Bio 220: Antimicrobial Agents

Front 1

What factors effect the killing of organisms?

Back 1

1. # of organisms present
2. # of molecules (bullets of chemical agents)
3. time
4. temperature
5. pH
6. Media/Environment
7. Type of Organism
8. Physiologic State and/or age of organisms

Front 2

Killing of Organisms
# of organisms present

Back 2

it is harder to kill a larger population of cells

Front 3

Killing of Organisms
# of molecules (bullets of chemical agents)

Back 3

a more concentrated killing chemical agent may be more effective

Front 4

Killing of Organisms
time

Back 4

more exposure time, more death.

Front 5

Killing of Organisms
temperature

Back 5

increased temperature results in increased killing rate. The molecules are moving faster.

Front 6

Killing of Organisms
pH

Back 6

altered pH can alter the potency. Depends upon the specific killing agent.

Front 7

Killing of Organisms
Media/Environment

Back 7

the more organic material present, the less susceptible to killing. Organic material can combine the chemical causing inactivation. Organic material can cause the chemical causing inactivation. Organic material can cause the chemical agent to precipitate out of solution. Organic material can coat the microorganism and protect it.

Front 8

Killing of Organisms
Type of Organism

Back 8

There are differences between bacteria, fungi and other infective agents. It is harder to destroy Gram negative bacteria then Gram positives. Bacterial spores are hardier and more resistant than fungal spores.

Front 9

Killing of Organisms
Physiologic State and/or Age of Organisms

Back 9

Younger bacterial cells reproduce more and are easier to kill than older cells. Chemicals have a greater chance of reacting with cells actively engaged in metabolic (growth) activities.

Front 10

What are examples of Physical Agents?

Back 10

1. Dry Heat
2. Moist Heat
3. Cold Temperatures
4. Lyophilization
5. Pasteurization
6. UV radiation
7. X-rays and gamma rays
8. Cathode rays
9. Filtration
10. Osmotic Pressure
11. Sonic Vibration (cavitation)

Front 11

Physical Agents
Dry Heat

Back 11

conventional ovens set at 300 to 320 degrees F. for a period of 2 hours can be used for sterilizing pipettes with cotton for example. Open flames can be used to incinerate organism on inoculation loops.

Front 12

Physical Agents
Moist Heat

Back 12

a. Boiling water - will kill vegetative bacterial cells and eukaryotic spores (fungal). Will destroy most viruses. High altitude boiling occurs at a lower temperature and is less effective. Bottom line, boiling does not sterilize even with extended periods of boiling (ex. 30 min). Can be better than nothing.
b. Autoclaving - 121 degrees C for 15 min @15 pounds of pressure per square inch. Coagulates proteins and essential enzymes. Kills all forms of life. More volume requires more time in the autoclave as different volumes are not heated equally. Steam will not penetrate oil and oil based products. Test the effectiveness of the autoclave by using spore strips that are impregnated with Bacillus subtilis spores.
c. Tyndalization - intermittent periods of heating followed by incubation. This is a method used to rid cultures of spores. Uses free flowing steam that is not under pressure.

Front 13

Physical Agents
Cold Temperatures

Back 13

does not kill many bacteria. It is bacteriostatic . Used to preserve bacteria and viruses.

Front 14

Physical Agents
Lyophilization

Back 14

(freeze-drying or cryodessication)
a method of preservation

Front 15

Physical Agents
Pasteurization

Back 15

63 degrees C for 30 min- kills human pathogens and serves to increase shelf life of a product by destroying many of the spoilage bacteria. Does not kill spore formers or destroy spores. Can use HTST (High temperature, short time) method commercially. Milk is heated at 71 degrees C. for 15 sec. Increase temperature and decrease the time for more fat content. (82.2 degrees C. for 1 sec. for cream). Beer, wine and fruit juices can also be pasteurized. Generally, pasteurization times are dependent upon the materials, potential pathogens and contaminants present.

Front 16

Physical Agents
UV radiation

Back 16

creates thymine dimers. Most lethal at 260-270 nm. Does not penetrate well. UV intensity is inversely proportional to the square of the distance from its target. Used mostly for aerial contaminants. Effectively used in areas where cultures are transferred, restaurants, operating rooms and meat storage areas. A visible light repair mechanism and a two step dark repair mechanism can reverse the damage.

Front 17

Physical Agents
X-rays and gamma rays

Back 17

hyperactive ions that ultimately cause protein, DNA and other damage. Destructive peroxide and super oxide formation occurs. Ionizing radiation is used to sterilize prepackaged dog food as well as increasing the shelf life of fruits and cold vegetables.

Front 18

Physical Agents
Cathode rays

Back 18

consist of a beam of electrons. Used for research purposes.

Front 19

Physical Agents
Filtration

Back 19

use filter grids that range from 0.2 to 0.45 micrometers for filtering out bacteria. Commonly used to sterilize thermo labile fluids such as human and animal serum and plasma. This is a slow and costly method. The filters oftentimes clog too easily. Urea in urea agar is sterilized using filtration.

Front 20

Physical Agents
Osmotic pressure

Back 20

use high concentrations of salt and sugar to create hyper osmotic environments and therefore osmotic imbalances. 10 to 15% salt concentrations will inhibit most bacteria and microorganisms. 50% sugar concentrations will inhibit most bacteria and microorganisms.

Front 21

Physical Agents
Sonic vibration (cavitation)

Back 21

differential pressures created by targeting one side of a bacterial cell will cause the cell to burst. Used in research. A method for obtaining cell free extracts.

