Microbiology Chapter 18

Adequate sterilization depends on __ and ___.

temperature; length of time Higher temperatures require shorter treatment times.

Two Common forms of Surfactants

soaps & detergents

Radiation

energy that is omitted from atomic activities

Target Cell wall

-maintains the integrity of the cell. -Damaging the cell wall makes a cell susceptible to lysis. -Several chemical agents can damage this barrier.

Endospore formation

-may be the most important factor Spore-forming organisms are not susceptible to most chemical agents. (This is especially important for nosocomial infections.)

Two types of Radiation

Ionizing radiation Non-ionizing radiation

glutaraldehyde.

-Research lasbs use 2% This effectively kills bacteria, viruses, and fungi. Treatment for 10 minutes will disinfect most objects. Treatment for 10 hours will sterilize.

Plasma Membrane

-Responsible for enclosing the cytoplasm -Involved with DNA replication -Involved in ATP Production

Selecting an Anti-microbial Agent

-Some chemical agents are better for certain uses than others. -Qualities to be considered include: Is it reactive against all types of organisms without destroying tissue. Is it effective in the presence of organic material. Is it stable and, if possible, inexpensive.

Alcohols

- are bacteriocidal, fungicidal, and virucidal for enveloped viruses. -have no effect on fungal spores and bacterial endospores. -are intermediate-level disinfectants. -Alcohol is often used to carry other antimicrobial chemicals.

Phenol and Phenolic Compounds

- are derived from phenol. -Many have greater efficacy and fewer side effects than phenol. -Phenols and phenolic compounds are low-level to intermediate-level disinfectants and antiseptics -They are commonly used as disinfectants in health care settings and laboratories.

Sanitization

- disinfection of places or things used by the public -Used to reduce the number of pathogens to meet accepted public health standards

Pressurized Steam

- gives the highest temperature. -Sterilization occurs when steam condenses to liquid water on the object. Hot water gradually raises the object's temperature. -Autoclaves are superior for sterilizing heat-resistant materials. -They are not useful for substances that repel water.

Temperature

- the lower the temperature, the longer it will take to kill

Sterilization

- the removal of all microbes Sterilization does not destroy prions.

The Disk method

- uses tiny disks of filter paper soaked in the agent. -An agar plate is inoculated and the disks are placed at various positions. -Inhibition of growth around the disk is called the zone of inhibition. (Sizes of the zones are not comparable.) (Sizes may reflect differences in concentration and diffusion rates.) -This method cannot distinguish between microbicidal and microbistatic.

Destroying replication or gene expression

-Alkylating agents e.g. ethylene oxide and nitrous acid, act as mutagens for viral nucleic acid. -This inhibits replication and proliferation.

halogens are effective against

-Bacterial and fungal cells -Fungal spores -Some bacterial endospores -Protozoan cysts -Many viruses

Types of Chemical agents

-Chemical agents are used more than physical means for disinfection, antisepsis, and preservation. - affect cell walls, plasma membranes, proteins, or nucleic acids. - destroy or inhibit growth of enveloped viruses, bacteria, fungi, and protozoans. They are ineffective against protozoan cysts and bacterial endospores.

Destroying the virions

-Detergents, alcohols, and other denaturing agents can affect capsid proteins. -Viral envelopes are susceptible to agents that act on lipids.

Two ways of Pasteurization

-Flash method - temperature of 71.6˚C for 15 seconds -Batch method - temperature of 63-66˚C for 30 minutes It does not affect endospores, non-pathogenic lactobacilli, micrococci, or yeasts

Lyophilization

-Freeze dying -used to preserve cells by removal of water -used for long-term sgtorage

Gaseous agents chararcteristics

-Gases rapidly penetrate and diffuse into any space. -Over time, they can denature proteins and DNA. -They kill everything they come in contact with and cause no damage to inanimate objects.

Forms & Types of Disinfectants

-Heat -Alcohol -Ultraviolet radiation Some disinfecting agents do not affect spores

Nucleic Acid Synthesis

-Nucleic acids are required for cell survival. -Some agents can disturb this synthesis by binding irreversibly to DNA. This prevents gene expression. -Others are mutagenic and cause lethal mutations.

Soap

-One end of a soap molecule is ionic and is hydrophilic. The other end is a fatty acid and is hydrophobic. - good de-germing agents but poor antimicrobial agents. - can be made more potent by adding antimicrobial triclosan.

Oxidizing Agents; Ozone

-Ozone is a very reactive form of oxygen. -It is generated when O2 is exposed to electrical discharge. -Some cities use ozone for water treatment. It is expensive to produce. It is difficult to maintain at the proper concentration.

