Lect.6 (chapter 5 microbial control)

Dr. Adel Ahmed Ali El-Morsi
B.Sc. (General BotanyB.Sc. (General Botany , (, ( MansouraMansoura University, 1995University, 1995
M.Sc. Microbiology (virology), Mansoura University, 1999M.Sc. Microbiology (virology), Mansoura University, 1999
Ph.D. Microbiology (virology), Mansoura University, 2005Ph.D. Microbiology (virology), Mansoura University, 2005
General MicrobiologyGeneral Microbiology
B204B204

General Microbiology (General Microbiology (B204B204))
Prepared by Dr. Adel Ahmed Ali El-MorsiPrepared by Dr. Adel Ahmed Ali El-Morsi

Controlling Microorganisms
 Reduce or destroy undesirable microbes in a given area
Effected in two basic ways:Effected in two basic ways:
1. By Killing Microorganisms
2. By inhibiting the Growth of Microorganisms
Usually involves the use of:Usually involves the use of:
1. Physical Agents
2. Chemical Agents

Microbial Control
AA
BB
CC
DD
EE

 SterilizationSterilization
 Sterile
• Inanimate objects free of all life
• Sterilization is the removing of all
microorganisms in a material or on the
surface of an object (an extreme level of cleanliness)
• A surface or an object is either sterile or it is
not sterile, there are no gradations in sterility
• Typically the last things to die when one
attempts sterilization is the highly heat- (and
chemical-, etc.) resistant endospores
• Once something is sterilized, it will remain sterile
if properly sealed.
SterilizationSterilization –– a process that destroys all viablea process that destroys all viable
microbes, including viruses and endosporesmicrobes, including viruses and endospores
Terminology of Microbial ControlTerminology of Microbial Control
 PreventionPrevention
Control of Growth to Prevent Infection and Spoilage

 Disinfection/disinfectantsDisinfection/disinfectants
•Disinfection means reducing the number of viable
microorganisms present in a sample
•Disinfection / Reducing Growth Nonliving Surfaces
•A disinfectant is a chemical or physical agent that is
applied to inanimate objects to kill microbes
•Not all disinfectants are capable of sterilizing, but, of
course, all disinfectants are employed with the hope of
disinfecting.
•While disinfection is not as extreme as sterilization, but it
is considered to be an adequate level of cleanliness for
most situations.
 DisinfectionDisinfection
destroy vegetative pathogens not endospores
Disinfection – a process to destroy vegetativeDisinfection – a process to destroy vegetative
pathogens, not endospores; inanimate objectspathogens, not endospores; inanimate objects
Examples of disinfectants include iodine solution,
copper sulfate, ozone, and chlorine gas.

 Sepsis refers to microbial contamination.
 Asepsis is the absence of significant contamination.
• Aseptic surgery techniques prevent microbial
contamination of wounds.
 Antisepsis/antiseptic Antiseptic
• Typically an antiseptic is a chemical agent that is
applied to living tissue to kill microbes
• Note that not all disinfectants are antiseptics because
an antiseptic additionally must not be so harsh that it
damages living tissue
• In general antiseptics are either not as cheap or not as
effective at killing microbes as disinfectants
Antiseptic – disinfectants applied directly to exposed body surfaces
AntisepsisAntisepsis
• Antisepsis / Reducing Growth on Living Tissue
• Antiseptics are generally less toxic than disinfectants because they
must not cause too much damage to the host living tissue.
• Examples:iodine,70%ethanol,3%hydrogen peroxide.

 SanitizationSanitization
• cleansing technique that mechanically removes
microbes to safe levels
• Sanitization is the cleaning of
pathogenic microorganisms from public
eating utensils and objects such as that
done by the kitchen of a restaurant
• Subject to High Temperature Washing
(Dishwashers)
Sanitization – any cleansing technique that
mechanically removes microbes
 PasteurizationPasteurization
• not sterilization (removes unwanted organisms)
• Pasteurization uses mild temperatures (63°C for
30 minutes or 71°C for 15 seconds) to kill
pathogens and reduce levels of non-pathogenic
organisms that cause milk and other foods to
spoil

 Microbicidal agentsMicrobicidal agents (cidal means kill)
• Causes microbial death
• Bactericide, Sporocide, Fungicide,Viricide
• Suffix-cidal
• An antimicrobial that kills a microorganism (or, more
specifically, a bacterium) is said to be bactericidal
 MicrobistasisMicrobistasis (static means inhibition of growth and
multiplication)
• Prevents microbial growth
• Bacteriostatic, Fungistatic
• Suffix –stasis/-static
• Bacteriostatic means that the antimicrobial inhibits
bacterial growth but does not kill the bacteria;
consequently, removal or dilution of the antimicrobial
can result in a resurgence of bacterial growth
 DegermingDegerming ((Swab the Skin)
removing organisms from an object’s surface
Degermation – mechanically removing microbes
form surface (skin) such as surgical hand scrubbing,
or wiping skin with alcohol prior to venapuncture

 Sterilization vs. DisinfectionSterilization vs. Disinfection
• Sterilization: destroying all forms of life
• Disinfection: destroying pathogens or unwanted organisms
 Disinfectant vs. AntisepticDisinfectant vs. Antiseptic
• Disinfectant: antimicrobial agent used on inanimate objects
• Antiseptic: antimicrobial agent used on living tissue
Sanitization: Lowering of microbial counts to prevent
transmission in public setting (e.g., restaurants & public rest
rooms)
 cidal vs. staticcidal vs. static
• Bactericidal - kills bacteria
• Bacteristatic - inhibits bacterial growth
Fungicidal, Fungistatic, Algacidal, Algastatic
Terms for Microbial ControlTerms for Microbial Control

