Controlling Microbial Growth In Vitro — TE Microbial Growth Availability of Nutrients Moisture ‘Temperature pH Osmotic Pressure and Salinity Barometric Pressure Gaseous Atmosphere Part 2: Encouraging the Growth of Microbes In Vitro Culturing Bacteria in the Laboratory Bacterial Growth Cuture Media Inoculation of Culture Media Importance of Using “Aseptic Technique Incubation Bacterial Population Counts Bacterial Population Growth Curve Culturing Viruses and Other Obligate Intracellular Pathogens in the Laboratory Culturing Fung! in the Laboratory CCulturing Protozoa in the Laboratory Part 3: Inhibiting the Growth of Microbes In Vitro Definition of Terms Steriization Disinfection, Pasteurization, Disinfectants, Antiseptics, and Sanitization Microbicidal Agents Microbistatic Agents Bacterial colonies on various types of solid culture media. Sepsis, Asepsis, Aseptic Technique, Antisepsis, and Antiseptic Technique ‘Sterile Technique Using Physical Methods to Inhibit Microbial Growth Heat Cold Desiccation Radiation Uttrasonic Waves Fittration Gaseous Atmosphere Using Chemical Agents to Inhibit Microbial Growth Disinfectants Antiseptics Controversies Relating to the Use of Antimicrobial Agents in Animal Feed and Household Products 129 Scanned with CamScannerTE een ‘After studying this chapter, you should he oie a Name three ee a eater * Ust sveal coe that affect the eae i tare ‘sites where microbial grey stg Soe eaten bameen toe en oe . tale Se casa ohare microbial growth is «_ Safarentiate between bactericidal and bactergg, + Bitorentato among enriched, cote. ooh ; Sonn tno processes of pasteurization ang diterotal media, and cto two eA ET ig Seal physical methods used to inhibit the + Explain the importance of using “asepti microbiology laboratot «Deserve tho treo types ‘of incubators that are used in the microbiology laboratory + Draw a bacterial growth curve an phases + Cite two reasons why bacteria die durin phase d label its four Wg the death INTRODUCTION In certain locations, such as within microbiology laborato- ries, the growth' of microbes is encouraged; in other words, scientists ant them to grow. In other locations—such as on hospital wards, in intensive care units, in operating rooms, in kitchens, bathrooms, and restaurants—itis necessary or desirable to inbibie the growth of microbes. Both concepts, encouraging and inhibiting the in vitro growth of microbes, are discussed in this chapter. (Recall from Chapter 1 that, as, ‘used in this book, in vitro refers to events that occur outside the body, whereas in vivo refers to events that occur inside the body.) Before discussing these concepts, however, various factors that affect the growth of microbes are examined. PART 1: FACTORS THAT AFFECT MICROBIAL GROWTH Microbial growth is affected di i mental acorn incleding the salabibey fener moisture, temperature, pH, osmotic pressure, barometric pressure, and composition ofthe atmosphere. These envi. ronmental factors affect microorganisms in our daily lin 7 and play important roles inthe control of microorganisms in laboratory, industrial, and hospital settings. Whe ther scents wish to encourage or inhibi the growth ofmi croorganisms, they must first: i eroorganis hey understand the fundamental “The word “growth” is used in this chay 7 ‘or multiplication. ipter to mean proliferation growth of microorganisms «ote three ways in which disinfectants kil microorga. «eee tity several factors that can influence he ne effectiveness of disinfectants «Explain briefly why the use of antibiotics in animal gg, and household products is controversial Availability of Nutrients ‘As discussed in Chapter 7, all living organisms requiy utrients—the various chemical compounds that orgies use to sustain life. Therefore, to survive in a particulares. vironment, appropriate nutrients must be available, Maxy nutrients are energy sources; organisms will obtain ene from these chemicals by breaking chemical bonds. Ni trients also serve as sources of carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur as well as other elements (eg. sodium, potassium, chlorine, magnesium, cicua, and trace elements such as iron, iodine, and zinc) thatae usually required in lesser amounts. About two dozen of the approximately 92 naturally occurring elements essential to life. Moisture On Earth, water is essential for life, as we know it. consist of anywhere between 70% and 95% water: ving organisms require water to carry out their normal mea! Processes, and most will die in environments cont too little moisture. There are certain microbial stages" bacterial endospores and protozoan Water is essential 23%) however, that can survive sae | ication) The eying process (des: | Coig are como Within ee’ organisms contained 770% 1095% | remiorone {ore dormant or resting state; if they "Sends con ona ntinue to debate the actual number of ME jes Between 9 sna but most would agree that the ™ Scanned with CamScannerJaced in a moist, nutrient-rich enviro: i frow and reproduce normally aera Temperature very microorganism has an optimum growth temperature— the temperature at which the organism grows best. Every microorganism also has a minimum growth temperature below which it ceases to grow, and a maximum growth temperature, above which it dies. The temperature range (.e,the range of temperatures from the minimum growth temperature to the maximum growth temperature) at which an organism grows can differ greatly from one microbe to another. Toa large extent, the temperature and pH ranges cover which an organism grows best are determined by the enzymes present within the organism. As discussed in Chapter 7, enzymes have optimum temperature and pH ranges at which they operate at peak efficiency. Ifan organism's enzymes are operating at peak efficiency, the organism will be metabolizing and growing at its maximum rate, ‘Table 8-1 contains information regarding temperatures at which various organisms live in bodies of water at Yellowstone National Park. ‘Microorganisms that grow best at high temperatures ate called thermophiles (.e., organisms that love heat). ‘Thermophiles can be found in hot springs, compost pits, and silage as well as in and near hydrothermal vents at the bottom of the ocean (check thePoint for “A Closer Look at Hydrothermal Vents"). Thermophilic cyanobacteria, certain other types of bacteria, and algae cause many of the colors observed in the near-boiling hot springs found in Yellowstone National Park (Fig. 8-1). Organisms that favor tempera- tures above 100°C are referred to as hyperthermophiles (or extreme thermophiles). The highest temperature at which a Every microorgan- ism has an optimal, aminimum, and a ‘maximum growth ‘temperature. Thermophiles are or- : vdlivingis isms that "lover | bacterium has been found living oh terpenes” | around 113°C; iewas an archaeon - named Pyrolobus fumarii. (oo: Rees Poin ect Romer oir) EXCISE tar i ene ert Temperature 188° (73°) or ower TSE 62°) or tower 40°F (60°) o omer pad (56°0) or tower ©) or lower 20°F 27°C) or ower Chapter 8 + Controling Microbial Growth In Vitro. 134 Figure 8-1, Colortul thermophiles living In geothermal {feature at Yellowstone National Park, WY. (Provided by Biomed Ed, Round Rock, TX) Microbes that grow best at moderate temperatures are called mesophiles. This group includes most of the species that grow on plants and animals and in warm soil and water. ‘Most pathogens and members of the indigenous microbi- ota are mesophilic because they grow best at normal body temperature (37°C). Psychrophiles prefer cold temperatures. They thrive in cold ocean water. At high altitudes, algae (often pink) can be seen living on snow. Biologists studying microbial life in the Antarctic have reported finding bacteria in a lake that hhas been iced over for atleast 2,000 years.