Peracetic acid

-Peracetic acid is the peroxide form of acetic acid and an extremely effective sporicide. -It is used to sterilize surfaces and medical and food processing equipment. -It is not affected by organic contaminants. -It leaves no toxic residue.

Protein Structure and Function

-Proteins are important molecules in the microbial cell. -They have a 3-D shape directly related to the function. Inhibition or elimination of function can result in cell death.

Most Efficient way to determine Microbial Death

-The most efficient is to determine whether an organism can reproduce. (Move it from the antimicrobial environment to fresh media. If there is no growth, the organism is dead.)

Evaluation for Disinfectants and Antiseptics

-There is no completely satisfactory method for evaluating antimicrobial chemical agents. There are several tests including: 1. Comparison of the agent with phenol - the phenol 2.coefficient 3. Disk method 4.Use dilution method

Gaseous agents Disadvantage

-They are explosive, poisonous, and potentially carcinogenic. -Disinfection with gaseous agents takes considerable time. -There is a need for continuous cleanup

Chemicals that are used for disinfection and antisepsis are grouped into three types:

-Those affecting proteins -Those affecting membranes Those affecting viruses

Iodine

-a well-known antiseptic. -used medically as a tincture or as an iodophor. -Iodophors are longer lasting and non-irritating. -routinely used to prepare skin for surgery. - used to treat burns

Detergents

-are positively charged organic surfactants that are more soluble in water than soap. -The most popular are quaternary ammonium compounds (QUATS).

Moist Heat

-can be as effective as dry heat in a much shorter time at lower temperature. -It quickly denatures proteins which halts microbe metabolism and causes death.

Chemicals

-can kill microbes. -can be harmful to humans. -If they are to be useful as disinfectants, they must also be safe to use.

QUATS

-contain ammonium cations. - are low-level disinfectants/antiseptics. -Their advantage is being odorless, tasteless, and harmless to humans. -used in many industrial and medicinal applications including mouthwash

Dry Heat

-dehydrates microbial cells. The absence of water inhibits metabolism. -Protein denaturation takes a long time.

Boiling

-easy but does not kill heat-resistant cells. - effective for disinfection but not sterilization. Boiling for 30 minutes kills most non spore-forming pathogens.

Chlorine

-found in drinking water, swimming pools, and waste-water. -major ingredient in disinfectants such as chlorine bleach. -used to disinfect kidney-dialysis equipment.

Denaturation

-involves breaking of hydrogen and other bonds. -These hold the 3D shape. -When broken, the protein unfolds and is inactivated.

UV radiation disadvantages

-poor penetration -retinal damage, cancer, skin wrinkling

The Use Dilution Method

-time-consuming. - can tell whether the agent is microbistatic or microbicidal. -A series of solutions of different concentrations of the disinfectant are prepared -Cultures of the test organism are dried down on stainless steel cylinders

Filtration

-used for sterilizing liquids -involves passing the liquid through membrane filters -used for sterilization of growth media, drugs, and vitamins -for fecal coliform contamination -purify air

Pasteurization

-using heat to kill pathogens -Does not sterilize but is used to reduce number of pathogens - reduces the number of organisms that can cause spoilage

Two types of Heat Used

-usually lethal to most pathogenic microbes. Moist heat - from hot water, boiling water, or steam Dry heat - from hot air with low moisture e.g. from ovens

Surfactants

-very effective for disrupting the plasma membrane. -Made of polar molecules with hydrophobic and hydrophilic regions -They bind to and penetrate the phospholipid bilayer. (This causes openings to form.) -They also affect virus envelopes. )Damage to the envelope causes the loss of capacity to infect)

Heavy Metals: Mercury and Silver

-were formerly used in clinical situations. -Mercury has been abandoned. -Silver is still occasionally used in: Surgical dressings Burn creams Catheters

For most chemical agents;

..... increase in concentration = increase in its potency. -This is not true for alcohol. -Increase in alcohol concentration actually hinders killing. Alcohol must have some water associated with it. -This makes for better penetration and denaturation of proteins.

Factors that affect the rate of microbial death

1. Numbers 2. Duration of exposure 3. Temperature 4. The environment 5. Endospore formation

Four members of the Halogens chemical family that have antimicrobial activity

1.Iodine 2.Chlorine 3.Bromine 4.Fluorine They are all intermediate-level antimicrobial chemical agents.

Same for changes in pH

Changes in pH can increase or decrease an agent's potency.

Phenols and phenolic compounds are low-level to intermediate-level disinfectants and antiseptics

Denature proteins Disrupt the plasma membrane Remain very effective in the presence of organic material Remain active for prolonged periods

Physical Methods for Controlling Microbial Growth

Drying Heating Cold Filtration Osmotic pressure Radiation

Most frequently used gaseous agents

Ethylene oxide . It is used in hospitals and dentists' offices for sterilizing instruments and equipment.

