA:
Damage to nucleic acids by applying different agents are lethal for cell. Cell can
no longer replicate, nor carry out metabolic function.

❖Damage Protein:
Enzyme are vital for metabolic processes. With out metabolic processes an
organism cannot survive.
Important Terminology
Biocide: to mean all antimicrobial agents that can be used to
control microorganisms. In general, to control microorganisms a
biocide must be evaluated so as to determine the specific parameters
under which it will be effective.

Cide - a suffix indicating that the agent will kill the kind of
organism in question. e.g., bactericide- killing of bacteria.

Static - a suffix indicating that the agent will prevent the growth
of the type of organism in question. e.g., bacteriostatic-prevention
of bacterial growth.
 MICROBIAL DEATH
⮚ When bacterial populations is treated with any
target control they usually die at a constant rate.

⮚ For example, suppose a population of 1 million

microbes has been treated for 1 minute, and 90% of
the population has died.
⮚ We are now left with 100,000 microbes.
⮚ If the population is treated for another 1 minute,
90% of those microbes die.
⮚ We are left with 10,000 survivors.

⮚ In other words, for each minute the treatment is

applied, 90% of the remaining population is killed.
 If the death curve is plotted
logarithmically, the death
rate is constant, as shown by
the straight line
❖It is essential to have a precise measure of an agent’s killing efficiency. One such measure is the D
value.

❑Decimal reduction time (D value): This is the time in minutes to reduce the population by 90%, or
it is the time in minutes for the thermal death-time curve to pass through one log cycle.
❖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.
 Conditions Influencing Antimicrobial
Activity
• Several factors play key roles in determining the effectiveness of an antimicrobial
agent, including:
• Population size
• Types of organisms
• Concentration of the antimicrobial agent
• Duration of exposure
• Temperature
• pH
• Organic matter
• Biofilm formation
 Mechanical Removal Methods Rely on Barriers
Physical removal of microbes and dust particles from solutions and
gasses; often used to sterilize heat-sensitive solutions/to provide a
sterilized air flow.

❖HEPA filters: High efficiency particulate air filters used in

biosafety cabinets that let air move freely but restrict
microorganisms.
• HEPA filters are available that remove viruses that are 0.1 μm and
smaller; they are used to sterilize air.

❖Membrane filters: has pores that are uniform and specific

predetermined size (0.45 um). That trap/retain the microorganism
whereas solution pass through. eg. Nitrocellulose, nylon.
Physical Methods
1. Heat
▪Moist Heat
▪Autoclave
▪Boiling
▪Pasteurization
▪Dry Air Heat
2. Radiation
▪Ionizing radiation
 Heat
Heat is most widely applicable, effective, economical and easily controllable agent to
kill microbes.
❖Moist heat: kills microbes by coagulating & denaturing of proteins (Presence
of H2O quickens the process).

❖Autoclave: is a ‘double-jacketed’ pressurized device designed to heat aqueous

solutions above their boiling point to achieve sterilization.
⮚ Autoclaving is used to sterilize material that can not be damaged by heat/moisture.
⮚ Generally, the autoclave is operated at a pressure of app. 15 lb/in² (at 121°C).
⮚ The time of operation depends on the nature & volume of the material & type of the
container.
AUTOCLAVE

Autoclaving is used to sterilize culture

media, instruments, dressings,
intravenous equipment, applicators,
solutions, syringes, transfusion
equipment.
❖Boiling water: Exposing instruments to boiling water for short periods of time. All
vegetative cells will be destroyed within minutes. However, some bacterial spores can
withstand this condition for many hours.
❖Pasteurization: slow heating at lower temp 55- 60oC to destroy unwanted microbes that
can spoil food and beverage is known as pasteurization. Many substances are sensitive to high
temp. they can be pasteurized with temp. below boiling.
⮚ It kills the vegetative cells of most microorganism but cannot kill spores.
⮚ Milk is pasteurized at 62.8°C for 30 min.

❖Dry air heat: is less effective than moist heat. However, dry heat has
some definite advantages.
• It does not corrode glassware and metal instruments as moist heat Boiling
does.
• It can be used to sterilize powders, oils, and similar items.
• Despite these advantages, dry heat sterilization is slow and not suitable
for heat-sensitive materials such as many plastic and rubber items.
 Radiation
❖Ultraviolet radiation: DNA absorbs UV radiation at
260 nm wavelength. This causes damage to DNA in the
form of thymine dimer (T=T) mutations.
▪ Useful for continuous disinfection of work surfaces, e.g. in
biological safety cabinets.