* These microbes thrive in an environment that is —13°C, has 20% salinit and contains high concentrations of ammonia and sulfur. Ironically, the optimum growth temperature of one ‘group of psychrophiles (called psychrotrophs) is refrigerator temperature (4°C); perhaps you encountered some of these microbes (¢g., bread moulds) the last time you cleaned out your refrigerator. Microorganisms that prefer warmer temperatures, but can tolerate or endure very cold temperatures and can be preserved in the frozen state, are known as psychroduric organisms. Refer to Table 8-2 for the temperature ranges of psychrophilic, mesophilic, and thermophilic bacteria. pH ‘The term “pH” refers to the hydrogen ion concentration of ‘a solution and thus the acidity or alkalinity of the solution (ee Appendix 3 on thePoint: “Basic Chemistry Concepts”). ‘Most microorganisms prefer a neutral or slightly alkaline ‘medium (pH 7.0-7.4), but acidpbilic microbes (aci- dophiles), such as those that can live in the human stomach, ‘and in pickled foods, prefer a pH of 2 to 5. Fungi prefer acidic environments. Acidophiles thrive in highly acidic environments, such as those created by the production of Seww-pnas.org/cgi/doi/10.1073/pnas.1208607109 Scanned with CamScannerjicrobes: 432 Section IV + Controlling the Growth of Mi Table 8-2 $5 Maximum Growth = mn optimum Graney Temperature (°C) imum Grow perature Temporawree) 113 Category 22 11 = 25 2 bs Maso Ne 10-20 Payehropites = ° the cell to swell. If sufficient ; thin the cell causes ae is \d hot springs as Wi ill burst (lyse). In the case of sulfrous gases in hydrothermal ven 36 1! ples ners the Cel WT molysis fa bacterial els peed Sine debes produced from coal mining. Allaiphiles °° 5 called Fabactes refer an alkaline environment (pH > 8.5), such 35 this partonic solution (such a8 distilled water), the el Pp found inde tie — oe may not burst (because of the gid eval but the fui 73), insoil aden wit ath increases greatly. This i fccophies peter | Zo''n he sovaled soda lakes. pressure within the increased acicic environments, | Ybris cholerae—the bacterium that causes cholera—is the only human pathogen that grows well above pHi 8. whereas alkaliphiles prefer alkaline environments. Osmotic Pressure and Salinity Osmotic pressure is the pressure exerted on a cell mem- brane by solutions both inside and outside the cell. When cells are suspended in a solution, the ideal situation is that the pressure inside the cell is equal to the pressure of the solution outside the cell. Substances dissolved in liquids are referred to as solutes. When the concentration of solutes in the environment outside a cell is greater than the concentration of solutes inside the cell, the solution in which the cell is suspended is said to be hypertonic. In such a situation, whenever possible, water leaves the cell by osmosis in an attempt to equalize the two concentrations. Osmosis is defined as the movement ofa solvent (eg., water), through a permeable membrane, from a solution having a lower concentration of solute toa solution having a higher concentration of solute. Ifthe cell is a human cell, such asa red blood cell (erythrocyte), the loss of water causes the cell to shrink; this shrinkage is called erenation, and the cell is said to be crenated (Fig. 8-2). If the cell isa bacterial cell, having a rigid cell wall, the cell does not shrink. Instead, the cell membrane and cytoplasm shrink away from che cell wall. This condition, known as plasmolysis, inhik bacterial cell growth and multiplication. Salts and ements are added to certain foods e's ‘way of preserving them. Bacte- ria that enter such hypertonic environments will die as a result in which the cell is suspended is sad sucha situation, whenever possible, water: i an attempt to equalize the two concenaige ee eelin a human cell, sch as an he ace thecelis water pressure occursin cells having rigid Pratl, such as plant cells and Cells swell up, and bacteria. If the pressure becomes sometimes burst so great that the cell ruptures, the | when placed into a escape of cytoplasm from the cell | hypotonic solution js referred to as plasmoptysis. ‘When the concentration of solutes outside a cell equals the concentration of solutes inside the cell, the solution is said to be isotonic. In an isotonic environment, exces water neither leaves nor enters the cell, and thus, no plasmolysis or plasmoptysis occurs; the cell has normal turgor (fullness). Refer to Figure 8-3 for a comparison of the effects of various solution concentrations on bacter cells and red blood cells. Sugar solutions for jellies and pickling brines (st solutions) for meats preserve these foods by inhibiting the ie ge : * eo; . ees, © e*y ° } é ¢@ ® e- a b 2 aor eo. : Figure 8-2. = Stained peripheral blood smear showing ‘88 acanthocyt red blood cells, which are also known, number of ceh ee? cenated red Blood calls have dove? or ede Sopearanse actos thus giving the calls =e Scanthocytes ear roe, Acanthocytosis—the formation Giseaso be indicative ofa numberof homstO9,« Rt api larger purple-stained cell in the oO" is a white bi by 2am to ite Blood al. Provided by 25 Scanned with CamScannerIsotonle solution Red blood call —!. = PL mca a Hypotonle solution Hypertonic solution mm qj a Hhemotysist kh Chapter 8 + Controlling Microbial Growth In Vitro 133 Piezophiles, capable of living in the deepest parts of the ‘ocean cs thePoint.for “A Closer Look at Barometric sure”). Gaseous Atmosphere As discussed in Chapter 4, microorganisms vary with respect to the type of gaseous atmosphere that they require. For example, some microbes (obligate aerobes) prefer the same atmosphere that humans do (i.e., about 20%-21% oxygen and 78%-79% nitrogen, with all other atmospheric gases ‘combined representing <1%). Although microaerophiles also require oxygen, they require reduced concentrations of oxygen (around 5%). Obligate anaerobes are killed by the presence of oxygen. Thus, in nature, the types and ‘concentrations of gases present in a particular environment determine which species of microbes are able to live there. ‘To grow a particular microorganism in the laboratory, itis necessary to provide the atmosphere that it requires. For example, to obtain maximum growth in the laboratory, capnophiles require increased concentrations of carbon dioxide (usually from 5% to 10%). Figure 8-3. Effects of changes in osmotic pressure. No change in pressure occurs within the cell in an isotonic solution internal pressure is increased in a hypotonic solution, resulting in swelling of the cell. Internal pressure 's decreased in a hypertonic solution, resulting in shrinking of the cal. (Arows indicate the direction of water flow. ‘The larger the arrow, the greater the amount of water lowing In that direction) ‘growth of most microorganisms. However, some types of ‘moulds and bacteria can survive and even grow in a salty ‘environment, ‘Those microbes that actually prefer salty environments (uch s the concentrated salt water found in the Great Sale Lake and solar salt evaporation ponds) are called halophilic prganisms; halo referring to “salt” and philic meaning “to [e¥e” Microbes that live in the ocean, such as V cholerae (pentioned earlier) and other Vibrio species, are halophilic. atatisms that do not prefer to live in salty environments are capable of surviving there (such as Staphylococcus ‘ureus) are referred to as haloduric organisms. Barometric Pressure Most bacteria are not affected by minor changes in baromet~ “Pressure. Some thrive at normal atmospheric pressure (about 14.7 psi). Others, known as piezophiles, thrive deep in the ocean and in oil wells, where the atmospheric pressure is very high. Some archaea, for example, are Microor ganisms that Preto salty envi- fonments are call halophies, et a STUDY AID ~ -Phile SN ‘The suffix -phile means to love wr ‘something. For example, acidophiles & are organisms that love acidic conditions; therefore, they live in acidic environments. Alkaliphiles live in alkaline environments. Halophiles live in salty environments. Piezophiles (formerly called barophiles) live in envi- ronments where there is high barometric pressure, ‘such as at the bottom of the ocean. Thermophiles prefer hot temperatures. Mesophiles prefer moderate ‘temperatures. Psychrophiles prefer cold tempera- tures. Microaerophiles live In environments containing reduced concentrations of oxygen (around 5% ‘oxygen). Capnophiles grow best in environments rich in carbon dioxide. PART 2: ENCOURAGING THE GROWTH OF MICROBES IN VITRO ‘There are many reasons why the growth of microbes is encouraged in microbiology laboratories. For example, technologists and technicians who work in clinical microbi- ology laboratories must be able to isolate microorganisms from clinical specimens and grow them on culture media so that they can then gather information that will enable identification of any pathogens that are present. In mi- crobiology research laboratories, scientists must culture Scanned with CamScannerial agents; rured in gen earories of certain