Chemical Control of Microorganisms
1. Distinguish between " - cidal" and "-static"
2. Discuss and give examples of the role of halogens, phenolics, heavy metals, and alcohols, in disinfection.
3. Define the roles of formaldehyde, ethylene oxide, glutaralydehyde, hydrogen peroxide, and detergents.
Oliver Wendel Holmes- contagious nature of puerperal fever (Strephococcus) after childbirth.
Ignaz Semmelweis- soap and water handwashing between patients, calcium hypochlorite disinfectant.
Tincture of iodine- first used widely as an antiseptic during the Civil War
Paul Erlich- use of Salvarsan (mercury compounds = “magic bullet”) to treat syphilis
Joseph Lister- first used phenol (carbolic acid) on surgical sites and to disinfect operating room air.
I. Principles of chemical control-
A. Sterilization- destruction of all forms of life including spores (there are no "degrees" of sterilization). Chemical agents rarely achieve true sterilization.
B. Disinfection- destruction of pathogens on an object/surface (reduce numbers below level of any threat
1. Disinfectant- used on inanimate objects. Disinfectants are usually cidal (bactericidal- kills bacteria.
2. Antiseptic- used on living skin/tissue. Antiseptics are usually static (bacteriostatic- only prevents multiplication in the presence of the agent, inhibits growth but doesn't kill).
Some compounds (iodine) can be used for both, but formulas and dilutions are different.
C. Selecting antiseptics/disinfectants- ideal properties
1. Fast acting
2. Broadly effective- at different temperatures, pH, and many types of microbes killed.
3. Good penetration/with no damage to surfaces (disinfectant), or nontoxic to humans/animals (antiseptic).
4. Easy to prepare and stable (long shelf life)
5. Soluble in water and works well when dilute
6. Smells good (or at least doesn’t stink).
7. High phenol coefficient- phenol used to be the standard all other compounds were compared to against S. aureus (G+) and S. typhi & P. aeruginosa (G-) under identical circumstances and dilution. They measured the concentration that kills in 10 minutes, but not in five minutes. Coefficient >1 if more effective than phenol (the higher the better), <1 if less effective than phenol. Not used any more because didn’t take into account 1) tissue toxicity, 2) temperature, and 3) activity in presence of organic matter (blood, feces)
D. How Disinfectants/Antiseptics Work-
1. Denatures proteins- permanently alters secondary and quaternary structure by disrupting H bonds. Also permanently alters tertiary structure by disrupting disulfide bridges (sulfhydryl groups). Generally cidal, examples include peroxide, halogens, heavy metals.
2. Causes leaks in cell membranes- solublize or disrupt lipids with surfactants (such as alcohols, detergents, quaternary ammonium compounds).
3. Inactivation of viruses- must destroy viral nucleic acid or protein (alkylating agents, ethylene oxide gas, nitrous acid, hydroxylamine)
II. Important Chemical Agents (Table 7.8)-
A. Halogens- not sporocidal usually - ~5% NaOCl
1. Chlorine- germicidal action due to hypochlorous acid (HOCl, oxidizing agent) formation whenever Cl2 and H2O are mixed.
a. Chlorine gas- used alone or with lime (calcium hypochlorite)- Ca(OCl)2 as tertiary treatment for municipal drinking water treatment plants. Chlorine gas combines with water ions- need enough left over (free) to have antibacterial activity (0.2 -1.0 ppm).
b. Sodium hypochlorite- (NaOCl, Chloroxâ is 5-10%)- very useful in treating your own drinking water 2 drops/liter (4 drops if cloudy) treat for 30 minutes.
c. Chloramines (Cl2 + ammonia)- release Cl more slowly
d. U. S. Military gets Chlor-Floc (sodium dichloroisocyanurate) tablets to purify water and settle (clarify) suspended solids for drinking.
2. Iodine- more reactive and germidical than chlorine (but won't sterilize skin), takes several minutes to act. Mode of Action- combines with certain amino acids (inactivating proteins) and acts as oxidizing agent. Also used in tablet and liquid (Polarpure)forms to treat drinking water.
a. Tincture of iodine- 2% in water and alcohol mix (can treat clear water with 5 drops/liter for 30 minutes = safe to drink.
b. Iodophor- iodine-detergent (organic) complex. Includes the antiseptics Wescodyne and Betadine (providone ioline)- slow release, doesn't stain as badly as iodine, loosens organic mater (due to detergent wetting agent).
