Post on 21-Apr-2022
Microbial Control
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Source : Prescot et al Microbiology,
Microbiology by Pelczar, Brock et
al Microbiology
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Microbial deathThe permanent loss of reproductive capability, even under
optimum growth conditions is the accepted microbial definition of death
Factors affecting microbial death rate
a) Number of microorganisms
b) Nature of the microorganisms in the target population
c) Temperature and pH of the environment
d) Concentration of the agent
e) Mode of action of the agent
f) Presence of solvents, interfering organic matter, and inhibitors
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Practical Concerns in Microbial
Control
Selection of method of control depends on circumstances:
• 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 and is it safe?
Basic Principles of
Microbial Control
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The Agents Versus the Processes• –cide: to kill
– Bactericide: chemical that destroys bacteria (not endospores)
– Fungicide: a chemical that can kill fungal spores, hyphae, and yeasts
– Virucide: a chemical that inactivates viruses
– Sporicide: can destroy bacterial endospores
– Germicide and microbicide: chemical agents that kill microorganisms
• Stasis and static: to stand still
– Bacteristatic: prevent the growth of bacteria
– Fungistatic: inhibit fungal growth
– Microbistatic: materials used to control microorganisms in the body, for example
Relative susceptibilities of microbes to antimicrobial agents
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Targets to Control Microbial Presence
• Injure cell wall
• Injure cell membranes
• Interfere with nucleic acid synthesis
• Interfere with protein synthesis
• Interfere with protein function
Which of the above would effect our cells too?
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Bacterial populations die at a constant logarithmic rate
The Selection of Microbial Control Methods
• Factors Affecting the Efficacy of Antimicrobial Methods
▫ Site to be treated
Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects
Method of microbial control based on site of medical procedure
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The Selection of Microbial Control Methods
• Factors Affecting the Efficacy of Antimicrobial Methods
▫ Relative susceptibility of microorganisms
Germicides classified as high, intermediate, or low effectiveness
High-level kill all pathogens, including endospores
Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria
Low-level kill vegetative bacteria, fungi, protozoa, and some viruses
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Physical Methods of Microbial Control
• Heat-Related Methods▫ Effects of high temperatures
Denature proteins
Interfere with integrity of cytoplasmic membrane and cell wall
Disrupt structure and function of nucleic acids
• Heat▫ Thermal death point (TDP): Lowest temperature at which all cells
in a culture are killed in 10 min.
▫ Thermal death time (TDT): Time to kill all cells in a culture
▫ Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature
▫ z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions
▫ F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores
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Physical Methods: Moist Heat
• Measurements of killing by moist heat (cont.)
▫ Decimal reduction time (D value): The time required to reduced a population of microbes by 90% (a 10-fold, or one decimal, reduction) at a specified temperature and specified conditions
▫ z value: The change in temperature, in ºC, necessary to cause a tenfold change in the D value of an organism under specified conditions
▫ F value: The time in minutes at a specific temperature (usually 121.1°C or 250 °F) needed to kill a population of cells or spores
Physical Methods of Microbial Control
• Heat-Related Methods▫ Moist heat
Used to disinfect (kill organisms and remove spores), sanitize (kill organisms but not necessarily their spores), and sterilize (kill all organisms and spores)
Denatures proteins and destroys cytoplasmic membranes
More effective than dry heat Methods of microbial control using moist heat
Boiling
Autoclaving
Pasteurization
Ultrahigh-temperature sterilization
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Physical Methods of Microbial Control
• Heat-Related Methods
▫ Moist heat
Boiling
Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses
Boiling time is critical
▫ Different elevations require different boiling times
Endospores, protozoan cysts, and some viruses can survive boiling
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Physical Methods of Microbial Control
• Heat-Related Methods
▫ Moist heat
Autoclaving
Pressure applied to boiling water prevents steam from escaping
Boiling temperature increases as pressure increases
Autoclave conditions – 121ºC, 15 psi, 15 min
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The relationship between temperature and pressure
Figure 9.5
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• Moist heat denatures proteins
• Autoclave: Steam under pressure
• 15 min at 121oC at 15 psi
Autoclaving
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Nonpressurized Steam
• Tyndallization – intermittent sterilization for substances that cannot withstand autoclaving
• Items exposed to free-flowing steam for 30 –60 minutes, incubated for 23-24 hours and then subjected to steam again
• Repeat cycle for 3 days.