food well as other industries: Many dierent eso an ey oe : in vito, a pai i placed OP oy rill Euleingothertypesof microbes ism tory cutturing Bacteria in the Laborat see In many ways, moder microbiology borat se aontes {hove of 50, 100,or even 150 years ago, Todays ANT gin tl use many of the same basic tools that an the pas-For example, microbiologists stil use Fong light microscopes, Pet dishes containing sold eoNTT sedia, cubes containing liquid culture media, wire of plastic inoculating loops, bottles of staining reagen's Incubators. However, a closer inspection will reveal many modern, commercially available products and instruments that would have been inconceivable in the days of Louis Pasteur and Robert Koch. Bacterial Growth fer ‘With respect to humans, the term growth refers to an increase in size, for example, going from a tiny newborn baby toa large adult. Although bacteria do increase in size before cell division, bacterial growth refers to an increase in the mumber of organisms rather than an increase in their size. Thus, with respect to bacteria, growth refers to their proliferation or multiplication. When each bacterial cell eaches its optimum size, it divides by binary fasion (bi ‘meaning “rwo" into two daughter cells (.e, each bacterium ‘imply splits in halfto become two identical eel). (Resa HISTOR| A Nowe OAL Culturing Bacte, Laboratory rain The earliest successiy ay tempts to culture mic, isms in a laboratory ser® were made by Ferdnang? Cohn (1872), Joseph So, eter (1875), and Oscar Brefeld (1875). Robert koe, described his culture techniques in 1881. int, Koch used slices of boiled potatoes on which i, ie ja, but he later developed both liquid ang solid forms of artificial media. Gelatin was init used as a solidifying agent In Koch's culture madi, but in 1882, Fanny Hesse, the wife of Dr. Walther Hesse—one of Koch's assistants—suggested the, of agar. Frau Hesse (as she is most commonly caes had been using agar in her kitchen for many yeanas_| a solidifying agent in fruit and vegetable jelies, An. other Koch's assistant, Richard Julius Petri, invent glass Petri dishes in 1887 for use as containers fo solid culture media and bacterial cultures. The Peri es in use today are virtually unchanged from tte design, except that most of today’s labora: ries use plastic, presterilized, disposable Petr dita In 1878, Joseph Lister became the first person to obtain a pure culture of a bacterium (Streptococaus lactis) in a liquid medium. As a resutt of their abilty to obtain pure bacterial cultures in their laboratorss | Louis Pasteur and Robert Koch made significantoot tributions to the germ theory of disease. the abt? allow and ende wh cengent® are depleted or the prescettion of cellular waste snot Saphir loco ert The tne in Figure 2-1), $ Aion eatetS2 One cel to become wocdst ey © Beneration time. The gene”™ bacteria. Scanned with CamScannervaries from one bacterial speci to another, In the laboratory, under ideal growth conditions Escherichia coli, V. cholerae, Staph. lococcus spp., and Streptocoreus ‘pp. all have a generation time of about 20 minutes, whereas some Becteria multiply pybinary fission. ‘The time taken by ‘a particular bacte- fil species to un- ergo binary fission iscalled the gen- eration time of that | Pseudomonas and Clastridivam spp. cate’ may divide every 10 minutes, and ‘tuberculosis may divide every 18 to 24 hours. Bacteria with short generation Soa sare refered 0 as rapid growers, whereas hese oh Jong generation times as slow growers. “The growth of microorganisms in the body, in nature, or in the laboratory is greatly influenced by temperature, pH, ‘moisture content, available nutrients, and the sti ofother organisms present. Therefore, the number of bac teria in nature fluctuates unpredictably because these factors vary with the seasons, rainfall, temperature, and time of day. In the laboratory, however, a pure culture of a single of bacteria can usually be maintained if the appro- priate growth medium and environmental conditions are provided. The temperature, pH, and proper atmosphere are quite easily controlled to provide the optimum conditions {for growth. Appropriate nutrients must be provided in the growth medium, including an appropriate energy and carbon source, Some bacteria, described as being fastidious, have complex nutritional requirements. Often, special mixtures ofvitamins and amino acids must be added to the medium to culture these fastidious organisms. Some organisms vill not grow at all on artificial culture media, including obligate intracellular pathogens, such as viruses, rickett- sis, and chlamydias. To propagate obligate intracellular Pathogens in the laboratory, they must be inoculated into live animals, embryonated chicken eggs, or cell cultures. Other microorganisms that will not grow on artificial media include Treponema pallidum (the bacterium that causes syphilis) and Mycobacterium leprae (the bacterium that causes leprosy). Microorganisms that are difficult to grow in the laboratory are Said to be fastidious. Culture Media media (sing., medium) that are used in microbiology ratories to culture bacteria are referred to as artificial i or synthetic media because they do not occur nat- wally; rather, they are prepared in the laboratory. There Xe a number of ways of categorizing the media that are =a to culture bacteria. fedien ine way to classify culture media is based on w! ogee contents ofthe rneda are mown A chemically knon®d medium is one in which all the ingredients +e liber this is because the medium was prepared in the the tery by adding a certain number of grams of each o tteeo g., carbohydrates, amino acids, and salts) “omplex medium is one in which the exact contents Chapter 8 + Controling Microbial Growth In Vitro 135 r | 8 Figure 8-5. Examples of solid and liquid cutture media used In a clinical microbiology laboratory. (Provided by Dr. Robert Fader and Biomed Ed, Round Rock, TX) are not known. Complex media contain ground-up or di- gested extracts from animal organs (eg., hearts, livers, and brains), fish, yeasts, and plants, which provide the necessary nutrients, vitamins, and minerals. Culture media can also be categorized as liquid or solid (Fig. 8-5). Liquid media (also known as broths) are contained in tubes and are thus often referred to as tubed media. Solid media are prepared by adding agar to liquid media and then pouring the media into tubes or Petri dishes, where the media solidifies. Bacteria are then grown on the surface of the agar-containing solid media. Agar isa complex polysaccharide thatis obtained from a red marine alga; itis used as a solidifying agent, much like gelatin is used as a solidifying agent in the kitchen. ‘An enriched medium is a broth or solid medium containing a rich supply of special nutrients that promotes the growth of fastidious organisms. Icis usually prepared by adding extra nutrients to a medium called nutrient agar. Blood agar (nutrient agar plus 5% sheep red blood cells) and chocolate agar (nutrient agar plus powdered hemoglobin) are examples of solid enriched media that are used routinely in the clinical bacteriology laboratory. Blood agar is bright red, whereas chocolate agar is brown (the color of chocolate). Although both of these media contain hemoglobin, chocolate agar is considered to be more enriched than blood agar because the hemoglobin is more readily accessible in chocolate agar. Chocolate agar is used to culture important, fastidious, bacterial pathogens, such as Neisseria gonorrboeae and Haemophilus influenzae, which will not grow on blood agar. ‘Asclective medium has added inhibitors that discourage the growth of certain organisms without inhibiting the groweh of the organism being sought. For example, MacConkey Blood agar and chocolate agar are examples of enriched media, Scanned with CamScanneree Se bes 4196 Section v + Controting the Grown of Microt ti ceria and agar inhibits the groweh of Gram-positive BST yy thus is selective for Gram-negative oa (CNA) agar sh FE and ci te oF ai nisaelective for Gram-positive bueteria, Thayer-Martin agar and Martin-Lewis agar (chocolate gars containing extra nutrient pls several antimicrobial gens) sre selective for N. gonorrbocae. frowth of the organ- | Only salt-tolerant (haloduric) se ceughe, | bacteria can grow on mannitol salt agar (MSA). 