Biology 120 lecture 5 2011 2012

CONTROL OF MICROORGANISMS

Lecture 5Thursday, September 15, 2011

LECTURE OUTLINEDefinition of Terms in Microbial Control

Pattern of Microbial Death

Conditions Influencing the Effectiveness of Antimicrobial Agents

Use of Physical Agents

Use of Chemical Agents

Thursday, September 15, 2011

The Control of Microbial Growth SEPSIS

microbial contamination

ASEPSIS absence of significant

contamination

The Control of Microbial Growth SEPSIS

microbial contamination

ASEPSIS absence of significant

contamination

The Control of Microbial Growth STERILIZATION

Removal of all microbial life

COMMERCIAL STERILIZATION Killing C. botulinum endospores

The Control of Microbial Growth STERILIZATION

Removal of all microbial life

COMMERCIAL STERILIZATION Killing C. botulinum endospores

The Control of Microbial Growth DISINFECTION

Removal of pathogens

ANTISEPSIS from living tissue

DEGERMING from a limited area

The Control of Microbial Growth DISINFECTION

Removal of pathogens

ANTISEPSIS from living tissue

DEGERMING from a limited area

The Control of Microbial Growth SANITATION

Lower microbial counts on eating utensils

BIOCIDE/GERMICIDE: Kills microbes

BACTERIOSTATS: Inhibiting, not killing, microbes

The Control of Microbial Growth SANITATION

Lower microbial counts on eating utensils

BIOCIDE/GERMICIDE: Kills microbes

BACTERIOSTATS: Inhibiting, not killing, microbes

Bacterial populations

die at a constant

logarithmic rate

PATTERN OF MICROBIAL DEATH

Bacterial populations

die at a constant

logarithmic rate

PATTERN OF MICROBIAL DEATH

The Control of Microbial Growth

HOW DO WE DECIDE WHETHER THEY ARE ACTUALLY DEAD?

“a microbe is defined DEAD if it does not grow when inoculated into culture medium

that would normally support its growth”Thursday, September 15, 2011

Conditions Influencing Effectiveness of

Antimicrobial Agent Activity1. Population size

Larger population requires a longer time to die

2. Population compositionMicroorganisms vary markedly on susceptibilityVegetative versus SporesYoung versus Mature cells

Antimicrobial Agent Activity1. Population size

Larger population requires a longer time to die

2. Population compositionMicroorganisms vary markedly on susceptibilityVegetative versus SporesYoung versus Mature cells

3. Concentration or Intensity of an Antimicrobial Agent

The more concentrated an agent the more rapidly microbes can be destroyedBUT sometimes an agent may be more effective at lower concentrations (e.g. 70% alcohol)

4. Duration of ExposureThe longer the exposure to an agent the more they will be killed

Antimicrobial Agent Activity

3. Concentration or Intensity of an Antimicrobial Agent

The more concentrated an agent the more rapidly microbes can be destroyedBUT sometimes an agent may be more effective at lower concentrations (e.g. 70% alcohol)

4. Duration of ExposureThe longer the exposure to an agent the more they will be killed

5. TemperatureAn increase in temperature at which a chemical acts often enhances it activityExample: acids used in high T = more effective

6. Local environmentpH, organic matter, etcControls or Protects the pathogen

5. TemperatureAn increase in temperature at which a chemical acts often enhances it activityExample: acids used in high T = more effective

6. Local environmentpH, organic matter, etcControls or Protects the pathogen

PHYSICAL METHODS: HEAT

Fire and boiling Sufficient to destroy vegetative

cells (10 minutes) Not high for killing endospores Disinfection but not

sterilization!Thursday, September 15, 2011

Fire and boiling Sufficient to destroy vegetative

cells (10 minutes) Not high for killing endospores Disinfection but not

sterilization!Thursday, September 15, 2011

Thermal Death Point (TDP)The lowest temperature at which a

microbial suspension in killed in 10 minutes

Thermal Death Time (TDT)The shortest time needed to kill all

organisms in a microbial suspension at a specific temperature and under defined conditions

Thermal Death Point (TDP)The lowest temperature at which a

microbial suspension in killed in 10 minutes

Thermal Death Time (TDT)The shortest time needed to kill all

organisms in a microbial suspension at a specific temperature and under defined conditions