• Used for some canned foods and laboratory media
• Disinfectant
Physical Methods of Microbial Control
• Heat-Related Methods▫ Moist heat
Pasteurization Used for milk, ice cream, yogurt, and fruit juices
Not sterilization▫ Heat-tolerant microbes survive
Pasteurization of milk▫ Batch method
▫ Flash pasteurization (High temp, short time)
▫ Ultrahigh-temperature pasteurization (very high temp, very short time)
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Pasteurization
63oC for 30 minutes
72oC for 15 seconds
140oC for 1 second
Pasteurization reduces spoilage organisms and pathogens
Pasteurization of milk
Batch method • The batch method uses a vat pasteurizer which consists
of a jacketed vat surrounded by either circulating water, steam or heating coils of water or steam.
• Batch method: Expose to 63°C to 66°C for 30 minutes (LTLT)
In the vat the milk is heated and held throughout the holding period while being agitated. The milk may be cooled in the vat or removed hot after the holding time is completed for every particle.
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Pasteurization of milk
Flash method• High Temperature Short Time (HTST)
• Milk is heated to 72°C (161.6°F) for at least 15 seconds.
• Used for perishable beverages like fruit and vegetable juices, beer, and some dairy products. Compared to other pasteurization processes, it maintains color and flavor better.
• It is done prior to filling into containers in order to kill spoilage microorganisms, to make the products safer and extend their shelf life. Flash pasteurization must be used in conjunction with sterile fill technology.
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Pasteurization of milk
Ultrahigh-temperature method
• Heating for 1-2 seconds at a temperature exceeding135°C (275°F), which is the temperature required to killspores in milk and then rapid cooling.
• Treated liquids can be stored at room temperature.
• The most common UHT product is milk, but the processis also used for fruit juices, cream, soy milk, yogurt,wine, soups, and stews.
• Can cause browning and change the taste and smell ofdairy products.
• UHT canned milk has a typical shelf life of six to ninemonths, until opened.
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Physical Methods of Microbial Control
• Heat-Related Methods▫ Dry heat
Used for materials that cannot be sterilized with moist heat
Denatures proteins and oxidizes metabolic and structural chemicals
Requires higher temperatures for longer time than moist heat
Incineration is ultimate means of sterilization
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• Dry Heat Sterilization kills by
oxidation
▫ Incineration
▫ Hot-air sterilization
Physical Methods of Microbial Control
Hot-air Autoclave
Equivalent treatments 170˚C, 2 hr 121˚C, 15 min
Dry Heat: Hot Air and Incineration
• Incineration
▫ Ignites and reduces microbes to ashes and gas
▫ Common practice in microbiology lab-incineration on inoculating loops and needles using a Bunsen burner
▫ Can also use tabletop infrared incinerators
Dry Oven
• Usually an electric oven
• Coils radiate heat within an enclosed compartment
• Exposure to 150°C to 180°C for 2 to 4 hours
• Used for heat-resistant items that do not sterilize well with moist heat
Physical Methods of Microbial Control
• Refrigeration and Freezing▫ Decrease microbial metabolism, growth, and
reproduction
Chemical reactions occur slower at low temperatures
▫ 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
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Physical Methods of Microbial Control
• Dessication and Lyophilization
▫ Dessication is drying (98% of the water is removed) inhibits growth due to removal of water
▫ Lyophilization (freeze-drying)
Substance is rapidly frozen and sealed in a vacuum
Substance may also be turned into a powder
▫ Used for long-term preservation of microbial cultures
Prevents formation of damaging ice crystals
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The use of dessication as a means of preserving
apricots
Figure 9.8
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• Effective for removing microbes from air and liquids
• Fluid strained through a filter with openings large enough for fluid but too small for microorganisms
• Filters are usually thin membranes of cellulose acetate, polycarbonate, and a variety of plastic materials
• Pore size can be controlled and standardizes
Decontamination by Filtration:
Techniques for Removing Microbes
Filtration equipment used for microbial control
Figure 9.9
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The role of HEPA filters in biological safety cabinets
Figure 9.10
High-Efficiency Particulate Arresting (HEPA) air filters are used in medical facilities, automobiles, aircraft, and homes. The filter must remove 99.97% of all particles greater than 0.3 micrometer from the air that passes through.