 Sterilization:Sterilization: Removal (destruction ) of all microbial life
Commercial Sterilization: Heat Treatment of Canned Foods
e.g. Killing C. botulinum endospores
 Disinfection:Disinfection: Removal of pathogens (destruction of
vegetative pathogens)
 Antisepsis:Antisepsis: Removal of pathogens from living tissue
(disinfection of living tissue)
 Sepsis: refers to microbial contamination.
 Degerming:Degerming: Removal of microbes from a limited area
 Sanitization:Sanitization: Lower microbial counts on eating utensils
 Biocide/GermicideBiocide/Germicide: Kills microbes
 Bacteriostasis:Bacteriostasis: Inhibiting, not killing, microbes
TerminologyTerminology

Microbial DeathMicrobial Death
 Involves permanent loss of reproductive capability
• even under optimum growth conditions
 Still alive, but can’t reproduce!!
• Not viable in environment
 Hard to detect
 Loss of movement.
BB

Rate of Microbial DeathRate of Microbial Death
 Bacteria usually die at a constant logarithmic rate
 Plotted Logarithmically This Will Give a Straight Line

 Any method (agent) which can reduce or destroy undesirable
microbes in a given area through killing and /or inhibiting using:-
• Physical
• Chemical
• Mechanical
CC

Cellular Targets of ControlCellular Targets of Control
 Mode of action of antimicrobials:
 Cell wallCell wall
 Cell membraneCell membrane
 Cellular synthetic processes (DNA, RNA)Cellular synthetic processes (DNA, RNA)
 Proteins (Proteins (Enzymes)Enzymes)
Targets of Antimicrobial AgentsTargets of Antimicrobial Agents
Control Agents Act ByControl Agents Act By
Mechanisms of action for Antimicrobial control agents
 Alternation of membrane permeability
 Damage to proteins
 Damage to nucleic acids
 Interfere with metabolic pathways

 Many types of chemical and physical microbial controls
 Modes of action fall into two basic categories
1.1. Alteration of cell walls or cytoplasmic membranesAlteration of cell walls or cytoplasmic membranes
2.2. Interference with protein and nucleic acid structureInterference with protein and nucleic acid structure
Action of Antimicrobial AgentsAction of Antimicrobial Agents
 Alteration of Membrane Permeability
- Susceptibility of membrane is due to its lipid and
protein composition
- Control Agents can alter permeability
 Damage to Proteins and Nucleic Acids
- Break hydrogen and covalent bonds in proteins
- Interfere with DNA, RNA, Protein Synthesis

 Cell wall maintains integrity of cell
• When disrupted, cannot prevent cell from bursting due to
osmotic effects
 Cytoplasmic membrane contains cytoplasm and controls passage
of chemicals into and out of cell
• When damaged, cellular contents leak out
 Viral envelope responsible for attachment of virus to target cell
• Damage to envelope interrupts viral replication
 Nonenveloped viruses have greater tolerance of harsh conditions
Alteration of Cell Walls and MembranesAlteration of Cell Walls and Membranes
 Protein function depends on 3-D shape
• Extreme heat or certain chemicals denature proteins
 Chemicals, radiation, and heat can alter or destroy nucleic acids
• Can produce fatal mutants
• Can halt protein synthesis through action on RNA
Damage to Proteins and Nucleic AcidsDamage to Proteins and Nucleic Acids

 Ideally, agents should be:Ideally, agents should be:
• Inexpensive
• Fast-acting
• Stable during storage
• Control all microbial growth while being
harmless to humans, animals, and objects
Selection of Microbial Control MethodsSelection of Microbial Control Methods
Practical Concerns for Microbial ControlPractical Concerns for Microbial Control
 Does the application require sterilization?
 Is the item to be reused?
 Can the item withstand heat, pressure, radiation, or chemicals?
 Is the method suitable?
 Will the agent penetrate to the necessary extent?
 Is the method cost- and labor-efficient & is it safe?

1. Nature of site to be treated
2. Degree of susceptibility of microbes involved
3. Environmental conditions that pertain
Factors Affecting the Efficacy ofFactors Affecting the Efficacy of
Antimicrobial methodsAntimicrobial methods
DD

 Harsh chemicals and extreme heat cannot be
used on humans, animals, and fragile objects
 Method and level of microbial control based
on site of medical procedure
Site to Be TreatedSite to Be Treated

Organismal differencesOrganismal differences
 Microorganisms differ in their
susceptibility to antimicrobial
agents."
 Often what fails to be killed by a
disinfectant are endospores
though certain viruses and some
vegetative bacteria are also
highly resistant to disinfection
 Also, the same organism may
differ in susceptibility depending
on growth phase with actively
growing organisms typically
more susceptible to disinfection
than not-growing cultures
Microbial Characteristics and ControlMicrobial Characteristics and Control

Level of ResistanceLevel of Resistance
1. Endospores
2. Mycobacteria
3. Fungal Spores
4. Small Non-enveloped Viruses
- Polio, Rotavirus, Rabies
5. Vegetative Fungal Cells
6. Enveloped Viruses
- Herpes, Hepatitis B & C, HIV
7. Vegetative Bacteria
Susceptibilities VarySusceptibilities Vary
 Endospores are Difficult to Kill