3 ‘A differential medium permits the differentiation of organisms that grow on the medium. For example, Mac- Conkey agar is frequently used to differentiate among Various Gram-negative bacilli that are isolated from fecal {pecimens. Gram-negative bacteria capable of fermenting Inctose (an ingredient of MacConkey agar) produce pink colonies, whereas those that are unable to ferment lactose produce colorless colonies (Fig. 8-6). Thus, MacConkey Agar differentiates between lactose-fermenting and nonlac- tose-fermenting Gram-negative bacteria. MSA is used to screen for S, aureus, S. aureus not only will grow on MSA ‘butalso curs the originally pink medium to yellow because ofits ability to ferment mannitol (Fig. 8-7). In a sense, blood agar is also a differential medium because it is used to determine the type of hemolysis (alteration or destruction of red blood cells) tat te bacesil ioe prods ~ ig. 8-8). {Ths various categories of media (enriched, selective, and differential) are not mutually exclusive, For example, Aselective medium is used to discour- age the growth of certain organisms without inhibiting the | Acitten ential med- ium allows one to | readiy ditferentiate ‘among the various types of organisms that are growing on { the medium Figure 8-7. Mannitol salt agar, a selective and citterentg Figdium, is used to screen for Staphylococcus aureus, Jay bacteria capable of growing in a 7.5% sodium chloride Ao contration will grow on this medium, but S. aureus wit {urn the medium yellow because of its ability to ferment the Trannitol in the medium. The organism growing on the upper Tection of the plate is unable to ferment mannitol, but the Sranism growing on the lower section Is a mannitol ermen- ter (From Koneman E, et al. Color Atlas and Textbook of Diagnostic Microbiology. Sth ed. Philadelphia, PA: Lippineet Wiliams & Wikins; 1997.) as just seen, blood agar is enriched and differential. Mic Conkey agar and MSA are selective and differential. PEA and CNA are enriched and selective: they are blood agus to which selective inhibitory substances have been added. “Thayer-Martin and Martin-Lewis agars are highly enriched and highly selective. ‘Thioglycollate broth (THIO) is a very, popular liquid medium for use in the bacteriology laboratory. THIO supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes. How is ths possible? Within the tube of THIO, there is a cone tration gradient of dissolved oxygen. The concentratio® Informat ‘the red blood calls In the 'n Appendix by about the Grows righator can ana Tone Dd From Winn We se ot at Renoman's al. s a i ‘Koneman’ COO incon Willams & whine soe om oo Pri Scanned with CamScanner‘oygen eae Obligate aerabes will grow will rere, there is 20-21% oxygen 18% 10% ites wil grow where set there Is about 5% oxygen, Obiigato anacrobes wil grow el whore there Is O% oxygen. Figure 8-9. Thioglycollate broth contains a concentra- tion gradient of dissolved oxygen, ranging from 20% to 21% O; at the top of the tube to 0% O; at the bottom of ‘the tube. A particular bacterium will grow only in that part of the broth containing the concentration of oxygen that it requires. of oxygen decreases with depth. The concentration of corygen in the broth at the top of the tube is about 20% 10 21%. At the bottom of the tube, there is no oxygen in the broth. Organisms will grow only in that part of the broth where the oxygen concentration meets their needs (ig.8-9). For example, microaerophiles will grow where there is around 5% oxygen, and obligate anaerobes will grow only at the very bottom of the tube where there is no oxygen. Facultative anaerobes can grow anywhere in the tube. (Recall that facultative anaerobes can live in the Presence or absence of oxygen.) Inoculation of Culture Media : In clinical microbiology laboratories, culture media are Toutinely inoculated with clinical specimens (.¢., speci~ ‘mens that have been collected from patients suspected of having infectious diseases). Inoculation ofa liquid medium involves adding a portion of the specimen to the medium. Inoculation of a solid or plated medium involves the use of, sterile inoculating loop to apply a portion of the spec- jmen to the surface of the medium; a process commonly referred to as streaking (Fig. 8-10). The proper method of inoculating plated media to obtain well-isolated colonies is described in Appendix $ on thePoint “Clinical Microbiology Laboratory Procedures.” Importance of Using “Aseptic Technique” Individuals working in a microbiology laboratory must Practice what is known as aseptic technique and = “understand its importance. Aseptic technique is practice 137 Chapter 8 + Controling Microbial Growth In Vitro Figure 8-10. Laboratory professional demonstrating the proper method of inoculating the surface of an agar plate. “The plate is held in the palm of one hand. The other hand Is used to lightly drag the inoculating loop over the surface Of the solid culture medium. The inoculating loop is held in much the same manner as a small camel-hair paintbrush is held by an artist when applying paint to the surface of a ‘canvas, (Additional information regarding the proper method Of “streaking” can be found on thePoint) (Provided by Dr. Robert Fader) to prevent (a) microbiology professionals from becoming infected, (b) contamination of their work environment, and (¢) contamination of clinical specimens, cultures, and subcultures, For example, when inoculating plated media, it is important to keep the Petri dish lid in place at all times, except for the few seconds that it takes to inoculate the specimen to the surface of the culture medium. Every additional second that the lid is off provides an opportunity for airborne organisms (e.g., bacterial and fungal spores) to land on the surface of the medium where they will then grow. Such unwanted organisms are referred to as, contaminants, and the plate is said to be contaminated. Of equal importance is to maintain the sterility of the media before inoculation and to avoid touching the agar surface with fingertips or other nonsterile objects. Inoculating media within (Aseptic technique a biologic safety cabinet (BSC) | is practiced in minimizes the possibility of | the microbiology contamination and protects the | laboratory to laboratory worker from becoming | Prevent infection infected with the organism(s) that | of individuals and heorshe is working with. BSCs | tamination of the ae raahet discussed in Appendix | Work environment, clinical specimens, and cultures. 4 on thePoint: “Responsibilities of the Clinical Microbiology Laboratory.” Incubation After media are inoculated, they must be incubated (i they must be placed into a chamber (called an incubator] that contains the appropriate atmosphere and moisture Scanned with CamScanner438 section V + Contoting the Growin of Microbes 1 and is set to maintain level The three tes ot the appropriate temperature). Incubators used in | “This is called incubation. To the microbiology ald ae laboratory are COz ‘culture me u cD Dorn Go, | the incubator is set at 35°C "0 Incubators, and anaer- | 37°C. Three pes ofins ors bic incubators dre used in a clinical micro ology laboratory. 5 (carbon dioxide) incubator is an incubator to ee tinder of CO; is atached. CO; is periodically Introduced into the incubator to maintain a CO; con ‘centration of about 5% to 10%. Such an incubator is ‘used to isolate capnophiles (organisms that grow best in atmospheres containing increased CO,). Itis important to keep in mind that a CO incubator contains oxygen (about 15%-20%) in addition to CO;. Thus, a CO; incubator is not an anaerobic incubator. 2. Anon-CO; incubator is an incubator containing room. ; thus, it contains about 20% to 21% oxygen. 