Control of Microorganisms
Microbiology 2314
Control of Microbial Growth
Effected in two basic ways:
1. By Killing Microorganisms
2. By inhibiting the Growth of Microorganisms
Usually involves the use of:
1. Physical Agents
2. Chemical Agents
Level of Resistance
1.
2.
3.
4.
Endospores
Mycobacteria
Fungal Spores
Small Non-enveloped Viruses
- Polio, Rotavirus, Rabies
5. Vegetative Fungal Cells
6. Enveloped Viruses
- Herpes, Hepatitis B & C, HIV
5. Vegetative Bacteria
Principles of Microbial Control
(Definitions)
• Prevention / Control of Growth to Prevent
Infection and Spoilage
Principles of Microbial Control
(Definitions)
• Sterilization / Complete Destruction
- No degrees of sterilization (All or Nothing)
- Utilizes
a. Heat
b. Radiation
c. Chemicals
d. Physical Removal
• Once something is sterilized, it will remain sterile
if properly sealed. Sterilization is performed on
surgical equipment, needles, and certain lab
equipment in order to prevent the spread of
microorganisms.
• Sterilization is an
extreme level of
cleanliness that is
usually not required
outside of a medical,
industrial, or laboratory
environment.
• In everyday
environments,
disinfection of objects
is considered a more
practical option.
Nano Disinfection Scanner
Principles of Microbial Control
• Commercial Sterilization / Heat
Treatment of Canned Foods
Principles of Microbial Control
• Disinfection / Reducing Growth Nonliving
Surfaces
• Disinfection may not necessarily eliminate
spores or all of the microorganisms from an
object or environment.
• While disinfection is not as extreme as
sterilization, but it is considered to be an
adequate level of cleanliness for most situations.
• Examples of disinfectants include iodine
solution, copper sulfate, ozone, and chlorine gas.
Principles of Microbial Control
• Antisepsis / Reducing Growth on Living Tissue
Principles of Microbial Control
• Antiseptics are generally less toxic than
disinfectants because they must not cause too
much damage to the host living tissue.
• Examples of antiseptics include iodine, 70%
ethanol and 3% hydrogen peroxide.
Principles of
Microbial
Control
• Degerming /
Swab the Skin
Principles of
Microbial
Control
• Sanitize
Subject to High
Temperature
Washing
(Dishwashers)
Principles of
Microbial
Control
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Cide – Suffix Meaning to Kill
Stat – Suffix Meaning to Inhibit
Sepsis – Bacterial Contamination
Asepsis – Lack of Bacterial Contamination
Rate of Microbial Death
• Bacteria Usually Die At a Constant Rate
• Plotted Logarithmically This Will Give a Straight Line
Time to Kill in Proportion to the
Population Size
• Large Numbers Require Greater Time
• Small Numbers Require Less Time
Susceptibilities Vary
• Endospores are Difficult to Kill
• Organic Matter May Interfere with Heat
Treatments and Chemical Control Agents
Control Agents Act By
• 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
Denatures Proteins
Physical Methods of Microbial
Control
Physical
Methods
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Heat
Filtration
Low Temperatures
Desiccation
Osmotic Pressure
Radiation
Heat
• 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.
• Moist Heat /
Denatures Enzymes
• Moisture improves
heat penetration,
making sterilization by
moist heat more
effective then dry heat.
• Includes boiling and
autoclaving.
• Rare types of bacteria are capable of growing at
high temperatures.
• These bacteria are classified as thermophiles
and hyperthemophiles.
• These organisms normally growth in unusually
hot environments, including hot springs and
deep-sea vents.
• Clearly sterilization by heat may not be the most
efficient method to eliminate these types of
bacteria, but they are almost never found in
common environments and they are not
pathogenic.
• 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
Point
Thermal 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 Time
• 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.
Autoclaving (Moist Heat Sterilization)
• Steam Under
Pressure
• 121° C for 15
Minutes at
15 lb/in2
• Heat-labile
Substances will
be Denatured
• Steam Must
Contact the
Material
Dry Heat
Sterilization
• Direct Flaming
• Incineration
• Hot-Air Sterilization (Oven)
Flaming the Loop
 Flaming the loop
helps to prevent
contamination of
the bacteria.
 When flaming the
loop, make sure
that all of the wire
has been heated to
redness.