B. Phenol and phenolics (Fig.7.7)-. All phenolics work by injuring plasma membrane of bacteria, some also denature proteins (inactivate enzymes). Good anti-Mycobacterial activity.
1. Phenol- since Lister (1847), phenol (carbolic acid) is not used as an antiseptic anymore due to tissue irritation and odor - - used in throat lozenges and throat sprays (Chloraseptic) as local anesthetic and antiseptic, but need to have >10% concentration for antibacterial effect. expensive and caustic to skin - amphyl, chlorhexidine gluconate (Hibiclens) and hexachloroaphene (a biphenol-see below)
2. Cresols- coal tar derivatives, also wood preservatives (creasote). Good surface disinfectants, have lower toxicity, greater germicidal activity than phenol. O-phenylphenol is active ingredient in Lysol, Staphene, and Amphyll.
3. Bisphenols- contain two phenol molecules
a. Hexachlorophene- found in Ipana toothpaste, Mum deodorant, Dial soap, Phisoderm and Phisohex (prescription only strength). Bacteriostatic and especially good for Staphylococcus and Streptococcus. If left on skin or scalp, can be absorbed and cause neurological damage.
b. Triclosan- used in a number of antibacterial soaps, at least one toothpaste, and even incorporated into plastic kitchenware (cutting boards and utensils). Especially effective against G+ bacteria and fungi. Widespread use has led to reports of resistance.
C. Biguanides- injure cell membranes and are cidal for vegetative cells, but ineffective against endospores, Mycobacterium, protozoal cysts, and most viruses.
1. Chlorhexidine- at 0.2% is good antiplaque and antigingivitis agent. Nontoxic to skin and mucous membranes, so makes a good surgical scrub (Hibiclens) when combined with detergent or alcohol. It is toxic to eyes.
2. Hexylresorcinol- mouthwash, Sucrets- decreases surface tension loosens bacteria (plaque) from oral surface
D. Heavy metals- large atomic weights, bind protein molecules together at their sulfhydryl groups to cause denaturation. Ineffective against endospores or Mycobacterium.
1. Mercury- Mercurochrome, Merthiolate- mercury is combined with a carrier so it is less toxic to skin. Mercury is also used as a preservative in vaccines and sera.
2. Copper- CuSO4 used as algicide for pools (inhibits the growth of any photosynthetic organism (chlorophyll). Combined with lime as an antifungal (Blue-white Bordeaux Mix) to pevent downy mildew of grapes. Both mercury and copper are added to paints along with zinc oxide as mildew inhibitors.
3. Silver- silver nitrate (AgNO3), for years was used as 1% solution in eyes of newborns to prevent blindness due to gonnococcal opthalmia (N. gonorrheae). Today use erythromycin ointment instead. Silver is also incorporated into wound dressings (slow release) good for antibiotic resistant bacteria, and to treat suture materials and catheters. It is also combined with sulfadiazine to create silver sulfadiazine (Silvadene), which is an important topical used on burns.
4. Zinc- zinc chloride common in mouthwashes and anti-cold virus lozenges, and zinc oxide as an antifungal in paint.
E. Alcohols- cidal for bacterial vegetative cells, fungi, enveloped viruses, but not effective for endospores and non-enveloped viruses. Good for local physical degermination (wiping) of skin, but not on wounds. Primarily work by denaturing proteins and solublizing lipids (disrupts cell membrane). Alcohols also increase the effectiveness of certain agents like iodine and zephiran when these are mixed in a tincture- mixture of alcohol and water.
1. Ethyl alcohol (ethanol)- a 2-carbon, consumable alcohol. 70% appears to work best, 50-80% will do.
2. Isopropyl alcohol (rubbing alcohol)- better than EtOH as an antiseptic/disinfectant, also cheaper and less volatile.
F. Surface active agents (soaps and detergents)- alone they don’t have any antimicrobial properties. They simply function to remove skin oils and grease by emulsifying lipids and anything trapped in them (serve as wetting agents). They can do this because they are amphipathic molecules (have a water-soluble portion AND a lipid-soluble portion (Remember, soaps are created by saponification- heating a fat with an alkali, such as NaOH or KOH). Many so-called “deodorant soaps” contain triclocarban as an antibacterial agent (especially effective against G+s).
