Chapter 7 Control Agents

supercritical fluid

CO2 with gas and liquid properties. Sterilzes medical implants from donor patients.

decimal reduction time

Minutes to kill 90% of a population at a given temperature

Moist Heat Sterilization

-Moist heat denatures proteins -Autoclave: Steam under pressure. Steam must contact item's surface.

radiation methods of controlling microbial growth

-ionizing: X rays, gamma rays and electron beams cause hydroxyl radicals. -non-ionizing/ UV causes thymine dimers. -microwaves kill by heat.

How many microliters are in a milliliter?

1,000. 100 uL= 0.1 mL

Principles of effective disinfection

1. Concentration of disinfectant 2. Organic matter 3. pH 4. Time

Describe the effects of microbial control agents on cellular structures.

1. Damage to cell membrane. Antimicrobial agents cause damage to the lipids or proteins of the plasma membranes by altering permeability and causes cellular contents to leak into the surrounding medium and interferes with the growth of the cell. 2. Damage to the cell wall. By either blocking its synthesis, digesting it, or breaking down its surface. 3. Inhibits protein and nucleic acid synthesis. Nucleic acids and proteins get damaged. Damage to nucleic acids by heat, radiation, or chemicals is lethal to cell. The cell can no longer replicate or carry out normal metabolic functions- synthesis of enzymes

Actions of Microbial Control Agents

1. alteration of membrane permeability. Causes leaking and inhibits growth/replication. 2. damage to proteins. Causes loss of metabolic function when proteins are denatured. And loss of protein structures. 3. damage to nucleic acids. Causes loss of ability to express proteins and loss of ability to grow/replicate.

ionizing radiation

1. ionizes water to release OH radicals (highly reactive hydroxyl radiacals which is toxic forms of oxygen from chapter 6). Hydroxyl radicals react with cellular components, but especially damages DNA.. X rays, gamma rays and electron beams

UVC wavelength

100-280 nm. shortest wavelength. most powerful & dangerous form of UV radiation, but is completely absorbed in the ozone layer. UVC lightbulbs can be dangerous.

UVB wavelength

290-320 nm. shorter wavelength/ medium wavelength. Causes DNA damage.

UVA wavelength

320-400 nm. longer wavelength. Important for vitamin D production in the skin, but also causes sunburn and cataracts.

equivalent treatments to pasteurization

63 C for 30 min. High temperature short time: 72 C for 15 seconds. Ultra-high temperature: 140 C for less than 1 second. Thermoduric organisms survive, but are less likely to cause spoilage or disease.

Filtration

A process that separates materials based on the size of their particles. Filter must be small enough to capture microbes.

Microwave

Basically somewhat ineffective against microbes. Only moist areas have vegetative pathogens affected. Solid foods heat unevenly and have been known to spread infection.

Why does alcohol require water?

Because it evaporates too quickly to maintain contact and be effective without water. Denaturation of proteins requires water. Dilution important.

Alcohols

Denature proteins, dissolve lipids. Cell membrane dissolved, then tit can enter cells to cause leakage and denature proteins. Requires water. Ethanol and isopropanol.

Gaseous Sterilants

Denature proteins. Use: heat-sensitive material Ethylene oxide

Disinfectant effect on endospores and mycobacteria

Endospores difficult to destroy, but fairly effected by Glutaraldehyde, a type of aldehyde that inactivates proteins by cross-linking with functional groups and is used for medical equipment. Mycobacteria best effected by phenolics, which disrupt plasma membranes. Mycobacteria easier to destroy than endospores.

disk diffusion method

Evaluates efficacy of chemical agents. Filter paper disks are soaked in a chemical and placed on a culture. Look for zone of inhibition around disks

Describe how filtration, low temperatures, high pressure, dessication, and osmotic pressure suppresses microbial growth.