Relative Resistance of MicrobesRelative Resistance of Microbes
 Highest resistanceHighest resistance
• Bacterial endospores & prions
 Moderate resistanceModerate resistance
• Pseudomonas sp.
• Mycobacterium tuberculosis
• Staphylococcus aureus
• Protozoan cysts
 Least resistanceLeast resistance
• most vegetative cells
• fungal spores
• enveloped viruses
• yeast
• protozoan trophozoites

Relative Susceptibility of MicroorganismsRelative Susceptibility of Microorganisms
 Effectiveness of germicides classified as
high, intermediate, or low
• High-levelHigh-level kill all pathogens, including
endospores
• Intermediate-levelIntermediate-level kill fungal spores,
protozoan cysts, viruses and pathogenic
bacteria
• Low-levelLow-level germicides kill vegetative
bacteria, fungi, protozoa, and some
viruses

Environmental conditions that pertainEnvironmental conditions that pertain
 Increasing temperatures increases the efficacy of
most chemical antimicrobials
 The converse of this statement is that relatively
cold temperatures result in relatively poor
disinfection

Environmental conditions that pertain (cont.(
 Concentration effects: Generally, the use of
more disinfectant provides better killing
than the use of less disinfectant
 The fewer organisms present, the shorter the time needed to
achieve sterility. Thoroughly cleaning objects before attempting
to sterilize them is a practical application of this principle.
Clearing objects of tissue debris and blood is also important
because such organic matter impairs the effectiveness of many
chemical agents.

Factors That Affect Death RateFactors That Affect Death Rate
1. Number of microbes
2. Nature of microbes in the population. (Microbial
characteristics e.g. glycocalyx, cell wall, resistance)
3. Environment. (organic matter, temperature, concentration,
biofilms & pH )
4. Concentration or dosage of agent.
5. Mode of action of the agent.
6. Presence of solvents, organic matter, or inhibitors.
7. Time of exposure (Time to Kill in Proportion to the
Population Size).
Effectiveness of antimicrobial treatment depends on:

1. Heat – Moist verse Dry (Exposure to extremes of heat)
2. Cold temperatures (Exposure to extremes of cold)
3. Desiccation
4. Radiation
5. Osmotic pressure
Physical Methods of Microbial ControlPhysical Methods of Microbial Control
 Not for use on living organisms
 Somehow, alter membrane permeability and / or
structure of proteins and nucleic acids

HeatHeat
 Most Frequent and Widely Used.
 Always Consider
1. Type of Heat
2. Time of Application
3. Temperature
 Endospores are the most heat resistant of all cells.
 Effects of high temperatures
• Denaturation of proteins
• Interference with integrity of cytoplasmic membrane
and cell walls
• Disruption of structure and function of nucleic acids

Thermal Death MeasurementsThermal Death Measurements
 Thermal death point (TDP):Thermal death point (TDP):
• lowest temperature required to kill all microbes in
a sample in 10 minutes
 Thermal death time (TDT):Thermal death time (TDT):
• shortest length of time required to kill all microbes
at a specified temperature
Heat
 Decimal reduction time (DRT):Decimal reduction time (DRT):
• Minutes to kill 90% of a population at a given temperature

Thermal Death PointThermal Death Point
 Thermal Death Point (TDP) /
Lowest Temp to Kill All the
Bacteria in a Broth in 10 Minutes.
• This aspect of thermal death is
useful in purifying water via
boiling.
 Boiling:
Kills Many Vegetative Cells and
Inactivates Viruses Within 10
Minutes (30 Minutes to be Safe)
but has no effect on spores

Thermal Death TimeThermal Death Time
 Thermal Death Time
(TDT) / Time Span
Required to Kill All the
Bacteria in a Broth at a
Given Temperature.
 It was developed for food
canning and has found
applications in cosmetics
and pharmaceuticals.

Decimal Reduction TimeDecimal Reduction Time
(DRT(DRT((
 Decimal Reduction
Time (DRT) / Length of
Time in Which 90% of a
Bacterial Population will
be Killed at a given
Temperature
 Reduces the number of
organisms to 1/10 the
initial level.
 Used in Commercial
Sterilization.

 Used to disinfect, sanitize, and sterilize
 Kills by denaturing proteins, destruction of DNA and
destroying cytoplasmic membranes.
 More effective than dry heat; water better conductor of heat
than air. Moist heat is also more penetrating than dry heat
 Methods of microbial control using moist heat
• Boiling
• Autoclaving
• Pasteurization
• Ultrahigh-Temperature Sterilization
Moist HeatMoist Heat

 Kills vegetative cells of bacteria and fungi,
protozoan trophozoites, and most viruses within
10 minutes at sea level
 Temperature cannot exceed 100ºC at sea level;
steam carries some heat away
 Boiling time is critical
 Water boils at lower temperatures at higher
elevations; requires longer boiling time
 Endospores, protozoan cysts, and some viruses
can survive boiling
BoilingBoiling
• Hepititis (20 min)
• Some spores may survive
boiling water for up to 20 hrs

AutoclavingAutoclaving
 Pressure applied to boiling water prevents steam
from escaping
 Boiling temperature increases as pressure increases
 Autoclave conditions – 121ºC, 15 psi, 15 minutes
AutoclaveAutoclave

 Pressure applied to boiling water prevents steam from escaping
 Boiling temperature increases as pressure increases
 Autoclave conditions – 121ºC, 15 psi, 15 minutes
 Volume, Contact, Wrapping, Testing
Autoclave Tape

 Steam Under Pressure
 121° C for 15 Minutes at 15 lb/in2
 Heat-labile Substances will be Denatured (proteins)
 Steam Must Contact the Material

 Pasteurization is the application of moist
heat of less-than boiling temperatures to
foods to prevent the growth of food-
spoiling organisms as well as various
heat-labile pathogens. without destroying
the food flavor or value
 batch method
63°C - 66°C for 30 minutes
 flash method
71.6°C for 15 seconds
Pasteurization
 Pasteur’s method
 Pasteurization is a process used in preserving heat sensitive
foods such as milk, ice cream, yogurt, fruit juices, beer, and
other beverages.

