1. Controlling Microbial Growth in the Environment
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Chapter 9

2. Terminologies
Sterilization: the removal or destruction of all microbes, including viruses and bacterial endospores, in or on an object.
Aseptic: An environment or procedure that is free of contamination by pathogens.
Disinfection: The use of physical or chemical agents known as disinfectants to inhibit or destroy microorganisms, especially pathogens.
Does not guarantee elimination of all pathogens and
Applies only to treatment of inanimate objects.
Antisepsis: When a chemical is used on skin or other tissue and the agent is an antiseptic.
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3. Degerming is the removal of microbes from a surface by scrubbing, whether that surface is human skin or a table top.
Sanitization is the process of disinfecting places and utensils used by the public to reduce the number of pathogenic microbes to meet acceptable public health standards.
Pasteurization is the use of heat to kill pathogens and reduce the number of spoilage microorganisms in food and beverages.
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4. Suffixes
–stasis or –static: Agents or techniques that inhibit the growth of microbes without necessarily killing them are indicated by the suffix. For example, refrigeration is bacteriostatic.
–cide or –cidal: Agents or methods that destroy or permanently inactivate a particular type of microbe. For example, fungicides kill fungal hyphae, spores, and yeasts.
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5. Table 9.1 Terminology of Microbial Control
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6. Microbial Death Rates
Microbial death: The permanent loss of reproductive ability under ideal environmental conditions.
Microbial death rate
Used to evaluate the efficacy of an antimicrobial agent
Usually constant over time for any particular microorganism under a particular set of conditions.
When the microbial death rate is plotted on a semilogarithmic graph, this constant death rate produces a straight line.
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7. Basic Principles of Microbial Control
The modes of action of antimicrobial agents fall into two basic categories:
Those that disrupt the integrity of cells by adversely altering their cell walls or cytoplasmic membranes
Cell wall maintains integrity of cell. When damaged, cells burst because of osmotic effects
Cytoplasmic membrane controls passage of chemicals into and out of cell. When damaged, cellular contents leak out.
***Non-enveloped viruses more tolerant of harsh conditions
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8. Action of Antimicrobial Agents, contd.
Those that interrupt cellular metabolism and reproduction by interfering with the structures of proteins and nucleic acids.
Protein function depends on 3-D shape
Extreme heat or certain chemicals denature proteins
Chemicals, radiation, and heat can alter/destroy nucleic acids
Produce fatal mutants
Halt protein synthesis through action on RNA
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9. The Selection of Microbial Control Methods
A perfect antimicrobial method or agent would be:
Inexpensive,
Fast-acting,
Stable during storage,
Harmless to humans, and
Effective against all types of microbes.
No single method or agent meets all these criteria
Several factors are considered when evaluating methods and agents.
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10. Factors Affecting the Efficacy of Antimicrobial Methods
Site to be treated
Harsh chemicals or intense heat cannot be used on human and animals tissues, as well as fragile objects.
Relative susceptibility of the microorganisms.
Generally, selected method should kill the hardiest microorganisms present, assuming that more fragile microbes will be killed as well.
High - kill all pathogens, including endospores
Intermediate - kill fungal spores, protozoan cysts, viruses, pathogenic bacteria
Low - kill vegetative bacteria, fungi, protozoa, some viruses
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11. Degrees of resistance of microbes:
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12. Factors Affecting the Efficacy of Antimicrobial Methods
The environmental conditions under which the antimicrobial agent is used, such as temperature and pH.
For example, since chemicals react faster at higher temperatures, warm disinfectants generally work better than cool ones
Figure 9.3 Effect of temperature on the efficacy of an antimicrobial chemical
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13. Methods for Evaluating Disinfectants and Antiseptics – Phenol Coefficient
Phenol was an antiseptic used during surgery in the late 1800s.
Phenol coefficient
Evaluates efficacy of disinfectants and antiseptics by comparing to phenol an agent’s ability to control microbes
Greater than 1.0 indicates agent is more effective than phenol
Example: Chloramine (Chlorine + Ammonia). Phenol Coefficient –
133 against S. aureus
100 against Salmonella enterica
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14. Methods for Evaluating Disinfectants and Antiseptics - Use-dilution test
Metal cylinders are dipped into broth cultures of bacteria
Cylinders are dried
Immerse cylinders into a different dilution of the disinfectants being evaluated.
After 10 minutes, remove cylinders and incubate.
The most effective agent is the one that entirely prevents microbial growth at the highest dilution.
Current standard test in the U.S.
New standard procedure being developed
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15. Methods for Evaluating Disinfectants and Antiseptics – Kelsey-Sykes Capacity Test
Alternative assessment to the use dilution test
Approved by the European Union
Determines the capacity of a given chemical to inhibit bacterial growth.
Test chemicals are added to broth cultures of bacteria, and the turbidity of the cultures is measured after 48 hours as an indication of growth inhibition.
Can establish minimum time required for disinfectant to be effective
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16. Methods for Evaluating Disinfectants and Antiseptics - In-use test
Provide accurate determination of an agent’s efficacy under realistic conditions
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 situation is important
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17. Summary of Microbial Control Methods17 17