Filltration- removes microbes by passage of liquid through a screen-like material with pores small enough to retain microorganisms. Low temperature- metabolic rate of most microbes is so reduced that they cannot reproduce or synthesize toxins. High pressure- denatures proteins. Desiccation (absence of water)- prevents metabolism. Osmotic pressure- causes plasmolysis

Summary of lab 10 chemical agents

Goal is to compare disinfectant effect on bacteria. Using Serratia Marcescens (Gram negative so LPS layer is extra protection, rod shaped bacillus, facultative anaerobe), and Bacillus Thuringiensis (Gram positive, endospore former, toxic crystal produced that kills insects). Independent Variable- Time of exposure to the selected disinfectant. Various types of disinfectants were used by the groups. Dependent variable- Growth is dependent on the effectiveness of the selected disinfectant based on the length of exposure time. Control plate- no disinfectant used to see natural uninhibited growth.

Summary of lab 10 UV light exposure

Goal is to see how UV light affects Serratia Marcescens. If sunscreen limits UV exposure, agar plates inoculated with S. Marcescens will have a greater population when protected with sunscreen compared to not protected with sunscreen. Negative Control- eliminate the independent variable to make the experiment fail. Positive control- check nothing went wrong. Use known/standard value, established controls with predictable results.

Dry heat vs. autoclave

Hot air at 170 C for 2 hours. Autoclave at 121 C for 15 minutes.

Chlorine

In bleach, is an oxidizing agent that causes Hypochloric acid HOCl in water. A strong oxdizing agent that prevents cellular enzymes from functioning. Semmelweis. A halogen.

How is low temperature a physical method of controlling microbial growth?

Inhibits growth/replication as metabolic activity is suspended

Why are gram-negative bacteria more resistant to chemical biocides than gram-positive bacteria?

LPS layer and the enzymes below give chemical protection.

Physical methods of microbial control

Low temperature High pressure Desiccation Osmotic pressure

Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat.

Moist heat: kills microorganisms by coagulating proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in their three-dimensional structure. steam must contact item's surface. Dry heat: kills by oxidation effects, flaming, incineration, hot-air sterilization. Water is a better conductor of heat compared to air, so an autoclave can use lower temperature for less time than dry heat.

Peroxygens

Oxidizing agents Used on Contaminated surfaces. Ozone O3, H2O2, peracetic acid.

Low temperature storage

Refrigeration inhibits growth of pathogens and spoilage organisms by slowing or stopping enzyme reactions. Psychrotrophs, psychrophilic organisms can still grow. Bacteriostatic effect. Freezing preserves by stopping all microbial growth. Some microbial cells killed by ice crystal formation, but many survive and can grow once thawed

Quaternary Ammonium Compounds (Quats)

Surfacant detergent are bactericidal, denature proteins, disrupt plasma membrane. Kill gram positive more than gram negative. Do not kill endospores

Differentiate halogens used as antiseptics from halogens used as disinfectants.

Surface disinfectants may be too harsh for use on tissue because the same effects would damage the host Antiseptic- removing pathogens from living tissue. Disinfectant- removing pathogens from surfaces. iodine is effective antiseptics- alters protein synthesis and membranes;chlorine is effective disinfectant- oxidizing agents

Explain why endospore forming bacteria are resistant to most control agents?

Their cell wall of endospore is neither gram positive or gram negative. The spore coat is a thick tough protective layer that makes it less likely for chemicals to penetrate the cell. The cell is dormant, so metabolically less active. In this state it is less vulnerable to chemicals because reactions that would affect the cell's homeostasis are not occurring.

Plasmolysis

This happens when a cell shrinks inside its cell wall while the cell wall remains intact.

thymine dimers

This is a type of damage often caused by ultraviolet radiation. directly effects DNA; then RNA and proteins would be affected and cell replication inhibited.

non-ionizing radiation

UV light Damages DNA. Causes thymine dimers for form, inhibiting cell replication because DNA has extra bonds between thymine bases. Little penetrating power so it must be directly exposed. Best wavelength is 260 nm for microbes. Air sterilization is possible.