3. An anaerobic incubator is an incubator containing an atmosphere devoid of oxygen. Once a particular species of bacteria has been isolated from a clinical specimen, it can be separated from any other organisms that were present in the specimen and can be grown as a pure culture. The term pure culture refers to the fact that there is only one bacterial species present. The changes in a bacterial population over an ‘extended period follow a definite predictable pattern that can be shown by plotting the population growth curve ona graph (discussed Inter in this chapter). Apure culture is a culture that contains only one species of organism. Bacterial Population Counts Microbiologists sometimes need to know how many bacteria are present in a particular liquid at any given time (e.g., to determine the degree of bacterial contam- ination in drinking water, milk, and other foods), The microbiologist may determine (a) the total number of through. As bacte turbid (cloudy) increases (.e, crease in equate the amount of concentration of organi: je uiually expressed as the number of ion. viable plate counts used to determine the ming of ane Trereria in a liquid sample, such as milk = vind food diluted in water, or a broth culture. ty 4 rocedure, serial dilutions of the sample are iat Fen 0.1- or 1-mL aliquots (portions) are inoculated gy, plates of nutrient agar. After overnight incubation, 4, Jumber of colonies is counted. (Usually, a plate contining 30-300 colonies is used.) To determine the concentrates of bacteria in the original sample, the number of colonies rust be multiplied by the dilution factor(s). For example if 220 colonies were counted on an agar plate that had been inoculated with a 1.0-mL sample of a 1:10,000 dilution, there were 220 X 10,000 = 2,200,000 bacteria/ml. of the origina material at the time the dilutions were made and cultured, If however, 220 colonies were counted on an agar plate tht had been inoculated with a 0.1-mL sample of a 1:10,000 dilution, there were 220 X 10 X 10,000 = 22,000,000 bc. teria/mL of the original material at the time the dilutions were made and cultured. In the clinical microbiology laboratory, a viable cell court is an important part of a urine culture. (The techniqueis described in Chapter 13.) The number of viable bacteria per milliliter of a urine specimen is used as an indicator of a urinary tract infection. As explained in Chapter 1, high colony counts may also be caused by contamination of the urine specimen with indigenous microbiota during specimen collection or failure to refrigerate the specimen between collection and transport to the laboratory. Bacteria Population Growth Curve 7 population growth curve for ‘icular species| bacterium may be determined by growing a purecltucal the organism in a liquid medium at a constant temperatut ‘Samples of the culture are collected at fixed intervals (> every 30 minutes), and the number of viable organismsi® cksamnle is desermined. ‘The data are then plowed ¢ rithmic graph paper. The graph in Figure 8-11 ¥ obtained by ploting the logarithns ae (logic) of the number of viable bacteria (on the vertical or y-axis) against the incubation time (on the horizontal or x-axis). (Fyou A bacterial popula: tion growth cuve consists of four phases: a lag phas® are not fmliar with logarithm, a8 ee ot refertoamath book) mene (death phase. ; ‘The growth curve consists of the following four pis** - The first phase of i dine ase ofthe groweh curve i the lag bacteria absorb nutrients, syathess enzymes, and prepare for cell division, The bactet2®” Rot increase in number durin, phase. Thes the lag phase. second phase of the growth curve k the logs growth phi ase (also known as the log phase or on itn om incr rateis the greats duing™™ a: hase). Inthe log phase, the bacteria Scanned with CamScannerStationary phase Log (exponential) phase 5 5 5 Figure 8-11. A population growth curve of living ‘organisms. The logarithm of the number of bacteria per rillter of medium is plotted against time (see text for details). {Redrawn from Harvey RA, et al. Lippincott's ilustrated Reviews: Microbiology. Srd ed. Philadelphia, PA: Lippincott Wiliams & Wilkins; 2013.) phase. The log phase is always brief, unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent removal of waste products. When plotted on logarithmic graph paper, the log phase appears asa steeply sloped straight line. 3, Because the nutrients in the liquid medium are used up and the concentration of toxic waste products from the metabolizing bacteria build up, the rate of division slows, such that the number of bacteria that are dividing equals the number of bacteria that are dying. The result is the stationary phase. It is during this phase that the culture is atts greatest population density. 4. Asovercrowding occurs, the concentration of toxic waste products continues to increase and the nutrient supply decreases. The mi isms then die at a rapid rate; this is the death phase or decline phase. The culture ‘may die completely, or a few mi isms may con- tinue to survive for months. If the bacterial species is a spore former, it will produce spores to survive this phase. When cells are observed in old cultures of bacteria in the death phase, some of them look different from healthy organisms seen in the log phase. As a result of unfavorable conditions, morphologic changes in the cells may appear. Some cells undergo involution and as- sume various shapes, becoming long, filamentous rods or branching or globular forms that are difficult to identify. Some develop without a cell wall and are refered om Protoplasts, roplasts, or ‘L-phase variants rms). When — forms are inoculated into a fresh ‘nutrient medium, they usually revert to the original shape f the healthy bacteria. ‘Many industrial and research procedures depend on the maintenance of an essential species of microorganism. ‘These are continuously cultured in a controlled environment ‘alled a chemostat (Fig. 8-12), which regulates the supply Chapter 8 + Controlling Microbial Growth In Vitro 139 »—— Forced — ‘Stopcock to ( sterile alr i Fritted glass disk ‘to break air into tiny bubbles: | Growth ‘chamber I Collection vessel Figure 8-12. Chemostat used for continuous cultures. Rates of growth can be controlled either by controlling the rate lat which new medium enters the growth chamber or by limiting ‘@ required growth factor in the medium. of nutrients and the removal of waste products and excess microorganisms. Chemostats are used in industries where yeast is grown to produce beer and wine, where fungi and bacteria are cultivated to produce antibiotics, where E.coli cells are grown for genetic research, and in any other process needing a constant source of microorganisms. Cutturing Viruses and Other Obligate Intracellular Pathogens in the Laboratory Recall from Chapter 4 that obligate intracellular pathogens are microbes that can survive and multiply only within living cells (called host cells). Obligate intracellular patho- gens include viruses and two groups of Gram-negative bacteria—rickettsias and chlamydias. Because obligate intracellular pathogens will not grow on artificial (synthetic) media, they presenta challenge to laboratorians when large numbers (Sp, ofthe organisms are required for | OuyOe nan ular diagnostic or research purposes | propagated in the (egudevelopmentofvaccinesand | faberarory using new drugs). To grow such organ- | embryonated fsmsin thelaboratory, they must_| chicken eggs, be inoculated into embryonated | laboratory animals, chicken eggs, laboratory animals, or cell cultures. In the virology laboratory, cell cultures are primarily used for the propagation of viruses. Because a given virus can only attach to and infect cells that bear appropriate or cell cultures. Scanned with CamScannerbes 140 sention1¥ + Coating te Growth of Micro intain several tors, it is necessary io mail a cal srs ee ins inte tO De RES ines are kidney cells FOm TION Examples Organ human and mink a a ich rabbits of hae afer appropriate cellsae inocula eafnial specimen suspected ofeonsining sane ice ofvi incubat or sever ar e Str tec sowe smicresphologic alterations tothe cells. Se alld optic effect (CPE), ‘amples es ide rounding, swelling, and shrin , CPE ina es, gy, aewlateds oF fsed (ilustrated in Fig, 13-23 in Chapter 13). Viruses can then deescatfed based upon the particular type of they cause in a specific cell line. Culturing Fungi in the Laboratory 5 including yeasts, moulds, and dimorphic fangi) will pen n various solid and liquid culeure media. There Erno one medium that is best for all medically important fangi. Examples of solid culture media used to grow fungi are brain-heart infusion (BHI) agar, BHI agar with blood, and Sabouraud dextrose agar (SDA). Antibacterial agents are often added to the media to suppress the growth of bacteria. ‘The low pH of SDA (pH 5.6) inhibits the growth of most bacteria; thus, SDA is selective for fungi. Laboratory per- sonnel must exercise caution when culturing fungi because the spores of certain fungi are highly infectious. Because of the potential danger, a Class II BSC must be used, and Petri plates are often sealed to prevent accidental exposure. Cutturing