Controlling Microbes (Sterilization & Disinfection)
Some Important Terms Defined:
sterilization – treatment to destroy all microbial life (even destroys bacterial endospores and fungal spores); there are no degrees of sterility!
disinfection (sanitation) – treatment to reduce the number of pathogens to a level at which they pose no danger of disease; disinfectants are used to kill microbes on inanimate objects (most disinfectants are too harsh for use on delicate tissue); most disinfectants do not kill spores.
antisepsis – kill microbes or inhibit their growth on skin or other living tissue; antiseptics are applied to living tissue.
sanitizer – typically used on food-handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards (may mean just washing with soap in some cases).
“-static” – treatments that inhibit rather than kill; ex. refrigeration. (bacteriostatic, fungistatic, etc.)
“-cidal” – treatments that kill. (bactericidal, fungicidal, viricidal, etc.) (germicidal is a more general)
chemotherapeutic agents – chemicals, incl. antibiotics, used to treat disease (discussed in Chap. 12)
I. Physical Controls
1. Advantages – simple, inexpensive, effective penetrates to kill microbes throughout the object; best method if material being treated is not damaged by heat.
2. Mode of Action – denatures proteins.
a. Dry Heat Sterilization – ex. flaming loops, tubes in lab & hot air ovens (171oC, 1hr., 160oC for 2 hr., 121oC for 16 hrs.); used to sterilize materials that can withstand high temps. & any materials damaged by moisture.
b. Moist Heat Sterilization – ex. boiling or in autoclaves; effective at a lower temperature than dry heat & it penetrates more quickly; disadvantages of boiling – does not kill thermophiles, endospores; autoclave is more effective than boiling- it uses pressure to raise the temperature above that of boiling (121oC, 15psi, for 20 min.); used to sterilize liquids and material easily charred; used in food canning & the lab to sterilize glassware & media.
c. Pasteurization – limits growth, but does not sterilize; used to slow spoilage of milk & dairy products, wine, beer; advantage: causes minimal damage to the product; developed by Louis Pasteur; standard treatment: heat to 63oC for 30 min. or 72oC for 15 sec.
1. Effect – microbiostatic; does not sterilize; slows down enzymes.
a. Refrigeration – preserves food because it stops the growth of most species of microbes (slows chemical reactions); most disease-causing microbes are mesophiles, not psychrophiles; an exception is Listeria spp., which causes listeriosis (food poisoning).
b. Freezing – kills most bacteria, but survivors can remain alive for long periods in the frozen state; bacteria cultures can be preserved by rapid freezing, sometimes with the addition of a compound called DMSO, milk, or glycerol to protect proteins.
1. Electromagnetic Spectrum – Radiation is classified by wavelength with ionizing and UV light radiation at the short-wavelength end, visible light in the middle, & radio waves at the long-wavelength end. The shorter the wavelength, the greater its energy, & the more lethal it is. Mode of Action: denatures DNA.
2. Two types of radiation that kill bacteria directly are UV (ultraviolet) Light & Ionizing Radiation. The effect of both is sterilization.
a. UV Light – bacteria actually have special enzymes that can correct some damage done by UV light!; in the lab mercury vapor lamps (germicidal lamps) are used; disadvantages: kills only on surfaces & these wavelengths can also be harmful to humans.
b. Ionizing Radiation – 2 forms; both cause a chain of ionizations by stripping electrons from atoms, resulting in cell death; disadvantages: technically complex; is being used to sterilize some produce, much to the public’s dismay.
1.) X rays
2.) Gamma rays
D. Membrane Filtration
1. Effect – physically removes cellular organisms (not viruses – they are too small).
2. Uses – in lab, used with media, antibiotics, & other heat sensitive materials; filtration is replacing pasteurization in some causes, because filtration causes even less damage; you may have heard of the new “cold filtered” beers.
1. Defined – the removal of water.
2. Two processes:
a. evaporation involving heat – effect – kills many microbes; rarely used in lab because the high heat causes chemical changes (denaturation); is used in food industry.
b. lyophilization [freeze drying] – removes water directly by converting water from a solid state (ice) to a gaseous state; materials are frozen & placed in a chamber to which a partial vacuum is applied; avoids the chemical changes caused by heat drying; effect – stops microbial growth by stopping most chemical reactions (just like regular freezing) frequently used in the microbiology lab to preserve perishable materials such as proteins, blood products, & reference cultures of microbes; used in food industry to make instant coffee, etc.; disadvantage – expensive.
