Chapter 7 Study Guide Control of Microbial Growth

Transcription

1 Chapter 7 Study Guide Control of Microbial Growth Note: you will not be tested on the following: use-dilution test. 1. Be able to define and use the following terms in context: sterilization, commercial sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bactericide, virucide, fungicide, bacteriostasis, sepsis, and asepsis. 2. Discuss the factors that influence the rate of microbial death. Be able to interpret a graph similar to that shown in Fig Discuss the 3 main ways in which microbial control agents kill or inhibit the growth of microbes. 4. Define Thermal Death Point, Thermal Death Time and Decimal Reduction Time. 5. Describe the application of the following physical methods of microbial control: heat, moist heat, autoclaving, pasteurization, dry heat sterilization, filtration, low temperature, high pressure, desiccation, osmotic pressure, radiation (ionizing, non-ionizing, and microwaves). Discuss the pros and cons of each method above. 6. Describe the factors related to effective disinfection, and the disk-diffusion method of evaluating a disinfectant. 7. Regarding the various types of disinfectants your book talks about, pay special attention to Table 8 ( Chemical agents used to control microbial growth ). You will need to know the following: a. The names of the chemical agents. b. The mechanism of action for each chemical. c. ONE preferred use of each chemical (don t worry about specific chemical names in the far right-hand side column). 8. Know the relative resistance of the major microbial groups to chemical biocides, and be able to explain why: a. Gram negative bacteria are generally more resistant than Gram positive bacteria b. Enveloped viruses are less resistant than non-enveloped viruses c. Endospores and prions are very resistant to chemical biocides.

2 Chapter 7 The Control of Microbial Growth I. Terminology of Microbial Growth. Table 1. a. Sterilization: Removal or destruction of all microbial life. i. Commonly done by heating, but filtration can sterilize liquids and gasses, also. b. Commercial sterilization. i. Concerned with killing Clostridium botulinum endospores in canned food, which produce fatal toxins. ii. Endospores of thermophiles survive; they do not grow at the temperatures at which canned foods are stored. 1. Incubation above ~45 C would result in significant food spoilage by these organisms in canned food. c. Disinfection. i. Normally refers to the destruction of vegetative pathogens that do not form endospores. ii. Can be accomplished by using chemicals (disinfectants), UV radiation, boiling water, or steam. d. Antisepsis. i. Removal of pathogens from living tissue. ii. Chemical used is called an antiseptic. e. Degerming. i. Mechanical removal of microbes from a limited area. ii. Generally does not kill microorganisms. f. Sanitization. i. Lowers microbial counts on eating utensils to safe public health levels. g. Biocide/Germicide. i. Kills microbes, but not usually endospores. ii. Bactericides kill bacteria. iii. Fungicides kill fungi. iv. Virucides inactivate viruses. h. Bacteriostasis. i. Inhibiting the growth of microbes without necessarily killing them. i. Sepsis refers to microbial contamination. j. Asepsis is the absence of significant contamination. k. Aseptic surgery techniques prevent microbial contamination of wounds. i. Lister, 1867, phenol (carbolic acid). II. Rate of Microbial Death. a. Bacterial populations die at a constant logarithmic rate when heated or treated with antimicrobial chemicals. Fig. 1, Table 2. b. Effectiveness of antimicrobial treatment depends on: i. Number of microbes present at the beginning of the treatment. ii. Environment (organic matter, temperature, biofilms). iii. Time of exposure. iv. Microbial characteristics. III. Actions of Microbial Control Agents. a. Alteration of membrane permeability. i. Damage to the plasma membrane s lipids or proteins can cause cellular contents to escape and interfere with normal metabolism. b. Damage to intracellular proteins. i. Denature proteins and enzymes by targeting hydrogen bonds or disulfide bridges. c. Damage to nucleic acids. i. Often kills the cell.