 Pasteurization is not a method of sterilization,
which is why pasteurized foods will eventually
spoil if given enough time.
 Pasteurization extends the shelf life of a
product and reduces the level of pathogens
in the product.
 Not sterilization; heat-tolerant and heat-
loving microbes survive
• These do not cause spoilage prior to
consumption
• These are generally not pathogenic
• kills non-spore-forming pathogens and
lowers overall microbe count
• does not kill endospores or many
nonpathogenic microbes

Ultrahigh-Temperature SterilizationUltrahigh-Temperature Sterilization
 A new method called ultrahigh temperature (UHT) sterilization
involves heating at 140°C for 1-3 seconds, then rapid cooling.
 Treated liquids can be stored at room temperature
 Milk that has been treated in this way can be kept at room
temperature for 2 months with only minimal changes in flavor.

 Used for materials that cannot be sterilized with or are
damaged by moist heat
 Denatures proteins and oxidizes metabolic and structural
chemicals
 Requires higher temperatures or longer time than moist heat
 Nevertheless, application of dry heat is cheap and easy
 higher temperatures than moist heat
Dry HeatDry Heat

Dry Heat
 1. Direct Flaming1. Direct Flaming
• Bunsen burner (1870o
C)
• Dehydrates cells and removes water
• Inoculating Loop and Needle 100% effective
 2. Incineration (2. Incineration ( <<12001200oo
C )C )
• Burns and Physically Destroys Organisms
combusts & dehydrates cells
• Loops: needles, inoculating wires (1500°C)
• Disposable wastes (paper cups, bags, dressings)
• Glassware
 3. Hot Air Sterilization (dry ovens)3. Hot Air Sterilization (dry ovens)
• coagulate proteins
• used on substances that would be damaged by
moist heat sterilization and objects that Won’t
Melt e.g. glassware, metal, dressings or powders
(i) 171ºC for at least one hour
(ii) 160ºC for at least two hours
(iii) 121ºC for at least 16 hours

22..Cold (Low) TemperaturesCold (Low) Temperatures
 Microbistatic (Not Bactericidal)
• slows the growth of microbes
 Decreasing Temperature
Decreases Chemical Activity
 refrigeration 0-15o
C
 freezing <0o
C
 used to preserve food, media
and cultures

 Refrigeration is a great short term solution, it decreases
microbial metabolism, growth, and reproduction i.e. it merely
slows the growth of organisms rather than preventing it
• Chemical reactions occur slower at low temperatures
• Liquid water not available
 Psychrophilic microbes can multiply in refrigerated foods
 Refrigeration halts growth of most pathogens
 Slow freezing more effective than quick freezing
 Organisms vary in susceptibility to freezing
Refrigeration and FreezingRefrigeration and Freezing
 For organisms that survive the freezing process, freezing
constitutes a reasonably good long-term preservation method
with prevention of deterioration increasing as temperatures are
lowered
 Lower temperatures result in greater long-term storage (-
20ºC, -80ºC, -180ºC)

33..DesiccationDesiccation
 Gradual removal of water from
cells (Dehydration)
 Leads to metabolic
disruption/inhibition
 Stops Growth / Microbes Are
Still Viable
 not effective microbial control
• many cells retain ability to
grow when water is
reintroduced
 Viruses and Endospores Can
Resist Desiccation

 Lyophilization used for long term preservation of
microbial cultures
• Prevents formation of damaging ice crystals.
• Freeze-drying involves freezing something and
then evacuating air so that boiling occurs at low
temperatures; this desiccates material thereby
preventing deterioration and spoilage.
• Liquid nitrogen or frozen carbon dioxide (dry
ice).
LyophilizationLyophilization (Freeze-drying(Freeze-drying((

Electromagnetic spectrum, energy without mass travelling in waves at
the speed of light, 3x105km/sec, Efficiency is Dependent on the
Wavelength, Intensity, and Duration (shorter wavelength means
great energy and can penetrate further).
 Radiation damages DNA
 Two types used:
• Ionizing radiation: Wavelengths shorter than 1 nm
(X rays, gamma rays, electron beams)
• Non-ionizing radiation: Wavelengths greater than 1 nm (UV)
• (Microwaves kill by heat; not especially antimicrobial)
44..RadiationRadiation
 Radiation described as ionizing or nonionizing according to effects
on cellular chemicals.

