Lecture Summary Microbial Control of Growth (CH5) This chapter covers the processes available to control microbial growth. These processes are divided into two groups, physical and chemical methods of control. In my lecture notes I begin this discussion with a look at some definitions of terms, terms that we will use again when we discuss antibiotics. Let s start with these terms.the definitions of the terms bacteriostatic and bactericidal will be important in our discussion of the methods of control. Bacteriostatic refers to something that inhibits bacterial growth while the term bactericidal refers to something that kills bacterial cells. And then there are the terms antiseptic and disinfectant which are important in this chapter. A chemical known as an antiseptic is one that is used on skin and mucous membranes, while a disinfectant is used on inanimate surfaces. The history behind microbial control is interesting and you should be familiar with the contributions of Ignatz Semmelweis and Joseph Lister to this topic. Before we look at specific methods of microbial control it is important to review the characteristics of organisms that would make them more resistant/more sensitive to treatments. In other words, are all organisms equally sensitive??? And the answer to this is No! Some organisms are more resistant due to features like endospores, others are more resistant because of the cell wall composition (Mycobacteria), cysts of protozoa are more resistant than the vegetative forms (Giardia lamblia), and gram negative are more resistant than gram positive. Perhaps the most interesting is that viruses without envelopes are more resistant than viruses with envelopes. In addition to evaluating the type of organism you are dealing with, you need to consider the environment that you want to treat or decontaminate. For instance, you would choose to use a different method of treatment to sterilize nutrient agar media than to sterilize a heat sensitive solution. Review section 5.2 in the text for more information on things to consider when selecting an antimicrobial procedure. The physical methods of control are summarized on p. 112 of the text in Table 5.1 and include heat, filtration, radiation, and pressure. Heat is the most useful method of microbial control because it is easy to use, doesn t introduce chemicals into the material being processed, and is inexpensive. There are two categories of heat that can be used to control microbial growth, dry heat and moist heat. Examples of dry heat include incineration (flaming the loop in lab) that works by burning all of the cell components to ashes, and using an oven at a temperature of 170 o C for hours to sterilize that works by destroying cell components and denaturing proteins. Examples of moist heat include boiling, pasteurization, and using steam under pressure in an autoclave and work by denaturing proteins. Heated water molecules help to denature proteins by bombarding the protein structure and disrupting the hydrogen bonds that hold it together. Of these three types of moist heat, only the autoclave is able to sterilize. When material is boiled it will kill vegetative bacteria and viruses but NOT endospores. Recall that endospores have a dehydrated core, and the surrounding cortex keeps water from gaining entry. The absence of water in the endospore core keeps moist heat from denaturing core proteins because there is no water to help denature them. Pasteurization is a process of using heat to reduce the number of viable cells in a solution, it is not a method to sterilize. There are different types of pasteurization, HTST used to pasteurize milk and UHT used to
pasteurize the containers of creamer used in restaurants. How do these two types of pasteurization differ? Review section 5.3 in the text and note how you determine if a product has been properly processed in the autoclave. What is the benefit of using a biological indicator instead of a chemical indicator? Any solutions that need to be sterilized but are heat sensitive can be filter sterilized. Membrane filters of.45um and.22um are the size most often used. There are filtration units used for backpackers that use special membranes to trap any bacteria or protozoa that are present in water so that it is safe to drink. The use of radiation is another way to control/kill organisms. Ionizing radiation (gamma rays, x-rays) is most often used to sterilize heat sensitive medical equipment, drugs, and foods such as spices, meat, fruit, and vegetables, and works by destroying DNA, damaging cytoplasmic membranes, and producing reactive molecules that go on to damage other molecules. Non-ionizing radiation or UV radiation is used to destroy microbes in the air, in drinking water, or to disinfect surfaces and works by damaging DNA, but has poor penetration thru materials. Actively growing microbes are most sensitive to UV light and endospores are the most resistant. Can you think of a clinical application of UV light in a hospital setting? The use of high pressure is another method to kill organisms present in products such as food. The application of high pressure does not change the food color or flavor, while the treatment damages the proteins and alters the permeability of the cell. The second part of the chapter focuses on chemicals used to control microbial growth. These chemicals, also called germicides, are categorized into the following groups according to their potency. 1. Sterilants (scalpels) -destroy all microbes including endospores and viruses -destruction of endospores requires 6-10 hour treatment 2. High-level disinfectants (GI endoscopes) -destroy all viruses and vegetative microbes, do NOT reliably kill endospores 3. Intermediate level disinfectants (stethoscopes) -destroy all vegetative bacteria, including Mycobacteria -destroy fungi and most viruses 4. Low level disinfectants -destroy fungi and vegetative bacteria EXCEPT Mycobacteria, enveloped viruses -do not kill endospores or naked viruses What to consider when choosing a chemical: 1. toxicity 2. activity in the presence of organic matter 3. compatibility with material being treated 4. residue 5. cost and availability 6. storage and stability 7. environmental risk
