Transcript Microbiology: A Systems Approach

LECTURES IN
MICROBIOLOGY
Control of Microbial Growth
LESSON 7
Sofronio Agustin
Professor
Lesson 7 Topics
Controlling Microorganisms:
 Physical Means
 Chemical Means
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Overview
Various Microbial Control Methods
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Antimicrobial Action
-static agents- inhibit growth of microbes
Ex: bacteriostatic, fungistatic
-cidal agents - destroy or kill microbes
Ex: bactericidal, fungicidal
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Terms
Sterilization
Disinfection
Antisepsis
Sanitation
Degerming
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Sterilization
 Complete removal or destruction of all
viable microbes including endospores.
 Application: Surgical instruments and
commercially packaged foods.
 Commercial sterilization: Killing C.
botulinum endospores.
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Disinfection
 Use of physical or chemical agents
(disinfectant) to kill vegetative bacteria
and other microbes except endospores.
 Example: 10% Bleach solution on
bench tops.
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Antisepsis
 Use of physical or chemical agents to
kill pathogens on living tissue.
 Example: Scrubbing surgical patients
with chemical agents.
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Sanitization
Reduction of microbial counts to
acceptable levels of public health
standards.
Example: Sanitization of eating
utensils in restaurants.
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Degerming
 Physical removal or reduction of
microbes from a limited area.
Ex: Washing of hands with germicidal
soap.
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Efficacy Depends On
 Number of microbes
 Target microbe characteristics
 Environmental factors temperature, pH, biofilms
 Concentration and mode of action
of agent
 Organic matter - sputum, blood,
feces
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Factors
Factors that influence the effectiveness of antimicrobial agents
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Mode of Action
Damage to cell wall
Alteration of membrane function
Damage to proteins
Damage to nucleic acids
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Damage to Cell Wall
Effects on bacteria and fungi:
Blockage of cell wall synthesis
Degradation of cell wall
components
Reduction of its stability and
integrity
Ex: Penicillin, detergents, alcohols
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Injury to Cell Membrane
Effects on microbes including
enveloped viruses:
Bind and penetrate membrane lipids
Loss of selective permeability resulting
in leakage of cytoplasmic contents.
Ex: Surfactants - surface active agents.
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Surfactant Action
The effect of surfactants on the cell membrane.
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Effects on Nucleic Acids
Irreversible binding to microbial DNA
results in:
Ceasation of transcription and translation
Mutations
Ex: Formaldehyde and ionizing radiation
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Effects on Proteins
Blockage of enzyme active sites
prevents binding of substrate.
Protein denaturation.
Example: Heat, acids, alcohols,
phenolics, and metallic ions.
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Effects on Proteins
Effects of heat, pH, and blocking agents on protein function.
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Physical Methods
Heat
Radiation
Filtration
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Effects of Heat
Moist heat:
Coagulation of proteins
Denaturation of proteins
Dry heat:
Dehydration
Denaturation
Oxidation (burning to ashes)
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Effects of Heat
Thermal death point (TDP):
Lowest temperature at which all cells in
a culture are killed in 10 min.
Thermal death time (TDT):
Time needed to kill all cells in a culture.
Decimal reduction time (DRT):
Minutes to kill 90% of a population at a
given temperature.
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Moist Heat
Boiling
Tyndallization
Pasteurization
Steam under pressure
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Boiling Water
Boiling at 100 ˚C for 10-30 minutes kills
most non-spore forming pathogens.
Ex:
Boiling of baby bottles and unsafe
drinking water
Hepatitis B virus needs at least 1 hour
of boiling to be killed.
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Tyndallization
Intermittent use of free-flowing steam for 30
to 60 minutes.
Ex:
Used on heat-sensitive media, canned
foods.
Note: This will not destroy spores.
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Pasteurization
 Pasteurization reduces spoilage caused by microbes and
kills pathogens.
 Thermoduric microbes survive
Methods :
 Classical: 63 ˚C for 30 minutes
 High Temperature Short Time: 72 ˚C for 15 seconds
 Ultra High Temperature: 140 ˚C for < 1 second
 Used in milk industry, wineries, breweries.
 Prevents transmission of milk-borne diseases caused by:
Salmonella, Campylobacter, Listeria, Mycobacteria.
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Steam and Pressure
 Used in media preparation and glassware