 Wavelengths shorter than 1 nm – electron
beams, gamma rays, and X rays.
 are more penetrating but are more difficult and
expensive to use.
 Ionizing radiation is radiation that ionizes
water; this temporarily turns water into an
oxidizing agent.
Ionizing RadiationIonizing Radiation
 Create ions by ejecting electrons from atoms they strike.
 Ions disrupt hydrogen bonding, oxidize double covalent bonds, and
create hydroxide ions; hydroxide ions denature other molecules (DNA).
Types of Ionizing RadiationTypes of Ionizing Radiation
 Electron beamsElectron beams – effective at killing but do not penetrate well
• Used to sterilize spices, meats, microbiological plastic ware,
and medical and dental supplies
 Gamma raysGamma rays – penetrate well but require hours to kill microbes
• Used to sterilize meats, spices, and fresh fruits and vegetables
 X-raysX-rays require too much time to be practical for growth control

 Food irradiation is a process whereby the
food is exposed to high levels of radiation in
order to kill insects, bacteria and mold, and
make the food last longer on the store
shelves.
 One potential application of ionizing
radiation is as an antimicrobial is in food
preservation and other industrial processes.
 Used on substances that could be damaged by heat
• plastic petri dishes, plastic syringes, catheters, surgical gloves

 Usually use cobalt-60 which has a half-life of 5 years.
 Materials which are sterilized using this type of radiation do not
become radioactive, and controversy exists on whether or not
irradiation of food changes its nutritional value.
 In some cases the taste of the food is changed, similar to how milk
changes taste once it is pasteurized.
 Studies have shown that irradiating microorganisms like
E. coli and Salmonella may give rise to even more
dangerous, radiation-resistant strains of bacteria.
 Under laboratory conditions scientists found that one
particular type of bacteria can survive a radiation dose
five times what the FDA will allow for beef.
 Radiation is completely ineffective against viruses, and does
absolutely nothing to clean the food of waste products and other
unsanitary matter often left on beef, chicken, and lamb as the
result of slaughterhouse conditions.
 India, children blood tests showed chromosome damage
after being fed freshly irradiated wheat for six weeks.

Nonionizing RadiationNonionizing Radiation
 Excites electrons and causes them to make new covalent bonds
• Affects 3-D structure of proteins and nucleic acids
 UV light causes pyrimidine (Thymine) dimers in DNA
•Interfere with replication

UV RadiationUV Radiation
 The most lethal type of radiation is ultraviolet radiation with
a wavelength of 260 nm. This is the wavelength most
actively absorbed by DNA.
 Due to its poor penetration, does not penetrate plastic, glass
or proteinaceous matter, UV radiation is only useful for
disinfecting outer surfaces of objects , transparent fluids,
air and liquids.
 Used to reduce microbial populations
• hospital rooms, nurseries and operating rooms
 This type of radiation is also harmful if someone is directly
exposed to it (for extended periods of time), as it may
damage the skin and eyes.
Aquarium lightAir and surface microbial controlEgg surface microbial controlEgg surface microbial control

MicrowavesMicrowaves
 Kill Microbes Indirectly with Heat
Do Not Microwave Baby BottlesDo Not Microwave Baby Bottles
 Inside milk may be hotter than outer bottle.
 Heating the bottle in a microwave can cause slight changes
in the milk: Inactivates Antibodies (Breast Milk),
Denatures Protein and Destroys Vitamins
 Microwaving changes food nutrients.
 Increases Leukocytes in Blood (Sign of
Infection and Poisoning)
 Decreases Erythrocytes and Iron Stores and
Results in Anemia.
 Increases Cholesterol.
 Causes Production of Radiolytic Compounds
(Mutated Compounds) Which Depress the
Immune System and are Carcinogenic.

55..Osmotic PressureOsmotic Pressure
 High concentrations of salt or sugar in
foods to inhibit growth (jam, jerky,
pickled food, salted fish); Sugar Curing /
Salting
 Cells in a hypertonic solution of salt or
sugar lose water Causes plasmolysis i.e.
shrinkage of cytoplasm (Water in cell is
drawn out by osmosis); cell desiccates
 Fungi have greater ability than bacteria to
survive hypertonic environments (May
Still Get Some Mold or Yeast Growth)

FiltrationFiltration
 The passage of a liquid or gas through a
filter with pores small enough to retain
microbes (25µm to less than 0.01µm).
• physical removal of microbes
• passing a gas or liquid through filter
• organisms above a certain size
trapped in the pores
 Especially important to sterilize
(Removes microorganisms)
solutions which would be denatured
by heat (culture media, enzymes,
vaccines, antibiotics, injectable
drugs, amino acids, vitamins.)
 used to sterilize heat sensitive liquids &
air in hospital isolation units & industrial
clean rooms
• Air can be filtered using a high-
efficiency particulate air (HEPA)
filter

HEPA FiltersHEPA Filters
 HEPA filters are High-Efficiency Particulate
Air filters designed for the filtration of small
particles. Certified HEPA filters must capture a
minimum of 99.97% of 0.3 microns
contaminants.
 Filtration is the primary method of eliminating pathogens from the air supply
i.e. used to filter the air flowing into aseptic environments ( e.g. operating
rooms) and out of potentially contaminated ones (e.g., containment facilities)
1. Operating Rooms
2. Burn Units
3. Fume Hoods
4. Isolation Rooms
5. Bio-cabinets
6. Pharmaceutical Manufacturing Facilities

Effectiveness of Chemical DisinfectantsEffectiveness of Chemical Disinfectants
1. Type of Microbe1. Type of Microbe
- G + More Susceptible to Disinfectants
- Pseudomonands Can Grow in Disinfectants and Antiseptics
- M. tuberculosis is Resistant to Many Disinfectants
- Endospores Most Resistant
2. Environment2. Environment
• Concentration of disinfectant
• Organic matter
• pH
• Time
• Contact with microbetime