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• Prepare liquids that can’t withstand heat
• Can decontaminate beverages without altering their flavor
• Water purification
• Removing airborne contaminants (HEPA filters)
Applications of Filtration
Physical Methods of Microbial Control
• Osmotic Pressure
▫ High concentrations of salt or sugar in foods to inhibit growth
▫ Cells in hypertonic solution of salt or sugar lose water
▫ Fungi have greater ability than bacteria to survive hypertonic environments
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• Radiation damages DNA
▫ Ionizing radiation (X rays, gamma rays, electron beams)
▫ Nonionizing radiation (UV)- surface sterilization only
▫ (Microwaves kill by heat; not especially antimicrobial)
Physical Methods of Microbial Control
Physical Methods of Microbial Control
• Radiation: Cold sterilization▫ Ionizing radiation
Wavelengths shorter than 1 nm Electron beams, gamma rays
Excites the electrons to the point that they are ejected from the molecule entirely causing the formation of ions
Ions disrupt hydrogen bonding, cause oxidation, and create hydroxide ions Hydroxide ions denature other molecules (DNA) Also causes lethal chemical changes in organelles
and the production of toxins Electron beams – effective at killing but do not
penetrate well Gamma rays – penetrate well but require hours to kill
microbes
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Physical Methods of Microbial Control
• Radiation
▫ Nonionizing radiation
Wavelengths greater than 1 nm
Excites electrons, causing them to make new covalent bonds
Leads to abnormal linkages and bonds within molecules
Affects 3-D structure of proteins and nucleic acids
UV light causes pyrimidine dimers in DNA
UV light does not penetrate well
Suitable for disinfecting air, transparent fluids, and surfaces of objects
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Ionizing Radiation• Food products
• Medical products (drugs and tissues)Potential problems include changing flavor and nutritional
value, and introducing undesirable chemical reactions
Potential danger to machine operators and possible damage to some materials
Nonionizing Radiation• Usually disinfection rather than sterilization
• Hospital rooms, operating rooms, schools, food prep areas, dental offices
• Treat drinking water or purify liquidsPoses threat to human tissue if overexposure occurs
Applications of Radiation
Increased shelf life of food achieved by ionizing
radiation
Figure 9.11
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Irradiation food Radura
• The word "Radura" is derived from radurization, combining "radiation" with the stem of "durus", the Latin word for hard, lasting.
Physical Methods of Microbial Control
Sound Waves (Sonication)a) Used high-frequency sound waves to disrupt cell structure.
b) Sonicator–water-filled chamber through which the sound waves become vibrations that can disrupt cell structure.
c) Application of ultrasound waves causes rapid changes in pressure within the intracellular liquid; this leads to cavitation, the formation of bubbles inside the cell, which can disrupt cell structures and eventually cause the cell to lyse or collapse.
d) Gram-negative bacteria are most susceptible.
e)Often used to clean debris from instruments before sterilization.
Not a reliable form of disinfection or sterilization.
Useful in the laboratory for efficiently lysing cells to release their contents for further research.