UV light affect on DNA

UV light is absorbed by a double bond in pyrimidine bases (such as thymine and cytosine in DNA) or purine bases (such as adenine and guanine), opening the bond and allowing it to react with neighboring molecules. If it is next to another pyrimidine/ purine base, the UV-modified base forms direct covalent bonds with it, forming a dimer. Many organisms including bacteria have enzymes capable of repairing certain kinds of damage to DNA. Light repair or dark repair may occur, but damage can be permanent if the exposure is too long and mutations accumulate. UV radiation is good for microbe control because single-celled organisms are easily penetrated by UV. UV light is non-ionizing radiation.

non ionizing radiation effect on DNA

UV radiation causes thymine dimers

UV radiation as microbial control agent

UV radiation is a microbial control agent because the small single cells are easily penetrated. Because UV passes easily through the air, slightly through liquids, and poorly through solids, direct exposure is needed. Serratia Marcescens was killed by UV exposure. Dots of resistant bacteria were seen. These CFUs likely had mechanisms for light or dark repair made possible by the presence of enzymes to repair thymine dimers. Or, maybe these CFUs were able to prevent thymine dimers.

light repair (photoreactivation)

When thymine dimers are exposed to visible light, photolyases are activated; these enzymes split the dimers, restoring the DNA to its undamaged state.

ionizing radiation effect on DNA

X rays, gamma rays and electron beams cause hydroxyl radicals that damage cell components but especially DNA

use-dilution test

a method of determining the effectiveness of a disinfectant using serial dilutions. Metal cylinders dipped into broth cultures of bacteria. Contaminated cylinder immersed into dilution of disinfectant. Cylinders removed, washed, and placed into tube of medium. Most effective agents entirely prevent growth at highest dilution.

Plasmas

a state of matter in which a gas is excited, in this case by an electromagnetic field, to make a mixture of nuclei with assorted electrical charges and free electrons. Sterilizing tubes.

asepsis

absence of significant contamination

Iodine

alters protein synthesis and membranes. Inhibits protein function. An oxidizing agent. Affects proteins, nucleotides, and fatty acids. A halogen.

Equivalent treatments

as the temperature is increased, much less time is needed to kill the same number of microbes

oligodynamic action

the ability of small amounts of a heavy metal compound to exert antimicrobial activity

calculate the number of bacteria

bacteria per mililiter = CFU/ TDF amount in mL

why is microbial death measured in log 10?

because it is easier to see the pattern of microbial death rate as the rate is a straight line

Explain how the type of microbe affects the control of microbial growth.

biocides tend to be more effective against gram-positive bacteria. external lipopolysaccharide layer of gram-negative bacteria, makes them resistant;. mycobacteria are acid-fast and have a cell wall that is waxy, lipid-rich and gets protection by not being easily penetrated by chemicals. Slow metabolic activity due to harder to transport chemicals in and out. Viruses resistance depends on the presence or absence of an envelope. The lipid envelope ones expose the virus and because there is no metabolic activity to maintain homeostasis it is destroyed. Endospores from gram positve have thick protective spore coats that do not let many chemicals pass. Low metabolic activity, low water content and low or no enzyme activity, dormant life bearing structures resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying. peptidoglycan cross linked in the cortex, calcium-dipicolinate, desiccation, DNA repairing enzymes

Aldehydes

cause cross-linking of functional groups that inactivates proteins and nucleic acids. Used on medical equipment. Glutraldehyde and formaldehyde.

osmotic pressure

causes plasmolysis

List factors related to effective disinfection.

concentration of disinfectant organic matter pH time

population death rate

constant

Surface-active agents

decrease surface tension among molecules of a liquids. Acid-anionic detergent to sanitize. Quaternary ammonium compounds are bactericidal, denature proteins, disrupt plasma membranes.

Heavy Metals

denature proteins. Releases ions that are effective in even small amounts. Oligodynamic action.

High Pressure

denatures proteins

Biguanides

disrupt plasma membranes Chlorhexadine

phenols and phenolics

disrupt plasma membranes, causing leakage of cell contents. Phenols are irritating. Phenolics less so.