❖Ionizing radiation: has sufficient energy to remove electrons from atoms or molecules,
producing reactive free radicals. The free radicals react with nearby matter to weaken or destroy
it. Penetrates and damages both DNA and protein.

▪Powerful sterilizing agent. Effective against both vegetative cells and spores.

▪Sterilize heat-sensitive materials e.g. plastic lab-ware, petri dishes, antibiotics, hormones.
Chemical Methods

1.Phenol & Phenolic

2.Alcohol
3.Halogen
4.Detergent
5.Aldehyde
6.Gaseous Agents
❖Phenol & Phenolic
• Effective disinfectant and antiseptic. A 5% aqueous solution of phenol
rapidly kills the vegetative cells but not spore.
▪Mode of Action:
1. Disruption of cell membrane,
2. Leakage of amino acids from the cells.
3. Protein denaturation & enzymes inactivation

Advantages: phenolics are tuberculocidal, effective in the presence

of organic material, and remain active on surfaces long after
application.
Disadvantages: disagreeable odor and skin irritation.
❖Alcohols: most widely used disinfectants, antiseptics, and sanitizers.
▪Bactericidal and fungicidal but not sporicidal. some enveloped viruses
are also destroyed.
▪Popular alcohol germicides are ethanol and isopropanol, usually used
60 to 80% concentration.
▪Mode of Action: denaturing membrane proteins and dissolving
membrane lipids.
▪Use:
1. A 10- to 15-minute soaking is sufficient to disinfect small instruments.
2. Rubbing hands with specially formulated alcohol sanitizes them by killing
many pathogens.
 ❖Halogen:
Any of the five elements (fluorine, chlorine, bromine, iodine, and astatine).
Iodine and chlorine are important antimicrobial agents.

❖Iodine: At higher concentrations, it may even kill some spores.

❖Use:
▪ Used as a skin antiseptic.
▪ Disadvantages: Although iodine is an effective antiseptic, the skin may be
damaged, a stain is left, and iodine allergies can result.
▪ Iodophor: Iodine can be complexed with an organic carrier to form an
iodophor.
▪ Iodophors are water soluble, stable, and non-staining, and release iodine
slowly to minimize skin burns and irritation.
▪ Used in hospitals and laboratories for disinfecting.
❖Mode of Action: kills by oxidizing cell constituents and iodinating cell proteins.
❖Chlorine: 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), antimicrobial action comes
through hypochlorous acid.

Cl2 + H2O → HCl + HOCl

NaOCl + H2O → NaOH + HOCl
Ca(OCl)2 + 2H2O → Ca(OH)2 + 2HOCl
▪Mode of Action: Oxidizes cellular components and destroy microorganisms.
 Quaternary Ammonium Compounds are Cationic Detergents

❖Detergents: are organic cleansing agents that are amphipathic. Both

hydrophilic & hydrophobic component is present.
❖Cationic Detergent: The hydrophilic portion is a positively charged
quaternary nitrogen; thus they are called cationic detergents. Bactericidal
not sporicidal.
Mode of Action: disrupt microbial membranes & denature proteins
Use: disinfectants for food utensils and small instruments, and skin
antiseptics.
Advantage: stable and nontoxic
Disadvantage: inactivated by hard water and soap
 Aldehyde
Formaldehyde & Glutaraldehyde
❖Formaldehyde: dissolved in water or alcohol before use.
❖Glutaraldehyde: a 2% buffered solution of it is an
effective disinfectant. Usually disinfects objects within 10
min but may require 12 hrs to destroy all spores.
▪Mode of Action: are highly reactive molecules that
combine with nucleic acids and proteins, inactivate them, by
cross-linking & alkylating molecules.
▪Use: sporicidal & used as chemical sterilants.
 Gaseous Agent
Ethylene oxide
❖Ethylene oxide (EtO): microbicidal and sporicidal.
▪Use: heat-sensitive items such as disposable plastic Petri
dishes and syringes, heart-lung machine components,
sutures, and catheters are sterilized. It is a particularly
effective sterilizing agent because it rapidly penetrates
packing materials, even plastic wraps.
▪ Mode of Action: It is a very strong alkylating agent that
kills by reacting with functional groups of DNA and proteins
to block replication and enzymatic activity.
Thank You For the
attention !!!
Any Question?