Incineration
• Burns and Physically Destroys Organisms
• Used for
a. Needles
b. Inoculating Wires
c. Glassware
d. Body Parts?
Dry Heat (Hot Air Oven)
• 160° C for 2 Hours or 170° C for 1 hour
• Used for
a. Objects That Won’t Melt
b. Glassware
c. Metal
Pasteurization
• A High Temperature
Is Used For a Short
Time
• Batch Method
63 °C for 30 Minutes
• Pasteurization is a process used in preserving
heat sensitive foods such as milk, beer, and
other beverages.
• 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
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.
• A new method called ultrahigh temperature (UHT)
sterilization involves heating at 140°C for 3
seconds.
• Milk that has been treated in this way can be kept
at room temperature for 2 months with only
minimal changes in flavor.
Filtration
• The passage of a liquid or gas through a filter with pores
small enough to retain microbes.
• Especially important to sterilize solutions which would
be denatured by heat (antibiotics, injectable drugs,
amino acids, vitamins.)
HEPA 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.
HEPA Filters
• Filtration is the primary method of eliminating
pathogens from the air supply.
1. Operating Rooms
2. Burn Units
3. Fume Hoods
4. Isolation Rooms
5. Bio-cabinets
6. Pharmaceutical Manufacturing Facilities
Low Temperatures
• Decreasing Temperature Decreases
Chemical Activity
• Low Temps are Not Bactericidal
Desiccation
• Disrupts Microbial Metabolism
• Stops Growth / Microbes Are Still Viable
• Freeze-drying / Dehydration
• Viruses and Endospores Can Resist Desiccation
Osmotic Pressure
• Plasmolysis
• Sugar Curing / Salting
• May Still Get Some Mold or Yeast Growth
Radiation
• Acts By Destroying DNA or Damaging It.
• Its Efficiency is Dependent on the
Wavelength, Intensity, and Duration
UV 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.
• It is useful for disinfecting surfaces, air and
liquids.
• Unfortunately, this type of
radiation does not
penetrate dirt, glass, water,
or other substances. If a
surface is dusty, then
complete inactivation of all
microorganisms may not
occur.
• Due to its poor penetration,
UV radiation is only useful
for disinfecting outer
surfaces.
UV Radiation
• 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.
Ionizing Radiation
• Ionizing radiation (Gamma and X-rays) are more
penetrating but are more difficult and expensive
to use. They are however, finding application in
food preservation and other industrial processes.
• 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.
Ionizing Radiation
• 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, radiationresistant 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.
• In tests, scientists exposed
this bacterium to enough
radiation to kill a person
several thousand times
over; the bacteria survived.
• 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.
• In studies done on malnourished children by the
National Institute of Nutrition at the Council of
Medical Research in Hyderabad, India, blood tests
showed chromosome damage after being fed
freshly irradiated wheat for six weeks.
• Children fed a
similar but unirradiated diet did
not show damage.
• When the children
were taken off the
irradiated diet the
condition
gradually went
away.
Microwaves
• Kill Microbes Indirectly with Heat
History
• Microwave cooking
ovens were originally
researched and
developed by German
scientists to support
mobile operations
during the invasion of
the Soviet Union.
• After the war, the Allies discovered the medical
research and documentation concerning those
apparatuses.
• The papers and experimental microwave
equipment were transferred to the U.S. War
Department and classified for reference and
scientific investigation.
• The Soviet Union also retrieved some of the
devices and began an experiment on them
separately.
• They discovered that microwaving many foods
produces carcinogenic byproducts.
• The Russians – who have done the most diligent
research into the biological effects of microwave
ovens – OUTLAWED THEIR USE and issued
an international warning about the biological
and environmental damage that can result from
the use of this and similar-frequency electronic
apparatus:
• Over 90% of homes in the U.S. have
microwave ovens used for meal preparation.
Oklahoma
1991
• Lawsuit in 1991 in Oklahoma.
• A woman named Norma Levitt had hip surgery,
but was killed by a simple blood transfusion
when a nurse "warmed the blood for the
transfusion in a microwave oven!"
Do 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
- Destroys Vitamins
Research by Blanc and Hertel 1992
• 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.
