Chapter 7

Control of MOs by

Physical and Chemical Agents

(1). Destroy Pathogens and Prevent Transmission

(2). Reduce or eliminate MOs responsible for contamination of

water, food, and other etc.

Sterilization: (latin: sterilis) – unable to produce offspring - barren

• Process where all living cells, viable spores, viruses, and viroids are either destroyed or removed from an object or habitat.
• Totally free of viable mos, spores and other infectious agents.
• Sterilant: sterilization by a chemical agent.

Disinfection:

• The killing, inhibition, or removal of most hay may cause disease.
• Goal: destroy potential pathogens
• Reduces the total microbial population
• Disinfectants: agents, (usually chemicals)

Carry out disinfection, used on inanimate objects.

(a disinfectant does not necessarily sterilize an object bc viable spores and few mos may remain).

Sanitization:

• The microbial population is reduced to safety levels for the public health standards.
• The inanimate object is cleaned and partially disinfected.
• Example: eating utensils in restaurants.

Antisepsis:

§ Prevention of infection or sepsis and accomplished with antiseptics

§ Antiseptics: chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth.

-- Reduce the total microbial population

--(Not a toxic as disinfectants bc can not destroy too much

host tissue)

Suffix Denotation of Antimicrobial agents:

• Cide: substances that kill (Latin for cida-kill)
• Germicide: kills pathogens (and nonpathogens) but not necessarily endospores.

Disinfectants or antiseptic can be effective against a specific

Group: bactericide, fungicide, algicide or viricide.

• Static: do not kill, but prevent growth, if removed growth will resume.
• Bacteriostatic or Fungistatic.

Conditions Influencing the Effectiveness of

Antimicrobial Agent Activity

Antimicrobial agent

• An agent that kills mos or inhibits their growth

Six Factors:

1. Population Size
2. Population Composition
• Mos differ in susceptibility
• Endospores are more resistant than the vegetative form
• Younger cells are destroyed quicker than older cells
• Mycobacterium tuberculosis is more resistant than most bacteria
3. Concentration of microbial antimicrobial agent

§ Most often, the more concentrated a chemical agent, the more rapidly mos are destroyed.

§ Sometimes agent is more effective at lower concentrations: 70% ethanol is more effective than 95% ethanol, because its activity is more enhanced by the presence of water.

4. Duration of Exposure:

§ The longer the population is exposed to the a microbial agent, the more organisms are killed. To achieve sterilization, exposure should reduced population to10 ^-6 or less.

5. Temperature:

§ An increase in temperature at which the chemical acts often enhances its activity.

6. Local environment:

§ Population of mos will either be protect or aid in its destruction.

§ Organic matter can protect mos against heating and chemical disinfectants.

-- Biofilms: organic matter in a surface biofilm will protect the mos.

-- biofilm and mos are usually hard to remove.

n Example: clean, then disinfect or sterilize.

Syringes and medical and dental equipment

Should be cleaned then sterilized, bc presence of too much organic matter can increase the risk of pathogens by protecting the pathogen.

Drinking Water: the more organic matter, the more chlorine needed.

Four Most Frequently Employed Physical Methods/Agents in Control of Mos:

1. Heat

§ Either moist or dry, heat is still one of the most popular methods used to destroy mos.

§ Moist heat: kills viruses, bacteria, and fungi

§ Exposure to boiling water, 10 minutes – destroys vegetative cells and eukaryotic spores.

§ Boiling water (100° C or 212 °F) in not hot enough to destroy bacterial endopsores (which may survuve hours of boiling)

§ Boiling can be used for disinfection, but not sterilizing.

Thermal Death Time (TDT):

§ shortest time needed to kill all organisms in a microbial suspension at a specific temperature under defined conditions.

Decimal Reduction Time (D value):

§ The time required to kill 90% of the mos or spores in a sample at specific temperature.

§ D value is written with a subscript, indicating the temperature for which it applies.

§ More precise

§ Used to estimate the relative resistance of a mo to difference temp. through calculating the Z value.

Z Value:

• The increase in the temp. required to reduce D to 1/10 its value

F Value:

• Time in minutes at a specific temperature needed to kill a population of cells or spores.
• Usually 250°C or 121.1° C)

Steam Sterilization:

• Moist heat sterilization – carried out at temp above 100° C to destroy bacterial endospores
• Autoclave: Saturated Steam under pressure.
• Developed in 1884 by Chamberland
• Water is boiled to produce steam which is released into the jacket and into the autoclave’s chamber.
• Air in initially in chamber is forced out, until the chamber is filled with saturated steam and outlets are closed.
• Hot saturated steam will fill the chamber until it reaches 121° C and 15 pounds of pressure.