Classes of germicides I. Alcohols The mechanism of action for this group is protein denaturation, and the cell membrane or lipids in the cell can be denatured by alcohols. You typically only use this for intact skin, to sterilize the skin before an injection. Also need to have a portion of water in the mixture, so 70% solutions are ideal to promote the protein denaturation, etc. Target: 60-80% ethyl or isopropyl alcohol kill vegetative cells and fungi, NOT bacterial endospores and some naked viruses Action: acts by coagulating proteins, damaging lipid membranes Applications: used to sterilize the skin before injections. Treat instruments and surfaces tinctures: antimicrobial chemicals dissolved in alcohols, tincture of iodine application: used to prep skin before drawing blood or placing a IV line study done (2004) comparing the use of chlorhexidine tincture vs. tincture of iodine to prep skin before obtaining blood cultures found that the rate of contamination was 0.41% greater for the chlorhexidine prep skin (3.1%) vs tincture of iodine (2.7%) II Biguanides The one chemical that is in this group that I think is important is chlorhexidine. This is something that you will find in many antiseptic products. It is a compound that has a low toxicity, destroys a wide range or bugs, and adheres to the skin and mucous membranes. It is a chemical that is used in mouthwashes, and has even been added to products such as catheters. Application: Used in antiseptic type products (skin creams, disinfectants and mouth washes), found implanted in surgical mesh and catheters Action: Membrane disruption
III Halogens (Chlorine and Iodine) This group of compounds are found as common disinfectants (and in some cases antiseptics) that are thought to act by oxidizing proteins and other essential cell components. Chlorine is not used an as antiseptic but as a disinfectant. For example, sodium hypochlorite (bleach) is used to disinfect wastewater (500ppm), pools, and disinfect drinking water (0.5ppm) Action of chlorine: strong oxidizing agent; Chlorine can react with organics generating trihalomethanes which are potential carcinogens; Chlorine dioxide gas is a strong oxidizing agent that is used as a disinfectant and sterilant. Target: all types of microbes and viruses Iodine is used as an antiseptic found as an iodophore or tincture of iodine Iodophores: Betadine, Clinidine, Isodine Action of iodine: binds to the tyrosine residues in proteins damaging their structure; also alters cell membranes Target: effective against all bacteria, fungi, most endospores, and some viruses IV Phenolic Compounds Carbolic acid was the original form used. Now used as phenolics (derivatives of phenol) which have greater germicidal activity. Action: destroy cytoplasmic membranes, denature proteins; some are less-toxic: Examples: Triclosan and Hexachlorophene Hexachlorophene: has high activity against Gram-positive S. aureus. Triclosan: action is found to inhibit an enzyme needed for fatty acid synthesis Target: Kill vegetative cells and Mycobacterium tuberculosis, but NOT all groups of viruses Benefits to this group: wide range of activity, reasonable cost, and remain effective in presence of organic material, leave an antimicrobial residue
V. Quarternary Ammonium Compounds (Quats) This is a group of positively charged detergents that are non-toxic enough to be used as disinfectants on food surfaces. Since these compounds are positively charged (due to the ammonium ion), they react with membranes such as cell membranes, which are negatively charged. They do not kill endospores, Mycobacteria, or naked viruses. This class of compounds is also used for products such as mouthwashes, benzalkonium chloride or cetylpyridinium chloride are examples of quats. If you shake your mouthwash and it foams, there is a quat in it! VI. Heavy metals Most kill by binding to the sulfhydryl groups of proteins and interfere with their function. Metals such as silver is used in the form silver sulfadiazine for dressings in burn patients. Another form of silver is 1% silver nitrate which was used in the eyes of newborns to prevent eye infections from Neisseria gonorrhoeae, which can cause blindness. Now an antibiotic solution is used instead for newborns. Copper is used to keep down algal growth in fish tanks as copper sulfate. And mercury is found in paint products to reduce mold growth. VII. Soaps Regular soap just works to remove the organisms by reducing the surface tension. This is a very effective way to remove organisms that you have on hands, etc. Fancy antibacterial soaps are nice for use in the bathroom and kitchen area but we should really think twice about using them in the shower all over the skin. Remember, you need those healthy normal microbiota!!