Bisphenols

disrupt plasma membranes. Hexacholorphene, triclosan. Especially effective against gram positive, but do work on yeasts and gram negative.

dry heat sterilization methods

dry heat, direct flaming, incineration, hot air sterilization kills by oxidation.

bacteriostasis

inhibiting, not killing, microbes refrigeration

HEPA filter

high efficiency particulate air filter. removes microbes >0.3 micrometers

Describe the patterns of microbial death caused by treatments with microbial control agents

if the death curve is plotted logarithmically, the death rate is constant and a straight line. Factors that influence the effectiveness: 1. number of microbes. the more microbes there are, the longer it'll take. 2. Environmental influences: the presence of organic matter often inhibits the action of chemical antimicrobials. 3. Time of exposure: often require extended exposure to affect more-resistant microbes or endospores. 4. Microbial characteristics: affect the choice of chemical and physical control methods

Halogens

iodine and chlorine

Explain how radiation kills cells.

ionizing radiation- ionizes H2O water to release OH radials, damages DNA by oxidative effects. X rays, gamma rays and electron beams. nonionizing radiation- damages DNA by thymine dimers inhibiting replication. UV light. microwave- kill by heat; not especially antimicrobial

List the advantages of glutaraldehyde over other chemical disinfectants.

it is less irritating and more effective than formaldehyde. bactericidal, tuberculocidal, and virucidal in 10 minutes

biocide

killing microbes

dry heat sterilization

kills by oxidation

germicide

kills microbes

sanitation

lowering microbial counts on eating utensils

Dark repair

mechanism by which enzymes cut damaged DNA sections from a molecule, creating a gap that is repaired by DNA polymerase and DNA ligase. Can take place in presence or absence of visible light, but requires several enzyme-mediated reactions.

Examples of Heavy metals

mercury Hg, copper Cu, silver Ag silver nitrate for preventing gonorrhea blindness in newborns. Silver sulfadiazine as tropical burn cream. Copper sulfate as algicide in fish tanks. denatures proteins with oligodynamic action.

sepsis

microbial contamination

microbial death rate

number of dying cells per unit of time

Chemical food preservatives

organic acids- act to inhibit metabolism. nitrite- prevents endospore germination. antibiotics- prevent spoilage of cheese but resistance is a problem.

calculate percent survivors

percent survivors at a time point= (number of survivors at time point/ total number of bacteria per mL in the original sample) x 100 at 20 seconds, 4 CFUs in 0.2 mL plated on agar of 1/2 dilution. 4 CFU/ 0.5 x 0.2 = 40 cells/ 1mL

microbial death

permanent loss of reproductive capability

Identify chemical sterilizers.

plasma- free radicals destroy microbes, use on tubular instruments. supercritical fluids- CO2 with gaseous and liquid properties, use on medical implants. peroxygens- oxidizing agents, use on contaminated surfaces

Aseptic surgery techniques

prevent microbial contamination of wounds

Desiccation

prevents metabolism

Pasteurization

reduces spoilage organisms and pathogens.

membrane filtration

removes microbes >0.22 µm

sterilization

removing all microbial forms including viruses and endospores

Degerming

removing microbes from a limited area

Disinfection

removing pathogens

thermal death time

shortest length of time required to kill all test microbes at a specified temperature

Identify the appropriate uses for surface-active agents.

soap- degerming. Skin degerming and removal of debris. acid-anionic detergents- sanitizing utensils etc. quaternary ammonium compounds- bactericidal, denature proteins, disrupt plasma membrane. Used as antiseptic for skin and instruments. Soap- skin. acid anionic detergents- objects and surfaces. Quats- both skin and objects or surfaces.

commercial sterilization

sufficient heat treatment to kill endospores of Clostridium botulinum in canned food

Acid-anionic detergents

surface active agents /surfacants to sanitize dairy equipment.

thermal death point

the lowest temperature required to kill all microbes in a sample in 10 minutes

Calculate Dilution Factor

volume of sample/ (Volume of sample + volume of diluent) 3 mL of bacteria / (3 ml of bacteria + 3 ml of control agent) = 1/2 dilution factor

Why is moist heat more effective than dry heat?

water is a better conductor of heat than air

Chemical methods of microbial control

• Effectiveness of the disinfectant depends on: • Type of the chemical agent • Type of microbes • Concentration of a disinfectant • Time of contact • pH of the medium • Temperature -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