(saturated steams destroys all vegetative cells and endospores within 10 to 12 minutes - 15 minutes is the standard time to provide a margin of safety).

o Larger volumes will require longer autoclave times.

Moist heat kills (thought) by:

o Degrading nucleic acids

o Denaturing enzymes and other essential proteins.

o May disrupt Cell membrane.

Bacterial endospores are killed only if kept at 121 ° C for 10-12 minutes.

To ensure items have been autoclaved:

• Biological indicator

Culture tube containing sterile ampule of medium and a paper strip covered with Bacillus stearothermophilus or Clostridium PA3679, after autoclaving, ampule broken, and incubated for several days. (much longer process)

• Autoclave tape with an indicator strip that turns a different color or spell out the word autoclave.

Pasteurization:

• Controlled heating well below boiling.
• Does not sterilize a beverage, but does kill any pathogen present and drastically slows spoilage by reducing the level of nonpathogenic spoilage mos.
• Developed by Pasteur from wine spoilage study
• Observed wine, found bacteria responsible for lactic acid and acetic acid fermentation.
• Brief heat at 55 to 60° C destroyed the mos and preserved wine for longer periods.
• German Chemists V. H. and F. Soxhlet adapted technique for preserving milk and reducing milk transmissible disease.
• 1889 introduced into the US
• Milk, beer, and etc are pasteurized.

Milk Pasteurized:

Two Ways

1. 63° C for 30 minutes

2.

§ Flash Pasteurization (high temperature short term (HTST)

Large quantities of milk at 72° C for 15 seconds, then rapid cooling.

• Ultrahigh-temperature Sterilization (UHT)

o Dairy Industry

• Milk and Milk products are treated at 140 to 150 ° C for 1 to 3 seconds.
• Does not require refrigeration
• Example: small coffee creamers

Dry Heat Sterilization:

• Sterilization in the absence of water.
• Oven heated at 160 to 170 ° C for 2 to 3 hours.

Microbial Death results from:

o Oxidation of cell constituents and denaturation of proteins.

• DH is less effective than MH

Clostridium botulinum spores are killed in 5 minutes in MH and 2 hours after dry heating.

2. Low Temperatures

• Used by inhibiting growth and reproduction by freezing and refrigeration.
• Freezing at -20°C or lower stops microbial growth bc of the low temperature and the absence of liquid water.
• Some mos will be killed by ice crystal disruption of the cell membrane.
• Freezing is a very good method for long term storage of microbial samples.
• Refrigeration greatly slows microbial growth, but does not halt it completely.
• Most pathogens are mesophilic and do not grow well at temp above 4° C.
• Refrigerated items may be ruined by growth of psychrophilic and psychrotrophic mos, especially if water is present.
• Good for short term storage.

3. Filtration

• Reduces the microbial population in solutions that are heat-sensitive and sometimes can be used to sterilize solutions and media.
• Does not directly destroy mos, rather removes them.

Two types of filters:

1. Depth filters

• Fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter.
• Solution with mos are sucked (filtered) through this layer under vacuum and microbial cells are removed by entrapment and adsorption to the surface of the of filter materials.

2. Membrane filters

• Circular and porous membrane
• A little over 0.1mm thick
• Made of cellulose acetate ,or other synthetic material
• There are many different sizes, but 0.2um in diameter are used for the removal of most vegetative cells, but not viruses.
• Membrane filters remove mos by screening them out much as a sieve separate large particles from small ones.
• Have replaced depth filters.
• Used to: sterilize [pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics, and other heat-sensitive solutions.

Air Filtration:

§ Air can be sterilized by filtration

§ Example: surgical masks and cotton plugs on culture vessels.

§ Laminar flow biological safety cabinets:

Employs high-efficiency particulate air filters which

remove 99.97% of 0.3 um particles.

§ These cabinets force air through HEPA filters, then project a vertical curtain of sterile air across the cabinet opening.

§ Used to prevent contamination of the room.

§ Used when working with Mycobacterium tuberculosis, tumor viruses, and recombinant DNA.

4. Radiation

• Ionizing radiation: radiation if very short wavelengths or high energy.

1. X-rays - artificially produced

2. Gamma Rays - emitted during radioisotope decay

o Ionizing radiation is an excellent sterilizing agent and penetrates deep into the object.

o Destroys bacterial endospores and vegetative cells, both eukaryotic and prokaryotic, but not always effective against viruses.