However, such a destruction is logarithmic and it is theoretically NOT

POSSIBLE to “completely destroy” microbes in a sample

Decimal Reduction Time or D valueTime required to kill 90% of the

microorganisms or spores in a sample at a specified temperatureTime required for the line to drop by one

log cycle or tenfoldUsed to estimate the relative resistance

of a microbe to different temperaturesThursday, September 15, 2011

z ValueThe increase in temperature required to

reduce D to 1/10 its value or to reduce it by one log cycle

z ValueThe increase in temperature required to

reduce D to 1/10 its value or to reduce it by one log cycle

F valueTime in minutes at a specific temperature

needed to kill a population of cells or sporesUsually 121°C

APPLICATION: FOOD INDUSTRY

After a food have been canned, it must be heated to eliminate the risk of botulism arising from the presence of Clostridium spores

Example: (1012 to 100 spores) IF the D value = 0.204 minutes It would take 12D or 2.5 minutes to

reduce the spore number by heating at the specified temperature

If the z value for Clostridium spores is 10°C

If the z value for Clostridium spores is 10°C It takes a 10°C change in temperature to

alter the D value tenfold

THUS: if the cans are to be processed at 111°C rather than 121°C, the D value would increase by tenfold t 2.04 minutes

THUS: if the cans are to be processed at 111°C rather than 121°C, the D value would increase by tenfold t 2.04 minutes The 12D value = 24.5 minutes

PHYSICAL METHODS: HEAT (MOIST HEAT

STERILIZATION)

PHYSICAL METHODS: HEAT (MOIST HEAT

STERILIZATION)

Hot-air Autoclave

Equivalent treatments 170˚C, 2 hr 121˚C, 15 min

PHYSICAL METHODS: HEAT (Dry Heat Sterlization)

Hot-air Autoclave

Flaming

Hot-air Autoclave

Flaming

Hot-air Autoclave

Flaming

Incineration

Hot-air Autoclave

Flaming

Incineration

Hot-air Autoclave

Flaming

Incineration

Hot-air sterilization

Hot-air Autoclave

HOW DOES HEAT KILL MICROBES

MOIST HEAT Kill effectively by degradation of nucleic

acids and by denaturation of enzymes and other essential proteins May also disrupt cell membranes

DRY HEAT Microbial death results from the oxidation of

cell constituents and denaturation of proteins

PHYSICAL METHODS: FILTRATION

Applicable for heat-sensitive materials that needs sterilization

Types of filters Depth filters: consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter

Membrane filters: porous membranes; 0.2 µm pore sizes

Applicable for heat-sensitive materials that needs sterilization

Types of filters Depth filters: consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter

Membrane filters: porous membranes; 0.2 µm pore sizes

Laminar flow hood versus biological safety cabinets (HEPA filters)

High Efficiency Particulate Air Remove 99.97% particles 0.02 µm for sterilizing AIR

Laminar flow hood versus biological safety cabinets (HEPA filters)

High Efficiency Particulate Air Remove 99.97% particles 0.02 µm for sterilizing AIR

BIOSAFETY CABINETS

Class1 (from room=outside) protection: person and environment

Class 2 (Type A and B) ) Type A and B: product, person and

environment difference: type A air is recirculated back

to room, type B exhausted outside the building

Class 3: contained facility, higher level of protection and containment

BIOSAFETY CABINETS

Class1 (from room=outside) protection: person and environment

Class 2 (Type A and B) ) Type A and B: product, person and

environment difference: type A air is recirculated back

to room, type B exhausted outside the building

Class 3: contained facility, higher level of protection and containment

PHYSICAL METHODS: RADIATION

IONIZING RADIATION X rays, gamma rays, electron beamsExcellent as a sterilizing agent and penetrates deep into objects

NON-IONIZING RADIATION UV (about 260nm) Quite lethal but does not penetrate glass, dirt films, water and other substances very effectively

Microwaves: kill by heat not usually antimicrobial

PHYSICAL METHODS: RADIATION

IONIZING RADIATION X rays, gamma rays, electron beamsExcellent as a sterilizing agent and penetrates deep into objects

NON-IONIZING RADIATION UV (about 260nm) Quite lethal but does not penetrate glass, dirt films, water and other substances very effectively