Additional targets of the bacteria

Inhibition of protein synthesis Alteration in protein structure

2 Highly reactive aldehydes are used as antimicrobials

Glutaraldehyde - used in liquid form Formaldehyde - used in both liquid form and gaseous form

Formaldehyde

Health care workers use a 37% formaldehyde solution - formalin. -It is used to disinfect: Isolation rooms Exhausts Cabinets Surgical instruments Dialysis machines Formaldehyde is an irritant for mucous membranes and is also carcinogenic

Gaseous Agents

Many items cannot be sterilized with heat or chemicals. They can be sterilized using highly reactive antimicrobial and sporicidal gases. Ethylene oxide Propylene oxide β-propiolactone

Pasteurization

Pasteurization is used to reduce microbial load. It destroys pathogens. It preserves flavor and nutritive value in foods. It does not sterilize.

Accepted definition of Microbial death

Permanent loss of reproductive capability -Death rate is logarithmic

8 major categories of chemical agents

Phenol and phenolic compounds Alcohols Halogens Oxidizing agents Surfactants Heavy metals Aldehydes Gaseous agents

The Phenol Coefficient

Phenol coefficient of 1.0 = same effectiveness as phenol Greater than 1.0 = efficiency greater than phenol Less than 1.0 = efficiency less than phenol

Three ways of Using Moist Heat

Pressurized steam Boiling Pasteurization

Heavy Metals

The ions of heavy metals are inherently antimicrobial. -low-level bacteriostatic agents. Heavy metals include: -Arsenic -Zinc -Mercury -Silver -Copper

Target Plasma Membrane

The plasma membrane is composed of a phospholipid bilayer. When this is disrupted, the cell loses its selective permeability. This leads to the death of the cell.

Three most common Oxidizing Agents

The three most commonly used are: Hydrogen peroxide Ozone Peracetic acid Hydrogen peroxide is a common household antiseptic.

TDP

Thermal death point (TDP) is the lowest temperature needed to kill all organisms in 10 minutes.

TDT

Thermal death time (TDT) is the shortest length of time needed to kill all organisms at a specific temperature.

Aldehydes that cross-link to organic functional groups.

These reactions denature proteins and inactivate nucleic acids.

Tincture

alcohol used to carry other antimicrobial chemicals

Static

an agent that inhibits growth -In a bacteriostatic environment, numbers do not multiply but organisms are not dead.

Cidal

an agent that kills

Aseptic

an environment or procedure free from contamination

Antisepsis

associated with human body & skin

Disinfectants

associated with inanimate objects

Duration of exposure

can vary depending on: -Accessibility -Type of microbe -This is important to consider when using radiation treatment.

Ionizing radiation

causes electrons to be ejected from other atoms -can penetrate liquids and most solid materials

Bacteria's targets

cell wall and plasma membrane

Three major methods for controlling microbial growth

chemical physical mechanical removal

Chloramines

combinations of chlorine and ammonia. -Used in wound dressings, skin antiseptics, water supplies. -Less effective than chlorine as disinfectants/antiseptics. -Release their chlorine atoms more slowly therefore last longer.

Aldehydes

compounds containing a terminal -CHO group. -cross-link to organic functional groups.

Ultraviolet radiation

disrupts cells by generating free radicals -used for disinfection

____ break hydrogen bonds, which can result in __.

heat and solvents; total denaturation and coagulation

Chemical agents control viral pathogens by

inactivating the ability of the virus to infect or replicate. -This can be accomplished in two ways: Destroying the virion. Destroying replication or gene expression.

Death rate using a chemical agent

is accelerated by increasing the temperature. An increase of 10˚C doubles the rate of chemical reaction.

In a bacteriostatic environment_______.

numbers do not multiply, but organisms are not dead

Denatured

protein with a changed shape

Cold temperatures

retard the growth of microorganisms. They slow the rate of enzymatic reactions. They do not kill.

Sanitization is not sterilization but the same techniques can be used.

steaming high-pressure high temperature washing and scrubbing

Numbers

the greater the number of organisms, the longer it will take to kill If large numbers of organisms are present, it takes time for the agent to reach them all.

If mild treatments are used and removed

the protein will refold

De-germing

the removal of microbes from a surface by mechanical means

Disinfection

the use of chemical or physical agents to kill or inhibit the growth of microorganisms

Factors to consider when evaluating effectiveness are:

time temperature concentration

Refrigeration

used to delay spoilage of food

Most antimicrobial chemical agents used in proper concentration

will guarantee permanent denaturation of proteins