Protozoa in the Laboratory Most clinical microbiology laboratories do not culture protozoa, but techniques are available for culturing pro- toz0a in reference and research laboratories. Examples of protozoa that can be cultured in vitro are amebae (eg, Acanthamoeba spp., Balamutbia spp., Entamoeba histolytica, sad Nagle facler), Giardia lamblia, Leitemania spp., cre Of tee proton ig ond Toe ., it is of greatest it cxltuteAcnthamzeta Balimutia,andN oolong ie, microbiology laboratory. These amebac can cause seat (often fatal) infections of the central ne infections that are diffi jaaeeoeis io cult to di Parasitic protozoa are further decane AC PART 3: INHIBITING THE MICROBES IN VITRO Dein In certain environments, its itis osm of micobe nel inhibiethe growhofpalec atPe ts necesary ; ns : Patients, saffmembers,or tsitons one will notinfese ‘environments in ‘or desirable to inhibit » Hursing homes, and : desirable to inhibit microbial, which: itis ne erage processing plants, reat [dtchens, and bathrooms. Definition of Terms on jccussing the various methods used to di Bee ing ee numberof a iannderstood as they apply to microbiology, rilization : : Seeiization involves the destruction or elimination of erobes, including cells, spores, and viruses, So sale it is devoid of microbial life. In hea core facilities, sterilization of objects can be accomplished by physical or chemical methods. Dry hea, avtocaving ica under pressure), ethylene oxide gas, and vars liguid chemicals (uch as formaldehyde) are the princpl sterilizing agents in health care facilities. In some situations, certain of radiation (e.g., ultraviolet [UV] light and gamma rays) are also used. These techniques are discussed later in this chapter. Sterilization involves the destruction or | elimination of all microbes. Disinfection, Pasteurization, Antiseptics, and Sanitization Disinfection describes the elimination of most or all patho- gens (except bacterial spores) from nonliving objects. In health care settings, objects are usually disinfected by igid chemicals or wet pasteurization. The heating process deve- oped by Pasteur to kill microbes in wine—pasteurization— jisinfectants, isamethod of disinfecting liquids. Pasteurization is used today to (> inrection mvones eliminate pathogens from milkand | {hs simination of most other beverages. It should be remembered that pasteurization | TOSt or al patho: is nota sterilization procedure recause not all mi Seseuse not all microbes are pedaittmical used to disinfect inanimate objects 948 piaside equipment and operating rooms, are called dé. ctants. Disinfectants do not kill spores (ie., they at¢0% ‘Poricidal), Because they are strong chemical subse sre eeptants cannot be used on living tissue. AntisepO Sa solutions used to disinfect skin and other living ts gens (except bac terial spores) from nontiving objects- Microbicid: gents ‘The ‘ec = homide sae OF idl refers to “killing,” asin the VO, agents (germich nd genocide. General rms ie ay crocs, dal agents (biocides) i cro rebiies) are disinecnsse oe eps bacteria, burnett ABents (bacericides) specifica + But not necessarily bacterial endospores- Bee Scanned with CamScannercoats are thick and resistant to the effects o ieee sporicidal agents are required to kill ea endospores. Fungicidal agents (fungicides) kill fangi, including fungal spores. Algicidal agents (algicides) are used to kill algae in swimming pools and hot tubs, Viricidal agents (or virucidal agents) destroy viruses. Pseudomonicidal agents kill Pseudomonas species, and tuberculocidal agents kill 1M. tuberculosis, t3, ean ix -cidal” kil eens Sgents having the sfx “static” merely inhibit ther growth and reproduction. Microbistatic Agents “Amicrobistatic agent is a drug or chemical that inhibits reproduction of microorganisms, but does not necessarily, Lil them. A bacteriostatic agent is one that specifically inhibits the metabolism and reproduction of bacteria. Some of the drugs used to treat bacterial diseases are bac. terostatic, whereas others are bactericidal. Freeze drying (yophilization) and rapid freezing (using liquid nitrogen) ue microbistatic techniques that are used to preserve microbes for future use or study. Lyophilization is a process that combines dehydration (drying) and freezing. Lyophilized materials are frozen in a ‘acum; the container is then sealed to maintain the inactive state. This freeze-drying method is widely used in industry to preserve foods, antibiotics, antisera, microorganisms, and other biologic materials. It should be remembered that lyophilization cannot be used to Kill microorganisms, but rather is used to prevent them from reproducing and to store them for future use. Sepsis, Asepsis, Aseptic Technique, Antisepsis, and Antiseptic Technique Sepsis refers to the presence of pathogens in blood or {isues, whereas asepsis means the absence of pathogens. The two general categories of aseptic technique—medical and surgical asepsis—are described in detail in Chapter 12. Vasious techniques, collectively referred to as aseptic techniques, are used to eliminate and exclude pathogens. Earlier in this chapter, you learned of the importance of 'Sing aseptic technique in the microbiology laboratory when inoculating culture media. In other areas of the hospital, ‘septic techniques include hand hygiene’; the use of sterile Blores, masks, and gowns; sterilization of surgical instru- Tents and other equipment; and the use of disinfectan’s ‘iantiseptics, Antisepss is the prevention of infection. tiseptic technique, developed by Joseph Lister in 1867, fr to the use of antiseptics. Antiseptic technique is ¢ ‘We of aseptic technique. Lister used dilute carbolic sci “Tee tand pen” refers vo handwashing he of 100", forces the steam into the materials and being sterilized. Autoclaving ata ressure of 15 psi, ata temperature PF 121.5°C, for 20 minutes, kills Autoclaves should bbe set to run 20 minutes at a pres. °C, : ‘sure of 15 psi and vegetative microorBan nisms, bacterial | 3 temperature of endospores, and viruses, as long | 421.5°c, | as they are not protected by pus, feces, vornitus, blood, or other proteinaceous substan, Some types of equipment and certain materials, such x Figure at 14, DerRbert age ares. bbultt-n autoctave. Provides” Scanned with CamScannerFigure 6-15. Autoclave tape. Left: Appearance ofthe tape ‘autoclaving. Right: Dark lines appear on the tape after Peociaving. The dark lines indicate that the proper tempera- {tro was achieved. (Courtesy of Dr. Robert Fader) rubber, which may be damaged by high temperatures, can be autoclaved at lower temperatures for longer periods. The timing must be carefully determined based on the contents and compactness of the load, All articles must be properly packaged and arranged within the autoclave to allow steam to penetrate each package completely. Cans should remain open, bottles covered loosely with foil or coton, and instruments wrapped in cloth. Sealed containers should not be autoclaved. Pressure-sensitive autoclave tape Gg. 8-15) and commercially available strips or solutions containing bacterial spores (Fig. 8-16) can be used as quality control measures to ensure that autoclaves are functioning properly. Autoclave tape has diagonal markings that contain anink that changes color (usually from beige to black) after exposure to proper autoclave temperature (121.5°C).After the spore strips or solutions are used, they are examined tosee whether the spores were killed. Home canning conducted without the use of a pressure cooker does notdestroy the endospores of bacteria—notably the anaerobe, Clostridium botulinum. Occasionally, local ewspapers report cases of food poisoning resulting from the ingestion of C. botulinum toxins in improperly canned fruits, vegetables, and meats. Botulism food poisoning is preventable by properly washing and pressure cooking (sotoclaving) food. ___ Aneffective way to disinfect clothing, bedding, and dishes istouse hot water (>60°C) with detergent or soap and to agitate the solution around the items. This combination of heat, mechanical action, and chemical inhibition is deadly tomost pathogens. The best way to remove microbes from ‘kitchen sponge isto rinse it, wring it out, and microwave ‘it for 30 to 60 seconds.