F. Osmotic Strength
1. Method – high concentrations of salt or sugar.
2. Mode of action – microbes cannot grow if they are deprived of water; also, crenation or shrinkage can occur (you’re placing the microbes in a hypertonic environment).
3. Disadvantage – once added, solutes (such as salt or sugar) cannot be easily removed; not used in lab.
II. Chemical Control – The effectiveness of a chemical anitmicrobial agent is affected by time, temperature, pH, and concentration.
A. Testing Germicides – 3 ways:
1. Phenol coefficients: Germicides can be tested by comparing their effectiveness to phenol, a traditional germicide. It was phenol that Lister first used – he called it carbolic acid. The procedure involves preparing several dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen) or Staphylococcus aureus (a wound pathogen), incubating the tubes, and then checking for cloudiness in the tubes, indicating growth. The ratio of the effective dilution of the chemical agent to the dilution of phenol that has the same effect is the phenol coefficient. A disinfectant with a phenol coefficient of 1.0 has the same effectiveness as phenol. Less than 1.0 means it’s less effective. Greater than 1.0 means it’s more effective.
2. Paper disc method – paper discs are saturated with the chemical agent and placed on the surface of an agar plate inoculated with a test organism. Clear “zones of inhibition” appear around the discs if the chemical agent is effective.
3. Use-dilution test – The test microbe is added to dilutions of the chemical agent. The highest dilution that remains clear after incubation indicates a germicide’s effectiveness.
B. Mechanisms of Action
1. Affect Proteins – The alteration of protein structure is called denaturation. Denaturation can be permanent (bacteriocidal) or temporary (normal structure can be restored – bacteriostatic). Mechanisms of denaturation include:
a. Hydrolysis – breakdown of a molecule by addition of water
b. Oxidation – addition of oxygen or removal of hydrogen
c. Attachment of atoms or chemical groups – ex. heavy metals (mercury), alkylating agents (ex. –CH )
2. Affect Membranes
a. membrane proteins – denaturation (see above)
b. membrane lipids – can be dissolved.
3. Affect Cell Wall Formation
4. Affect Nucleic Acid Structure
5. Affect Metabolism
C. Types of Germicides
a. Structure – compounds with hydrophilic & hydrophobic parts.
b. Mode of action – Penetrate oily substances in water & break them apart into small droplets that become coated with surfactant molecules. The hydrophobic end of the surfactant stick into the droplets & the hydrophilic end is attracted to the water. The result is an emulsion, a fine suspension of oily droplets in water, which can now be rinsed away.
c. Effect of soaps & detergents – wash away microbes, but do not kill them.
d. Wetting agents are surfactants that are often used with other chemical agents to help the agent penetrate fatty substances. Surfactants are not germicidal by themselves!
e. Quaternary ammonium salts – four organic groups attached to a nitrogen atom. Effect: kill all classes of cellular microbes & enveloped viruses by disrupting membranes. Uses: nontoxic & widely used in the home, industry, labs, & hospitals. Their effectiveness is decreased in the presence of soap. Actually support Pseudomonas growth! Now being mixed with other agents to overcome some of these problems.
2. Phenol & Phenolics
a. Structure – compounds with hydroxyl groups (-0H) attached to a benzene ring.
Mode of Action – denature cell proteins, disrupt cell membranes.
b. Effect – kill most organisms; action is not impaired by organic materials (remain active even in the presence of blood, feces, etc.)
2.) Cresol – found in creosote; plant derivative used to prevent the rotting of wooden posts, fences, railroad ties.
3.) Hexachlorophene – chlorinated phenolic; effective as an antiseptic; once widely used as an ingredient in soaps & lotions; in 1970’s was found to increase risk of brain damage in babies; has now been replaced with chlorhexidine in hospitals – good agent for surgical scrubs.
a. Structure – compounds with a hydroxyl group (-OH).
b. Mode of Action – when mixed with water disrupt lipids in cell membranes & denature proteins.
c. Ethanol & Isopropanol – widely used as skin antiseptics; a 50 to 70% solution in water is the most effective concentration (one of the few exceptions to the rule: increase effectiveness by increasing concentration); does not sterilize skin because it evaporates quickly and does not penetrate deeply enough into skin pores.
d. Main disadvantage – do not kill endospores.
a. Mode of Action – inactivates enzymes by oxidation.