• Ultraviolet (UV) radiation: kills most bc of its short wavelength (10 to 400nm)
• The most lethal form has a wavelength of 260nm, wavelength most effectively absorbed by DNA
• Mechanism of Damage: formation of thymine dimmers in DNA. Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and function.
• Although UV at 260nm is lethal, it does not penetrate glass, dirt films, water, and other substances, therefore it can be used as a sterilizing agent only in a few specific situations.
• UV radiation burns the skin and damages the eyes

Use of Chemical Agents in Control:

Phenolics:

• First widely used antiseptic and disinfectant
• Lister (1867) used to reduce the risk of infections during operations.
• Phenol and phenolic (phenol deratives) are used in hospitals and laboratories as disinfectants.
• Cresols, xylenols, and etc.
• Act by denaturing proteins and disrupting cell membranes.

Alcohols:

• Among the most widely used disinfectants and antiseptics
• Bacterial and fungicidal , but not sporicidal
• Some lipid-containing viruses are destroyed.
• Two most important alcohol germicides are: ethanol and isoppropanol (70-80%)
• Act by denaturing proteins and possibly by dissolving membrane lipids.
• 10 to 15 minutes soaking is sufficient to disinfect thermometers and small instruments.

Halogens:

• Any of the five elements (fluorine, chlorine, bromine, iodine, and astatine) in group VIIA of the periodic table.
• Exit as diatomic molecules in the free state and form saltlike compounds with sodium and most other metals.
• Iodine and chlorine are important antimicrobial agent

• Iodine: skin antiseptic and kills by oxidizing cell constituents and iodinating cell protein.
• Higher concentrations kills spores

• Chlorine: disinfectant for water supplies and swimming pools, also used in dairy and food industry.
• May be used as a chlorine gas, sodium hypochlorite, or calcium hypochlorite all yielding hypochlorous acid (HClO) and then atmospheric oxygen.
• Damages by the oxidation of cellular materials and destruction of vegetative bacteria and fungi, Not spores.

Cl₂ + H₂0 à HCl + HClO

Heavy Metals:

• Mercury, silver, arsenic, zinc, and copper were used as germicides.
• Now there are other less toxic and more effective germicides.
• Many are bacteriostatic than bactericidal
• A 1% Silver nitrate is stilled used in the eyes of infants to prevent ophthalmic gonorrhea)
• Silver sulfadiazinc is used on burns
• Copper sulfate is an effective algicide in lakes and swimming pools.

Quaternary Ammonium Compounds:

• Detergents: organic molecules that serve as a wetting agents and emulsifiers because they have both polar hydrophilic and nonpolar hydrophobic ends
• Detergents solubilize otherwise insoluble residues and are very effective cleaning agents.
• Different than soap which is derived from lipids.
• Anionic detergents have some antimicrobial properties, only cationic detergents are effective disinfectants.
• Most popular disinfectants are quaternary ammonium compounds that have a positive charged quaternary nitrogen and a long hydrophobic aliphatic chain.
• Function to disrupt microbial membranes and may also denature proteins.

Aldehydes:

• Common – formaldehyde and glutaraldehyde – are highly reactive molecules that combine with nucleic acids and proteins and inactive them.

o Sporicidal and can be used as chemical sterilants.

o Glutaraldehyde an effective disinfectant in hospitals and laboratory equipment

Sterilizing Gases:

• Ethylene Oxide (EtO)
• Used on plastic petri dishes and syringes (heat sensitive, heart-lung machines components, sutures, and catheters.
• Both a microbial and sporicidal
• Kills by combining with cell proteins.
• Effective sterilizing agent bc rapidly penetrates materials

Evaluation of Antimicrobial agent Effectiveness:

Phenol Coefficient test:

• Best known disinfectant screening test
• Where the potency of a disinfectant is compared with that of phenol.
• A series of dilutions of phenol and the experimental disinfectant are inoculated with the test bacteria Salmonella typhi and Staphylococcus aureus, then placed in a 20 or 37º C water bath.
• The inoculated disinfectant tubes are next subcultures to fresh medium at 5 minute intervals and the subcultures are incubated for two or more days.
• The highest dilution that kills the bacteria after 10 minutes exposure, but not after 5 minutes are used to calculate the phenol coefficient.
• The reciprocal of the appropriate test disinfectant dilution is divided by that of phenol to obtain the coefficient.
• The higher the phenol coefficient value, the more effective the disinfectant.
• A value greater than 1 means that the disinfectant is more effective than phenol.