sterilization.
Ex: Autoclave and pressure cooker
 Autoclave setting at 121 ˚C, 15 p.s.i. pressure for
at least 15 minutes effectively destroys spores.
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Autoclave
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Dry Heat
Flaming
Hot air
Incineration
Requires greater temperature and
exposure time than moist heat
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Hot Air
Hot air oven
Effective at 170˚C for 2 hrs
Useful for sterilization of glasswares
and oils
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Incineration
Destroys microbes to ashes or gas
Bunsen flame - up to 1870˚C
Ex: Sterilization of loops and needles.
Furnace - 800˚C to 6500˚C
Ex: Incineration of animal carcasses.
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Incinerator
An infrared incinerator using flame to burn or oxidize materials into ashes.
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Radiation
Ionizing radiation:
X-rays
Gamma rays
Electron Beams
Nonionizing radiation:
Ultraviolet light
Microwave -kills by heat, not
especially microbicidal.
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Radiation
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Radiation Effects
Ionizing radiation:
Ejects orbital electrons from an atom
High energy - penetrates liquids and
solids effectively.
Nonionizing radiation:
Raises atoms to a higher energy level
Low energy - less penetrating
UV - formation of pyrimidine dimers
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Cellular Effects of Radiation
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Ultraviolet Radiation
UV radiation cause the formation of pyrimidine dimers on DNA.
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Applications of Radiation
Ionizing radiation:
Alternative sterilization method
Materials sensitive to heat or chemicals
Some foods (fruits, vegetables, meats)
Nonionizing radiation:
Alternative disinfectant
Germicidal lamp in hospitals, schools, food
preparation areas (inanimate objects, air, water)
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Gamma Irradiation
(a) Gamma radiation machine used to sterilize fruits,
vegetables, meats, fish, and spices (b) Radora symbol
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UV Irradiation
UV treatment system used to disinfect water.
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Filtration
Removes microbes and spores from liquids
and air
Perforated membrane of varying pore sizes
(0.22 to 0.45 um)
Applications:
Liquids that are sensitive to heat (e.g.
serum,vaccines, media)
HEPA filtration of operating rooms etc.
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Membrane Filter
(a) Membrane filtration system.
(b) Membrane filter close-up.
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Cold and Desiccation
Cold temperatures - reduce microbial
activity except psychrophiles.
Refrigeration
Freezing
Desiccation - dehydration stops microbial
metabolism.
 Lyophilization – freeze drying in a vacuum
preserves microbes and vaccines.
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Classes of Chemical Agents
Phenols and Phenolics
Biguanides
Halogens
Alcohols
Heavy metals
Surfactants - detergents and soaps
Aldehydes
Peroxygens
Gases
Dyes, acids, and alkali
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Phenol and Phenolics
 Phenol - carbolic acid
 Phenolics- Lysol
 Bisphenols

Hexachlorophene
Triclosan
Disrupt plasma
membranes
Ingredients in cutting
boards, kitty litter
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Biguanides
Used as surgical hand scrubs and
preoperative skin preparation
Strong binding affinity to skin and
mucus membranes
Disrupt plasma membranes
Ex: Chlorhexidine
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Halogens
 Oxidizing agents
 Disrupt sulfhydryl groups in amino acids
Iodine - topical antiseptic
Tincture or Iodophor
Chlorine -disinfectant and antiseptic
Hypochlorous acid (HOCl) -germicidal
Calcium hypochlorite used in dairy and
restaurant industries
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Alcohols
Dissolve membrane
lipids, denatures
proteins.
Used for skin
degerming.
Ethanol, Isopropanol
70% concentration most effective
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Soaps and Detergents
Quaternary ammonium (quats):
Cationic
Denature proteins and disrupt cell
membrane
Low-level disinfectant in the clinical setting
Soaps and detergents:
Fatty acids, oils, sodium or potassium salts
Sanitizing and degerming agents
More effective if mixed with germicides
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Detergent Action
Positively charged region of the detergent binds with bacteria
and the uncharged region integrates into the cell membrane
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Hand Scrubbing
Comparison of efficacy between nongermicidal and
germicidal soaps used in hand scrubbing.
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Heavy Metals
Ag, Hg, Cu
Denature proteins
Oligodynamic action
Preservatives in cosmetics and
ophthalmic solutions.
Silver nitrate - Ophthalmia neonatorum
prophylaxis.
Silver sulfadiazine - burn patients and
catheter tips.
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Oligodynamic Action
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Aldehydes
Crosslink with functional groups of proteins
on the cell surface (-NH2, -OH, -COOH, -SH)
Formaldehyde, Glutaraldehyde
Sterilants for surgical and dialysis
instruments
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Aldehyde Effect
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Gases
Denature proteins and affect functional
groups of DNA.
Sterilizes plastic materials
Example: Ethylene oxide
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Gas Sterilization
Ethylene oxide sterilization chamber
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Peroxygens
Oxidizing agents - form hydroxyl free radicals
Effective against anaerobes
Examples:
H2O2 - skin and wound cleaner
Peracetic acid - quick method for sterilizing
medical equipment
O3 - water treatment
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Food Preservatives
Prevents spore germination
Inhibits metabolism
Control molds and bacteria in food and
cosmetics
Examples:
Organic acids - sorbic, benzoic, acetic
acids
Calcium propionate - bread
Nitrite prevents endospore formation in
meat products.
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Summary of Chemical Methods
Chemical agents commonly used in healthcare
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Efficacy of Disinfectants
Principles of effective disinfection
Concentration of disinfectant
Presence of organic matter
Temperature
pH
Time
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Evaluation of Efficacy
Use-Dilution Test:
Metal rings dipped in test bacteria
are dried.
Dried rings placed in disinfectant for
10 minutes at 20oC.
Rings transferred to media to
determine bacterial death/survival.
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Evaluation of Efficacy
Disk-Diffusion Method (Kirby-Bauer)
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Efficacy of Chemical Agents
Chemical agent
Phenolics
Quats
Chlorines
Alcohols
Glutaraldehyde
Effectiveness against
Endospores
Poor
None
Fair
Poor
Fair
Mycobacteria
Good
None
Fair
Good
Good
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Antimicrobial Resistance
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Antimicrobial Resistance
 Highest resistance - bacterial spores and prions.
 Moderate resistance - some bacteria, protozoan
cysts, fungal (sexual) spores, naked viruses.
 Least resistance - most bacteria, fungal
(asexual) spores and hyphae, enveloped viruses,
yeast, protozoan trophozoites.
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Comparative Resistance
Relative resistance of bacterial endospores and vegetative cells
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