Outside the laboratory, sonication is used for cleaning surgical instruments, lenses, and a variety of other objects such as coins, tools, and musical instruments.
Physical Methods of Microbial Control
• Biosafety Levels▫ Four levels of safety in labs dealing with
pathogens Biosafety Level 1 (BSL-1)
Handling pathogens that do not cause disease in healthy humans
Biosafety Level 2 (BSL-2) Handling of moderately hazardous agents
Biosafety Level 3 (BSL-3) Handling of microbes in safety cabinets
Biosafety Level 4 (BSL-4) Handling of microbes that cause severe or fatal disease
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A BSL-4 worker carries
Ebola virus cultures
Figure 9.12
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Chemical Methods of Microbial Control
• Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA
• Effect varies with differing environmental conditions
• Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa
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Acids and Alkalis
• Very low or high pH can destroy or inhibit microbial cells
• Limited in applications due to their corrosive, caustic, and hazardous nature
Chemical Methods of Microbial Control
Chemical Methods of Microbial Control
• Phenol and PhenolicsPhenol (carbolic acid) was first used by Lister as a disinfectant.
Rarely used today because it is a skin irritant and has strong odor.
Phenolics are chemical derivatives of phenol
Cresols (Lysol): Derived from coal tar.Biphenols: Effective against gram-positive staphylococci
and streptococci. Excessive use in infants may cause neurological damage.
Destroy plasma membranes and denature proteins.Advantages: Stable, persist for long times after applied, and
remain active in the presence of organic compounds.
▫ Intermediate- to low-level disinfectants
▫ Remain active for prolonged time
• Commonly used in health care settings, labs, and homes
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Chemical Methods of Microbial Control
• Alcohols
▫ Intermediate-level disinfectantsKill bacteria, fungi, but not endospores or naked viruses.
Act by denaturing proteins and disrupting cell membranes.
Used to mechanically wipe microbes off skin before injections or blood drawing.
Not good for open wounds, because cause proteins to coagulate.
Ethanol: Drinking alcohol. Optimum concentration is 70%.
Isopropanol: Rubbing alcohol. Better disinfectant than
ethanol. Also cheaper and less volatile.
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Chemical Methods of Microbial Control
70% vs 100% Ethanol
Pure alcohol (100%) coagulates protein in contact. Suppose the purealcohol is poured over a single celled organism. The alcohol will gothrough the cell wall of the organism in all direction, coagulating theprotein just inside the cell wall. The ring of the coagulated protein wouldthen stop the alcohol from penetrating farther from the cell, and nomore coagulation would take place. At this time the cell would becomeinactive but not dead. Under the favorable conditions the cell wouldthen begin to function. If 70 % of alcohol is poured to a single celledorganism, the diluted alcohol also coagulates the protein, but at aslower rate, so that it penetrates all the way through the cell beforecoagulation can block it. Then the entire cell is coagulated and theorganism dies.
Chemical Methods of Microbial Control
Halogens• Chlorine – Cl2, hypochlorites (chlorine bleach), chloramines
▫ Denaturate proteins by disrupting disulfide bonds
When mixed in water forms hypochlorous acid:
Cl2 + H2O ------> H+ + Cl- + HOCl
▫ Intermediate level▫ Unstable in sunlight, inactivated by organic matter ▫ Water, sewage, wastewater, inanimate objects
• Iodine - I2, iodophors (betadine) Iodine tincture (alcohol solution) was one of first antiseptics used.
▫ Denature proteins▫ Intermediate level▫ Milder medical & dental degerming agents, disinfectants,
ointments
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Chemical Methods of Microbial Control
Oxidizing Agents
Oxidize cellular components of treated microbes.
Disrupt membranes and proteins.
A. Ozone:
Used along with chlorine to disinfect water.
Helps neutralize unpleasant tastes and odors.
More effective killing agent than chlorine, but less stable and more expensive.
Highly reactive form of oxygen.