Microwaves: kill by heat not usually antimicrobial

CHEMICAL AGENTS

PHENOLICS

ALCOHOLS

HALOGENS

HEAVY METALS

QUATERNARY AMMONIUM COMPOUNDS

ALDEHYDES

STERILIZING GASES

CHEMICAL METHODS

PHENOLICS

ALCOHOLS

HALOGENS

HEAVY METALS

QUATERNARY AMMONIUM COMPOUNDS

ALDEHYDES

STERILIZING GASES

CHEMICAL METHODS

Chemical agent Effectiveness againstEffectiveness againstEndospores Mycobacteria

Phenolics Poor GoodQuats None NoneChlorines Fair FairAlcohols Poor GoodGlutaraldehyde Fair Good

CHEMICAL METHODS

Chemical agent Effectiveness againstEffectiveness againstEndospores Mycobacteria

Phenolics Poor GoodQuats None NoneChlorines Fair FairAlcohols Poor GoodGlutaraldehyde Fair Good

First widely used antiseptic and disinfectant

Joseph Lister (1867): reduced the risk of infection during operations

Example: LYSOLR

Act by denaturing proteins and disrupting cell membranes

CHEMICAL METHODS: Phenolics

First widely used antiseptic and disinfectant

Joseph Lister (1867): reduced the risk of infection during operations

Example: LYSOLR

Act by denaturing proteins and disrupting cell membranes

Disruption of Cell Membranes

ADVANTAGES: effective in the presence of organic material and remain active on surfaces long after application

DISADVANTAGE: disagreeable odor and can cause skin irritation and in some instances brain damage (hexachlorophene)

ADVANTAGES: effective in the presence of organic material and remain active on surfaces long after application

DISADVANTAGE: disagreeable odor and can cause skin irritation and in some instances brain damage (hexachlorophene)

Widely used disinfectant and antiseptics

Bactericidal and fungicidal but not sporicidal

May not destroy lipid-containing viruses

CHEMICAL METHODS: Alcohols

Widely used disinfectant and antiseptics

Bactericidal and fungicidal but not sporicidal

May not destroy lipid-containing viruses

DENATURES PROTEINS, DISSOLVES

LIPIDS

Example: ethanol and isopropanol (70-80% concentration)

Act by denaturing proteins and possibly by dissolving membrane lipids

10-15 soaking in alcohol is sufficient to disinfect thermometers and small instruments

Example: ethanol and isopropanol (70-80% concentration)

Act by denaturing proteins and possibly by dissolving membrane lipids

10-15 soaking in alcohol is sufficient to disinfect thermometers and small instruments

Iodine Kills by oxidizing cell constituents

and iodinating cell proteins

Kill spores at high concentrations

DISADVANTAGE: a stain may be left (answer = iodophor)

CHEMICAL METHODS: Halogens

Iodine Kills by oxidizing cell constituents

and iodinating cell proteins

Kill spores at high concentrations

DISADVANTAGE: a stain may be left (answer = iodophor)

Chlorine Usually for water supply

Kills by oxidation of cellular materials and destruction of vegetative bacteria, fungi

Will not kill spores

Death within 30 minutes

Chlorine Usually for water supply

Kills by oxidation of cellular materials and destruction of vegetative bacteria, fungi

Will not kill spores

Death within 30 minutes

Mercury, Arsenic, Zinc, Copper Used as germicides How do they Kill:

Heavy metals combine with proteins, often with their sulfhydryl groups and inactivate them May also precipitate cell proteins

CHEMICAL METHODS: Heavy Metals

Mercury, Arsenic, Zinc, Copper Used as germicides How do they Kill:

Heavy metals combine with proteins, often with their sulfhydryl groups and inactivate them May also precipitate cell proteins