1. Anionic (acid) detergents- In general, not as effective against bacteria as cationic detergents. They are important in the cleaning of dairy equipment and utensils.
2. Cationic (basic) detergents- in general, more effective than anionic detergents. The cations, such as ammonium react with the negatively charged cell membrane to make it leaky. The most widely used are the quaternary, ammonium compounds (quats, Fig.7.9). Quats are effective against bacterial vegetative cells [better against G+s than G-s], protozoa, fungi, and enveloped viruses, but ineffective against endospres, Mycobacterium, and many Pseudomonas. Quats are very potent antimicrobials, are colorless, odorless, tasteless, stable, easily diluted and non-toxic to tissue. But they lose effectiveness when mixed with organic matter or other soaps, detergents, especially anionic ones. Two of the most widely used quats are Zephiran (benzalkanium Cl) and Cepacol (cetypyridinium Cl). If your mouthwash foams when shaken, it most likely contains a quat.
G. Alkylating agents- work by denaturing proteins (form covalent cross-links between several functional groups), and alters DNA structure. Formaldehyde is a common one (formalin is 37% formaldehyde). Gluteraldehyde is even more effective (bacteriocida [including TB] and virucidal in 10 min., sporicidal in 3-10 hr.) and is used as a 2% solution (Cidex) to treat surgical instruments (must pre-clean) like delicate fiber optic endoscopes without damage. Both are also used as embalming fluids.
H. Oxidizing agents (peroxygens)- inactivate various cellular components, especially effective against anaerobes.
1. Hydrogen peroxide (H2O2)- usually used as a 3% aqueous solution (light sensitive). Actually better for surface disinfection (sporicidal) than as a wound antiseptic (inactivated by catalase from our cells and slows down healing). New method of sterilization called plasma gas sterilization uses free radicals formed by treating hydrogen peroxide with radio or microwaves.
2. Ozone- very reactive form of oxygen created by exposing oxygen to high voltage (can smell after lightning storm or UV light use. Often used along with chlorine to treat drinking water commercially.
3. Benzoyl peroxide- used to kill anaerobes in deep wounds and to treat acne.
4. Peracetic acid- very effective (vegetative cells/fungi in 5 min., endospores in 30 min., not affected by organic matter, used on food processing and medical equipment.
I. Gaseous chemosterilizers- Ethylene oxide (highly explosive), propylene oxide and B- propiolactone (all are toxic and suspected carcinogens). Used to sterilize paper products, plastics, leather, wood, metal, etc. Work by alkylating amino, hydroxyl, sulflydryl, carboxy, groups of proteins and altering nucleic acids. Sporicidal, but require long exposure (4-18 hr.)
J. Organic acids- used as food preservatives. Lactic/acetic acid are used in cheese, sauerkaut, pickle products. Propionic acid (calcium propionate) is used in breads. Other preservatives include sorbic acid (K+ sorbate), benzoic acid (Na+ benzoate), sulfur dioxide (SO2 and sulfites) in wine, and nitrates/nitrites in meats (prevent C. butulinum growth and maintain pink color, but generate carcinogenic nitrosamines when heated).
K. Dyes- Venetian violet or crystal violet can be used to treat oral thrush.
III. Evaluation of Disinfectants- use a standard set of 3 bacterial and one viral reference strains.
Salmonella cholera-suis G-
Pseudomonas aeruginosa G-
Staphylococcus aureus G+
Newcastle Disease Virus (infects wild and domesticated bird)- to test virucidal activity
A. Use Dilution Test: This is the most widely used method. Steel carrier rings are dipped in cultures, air dried, and placed in recommended concentration of disinfectants for ten minutes at 20°C, then placed in liquid growth medicum to see if all micorganisms killed or not.
B. Filter Paper Disk Diffusion Method: Easier to perform, used in teaching labs. Soak sterile paper disk in recommended concentration of disinfectant/antiseptic, place on an agar plate swabbed with test bacterial strain, incubate for 18-24 hr., then measure zone (diameter) of inhibition.