Control of Microorganisms

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Control of Microorganisms

• Microbiology 2314

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Control of Microbial Growth

• Effected in two basic ways
• By Killing Microorganisms
• By inhibiting the Growth of Microorganisms
• Usually involves the use of
• Physical Agents
• Chemical Agents

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Level of Resistance

• Endospores
• Mycobacteria
• Fungal Spores
• Small Non-enveloped Viruses
• - Polio, Rotavirus, Rabies
• Vegetative Fungal Cells
• Enveloped Viruses
• - Herpes, Hepatitis B C, HIV
• Vegetative Bacteria

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Principles of Microbial Control (Definitions)

• Prevention / Control of Growth to Prevent
  Infection and Spoilage

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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

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• 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.

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• 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
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Principles of Microbial Control

• Commercial Sterilization / Heat Treatment of
  Canned Foods

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Principles of Microbial Control

• Disinfection / Reducing Growth Nonliving Surfaces

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• 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.

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Principles of Microbial Control

• Antisepsis / Reducing Growth on Living Tissue

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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.

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Principles of Microbial Control

• Degerming / Swab the Skin

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Principles of Microbial Control

• Sanitize
• Subject to High Temperature Washing (Dishwashers)

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Principles of Microbial Control

• Cide Suffix Meaning to Kill
• Stat Suffix Meaning to Inhibit
• Sepsis Bacterial Contamination
• Asepsis Lack of Bacterial Contamination

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Rate of Microbial Death

• Bacteria Usually Die At a Constant Rate
• Plotted Logarithmically This Will Give a Straight
  Line

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Time to Kill in Proportion to the Population Size

• Large Numbers Require Greater Time
• Small Numbers Require Less Time

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Susceptibilities Vary

• Endospores are Difficult to Kill
• Organic Matter May Interfere with Heat Treatments
  and Chemical Control Agents

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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

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Denatures Proteins
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Physical Methods of Microbial Control
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Physical Methods

• Heat
• Filtration
• Low Temperatures
• Desiccation
• Osmotic Pressure
• Radiation

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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.

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• Moist Heat / Denatures Enzymes
• Moisture improves heat penetration, making
  sterilization by moist heat more effective then
  dry heat.
• Includes boiling and autoclaving.

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• 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.

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Thermal 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

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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.

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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.

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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

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Dry Heat Sterilization

• Direct Flaming
• Incineration
• Hot-Air Sterilization (Oven)

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Incineration

• Burns and Physically Destroys Organisms
• Used for
• a. Needles
• b. Inoculating Wires
• c. Glassware
• d. Body Parts?

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Dry Heat (Hot Air Oven)

• 160 C for 2 Hours or 170 C for 1 hour
• Used for
• a. Objects That Wont Melt
• b. Glassware
• c. Metal

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Pasteurization

• A High Temperature
• Is Used For a Short Time
• Batch Method
• 63 C for 30 Minutes

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• Pasteurization is a process used in preserving
  heat sensitive foods such as milk, beer, and
  other beverages.

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• Pasteurization uses mild temperatures (63C for
  30 minutes or 71C for 15 seconds) to kill
  pathogens and reduce levels of non-pathogenic
  organisms that cause milk and other foods to spoil

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• Pasteurization is not a method of sterilization,
  which is why pasteurized foods will eventually
  spoil if given enough time.

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• Pasteurization extends the shelf life of a
  product and reduces the level of pathogens in the
  product.

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• A new method called ultrahigh temperature (UHT)
  sterilization involves heating at 140C 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.

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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.)

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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.

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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

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Low Temperatures

• Decreasing Temperature Decreases Chemical
  Activity
• Low Temps are Not Bactericidal

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Desiccation

• Disrupts Microbial Metabolism
• Stops Growth / Microbes Are Still Viable
• Freeze-drying / Dehydration
• Viruses and Endospores Can Resist Desiccation

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Osmotic Pressure

• Plasmolysis
• Sugar Curing / Salting
• May Still Get Some Mold or Yeast Growth

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Radiation

• Acts By Destroying DNA or Damaging It.
• Its Efficiency is Dependent on the Wavelength,
  Intensity, and Duration

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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.

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• 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.

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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.

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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.

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• 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.

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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.

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• Studies have shown that irradiating
  microorganisms like E. coli and Salmonella may
  give rise to even more dangerous,
  radiation-resistant strains of bacteria.

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• 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.

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• 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.

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• 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.

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• Children fed a similar but un-irradiated diet did
  not show damage.
• When the children were taken off the irradiated
  diet the condition gradually went away.

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Microwaves

• Kill Microbes Indirectly with Heat

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History

• Microwave cooking ovens were originally
  researched and developed by German scientists to
  support mobile operations during the invasion of
  the Soviet Union.

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• 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.

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• 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.

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• 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

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• Over 90 of homes in the U.S. have microwave
  ovens used for meal preparation.

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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!" 

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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

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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.

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Chemical Control Methods

• Phenols and Phenolics
• Biguanides
• Halogens
• Alcohols
• Heavy Metals and Their Compounds
• Surface-Active Agents
• Quaternary Ammonium Compounds
• Chemical Food Preservatives
• Aldehydes
• Antibiotics

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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

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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.)

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• 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. )

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In Lab We Use Soaked Filter Papers and Measure
the Zone of Inhibition
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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

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• Phenolics are Long Lasting, Good for Blood and
  Body Fluids
• No Effect on Spores

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• 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

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• Phenols can be toxic to pets especially cats and
  pigs.

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Types of Disinfectants

• Biguanides
• - Damage Plasma Membranes
• - Caution Can Damage Eyes Avoid Splashing
• - Only Operates in Narrow pH Range (5-7)
• - Example / Chlorhexidine

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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

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• 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

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• 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

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• Never Mix with Other Cleaning Agents!

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Types of Disinfectants

• Halogens
• - Iodine combines with Amino Acids
• a. Inactivates Enzymes
• b. Tincture / Alcohol
• c. Iodophor / Organic Molecule / Betadine

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• Iodine is normally considered to be the least
  toxic of the disinfectants.
• Iodine products can stain clothing and porous
  surfaces.

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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

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Types of Disinfectants

• Heavy Metals and Their Compounds
• - Used for Burn Treatment
• - Prevents Neonatal Gonorrheal Opthalmia
• - Denature Proteins
• - Example / Silvadene Ointment / Silver

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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

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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

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• 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.

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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

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Types of Disinfectants

• Aldehydes
• - Formaldehyde
• - Glutaraldehyde
• - Most Effective of all Chemical
• Disinfectants
• - Carcinogenic
• - Oxidize Molecules Inside Cells

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Types of Disinfectants

• Oxidizing Agents
• Hydrogen peroxide and other oxidizing agents are
  active against bacteria, bacterial spores,
  viruses, and fungi at quite low concentrations.

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Types of Disinfectants

• Antibiotics
• - Used to Preserve Cheese
• - Used in Feed Given to Food Animals
• - Nisin
• - Natamycin

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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

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MRSA
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•  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.

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