 Phenol Coefficient
 Use-Dilution Test
• Metal cylinders (rings)
• Serial dilution (test tubes)
 Diffusion Method
• Disk diffusion
• Cup plate diffusion
 In-Use Test
Methods for Evaluating Antimicrobial agentMethods for Evaluating Antimicrobial agent
Testing for Drug SusceptibilityTesting for Drug Susceptibility

 Old Standard Test
 The phenol coefficient is the value obtained
by dividing the highest dilution of the test
solution by the highest dilution of phenol
that sterilizes the given culture of bacteria
under standard conditions of time and
temperature.)
 Greater than 1.0 indicates that agent is more
effective than phenol
 Has been replaced by newer methods
Phenol CoefficientPhenol Coefficient

 Especially useful for determining the ability of disinfectants
to kill microorganisms dried onto a typical clinical surface
(stainless steel)
 Metal cylinders dipped into broth cultures of bacteria and
dried
 Contaminated cylinder immersed into dilution of
disinfectant for 10 minutes
 Cylinders removed, washed, and placed into tube of
medium for 48 h
 Most effective agent entirely prevents growth at highest
dilution
Use-Dilution TestUse-Dilution Test
Three microbes are used:Salmonella choleraesuis,
Staphylococcus aureus andPseudomonas aeruginosa

Dilution testsDilution tests
 Minimum inhibitory concentration (MIC)
• smallest concentration of drug that visibly inhibits growth
 In vitro activity of a drug is not always correlated with in vivo
effect
• If therapy fails, a different drug, combination of drugs, or different
administration must be considered
 Best to chose a drug with highest level of selectivity but
lowest level toxicity

Diffusion MethodDiffusion Method
 A method that requires less manipulation to judge the
efficacy of disinfectants.
 Here filter paper is soaked with disinfectant and then simply
placed on the agar surface of a petri dish that has been
inoculated with a lawn of test organism.
 The clear area around the disk following incubation is used
as an indication of disinfectant efficacy.
 Essential for groups of bacteria commonly showing resistance
 Kirby-Bauer disk diffusion test

 Swabs taken from objects before and after
application of disinfectant or antiseptic
 Swabs inoculated into growth medium and
incubated
 Medium monitored for growth
 Accurate determination of proper strength
and application procedure for each specific
situation
In-Use TestIn-Use Test

Chemical Control MethodsChemical Control Methods (Microbial Agents(
 Major Categories
 Phenols and Phenolics
 Biguanides (Chlorhexidine)
 Halogens
 Alcohols
 Oxidizing agents
 Heavy Metals and Their Compounds
 Surface-Active Agents (Surfactants)
• Detergents & soaps
• Quaternary Ammonium Compounds
 Chemical Food Preservatives
 Aldehydes
 Gaseous Agents and aerosols
 Antibiotics (Antimicrobics)

Phenol and Phenolics
- Another Name for Carbolic Acid / Lysol / Pine-Sol
- Joseph Lister
- Exert Influence By
1. Injuring Plasma membranes
2. Inactivating Enzymes
3. Denaturing Proteins
 Phenolics are Long Lasting, Good for Blood and Body Fluids
 Phenols are effective antibacterial agents,
and they are also effective against fungi and
many viruses but No Effect on Spores
 Phenols have a characteristic pine-tar odor and turn milky in water.
 Phenols can be toxic to pets especially cats and pigs.

BiguanidesBiguanides
- Damage Plasma Membranes
- Caution: Can Damage Eyes – Avoid
Splashing
- Only Operates in Narrow pH Range (5-7)
- Example / Chlorhexidine
ChlorhexidineChlorhexidine
A surfactant & protein denaturant with broad
microbicidal properties
Not sporicidal
Used as skin degerming agents for preoperative
scrubs, skin cleaning & burns

HalogensHalogens ((Intermediate-level antimicrobial chemicals))
- Can be Used Alone or in Solution
- Inactivated by Sunlight
- They damage enzymes
- Can be Corrosive to Metal
- Can Irritate Mucus Membranes
ChlorineChlorine -- Purifies Drinking Water
 Cl2, hypochlorites (chlorine bleach), chloramines
 Denaturation of proteins by disrupting disulfide bonds
 Can be sporicidal
 Forms an Acid Which is Bactericidal
 Acts as a Disinfectant in Gaseous Form or in Solution as Calcium Hypochlorite
Chlorine compounds are good disinfectants on clean
surfaces, but are quickly inactivated by dirt.
Chlorine can be irritating to skin and corrosive to metal.
Inexpensive / Chlorox
Never Mix with Other Cleaning Agents!

HalogensHalogens
IodineIodine – combines with Amino Acids
 I2 /Iodophor/Organic Molecule/Betadine
 Inactivates Enzymes / Denature proteins
 Can be sporicidal
Iodine is normally considered to be the least toxic of the disinfectants.
Milder medical & dental degerming agents, disinfectants, ointments
Iodine products can stain clothing and porous surfaces.