Made by exposing oxygen to electricity or UV light
B. Hydrogen Peroxide:
Not useful for treating open wounds due to catalase activity: the tissues convert it into H2O and 0xygen bubbles.
Effective in disinfection of inanimate objects
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Chemical Methods of Microbial Control
• Surfactants▫ “Surface active” chemicals
Reduce surface tension of solvents
▫ Soaps and detergents Soaps have hydrophilic and hydrophobic ends
Good degerming agents but not antimicrobial
Detergents are positively charged organic surfactants
▫ Quats (Quaternary ammonium cations) Low-level disinfectants; disrupts cell membranes Ideal for many medical and industrial applications Good against fungi, amoeba, and enveloped viruses,
but not endospores, Mycobacterium tuberculosis and non-enveloped viruses.
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Chemical Methods of Microbial Control
• Heavy Metals▫ Heavy-metal ions denature proteins
▫ Low-level bacteriostatic and fungistatic agents
▫ Include copper, selenium, mercury, silver, and zinc.
▫ Very tiny amounts are effective.
A. Silver:
1% silver nitrate used to protect infants against gonorrheal eye infections, now has been replaced by erythromycin.
B. Mercury
Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds.
C. Copper
Copper sulfate is used to kill algae in pools and fish tanks.
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Chemical Methods of Microbial Control
• AldehydesInclude some of the most effective antimicrobials.Inactivate proteins by forming covalent crosslinks with
several functional groups (–NH2, –OH, –COOH, —SH).
A. Formaldehyde:Excellent disinfectant, 2% aqueous solution.
Commonly used as formalin, a 37% aqueous solution.Formalin was used extensively to preserve biological
specimens and inactivate viruses and bacteria in vaccines.
Irritates mucous membranes, strong odor.
B. Glutaraldehyde:
Less irritating and more effective than formaldehyde.
Commonly used to disinfect hospital instruments.
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Chemical Methods of Microbial Control• Gaseous Agents
▫ Microbicidal and sporicidal gases used in closed chambers to sterilize items
▫ Denature proteins and DNA by cross-linking functional groups▫ Used in hospitals and dental offices Disadvantages
Can be hazardous to people Often highly explosive Extremely poisonous Potentially carcinogenic
Ethylene Oxide: Kills all microbes and endospores, but requires exposure of 4 to 18
hours. ETO is a colorless gas that is flammable and explosive but it is liquid at
temperatures below 10.8oC. Alkylation reactions with organic compounds such as enzymes (eg
inactivation of enzymes having sulfhydryl group) and other proteins. It has high penetrating power and passes through and sterilizes
large packages of materials, bundles of cloth, and even certain plastics.
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Chemical Methods of Microbial Control
• Enzymes▫ Antimicrobial enzymes act against microorganisms▫ Human tears contain lysozyme
Digests peptidoglycan cell wall of bacteria
▫ Enzymes to control microbes in the environment Lysozyme used to reduce the number of bacteria in
cheese Prionzyme can remove prions on medical
instruments
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Evaluating the Effectiveness
• Phenol Coefficient Test
▫ A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhiand Staphylococcus aureus and incubated at either 20°C or 37°C
▫ Samples are removed at 5 min intervals and inoculated into fresh broth
▫ The cultures are incubated at 37°C for 2 days
▫ The highest dilution that kills the bacteria after a 10 min exposure, but not after 5 min, is used to calculate the phenol coefficient
Chemical Methods of Microbial Control
Evaluating the Effectiveness
• Phenol Coefficient Test (cont.)▫ The reciprocal of the maximum effective dilution for the test
disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient
▫ For example:Suppose that, on the test with Salmonella typhiThe maximum effective dilution for phenol is 1/90The maximum effective dilution for “Disinfectant X” is 1/450The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5
▫ Phenol coefficients are useful as an initial screening and comparison, but can be misleading because they only compare two pure strains under specific controlled conditions
Chemical Methods of Microbial Control