1.) Iodine – antiseptic
a.) Tincture – iodine in a dilute alcohol solution; one of first skin antiseptics.
b.) Iodophor – mixture of iodine and surfactants; ex. Betadine and Isodine (used for surgical scrubs and to prepare skin for surgery)
2.) Chlorine – disinfectant; ingredient in household bleach; added to drinking water and swimming pools; inactivated by the presence of organic materials.
5. Hydrogen peroxide
a. Mode of Action – oxidizing agent (denatures proteins)
b. Uses of H2O2: antiseptic for cleaning wounds, disinfect medical instruments & soft contact lenses. When H2O2 comes into contact with tissue, it bubbles producing oxygen gas. This is because all aerobes (incl. eukaryotes) produce the enzymes catalase & peroxidase which decompose H2O2 into oxygen & H2O. H2O2 generally kills microbes before it is destroyed by catalase or peroxidase. (You can differentiate between Staphylococcus & Streptococcus using H2O2; Staph is relatively resistant to H2O2 because of the large amounts of catalase & peroxidase it produces.) May be used to clean deep puncture wounds, because the oxygen produced kills obligate anaerobes present in the wound (ex. Clostridium).
6. Heavy Metals
a. Mode of Action – heavy metals (mercury, copper, silver) react with the sulfhydryl groups of proteins (denaturation)
b. Effect – kills many microbes.
1.) Mercuric chloride – once widely used as an antiseptic; highly toxic; now merthiolate & mercurochrome are used (less toxic); merthiolate is prepared as a tincture; use – basic first aid kit supplies for disinfecting skin & mucous membranes.
2.) Silver Nitrate – once applied to eyes of newborns to prevent gonorrhea; the trend for a while was toward using antibiotics instead, but the development of antibiotic-resistant strains has necessitated the use of silver nitrate again.
3.) Selenium sulfide – kills fungi, including spores; commonly used to treat fungal skin infections; included in dandruff shampoos (dandruff is often caused by a fungus).
7. Alkylating Agents
1. Mode of action – they alkylate (attach short chains of carbon atoms) to proteins and nucleic acids. Must not be used where they may effect human cells (these agents are carcinogenic).
2. Formalin – 37% solution. of formaldehyde used to preserve tissues & to embalm; kills all microbes, including spores; lower concentrations are used to inactivate microbes for killed vaccines.
3. Glutaraldehyde – used to sterilize surgical instruments if equipment for heat sterilization is not readily available.
4. Ethylene oxide – gas; advantages: disappears from the object after treatment; disadvantage: extremely toxic to humans so must be used in a sealed chamber; kills all bacteria, including endospores; used to sterilize materials destroyed by heat (plastic, rubber gloves, animal feed, mattresses, telephones).
8. Dyes – Ex. Crystal violet blocks cell wall synthesis. It effectively inhibits growth of G(+) bacteria in cultures and in skin infections. It can be used to treat yeast infections.
III. Food Preservation
A. Temperature – Environmental factor most often used to preserve food. Canning is the oldest method. Two factors, time & temp., determine safe heat treatments for canning. Refrigeration is low enough to stop the growth of most microbes. Psychrophilic (ex. Listeria) microbes are the exception. See pg. 1 of this handout for more info. on temp.
B. pH – Acidity (low pH) prevents the growth of most microbes, especially in an anaerobic environment. Ex. adding vinegar (acetic acid) to foods. Low pH also increases the effectiveness of heat treatments (ex. acidic foods like tomatoes can be canned merely by boiling).
C. Drying – drying & salting do not sterilize but preserve food by making it unable to support microbial growth for lack of water, an essential nutrient. See pg. 1 & 2 of this handout for more info. on drying & freeze drying.
D. Chemicals – Various chemical preservatives are added to commercially prepared foods. Ex.:
1. calcium propionate – antifungal agent added to bread.
2. sorbic acid – antifungal agent added to soft drinks, salad dressings, cheeses.
4. sodium benzoate – antifungal agent added to soft drinks, salad dressings, cheeses.
5. sodium nitrate (nitrite) – antibacterial agent that prevents germination of Clostridium botulinum spores when added to bacon, ham, hot dogs.