Front 22

What are Desirable Qualities of Chemical Agents?

Back 22

1. Selective toxicity
2. Non-toxic to man, domestic animals and environment
3. Solubility in water
4. Bactericidal within 10 to 60 minutes
5. Penetrate through oils and/or organics

Front 23

Phenol coefficient test for affectivity

Back 23

Test organisms

1. Salmonella typhi
2. Staphylococcus aureus
3. Pseudomonas aeruginosa

Use dilutions of test chemical and phenol where killing occurs in 5 to 10 minutes

Example
Chemical X-diluted 1:450

Phenol-diluted 1:90

Chemical X has a phenol coefficient of 5 – It is 5 times more potent then phenol as it had to be diluted 5 times more to achieve the same killing as phenol

Front 24

What are examples of Chemical Agents?

Back 24

1. Acids and bases
2. Phenols
3. Alcohols
4. Halogens
5. Heavy metals
6. Dyes or biological stains
7. Quaternary ammonium compounds ("quats")
8. Glutaraldehyde ("cidex")
9. Ozone and Hydrogen peroxide (oxidizing agents)
10. Formaldehyde
11. Ethylene oxide

Front 25

Chemical Agents
Acids and bases

Back 25

cause hydrolysis and coagulation of proteins. So toxic that they are rarely used.

Front 26

Chemical Agents
Phenols

Back 26

disrupt membranes and denature proteins. Active against vegetative cells. Not generally effective against spores. Stable to heat and can survive temperature extremes. Used by Lister.
a. phenol
b. hexachlorophene - 2 phenols with 6 chlorines. Good for skin antisepsis.
c. hexylresorcinol - phenol compound with sugar. Used in some soap.
d. cresols (creosote is a derivative) - phenol compound with additional methyl groups. Good in presence of organic matter, but toxic to the skin. Used as a wood preservative against fungi.

Front 27

Chemical Agents
Alcohols

Back 27

the longer its carbon chain, the better its killing effect.
a. one carbon-methyl alcohol
b. two carbons-ethyl alcohol
c. three carbons-propyl alcohol
d. four carbons-butyl alcohol
Longer chains enter the cell more readily, shorter chained alcohols tend to get bound during entry. Alcohol denatures proteins in cell. Good for skin antisepsis. Optimally active at 70% concentrations. At higher concentrations the improved dehydration effect actually inhibits the alcohol during cell entry. Alcohols are good against Gram positive and Gram negative bacteria, fungal spores and enveloped viruses. They are not effective against bacterial spores and non-enveloped viruses.

Front 28

Chemical Agents
Halogens

Back 28

work by combining with proteins and rendering them ineffective. Active against spores, viruses and fungi. Chlorine and Iodine compounds predominantly

Best agent is bleach - active ingredient is 5.25% sodium hypochlorite (NaOCl). Iodine compounds are used for skin antisepsis prior to medical procedures. Iodine can also be used for small scale water purification.

Others include Bromine and Fluorine. Methyl bromine is used as an anti fungal gas on vineyards to kill oak root fungus. Compound can get into the water table and cause neurotoxicity when consumed.

Front 29

Chemical Agents
Heavy metals

Back 29

combine with proteins
a. mercuric chloride - very toxic. Was used prior to antibiotics to treat syphilis.
b. silver nitrate - placed in eyes to protect newborn from Neisseria gonorrhoeae.
c. mercurochrome - used as a topical antiseptic. Not widely available in the U.S. any longer due to mercury content
d. methiolate - used as a vaccine preservative
e. copper sulfate - used to reduce fungal contamination of grapes. Also used as an algaecide in swimming pools.

Front 30

Chemical Agents
Dyes or biological stains

Back 30

combine with proteins
a. crystal violet (gentian violet) - inhibits gram positive bacteria in cultures. Also used as an antifungal agent.
b. potassium permanganate- acts as an oxidizer. Used against fungal infections of the hands and feet. Also used in water disinfection.

Front 31

Chemical Agents
Quaternary ammonium compounds

Back 31

disrupts cell membranes. Also denatures proteins. Chemically central nitrogen is attached to four side chains. This nitrogen compound is positively charged. This compound is linked to a chloride anion. Is used in disinfection of utensils in dairies and restaurants. Quats have no effect on spores. Benzalkonium chloride is an example of a quat.

Front 32

Chemical Agents
Glutaraldehyde

Back 32

(“cidex”)
acts as an alkylating agent. Chemically these consist of two aldehyde (CHO) groups linked together. These agents replace labile hydrogens. Used primarily on inanimate objects and work against all forms of microbial life. Used to clean medical equipment in respiratory therapy.

Front 33

Chemical Agents
Ozone and Hydrogen peroxide

Back 33

(oxidizing agents)
ozone treatment can be used to replace chlorination of hot tubs. Hydrogen peroxide is used in cleansing of wounds.

Front 34

Chemical Agents
Formaldehyde

Back 34

acts as an alkylating agent. Used in vaccine preparation and in specimen preservation. Formaldehyde will inactivate viral particles for use in vaccines. Chemically formaldehyde is built from one aldehyde group.

Front 35

Chemical Agents
Ethylene oxide

Back 35

an alkylating agent that is a gaseous chemosterilant commonly used in hospitals. A ringed structure built from two carbons and oxygen that is a highly instable chemical. It is used in the sterilization of heat-labile materials (plastic petri dishes used in lab.). Effective against vegetative bacteria, spores and viruses.