® — * © 2m more about microbes in our food and Kitchens, st TP Takia e2ok at Microbes in Our Food” and “A Closer ine biting the Growth of Pathogens in Our Kitchens” on the Chapter 8 + Controling Microbial Growth In Vito 143 | | \ | | | | Figure 8-16. Biologic indicator used to monitor the effec- tiveness of autoclaving. Sealed ampules containing bacterial spores: in a growth medium are placed within the load to be sterilized. Following sterilization, the ampules are incubated at 35°C. If the spores were killed, there will be no ‘change in the color of the medium; it will remain purple. If the spores were not killed, germination will occur, and acid ‘production by the bacteria will cause the pH Indicator in the medium to change from purple to yellow. (Provided by Fisher Scientific.) Cold ‘Most microorganisms are not killed by cold temperatures and freezing, but their metabolic activities are slowed, greatly inhibiting their growth. Refrigeration merely slows the growth of most microorganisms; it does not completely inhibit growth. Slow freezing causes ice crystals to form within cells and may rupture the cell membranes and cell Walls of some bacteria; hence, slow freezing should not be ‘sed as a way to preserve or store bacteria. Rapid freezing, using liquid nitrogen, is a good way to preserve foods, biologie specimens, and bacterial cultures. It places bac- teria into a state of suspended ‘ation. Then, when the { Petigeration cannot ‘Emperatureisraised above the | berelied upon to kl freezing point, the organisms’ gars retabolie reactions speed up, | Morey Sows Me and the organisms begin tO | thor rato of growth. juce again. sree eho are invalvedin the preparation and preservation of foods must be aware Scanned with CamScanner444 Section V + Controling the Growth of Microbes that thawing foods allows bacterial spores in hiss to germinate and microorganisms to reume BUTE (2 Consequently, refreezing of thawed foods is an. te practice because it preserves the millions of mcr OTe that might be re a endospore f 1 food when it is rethawed. Also, Oi poatinuns or Cloeidium perfringens were presents she viable bacteria would begin to produce the toxins cause food poisoning. ion . Peers foods have been preserved by drying. ‘However, even when moisture and nutrients are lacking, many dried microorganisms remain viable, although they cannot reproduce. Foods, antisera, toxins, antitoxins, antibiotics, and pure cultures of microorganisms are often preserved by lyophilization—the combined use of freezing and drying (discussed previously). In the hospital or clinical environment, health care professionals should keep in mind that dried viable patho- ‘gens may be present in dried matter, including blood, pus, fecal material, and dust that are found on floors, in bed- ding, on clothing, and in wound dressings. Should these dried materials be disturbed, such as by dry dusting, the microbes would be easily transmitted through the air or by contact. They would then grow rapidly if they settled in a suitable moist, warm nutrient environment, such as a wound or a burn. Therefore, nahosptareee >) important precautions that must se eietimta | beobserved incinde ‘wet mopping specimens and dust | of floors, damp dusting of furni- may contain viable | ture, rolling bed linens and towels carefully, and proper disposal of wound dressings, Radiation ‘The sun is not a particularly reliable disinfecting becauseitils only those microorganisne thesaee eee seit kills, rganisms that are exposed to direet sunlight. The rays of the sun include the lox infrared (heat) rays, the visible light mye sade ge , penetrate cells and th cause damage to DNA. When th F bees severely damaged that the Saaees Peal ay beso microorganisms) ori drastically, ___Inpractice,a UV lamp (often is useful for reducing the number air. Its main component is a lor tube Sach imps ate found in Fooms, elevators, entryways, caferer where they are in ines designed to radiate UV li (especially uni — : ining instruments, paper and clo cabinet-containing To animate articles Many yo°?- ment, liquid, and ot n rucles. Many bi terials, such as sera, antisera, toxins, and vacxin, & sterilized with UV rays. “Those whose work involves the use of UY jan, must be particularly careful not to expose their eye,” skin to the rays because they can cause serious bus Snd cellular damage. Because UV rays do not penene loth, metals, and glass, these materials may be used, protect persons working in a UV environment. It his been shown that skin cancer can be caused by ercesine exposure to the UV rays of the sun; thus, extensive sg tanning is harmful. ‘X-rays and gamma and beta rays of certain wavelengy from radioactive materials may be lethal or cause mutation in microorganisms and tissue cells because they DNA and proteins within those cells. Studies performed in radiation research laboratories have demonstrated that these radiations can be used for the prevention of food spoilage, sterilization of heat-sensitive surgical equipment, preparation of vaccines, and treatment of some chronic diseases such as cancer, all of which xe very practical applications for laboratory research. The USS. Food and Drug Administration approved the useaf gamma rays (fom cobalt-60) to process chickens andrel meat in 1992 and 1997, respectively. Since then, gamm rays have been used by some food processing plants © kill pathogens (such as Salmonella and Campylobacter bacteria) in chickens; the chickens are labeled “irradi ated” and marked with the green international subs! for irradiation (Fig. 8-17). Ultrasonic Waves In hospitals, medical clinics, and dental clinis,ultasotit waves area frequently used means of cleaning de equipment. Ultrasonic cleaners consist of tanks cl> {? C \ Figure 8-17. for Inadtated weourternational symbol 0 Department ot agrcutre) Scanned with CamScanner‘with liquid solvent (usually water); the short so tre then passed through the liquid. The ed os mechanically dislodge organic debris on instruments and giassware, Glassware and other articles that have been Eieansed in ultrasonic equipment must then be washed to remove the dislodged particles and solvent and are then sterilized by another method before they are used. Following cleaning of their instruments, most dental jonals sterilize them using steam under pressure {autoclave), chemical (formaldehyde) vapor, or dry heat (gs 160°C for 2 hours). Filtration Filters of various pore sizes are used to filter or separate cells, larger viruses, bacteria, and certain other mi i from the liquids or gases in which they are suspended. Filters with tiny pore sizes (called micropore filters) are used in laboratories to filter bacteria and viruses out of liquids. The variety of filters is large and includes sintered glass (in which uniform particles of glass are fused), plastic films, unglazed porcelain, asbestos, diatomaceous earth, and cellulose membrane filters. Small quantities of liquid can be filtered through a filter-containing syringe, but large quantities require larger apparatuses. Acotton plug in a test tube, flask, or pipette is a good filter for preventing the entry of microorganisms. Dry gauze and paper masks not only prevent the outward passage of microbes from the mouth and nose but also protect the wearer ftom inhaling airborne pathogens and foreign particles that could damage the lungs. BSCs contain Microbes, even those as small ‘as viruses, can bbe removed from liquids using filters, having appropriate pore sizes. Effectiveness of antimicrobial Chapter 8 * Controlling Microbial Growth In Vitro 145, high-efficiency particulate air (HEPA) filters to protect workers from contamination. HEPA filters are also located in operating rooms and patient rooms to filter the air that ‘enters or exits the room. Gaseous Atmosphere In limited situations, itis possible to inhibit the growth of microorganisms by altering the atmosphere in which they are located. Because aerobes and microaerophiles require oxygen, they can be killed by placing them into an atmosphere devoid of oxygen or by removing oxygen from the environment in which they are living. Conversely, obligate anaerobes can be killed by placing them into an atmosphere containing oxygen or by adding oxygen to the environment in which they are living. For instance, ‘wounds likely to contain anaerobes are lanced (opened) to, expose them to oxygen. Another example is gas gangrene, a deep wound infection that causes rapid destruction of tissues. Gas gangrene is caused by various anaerobes in the genus Clostridium. In addition to debridement of the ‘wound (removal