18. Physical Methods of Microbial Control
Physical methods of microbial control include exposing the microbes to extremes of:
Heat and cold
Desiccation
Filtration
Osmotic pressure and
Radiation
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19. 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
Thermal death point: the lowest temperature that kills all cells in a broth in 10 minutes, and
Thermal death time: the time it takes to completely sterilize a particular volume of liquid at a set temperature.
Decimal reduction time (D) is the time required to destroy 90% of the microbes in a sample
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20. Heat-Related Methods – Moist Heat
More effective than dry heat because water is a better conductor of heat than air.
Used to disinfect, sanitize, and sterilize
Denatures proteins and destroys cytoplasmic membranes
Methods of microbial control using moist heat
Boiling
Autoclaving
Pasteurization
Ultrahigh-temperature sterilization
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21. Moist Heat – Boiling
Kills vegetative cells of bacteria and fungi, protozoan trophozoites, most viruses
Boiling time & atmospheric pressure is important.
The higher the atmospheric pressure, the higher the boiling point
Different elevations require different boiling times
Rate of boiling not important – bec. heat escapes
Boiling does not achieve true sterilization:
Endospores, protozoan cysts, and some viruses can survive boiling
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22. Moist Heat - Autoclaving
Pressure applied to boiling water prevents steam from escaping
Boiling temperature increases as pressure increases
Autoclaving – A device that consists of a pressure chamber, pipes, valves, and gauges that uses steam heat under pressure to sterilize chemicals and objects that can tolerate moist heat.
Autoclave conditions – 121ºC, 15 psi, 15 min
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23. Moist Heat - Pasteurization
A method of heating foods to kill pathogens and control spoilage organisms without altering the quality of the food.
Used for milk, ice cream, yogurt, and fruit juices
Not sterilization - Heat-tolerant microbes survive
Can be achieved by several methods:
Batch method at 63°C for 30 minutes,
Flash pasteurization at 72°C for 15 seconds, and
Ultrahigh-temperature pasteurization at 134°C for 1 second.
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24. Heat-Related Methods - Dry heat
Used for materials that cannot be sterilized with moist heat like powders and oils
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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25. Refrigeration and Freezing
Decreases microbial metabolism, growth, and reproduction
Chemical reactions occur more slowly at low temperatures
Liquid water not available
Refrigeration between 0°C and 7°C halts growth of most pathogens
Psychrophilic microbes can multiply in refrigerated foods
Slow freezing at temperatures below 0°C more effective than quick freezing
Organisms vary in susceptibility to freezing –
many vegetative bacterial cells, bacterial endospores, and viruses can survive subfreezing temperatures for years.
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26. Physical Method of Microbial Control: Desiccation & Lyophillization
Desiccation: Drying
Used to preserve such foods as fruits, peas, and yeast.
It inhibits microbial growth because metabolism requires liquid water.
Lyophillization: freeze-drying.
Preserves microbes and other cells for many years.
Microbial culture is frozen in liquid nitrogen or frozen carbon dioxide and then remove water via a vacuum.
Prevents the formation of large ice crystals, which would otherwise damage the culture; thus, enough viable cells remain to enable the culture to be reconstituted many years later.
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27. Filtration
The passage of air or a liquid through a material that traps and removes microbes.
Some membrane filters manufactured of nitrocellulose or plastic have pores small enough to trap the smallest viruses and even some large protein molecules.
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28. HEPA Filters
HEPA (High-Efficiency Particulate Air) filters remove microbes and particles from air.
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29. Physical Methods of Microbial Control: Osmotic Pressure
Use of high concentrations of salt or sugar in foods to inhibit growth
Examples: Pickles; Salted fish jerky; Jams; etc
Cells in hypertonic solution of salt or sugar lose water
Fungi have greater ability than bacteria to survive hypertonic environments
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30. Physical Methods of Microbial Control: Radiation
There are two types of radiation:
Particulate radiation consists of high-speed subatomic particles freed from their atoms
Electromagnetic radiation is atomic energy without mass traveling at the speed of light.
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31. Ionizing Radiation
Electromagnetic radiation with wavelengths shorter than 1 nm
Ejects electrons from atoms to create ions
Ions disrupt hydrogen bonding, oxidize double covalent bonds, and create hydroxide ions which can produce effects leading to the denaturation of important molecules and cell death.
Examples: electron beams, gamma rays, and X rays.
Electron beams – effective at killing but do not penetrate well
Gamma rays – penetrate well but require hours to kill microbes.
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32. Non Ionizing Radiation
Wavelengths greater than 1 nm
Excites electrons, causing them to make new covalent bonds
Affects 3-D structure of proteins and nucleic acids
Examples: Ultraviolet light, visible light, infrared light, and radio waves,
Only UV light has enough energy to be a practical antimicrobial agent
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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33. Biosafety Levels Handling microbes demands safety.
The CDC established four levels of guidelines for handling microbes in microbiology labs
Each level has guidelines for which precautions to take and which organisms can be safely handled under those conditions.
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34. Biosafety Levels BSL-1 is suitable for nonpathogenic microbes,
BSL-2 is intended for moderately hazardous microbes, and
BSL-3 is designed for containment of true pathogens and includes specifications for the physical facility as well as procedures.
BSL-4 is designed for the most dangerous pathogens and includes stringent guidelines for the design of the facility to contain the microbes.
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35. Chemical Agents in Microbial Control
Disinfectants, antiseptics, sterilants, degermers, and preservatives
Some desirable qualities of chemicals:
Rapid action in low concentration
Solubility in water or alcohol, stable
Broad spectrum, low toxicity
Penetrating
Noncorrosive and nonstaining
Affordable and readily available 35 35

