Control of Microorganisms Microbiology 2314 Control of Microbial Growth Effected in two basic ways: 1.By Killing Microorganisms 2.By inhibiting the Growth

Control of MicroorganismsControl of MicroorganismsMicrobiology 2314

Control of Microbial GrowthControl 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 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

5. Vegetative Bacteria

Principles of Microbial Control Principles of Microbial Control (Definitions)(Definitions)

• Prevention / Control of Growth to Prevent Infection and Spoilage

Principles of Microbial Control Principles of Microbial Control (Definitions)(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 ScannerNano Disinfection Scanner

Principles of Microbial ControlPrinciples of Microbial Control

• Commercial Sterilization / Heat Treatment of Canned Foods


• 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 ControlPrinciples of Microbial Control• 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 of antiseptics include iodine, 70% ethanol and 3% hydrogen peroxide.

Principles of Principles of Microbial Microbial ControlControl

• Degerming / Swab the Skin


• Sanitize

Subject to High Temperature Washing (Dishwashers)


• Cide – Suffix Meaning to Kill

• Stat – Suffix Meaning to Inhibit

• Sepsis – Bacterial Contamination

• Asepsis – Lack of Bacterial Contamination

Rate of Microbial DeathRate 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 Time to Kill in Proportion to the Population SizePopulation Size

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

Susceptibilities VarySusceptibilities Vary

• Endospores are Difficult to Kill

• Organic Matter May Interfere with Heat Treatments and Chemical Control Agents

http://www.amazon.com/gp/product/images/B000IFGSXU/ref=dp_image_0/105-1211790-6918835?ie=UTF8&n=3760901&s=hpc

Control Agents Act ByControl 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 ProteinsDenatures Proteins

Physical Methods of Microbial Physical Methods of Microbial ControlControl

Physical Physical MethodsMethods

• Heat

• Filtration

• Low Temperatures

• Desiccation

• Osmotic Pressure

• Radiation

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.

• 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 Thermal Death Death PointPoint

• 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 Decimal Reduction TimeReduction 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)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 Dry Heat SterilizationSterilization

• 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.

IncinerationIncineration

• Burns and Physically Destroys Organisms

• Used for

a. Needles

b. Inoculating Wires

c. Glassware

d. Body Parts?

Dry Heat (Hot Air Oven)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

PasteurizationPasteurization• 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.

FiltrationFiltration• 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 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.

HEPA FiltersHEPA 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 TemperaturesLow Temperatures

• Decreasing Temperature Decreases Chemical Activity

• Low Temps are Not Bactericidal

DesiccationDesiccation• Disrupts Microbial Metabolism• Stops Growth / Microbes Are Still Viable• Freeze-drying / Dehydration

• Viruses and Endospores Can Resist Desiccation

Osmotic PressureOsmotic Pressure

• Plasmolysis

• Sugar Curing / Salting

• May Still Get Some Mold or Yeast Growth

RadiationRadiation• Acts By Destroying DNA or Damaging It.

• Its Efficiency is Dependent on the Wavelength, Intensity, and Duration

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.

• 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 RadiationUV 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.

http://www.myspaceantics.com/image-myspace-graphic/funny-pictures/a-minor-sunburn.jpg.html

Ionizing RadiationIonizing 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 RadiationIonizing 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, 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.

• 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 un-irradiated diet did not show damage.

• When the children were taken off the irradiated diet the condition gradually went away.

MicrowavesMicrowaves• Kill Microbes Indirectly with Heat

HistoryHistory

• 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 Oklahoma 19911991

• 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 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

- Destroys Vitamins

Research by Blanc and Hertel 1992Research 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 MethodsChemical Control Methods• 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 Two Conditions Influence the Effectiveness of Chemical Effectiveness of Chemical

DisinfectantsDisinfectants1. 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 DisinfectantEvaluating 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 In Lab We Use Soaked Filter Papers and Measure the Zone of Inhibitionand Measure the Zone of Inhibition

Types of DisinfectantsTypes 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 Types of DisinfectantsDisinfectants

• Biguanides

- Damage Plasma Membranes

- Caution: Can Damage Eyes – Avoid Splashing

- Only Operates in Narrow pH Range (5-7)

- Example / Chlorhexidine


• 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!


• Halogens- Iodine – combines with Amino Acids

a. Inactivates Enzymesb. Tincture / Alcoholc. Iodophor / Organic Molecule /

Betadine

• Iodine is normally considered to be the least toxic of the disinfectants.

• Iodine products can stain clothing and porous surfaces.


• Alcohols

- Denature Proteins and Dissolve Lipids

- Evaporates

- Fast Acting, No Residue, Flammable

- Wet Disinfectants

a. Aqueous Ethanol (60% - 95%)

b. Isopropyl Alcohol


• Heavy Metals and Their Compounds

- Used for Burn Treatment

- Prevents Neonatal Gonorrheal Opthalmia

- Denature Proteins

- Example / Silvadene Ointment / Silver


• 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


• 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.


• Chemical Food Preservatives

- Sorbic Acid

- Benzoic AcidInhibitFungus

- Propionic Acid

- Nitrate and Nitrite Salts / Meats /

To Prevent Germination of Clostridium botulinum endospores


• Aldehydes

- Formaldehyde

- Glutaraldehyde

- Most Effective of all Chemical

Disinfectants

- Carcinogenic

- Oxidize Molecules Inside Cells


• Oxidizing Agents

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


• Antibiotics

- Used to Preserve Cheese

- Used in Feed Given to Food Animals

- Nisin

- Natamycin

Antibiotic ResistanceAntibiotic 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

MRSAMRSA

• 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.

Documents

Control of Microorganisms Microbiology 2314 Control of Microbial Growth Effected in two basic ways: 1.By Killing Microorganisms 2.By inhibiting the Growth