Dr. Adel Ahmed Ali El-MorsiB.Sc. (General BotanyB.Sc. (General Botany, ( , ( MansouraMansoura University, 1995University, 1995

M.Sc. Microbiology (virology), Mansoura University, 1999M.Sc. Microbiology (virology), Mansoura University, 1999Ph.D. Microbiology (virology), Mansoura University, 2005Ph.D. Microbiology (virology), Mansoura University, 2005

General MicrobiologyGeneral MicrobiologyB204B204

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

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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.

Sterilization Sterilization – – a process that destroys all viable a process that destroys all viable microbes, including viruses and endosporesmicrobes, including viruses and endospores

Terminology of Microbial ControlTerminology of Microbial Control Prevention Prevention 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.

DisinfectionDisinfectiondestroy vegetative pathogens not endospores

Disinfection – a process to destroy vegetative Disinfection – 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 agents Microbicidal 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

Degerming Degerming ((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.

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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

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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 MethodsPractical 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 of Factors Affecting the Efficacy of Antimicrobial methodsAntimicrobial methods

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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 Resistance1. 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:

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Why is prevention best?

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 temperatureHeat

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 Time (DRT)Decimal Reduction Time (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

AutoclavingAutoclaving

Autoclave Tape

AutoclavingAutoclaving 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 method71.6°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 (1870oC)

• Dehydrates cells and removes water

• Inoculating Loop and Needle 100% effective

2. Incineration ( 2. Incineration ( <<12001200ooC )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 0-15oC freezing <0oC 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).

Lyophilization Lyophilization (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 beams Electron beams – effective at killing but do not penetrate well

• Used to sterilize spices, meats, microbiological plastic ware, and medical and dental supplies

Gamma rays Gamma 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)

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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

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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 Methods Chemical 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

Halogens Halogens ((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 Agents Oxidizing 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 DisinfectantsHeavy 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

Surfactants Surfactants Surface-Active AgentsSurface-Active Agents

SoapDegerming

Acid-anionic detergentsSanitizing

Quarternary ammonium compounds Cationic detergents

Bactericidal, Denature proteins, disrupt plasma membrane

Quaternary Ammonium Compounds Quaternary 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

SaltsSugarDryOrganic Acids

• Inhibit metabolism

• Sorbic acid, benzoic acid, calcium propionate

• Control molds and bacteria in foods and cosmeticsNitrate and Nitrite Salts / Meats /To Prevent Germination of

Clostridium botulinum endospores Antibiotics. Nisin and natamycin prevent spoilage of cheese

Types of Types of DisinfectantsDisinfectants

AldehydesAldehydesCompounds 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 Resistance Drug 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