CHEMICAL METHODS: Heavy Metals

DETERGENTSAmphipathic (both polar and non-polar

ends)Kill by disrupting microbial membranes and

denature proteins

ADVANTAGE: stable, non-toxicDISADVANTAGE: inactivated by hard

CHEMICAL METHODS: Quats

DETERGENTSAmphipathic (both polar and non-polar

ends)Kill by disrupting microbial membranes and

denature proteins

ADVANTAGE: stable, non-toxicDISADVANTAGE: inactivated by hard

CHEMICAL METHODS: Quats

Surface-Active Agents or SurfactantsSoap Degerming

Acid-anionic detergents

Sanitizing

Quarternary ammonium compoundsCationic detergents

Bactericidal, Denature proteins, disrupt plasma membrane

FORMALDEHYDES Very reactive molecules that

combine with proteins and inactivate them

Sporicidal and can be used as sterilants

CHEMICAL METHODS: Aldehydes

FORMALDEHYDES Very reactive molecules that

combine with proteins and inactivate them

Sporicidal and can be used as sterilants

CHEMICAL METHODS: Aldehydes

EVALUATION OF ANTIMICROBIAL AGENT

EFFECTIVENESS PHENOL COEFFICIENT TEST

Best-known disinfectant screening test Potency of a disinfectant is compared

with that of phenol The highest dilution that killed bacteria

after a 10 minutes exposure are used to calculate phenol coefficient

EVALUATION OF ANTIMICROBIAL AGENT

EFFECTIVENESS PHENOL COEFFICIENT TEST

Best-known disinfectant screening test Potency of a disinfectant is compared

with that of phenol The highest dilution that killed bacteria

after a 10 minutes exposure are used to calculate phenol coefficient

The reciprocal of the appropriate test disinfectant dilution is divided by that for phenol to obtain the coefficient

Example: phenol dilution = 1/90 and the maximum effective dilution for disinfectant X = 1/450 Phenol coefficient = 5

CALCULATING PHENOL COEFFICIENTS

The reciprocal of the appropriate test disinfectant dilution is divided by that for phenol to obtain the coefficient

Example: phenol dilution = 1/90 and the maximum effective dilution for disinfectant X = 1/450 Phenol coefficient = 5

The higher the phenol coefficient value, the more effective the disinfectant under this conditions

DILUTION TESTS Metal rings dipped in test bacteria are dried

Dried cultures placed in disinfectant for 10 min at 20°C

Rings transferred to culture media to determine whether bacteria survived treatment

DILUTION TESTS Metal rings dipped in test bacteria are dried

Dried cultures placed in disinfectant for 10 min at 20°C

Rings transferred to culture media to determine whether bacteria survived treatment

DISK-DIFFUSION METHOD

CHEMOTHERAPEUTIC AGENTS

Antibiotics are medicines used to treat infections caused by bacteria only

Infections are usually caused by bacteria or viruses

Antibiotics, therefore, do not cure all infections

Many infections like the common cold, flu, mild sore throat or diarrhea are caused by viruses

Antibiotics are medicines used to treat infections caused by bacteria only

Infections are usually caused by bacteria or viruses

Antibiotics, therefore, do not cure all infections

Many infections like the common cold, flu, mild sore throat or diarrhea are caused by viruses

WHAT IF ANTIBIOTICS WERE USED INCORRECTLY?

No healing effect - If antibiotics are used for viral infections, there will be no effect on the illness

Antibiotic resistance - This occurs when one antibiotic no longer works on a specific type of bacteria

A stronger antibiotic will be needed to treat the infection caused by this resistant strain of bacteria

WHAT IF ANTIBIOTICS WERE USED INCORRECTLY?

No healing effect - If antibiotics are used for viral infections, there will be no effect on the illness

Antibiotic resistance - This occurs when one antibiotic no longer works on a specific type of bacteria

A stronger antibiotic will be needed to treat the infection caused by this resistant strain of bacteria

ANTIBIOTIC MECHANISMS

RESISTANCE

RESISTANCEThursday, September 15, 2011

DO YOU CONTRIBUTE TO RESISTANCE?

Another factor that contributes to resistance is that when patients are prescribed antibiotics for a just cause, many do not finish their medication

This allows resistant bacteria to survive more easily

The practice of saving unused medication to treat themselves or others at a later date can also lead to resistant strains

Also contributing to antibiotic resistance is the widespread use of antibiotics to promote weight gain and to control disease in cattle, pigs, and chickens

Forty to fifty percent of antibiotics produced are used in livestock feed

This leads to an increase of resistant bacteria in these animals, which is then spread to humans

ANY QUESTIONS???

NEXT MEETING: INTERACTIVE LECTURE/QUIZ ON METABOLISM

Biology 120 lecture 5 2011 2012

Technology

Transcript of Biology 120 lecture 5 2011 2012