• Denature Proteins (coagulating proteins of vegetative
bacterial cells and fungi)
• Dissolve Lipids and disrupt cytoplasmic membranes
• Evaporates both advantageous and disadvantageous
• Fast Acting, No Residue, Flammable
• Alcohols should not be applied to wounds since they can
cause tissue damage
• Swabbing of skin with 70% ethanol prior to injection
Wet Disinfectants
Ethyl, isopropyl in solutions of 50-90%
Act as surfactants
Not sporicidal
Good for enveloped viruses
AlcoholsAlcohols ((Intermediate-level disinfectants))

Oxidizing AgentsOxidizing Agents ((High-level disinfectants and antiseptics)
oxidizing agents are active against bacteria, bacterial spores,
viruses, and fungi at quite low concentrations.
Peroxides H2O2(skin), ozone O3 (swim pools), and peracetic acid
(dialysis equipment),bleach (sodium hypochlorite), bromine kill
by oxidation of microbial enzymes
Hydrogen peroxide can disinfect and sterilize surfaces of objects
Ozone treatment of drinking water
Peraceticacid effective sporocide used to sterilize equipment
Hydrogen PeroxideHydrogen Peroxide
Weak (3%) to strong (35%)
Produce highly reactive hydroxyl-free radicals that damage
protein & DNA while also decomposing to O2 gas
• toxic to anaerobes
Strong solutions are sporicidal in increasing concentrations

Types of DisinfectantsTypes of Disinfectants
Heavy Metals and Their CompoundsHeavy Metals and Their Compounds
Heavy Metals. Ag, Hg, Cu
• Oligodynamic action
Ions are antimicrobial because they alter the 3-D shape of proteins,
inhibiting or eliminating their function (Denature/inactivate proteins)
Denature proteins by breaking disulfide bonds
Low-level bacteriostatic and fungistatic agents i.e. Not sporicidal
Example Solutions of silver & mercury / Silvadene Ointment
1% silver nitrate to prevent blindness (antiseptic for eyes, wounds(
Zinc chloride (ZnCl2) mouthwashes
Thimerosal(mercury-containingcompound)used to preserve vaccines
Copper controls algal growth in reservoirs, fish tanks, swimming pools,
and water storage tanks; interferes with chlorophyll (CuSO(CuSO44 algicide)algicide)
• Indian tradition of storage of river
water in brass containers as a way to
prevent disease. The river water may
have up to 1 million fecal bacteria per
ml. That count could be reduced to
undetectable by 2 days of storage

Surfactants are substances that are soluble in water but are
able to dissolve lipids ie. “Surface active” chemicals that
reduce surface tension of solvents to make them more
effective at dissolving solutes
Soaps and detergentsSoaps and detergents
• Soaps have hydrophilic and hydrophobic ends; good
degerming agents but not antimicrobial
• Soaps Have Limited Germicidal Action but Assist in the
Removal of Organisms by Scrubbing
• Detergents are positively charged organic surfactants
• Acid-Anionic Detergents / Dairy
SurfactantsSurfactants Surface-Active AgentsSurface-Active Agents
Soap Degerming
Acid-anionic detergents Sanitizing
Quarternary ammonium
compounds Cationic detergents
Bactericidal, Denature proteins,
disrupt plasma membrane

Quaternary Ammonium CompoundsQuaternary Ammonium Compounds
(QUATS)(QUATS)
• Cationic Detergents Attached to NH4
+
• Ammonia compounds act as surfactants
• Most Effective on Gram-Positive Bacteria
• Mouthwashes and Sore Throat Remedies
QUATS are generally odorless, colorless,
nonirritating, and deodorizing.
Can be inactivated in the presence of some
soaps or soap residues and their antibacterial
activity is reduced in the presence of organic
material.
Disruption of plasma membrane, denaturation
of proteins
Examples Benzalkonium chloride (aka Zephiran),
Roccal: lab disinfectant and Cetylpyridinium chloride
(Cepacol)

Chemical Food PreservativesChemical Food Preservatives
Salts
Sugar
Dry
Organic Acids
• Inhibit metabolism
• Sorbic acid, benzoic acid, calcium propionate
• Control molds and bacteria in foods and cosmetics
Nitrate and Nitrite Salts / Meats /To Prevent Germination of
Clostridium botulinum endospores
Antibiotics. Nisin and natamycin prevent spoilage of cheese

Types ofTypes of
DisinfectantsDisinfectants
AldehydesAldehydes
Compounds containing terminal –CHO groups
e.g. Formaldehyde and Glutaraldehyde
- Most Effective of all Chemical
- Disinfectants
- Carcinogenic
- Oxidize Molecules Inside Cells
Cross-link with functional groups (–NH2, –OH, –COOH, —SH) amino, hydroxyl,
sulfhydryl, and carboxyl groups to denature proteins and inactivate nucleic acids
Glutaraldehyde both disinfects (short exposure) and sterilizes (long exposure), 2%
solution (Cidex) used as sterilant for heat sensitive instruments
Formaldehyde: Disinfectant to rooms and instruments,
preservative, toxicity limits use

Ethylene oxide not Ethyl oxide, propylene oxide, beta-
propiolactone & chlorine dioxide used in closed chambers to
sterilize items
Strong alkylating agents, sporicidal
Denature proteins and DNA by cross-linking functional groups
Use in sterilize hospital equipment, disposable lab plasticwareand
dental offices.
Can be hazardous to people, often highly explosive, extremely
poisonous, and are potentially carcinogenic
The most effective method of gas chemical sterilization presently
available is the use of ethylene oxide (ETO) gas. ETO gas
sterilization should be used only for material and supplies that will
not withstand sterilization by steam under pressure.
Gaseous Agents & AerosolsGaseous Agents & Aerosols