Chemical Control Methods
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Phenols and Phenolics
Biguanides
Halogens
Alcohols
Heavy Metals and Their Compounds
Surface-Active Agents
Quaternary Ammonium Compounds
Chemical Food Preservatives
Aldehydes
Antibiotics
Two Conditions Influence the
Effectiveness of Chemical
Disinfectants
1. 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. Environment
- Organic Matter and Increased Temp
Evaluating a Disinfectant
• Old Standard is the Phenol Coefficient Test
(FYI -- 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.)
• More Recently Have Moved to the Use Dilution
Test
(FYI -- An organism is dried to a rod made of
glass, stainless steel, polished porcelain or other
non-reactive material. The rod is then submerged
for 10 minutes or another established time in a
container with the disinfectant that is being
tested. There is very little clearance between the
side of the container and the rod that holds the
organisms. The rod can not touch the side of the
container. The rod is then raised and allowed to
drain. A Rodac plate with agar and the
appropriate nutrient is placed on the rod to remove
organisms for testing. The Rodac plate is
incubated for a predetermined amount of time. If
nothing grows, the disinfectant passes the test for
that organism. )
In Lab We Use Soaked Filter Papers
and Measure the Zone of Inhibition
Types of Disinfectants
• 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
• No Effect on
Spores
• Phenols have a characteristic pine-tar odor and
turn milky in water.
• Phenols are effective antibacterial agents, and
they are also effective against fungi and many
viruses
• Phenols can be toxic to pets especially cats and pigs.
Types of
Disinfectants
• Biguanides
- Damage Plasma Membranes
- Caution: Can Damage Eyes – Avoid Splashing
- Only Operates in Narrow pH Range (5-7)
- Example / Chlorhexidine
Types of
Disinfectants
• Halogens
- Can be Used Alone or in Solution
- Inactivated by Sunlight
- Requires Frequent Application
- Can be Corrosive to Metal
- Can Irritate Mucus Membranes
Chlorine -- Purifies
Drinking Water
- 2-4 Drops of
Chlorine per Liter /
30 Min
- 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 compounds are much more active in
warm water than in cold water.
• Chlorine solutions can be somewhat irritating to
skin and corrosive to metal.
• Inexpensive / Chlorox
• Never Mix with Other Cleaning Agents!
Types of Disinfectants
• Halogens
- Iodine – combines with Amino Acids
a. Inactivates Enzymes
b. Tincture / Alcohol
c. Iodophor / Organic Molecule / Betadine
• Iodine is normally considered to be the least
toxic of the disinfectants.
• Iodine products can stain clothing and porous
surfaces.
Types of Disinfectants
• Alcohols
- Denature Proteins and Dissolve Lipids
- Evaporates
- Fast Acting, No Residue, Flammable
- Wet Disinfectants
a. Aqueous Ethanol (60% - 95%)
b. Isopropyl Alcohol
Types of Disinfectants
• Heavy Metals and Their Compounds
- Used for Burn Treatment
- Prevents Neonatal Gonorrheal Opthalmia
- Denature Proteins
- Example / Silvadene Ointment / Silver
Types of
Disinfectants
• Surface-Active Agents
- Decrease Molecular Surface Tension
- Include Soaps and Detergents
- Soaps Have Limited Germicidal Action but
Assist in the Removal of Organisms by
Scrubbing
- Acid-Anionic Detergents / Dairy
Types of
Disinfectants
• Quaternary Ammonium
Compounds (QUATS)
- Cationic Detergents
Attached to NH4+
- Disrupt Plasma
Membranes
- 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.
Types of
Disinfectants
• Chemical Food Preservatives
- Sorbic Acid
- Benzoic Acid
InhibitFungus
- Propionic Acid
- Nitrate and Nitrite Salts / Meats /
To Prevent Germination of Clostridium
botulinum endospores
Types of
Disinfectants
• Aldehydes
- Formaldehyde
- Glutaraldehyde
- Most Effective of all Chemical
Disinfectants
- Carcinogenic
- Oxidize Molecules Inside Cells
Types of Disinfectants
• Oxidizing Agents
• Hydrogen peroxide and other oxidizing agents
are active against bacteria, bacterial spores,
viruses, and fungi at quite low concentrations.
Types of Disinfectants
• Antibiotics
- Used to Preserve Cheese
- Used in Feed Given to Food Animals
- Nisin
- Natamycin
Antibiotic Resistance
• 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
MRSA
• 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.