3 IV. Physical Methods of Microbial Control. a. Heat. i. Kills microorganisms by denaturing proteins and enzymes. ii. Thermal death point (TDP): Lowest temperature at which all cells in a liquid suspension are killed in 10 min. iii. Thermal death time (TDT): Minimum time required to kill all cells in a liquid at a given temperature. iv. Decimal reduction time (DRT = D value): Minutes to kill 90% of a population at a given temperature. v. Moist heat denatures (coagulates) proteins and enzymes. 1. Boiling liquid or flowing steam kills vegetative bacterial pathogens, most viruses, fungi and their spores within about 10 minutes; often much faster. a. Endospores and some viruses are not destroyed this quickly and some are able to survive for more than 20 hours in boiling water. 2. Autoclave: Steam under pressure. Fig. 2. a. Increasing pressure raises the boiling point of water. Table 3. b. Much higher temperatures can be achieved. c. Steam at a pressure of about 15 psi (121 C) will kill all organisms and their endospores in about 15 minutes. This is accomplished by denaturing proteins and enzymes. d. Sterilization of a surface requires that the steam directly contact it. vi. Pasteurization reduces the number of spoilage organisms and kills all pathogens. (Pasteur, 1857). 1. Mild heating that is sufficient to kill organisms that cause spoilage. This is accomplished by denaturing proteins and enzymes. 2. Thermoduric (heat-resistant) organisms survive. They are unlikely to cause disease or spoilage, however. 3. Equivalent treatments for milk pasteurization: a. 63 C for 30 min. b. High-temperature short-time (HTST) pasteurization: 72 C for 15 seconds. Note: Milk can also be sterilized using Ultra-high-temperature (UHT) treatment: 140 C for <1 second. Sterilized milk can be stored without refrigeration. vii. Dry heat sterilization kills by oxidation. 1. Flaming and incineration. 2. Hot-air sterilization. a. Items placed in an oven at ~170 C for 2 hours. b. Filtration removes microbes from liquids and gasses. Fig. 4. i. Pores in the filter allow the passage of the medium, but not the microbe. ii. Useful for sterilizing liquids that would be altered by heating. c. Low temperature inhibits microbial growth. i. Refrigeration (0-7 C). 1. Slows the metabolic rate of most microbes so that they cannot reproduce or synthesize toxins. 2. Psychrotrophs reproducing at a rate of three times per day will reach a population of more than 2 million within a week. ii. Slow freezing (in your refrigerator s freezer). 1. Allows ice crystals to form that can kill some species of bacteria. d. High pressure. i. When applied to a liquid, the pressure is transferred evenly throughout the sample.

4 ii. High pressure denatures proteins and alters carbohydrate structure, killing vegetative bacterial cells. iii. Used to preserve the color, flavor, and nutrient value of juices. iv. Endospores are relatively resistant to high pressure. e. Desiccation prevents metabolism. i. Cells can still remain viable for years. ii. Resistance to desiccation varies by species. f. Osmotic pressure causes plasmolysis. i. Plasma membrane pulls away from the cell wall and crenates. g. Radiation. Fig. 5. i. Ionizing radiation (X rays, gamma rays, electron beams) destroys DNA. 1. Short wavelengths. 2. Ionizes water, producing hydroxyl radicals that react with organic molecules, especially DNA. 3. Used on food, pharmaceuticals, and medical supplies. ii. Nonionizing radiation (UV) damages DNA. 1. Causes bonds to form between adjacent pyrimidine bases, usually thymines, in DNA chains. (Fig. 20, pg. 244, chpt. 8). a. These thymine dimmers inhibit correct replication of DNA during cellular reproduction. iii. Microwaves kill by heat; not especially antimicrobial. h. Table 5 summarizes the physical methods of microbial control. V. Chemical Methods of Microbial Control. a. Principles of effective disinfection. i. Concentration of disinfectant. ii. Organic matter. iii. ph. iv. Time. b. Evaluating a disinfectant. i. Use-dilution test. [NOT EXAMINABLE] 1. Metal rings dipped in test bacteria are dried. 2. Dried cultures placed in disinfectant for 10 min at 20 C. 3. Rings transferred to culture media to determine whether bacteria survived treatment. ii. Disk-Diffusion Method. Fig Paper discs soaked with a chemical or antibiotic are placed on a culture of a previously inoculated and incubated test organism. c. Types of Disinfectants. i. Phenol and Phenolics. Fig Lister (1867) was the first to use phenol to control surgical infections. 2. Phenol and phenolics damage the lipids of the plasma membrane, allowing cellular contents to leak. They also denature proteins and enzymes. 3. They remain active in the presence of organic compounds, are stable, and persist for long periods after application. 4. Suitable for disinfecting pus, saliva, and feces. ii. Bisphenols. 1. Work by disrupting plasma membranes and are used in disinfectant hand soaps and skin lotions. iii. Biguanides. 1. Chlorhexidine. 2. Disrupts plasma membranes, and is used to disinfect skin, especially in preparation for surgery.

