1. # of organisms present- it is harder to kill a larger population of cells.

Transcription

1 ANTIMICROBIAL AGENTS Killing of Organisms-Death depends upon 1. # of organisms present- it is harder to kill a larger population of cells. 2. # of molecules (bullets of chemical agents)- a more concentrated killing chemical agent may be more effective. 3. Time- more exposure time, more death. 4. Temperature- increased temperature results in increased killing rate. The molecules are moving faster. 5. ph- altered ph can alter the potency. Depends upon the specific killing agent. 6. Media/Environment- the more organic material present, the less susceptible to killing. Organic material can combine with 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.

2 7. Type of organism- 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. 8. Physiologic state and/or age of organisms- 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.

3 Physical Agents 1. Dry heat 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. 2. Moist heat 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 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

4 cultures of spores. Uses free flowing steam that is not under pressure. 3. Cold temperatures- does not kill many bacteria. It is bacteriostatic. Used to preserve bacteria and viruses. 4. Lyophilization (freeze-drying or cryodessication)- a method of preservation. 5. Pasteurization- 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. 6. UV radiation- creates thymine dimers. Most lethal at 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.

5 7. X-rays and gamma rays- 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. 8. Cathode rays- consist of a beam of electrons. Used for research purposes. 9. Filtration- 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. 10. Osmotic pressure- 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. 11. Sonic vibration (cavitation)- 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.

6 Desirable Qualities of Chemical agents 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 Phenol coefficient test for affectivity 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

7 Chemical Agents 1. Acids and bases- cause hydrolysis and coagulation of proteins. So toxic that they are rarely used. 2. Phenols- 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. 3. Alcohols-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

8 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. 4. Halogens- 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.

9 5. Heavy metals- 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. 6. Dyes or biological stains- 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. 7. Quaternary ammonium compounds ( quats )- disrupts cell membranes. Also denatures proteins. Chemically central nitrogen is attached to four side chains. This nitrogen compound is positively charged.

10 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. 8. Glutaraldehyde ( 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. 9. Ozone and Hydrogen peroxide (oxidizing agents)- ozone treatment can be used to replace chlorination of hot tubs. Hydrogen peroxide is used in cleansing of wounds. 10. Formaldehyde- 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. 11. Ethylene oxide- 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

11 of heat-labile materials (plastic petri dishes used in lab.). Effective against vegetative bacteria, spores and viruses.