36. Factors that Affect Germicidal Activity of Chemicals
Nature of the material being treated
Degree of contamination
Time of exposure
Strength and chemical action of the germicide 36 36

37. Nine major categories of antimicrobial chemicals used as antiseptics and disinfectants
Surfactants
Heavy Metals
Aldehydes
Gaseous Agents
Enzymes
Phenol and Phenolics
Alcohols
Halogens
Oxidizing Agents

38. Phenol and Phenolics
Compounds derived from phenol molecules that have been chemically modified by the addition of halogens or organic functional groups such as chlorine
Intermediate- to low-level disinfectants
Denature proteins and disrupt cell membranes
Effective in presence of organic matter
Remain active for prolonged time
Commonly used in health care settings, labs, and homes
Have disagreeable odor and possible side effects
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39. Alcohols Intermediate-level disinfectants –
Denature proteins and disrupt cytoplasmic membranes
Used either as 70–90% aqueous solutions Example: Isopropanol (rubbing alcohol) or
As a tincture - a combination of an alcohol and another antimicrobial agent. Ex. Tincture of Iodine
Intermediate-level disinfectants –
Effective as bactericidal, fungicidal, and virucidal against enveloped viruses; but
Not effective against fungal spores or bacterial endospores
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40. Halogens
Iodine, chlorine, bromine, and fluorine - four very reactive, nonmetallic chemical elements
Believed to damage enzymes via oxidation or by denaturation
Intermediate-level antimicrobial chemicals
Used alone or in combination with other elements in organic and inorganic compounds. Ex. Iodophore, Betadine
Widely used in numerous applications
Iodine tablets, iodophores, chlorine treatment, bleach, chloramines, and bromine disinfection
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41. Oxidizing Agents
Peroxides, ozone, and peracetic acid
Kill by oxidation of microbial enzymes due to production of oxygen radicals
High-level disinfectants and antiseptics
Hydrogen peroxide can disinfect and sterilize surfaces
Not useful for treating open wounds because of catalase activity
Ozone treatment of drinking water by some MUDs in Canada and Europe more effective and not carcinogenic
Peracetic acid is effective sporicide used to sterilize equipment.
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42. Surfactants Soaps have hydrophilic and hydrophobic ends
“Surface active” chemicals. Reduce surface tension of solvents. Types: Soaps and detergents
Soaps have hydrophilic and hydrophobic ends
Good degerming agents but not antimicrobial
Detergents are positively charged organic surfactants; disrupts cellular membranes
Quaternary Ammonium Compunds
Low-level disinfectants; not effective against mycobacteria, endospores, or nonenveloped viruses, and some pathogens
Ideal for many medical and industrial applications
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43. Heavy Metals Arsenic, silver, mercury, copper, and zinc
Heavy-metal ions denature proteins
Low-level bacteriostatic and fungistatic agents
1% silver nitrate to prevent blindness caused by N. gonorrhoeae
Thimerosal used to preserve vaccines
Copper controls algal growth
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44. Aldehydes Compounds containing terminal –CHO groups
Cross-link functional groups to denature proteins and inactivate nucleic acids
Glutaraldehyde disinfects and sterilizes
Formalin –
2% solution of glutaraldehyde and a 37% aqueous solution of formaldehyde
used in embalming and disinfection of rooms and instruments
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45. Gaseous Agents Can be hazardous to people Often highly explosive
Ethylene oxide, propylene oxide, and beta-propiolactone
Microbicidal and sporicidal gases used in closed chambers to sterilize items that cannot be sterilized easily with heat, water-soluble chemicals, or irradiation. Ex. Plastic laboratory ware, artificial heart valves, mattresses, and dried foods.
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. Kills everything they contact without harming inanimate objects
Potentially carcinogenic
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46. Enzymes
Naturally produced by organisms to inhibit or destroy microbes. Ex. Lysozyme in human tear which digests peptidoglycan
Scientists, food processors, and medical personnel are researching ways to use natural and artificial antimicrobial enzymes to control microbes in the environment, inhibit microbial decay of foods
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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47. Antimicrobials Antibiotics - produced naturally by microorganisms
Semisynthetics - are chemically modified antibiotics; and
synthetics, which are wholly synthetic antimicrobial drugs.
Typically used for treatment of disease
Some used for antimicrobial control outside the body
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48. Development of Resistant Microbes
Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health
Use of such products promotes development of resistant microbes
When susceptible cells die, they reduce competition for resources, allowing any remaining resistant cells to proliferate
Experts recommend limiting the use of such chemicals to food handling and health care involving high-risk patients and newborns
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