5 iv. Halogens. 1. Iodine (I 2 ) impairs protein synthesis and is a strong oxidizing agent. a. Available as a tincture (in solution with aqueous alcohol) and as an iodophor (combined with an organic molecule, from which iodine is released slowly). 2. Chlorine (Cl 2 ) is widely used, as a gas or in combination with other chemicals. Its germicidal action is due hypochlorous acid (HOCl), a strong oxidizing agent that disrupts cellular enzyme functioning. a. Chlorine gas is used to disinfect water. b. Chlorine compounds are used to disinfect a variety of surfaces. c. Sodium hypochlorite = bleach d. Chloroamines - consist of chlorine and ammonia i. used as disinfectants, antiseptics and sanitizing agents. 3. Iodine and chlorine are effective antimicrobial agents, either alone or as components of other compounds. v. Alcohols. 1. Kill bacteria and fungi, but not endospores or non-enveloped viruses. 2. Act by denaturing proteins and enzymes, and by dissolving lipids (plasma membrane, viral envelope). 3. Ethanol at 100% is less effective than at many lower concentrations (Table 6) vi. Heavy Metals. Fig I.e.: Ag, Hg, Cu. 2. Can be biocidal or antiseptic. 3. Denature proteins and enzymes by combining with sulfhydryl groups. vii. Surface-Active Agents or Surfactants. 1. Soaps. a. Emulsifies oils and are used to degerm surfaces (like the skin) by floating off contaminants with a solvent (water). 2. Acid-anionic detergents. a. Uncertain mechanism of action; thought to involve enzyme inactivation or disruption. Used as a sanitizer in the dairy and food processing industries. 3. Quaternary Ammonium Compounds (Quats). Fig. 9. a. Disrupt plasma membranes and denatures enzymes and proteins. Used as an antiseptic for skin, instruments, utensils, and rubber goods. viii. Chemical Food Preservatives. 1. Added to food to retard spoilage; inhibits metabolism (of molds, mostly). 2. Organic acids or salts of organic acids that the body can metabolize and are judged to be safe in food. ix. Antibiotics. 1. Injected or ingested to treat disease. x. Aldehydes. 1. Inactivate proteins and enzymes by cross-linking with functional groups such as NH 2, OH, COOH, SH. 2. Glutaraldehyde, formaldehyde. xi. Gaseous chemosterilizers. 1. Denature proteins and enzymes. 2. An object is placed in a chamber and the air is replaced with a gas like ethylene oxide. 3. Good for sterilizing objects that would be damaged by heat or liquids (mattresses, medical supplies, spacecraft electronics). xii. Peroxygens (Oxidizing agents).

6 1. Ozone (O 3 ) hydrogen peroxide (H 2 O 2 ) and peracetic acid. 2. Oxidize cellular molecules. d. Fig. 10 compares the effectiveness of various antiseptics. e. Table 8 summarizes the chemical agents used to control microbial growth. VI. Microbial Characteristics and Microbial Control. Fig. 11 and Table 7. a. Viruses. i. Enveloped viruses have an outer lipid envelope. ii. Antimicrobials that are lipid soluble are more likely to be effective against enveloped viruses. iii. Non-enveloped viruses, which have only a protein coat, are more resistant; fewer biocides are active against them. b. Prions. i. Infectious proteins. ii. Cause spongiform encephalopathies in animals, humans c. Fig. 11 shows the relative resistance of various microorganisms to chemical biocides.