Types of DisinfectantsTypes of Disinfectants
AntibioticsAntibiotics
- Used to Preserve Cheese
- Used in Feed Given to Food Animals
- Nisin
- Natamycin
Growing Problem
Indiscriminant and Inappropriate Use
Super Bugs
1. Methicillin Resistant S. aureus
2. Vancomycin Resistant Enterococcus
3. Multidrug Resistant M. tuberculosis
This is Why it is so Important to Order Sensitivities
In one study, three out of four patients seen in the emergency
room for skin infections had Staphylococcal aureus infections and
over 50% had MRSA infections.
That equates to 12 million MRSA infections each year in the USA.
MRSA

Origins of Antimicrobial DrugsOrigins of Antimicrobial Drugs
Antibiotics
• Common metabolic products of aerobic spore-forming
bacteria & fungi
• bacteria in genera Streptomyces & Bacillus
• molds in genera Penicillium & Cephalosporium
Inhibiting other microbes in the same habitat
• antibiotic producers have less competition for nutrients &
space
Typically used for treatment of disease …but
Some are used for antimicrobial control outside the body

Ideal Antimicrobial DrugIdeal Antimicrobial Drug…..…..
 Selectively toxic to microbe
 Not host cells
 Microbicidal, not microbistatic
 Soluble
 Potent
 No antimicrobial resistance
 Remains active
 Readily delivered to site of infection
 Expense
 Not allergen

ChemotherapyChemotherapy
 Antimicrobial
• Control infection
 Antibiotic
• Produced by the natural metabolic processes of microorganisms
• Can inhibit or destroy other microorganisms
 Semisynthetic
• Chemically modified drugs in lab
 Synthetic
• Synthesized compounds in lab
 Narrow spectrum
• Effective against limited microbial types
• Target a specific cell component that is found only in certain microbes
 Broad spectrum
• Effective against wide variety
microbial types
• Target cell components common
to most pathogens

Selectively ToxicSelectively Toxic
 Should kill or inhibit microbial cells without simultaneously
damaging host tissues
 Complete selective toxicity
• Difficult to achieve
• Characteristics of the infectious agent become more
similar to the vertebrate host cell
• More side effects are seen
 toxic dose of a drug
• The concentration causing harm to the host
 therapeutic dose
• the concentration eliminating pathogens in the host
 Together, the toxic and therapeutic doses are used to formulate
the chemotherapeutic index

Targets of Antimicrobial DrugsTargets of Antimicrobial Drugs
1. Inhibition of cell wall
synthesis
2. Inhibition of nucleic
acid synthesis, structure
or function
3. Inhibition of protein
synthesis
4. Disruption of cell
membrane structure or
function

11..Bacterial Cell WallBacterial Cell Wall
Most bacterial cell walls contain
peptidoglycan
Penicillin and cephalosporin block
synthesis of peptidoglycan
• Causes the cell wall to lyse
Penicillins do not penetrate the
outer membrane
• less effective against gram-
negative bacteria
Broad spectrum penicillins and
cephalosporins
• cross the cell walls of gram-
negative bacteria

22..Inhibit Nucleic Acid SynthesisInhibit Nucleic Acid Synthesis
May block synthesis of nucleotides, inhibit replication, or
stop transcription
sulfonamides and trimethoprim
• block enzymes required for tetrahydrofolate synthesis
• needed for DNA & RNA synthesis

33..Drugs that Block Protein SynthesisDrugs that Block Protein Synthesis
Ribosomes
• eukaryotes differ in size and
structure from prokaryotes
Aminoglycosides
(streptomycin, gentamicin)
• insert on sites cause
misreading of mRNA
Tetracyclines
• block attachment of tRNA and
stop further synthesis

44..Disrupt Cell Membrane FunctionDisrupt Cell Membrane Function
Damaged membrane
• dies from disruption in
metabolism or lysis
These drugs have specificity for a
particular microbial group
• based on differences in types
of lipids in their cell
membranes
Polymyxins
• interact with phospholipids
• cause leakage, particularly in
gram-negative bacteria
Amphotericin B and nystatin
• form complexes with sterols
on fungal membranes
• causes leakage

Drug ResistanceDrug Resistance (Development of Resistant Microbes(Development of Resistant Microbes((
Microorganisms begin to tolerate an amount of drug that
would ordinarily be inhibitory
• due to genetic versatility or variation
• intrinsic and acquired
Little evidence that extensive use of products containing
antiseptic and disinfecting chemicals adds to human or animal
health
The use of such products promotes the development of
resistant microbes

Mechanisms of Drug ResistanceMechanisms of Drug Resistance
Drug inactivation by acquired
enzymatic activity
• penicillinases
Decreased permeability to drug
or increased elimination of drug
from cell
• acquired or mutation
Change in drug receptors
• mutation or acquisition
Change in metabolic patterns
• mutation of original
enzyme

Side Effects of DrugsSide Effects of Drugs
5% of all persons taking antimicrobials will experience a
serious adverse reaction to the drug
• Toxicity to organs
• Allergic responses
• Suppression and alteration of microflora
Considerations in Selecting an Antimicrobial DrugConsiderations in Selecting an Antimicrobial Drug
1. Identify the microorganism causing the infection
• Specimens should be taken before antimicrobials initiated
2. Test the microorganism’s susceptibility (sensitivity) to various
drugs in vitro when indicated
3. Overall medical condition of the patient

What about virusesWhat about viruses?!?!??!?!?
Do not destroy their target pathogen
Instead they inhibit their development
• Inhibit virus before enters cell
• Viral-associated proteins
• Stop it from entering the cell
• Stop it from reproducing
• Prevent from exiting the cell

Lect.6 (chapter 5 microbial control)

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Lect.6 (chapter 5 microbial control)