of necrotic tissue) and the use of antibi otics, gas gangrene can be treated by placing the patient in a hyperbaric (increased pressure) oxygen chamber or in a room with high oxygen pressure. Asa result ofthe pressure, oxygen is forced into the wound, providing oxygen to the oxygen-starved tissue and killing the clostridia. Using Chemical Agents to Inhibit Microbial Growth Disinfectants Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens, either temporarily or permanently. The mechanism by which various disinfectants Scanned with CamScannerMicrobes: 46 Section IV * controling the Growth of ¥ ss from one 9 of 0 anothet i elie eee ere) oF CS sin tiene ors most be taken 7 es di i Thess Sines (®rst 8. feces include the following: 2 ee mm of the object or surface t© ced Rr ard thats presents tats the PT : organic mater (fees blood, vom Terials being treate w The bloburden; that the EyPE + The concentration ofthe disinfectant ae the + The contact time that is, the amout anisms disinfectant must remain ort for Closer Look at {n order to kill them (see thePoint for’ Time = ean at aeure ofthe objet being disinfected ‘nonth or rough surface crevices, and hinges) «= Temperature and pH rections for preparing the proper dilution of a dis- inform snst bellowed carefully because too weak oF too strong a concentration is usually less effective than the ‘proper concentration (see Appendix 6 on thePoint “Preparing Solutions and Dilutions”). The items to be disinfected must firstbe washed to remove any proteinaceous material in which pathogens may be hidden. Although the washed article may then be clean, itis not safe to use until t has been properly disinfected. Health care personnel need to understand an important limitation of chemical disinfection-that many disinfectants that are effective against pathogens in the controlled conditions of the laboratory may be ineffective in the actal hospital or clinical environment. Furthermore, the stronger and more effective antimicrobial chemical agents are of limited usefulness because of their destructiveness tohuman tissues and certain other substances. Almost all bacteria in the vegetative state, as well as fungi, protozoa, and most viruses, are susceptible to many disinfectants, although the mycobacteria that cause tuberculosis and leprosy, bacterial endospores, Pseudomonads (Preudomonas spp.), fungal spores, and and level of microbial Table 8-3 5) ne otably resistant (sce Table g. hepatitis viruses 27 disinfection should never}. Theres when it is possible to Use Proper physicr at . iques. rilization techniques. effective for éath tics, The sink Chemical gents wed ods must be Co A ent anol thetinotiotan we? ratory : eat oe nie iy a ee me bey din sputum iva a pe the oral and septs esto my erculosis, Species OF q ; 1M. tuberculosis; Sper us fungi that cause candidiasis, ba Streptococcus, the various fung? f asis, bs tomycosis, coccidioidomycosis, and histopl: i viruses. oA came most disinfection meth hos not destroy fal endospores that are present, any instrument or aed in the treatment of an infected wound ora disease cased by spore formers must be autoclaved arin cinerated, Gas gangrene, tetanus, one care cote Of diseases caused by spore formers that require the health care worker to take such precautions. Formaldehyde and ethylene oxide, when properly used, are highly destruce to spores, mycobacteria, and viruses. Certain articles ae heat sensitive and cannot be autoclaved or safely washed before disinfection; such articles are soaked for 24 hoursin astrong detergent and disinfectant solution, then washed, and finally sterilized in an ethylene oxide autoclave. The use of disposable equipment, whenever possible, in these situations helps to protect patients and health care personne ‘The effectiveness of a chemical agent depends to some extent on the physical characteristics ofthe article on whicht isused.A smooth, hard surface is readily disinfected, wheres a rough, porous, or grooved surface is not. Thought mustbe given to selection of the most suitable germicide for cleaning Patientrooms and all other areas where patients are te ‘The most effective antiseptic or disinfectant shoald be chosen for the specific purpose, environment, 3 pathogen(s) likely to be present. The characteristic of ideal chemical antimicrobial agent include the followin * Teshould have a wide or broad antimicrobial spect tis, it should kill a wide variety of microbes- old * Itshould be fast acti : ” be shore,” 5" #eting that is, the contact time * Tr should not be affected by the presence of org « Ate! 65 feces, blood, vomitus, and pus). : and na Oe Rontoxic to human tissues and rnoncorroee inaeondestructive to materials on which itis used being rif tincture (e.g, aleohol-water solusotl 2 19p eth evaporation ofthe alcohol solvent 29 concentration © etease to a 10% solution, and * Teshould leave i cause tissue damage). a a cate surface. ® residual antimicrobial film °° ust be : * Teshould be grees 8 Water and easy to app ale Specie deen PETE and easy to prepare with s™ Scanned with CamScanner« Iemust be stable both as a concentrate and as a workin, diluon,s0 tha itean be shipped and stored fr fcoer | iods. . Fiebould ‘be odorless. How do disinfectants kill microorganisms? Some disin- fectants (eg.,surface-active soaps and detergents, alcohols, phenolic compounds) target and destroy cell membranes. Byhers (c.g. halogens, hydrogen peroxide, salts of heavy ‘netals, formaldehyde, and ethylene oxide) destroy enzymes tnd structural proteins. Others attack cell walls or nucleic saids, Some of the disinfectants that are commonly used jn hospitals are discussed in Chapter 12. “The effectiveness of phenol as a disinfectant was demon- strated by Joseph Lister in 1867, when it was used to reduce the incidence of infections after surgical procedures.’ The effectiveness of other disinfectants is compared to that of phenol using the phenol coefficient test. To perform this testa series of dilutions of phenol and the experimental disinfectant are inoculated with the test bacteria, Salmonella typhi and S. aureus, at 37°C. The highest dilutions (lowest ee centrations) that kill the bacteria after 10 minutes are tsed to calculate the phenol coefficient. Antiseptics ‘Most antimicrobial chemical agents are too irritating and destructive to be applied to mucous membranes and skin, "Those that may be used safely on human tissues are called antiseptics, An antiseptic merely reduces the number of rims on a surface; it does not penetrate pores and hair follicles to destroy microorganisms residing there, To contaminating the surgical field, surgeons wear, sterile gloves on freshly scrubbed hands, ‘Antimicrobial chem- | and masks and hoods to cover ical agents that can | their face and hair. Also, an anti- salely be applied | septic is applied atthe sie of the skin are icis appli masses surgia incon destroy I Controversies Relating to the Use of Antimicrobial Agents in Animal Feed and Household Products Tehas been estimated that farmers and ranchers us¢ approximately 10 times the antibiotic tonnage ss employed in human medicine. The reason is obvio. cure or Prevent infectious diseases in farm animal: a that could lead to huge economic losses for farmers and ranchers, The problem is that when antibiotics are fe to an animal, the antibiotics kill any indigenous micro- biota organisms that are susceptible to the anebions But whee survives? Any organisms eat are resissans £2 ‘Leam more about Joseph Listerin a Historical Note in Chapter 12. infections Chapter 8 * Controling Microbial Growth In Vitro 147 the antibiotics. Having less competition now for space and nutrients, these drug-resistant organisms multiply and become the predominant organisms of the animal’ indigenous microbiota. These drug-resistant organisms are then transmitted in the animal's feces or food products (e.g., eggs, milk, and meat) obtained from the animal. ‘Many multidrug-resistant Salmonella strains—strains that cause disease in animals and humans—developed in this manner. The use of antibiotic-containing animal feed is quite controversial. Microbiologists concerned about ever-increasing numbers of drug-resistant bacteria have, for years, been attempting to eliminate or drastically reduce the practice of adding antibiotics to animal feed. In April 2012, the U.S, Food and Drug Administration announced a program calling for a voluntary halt to the use of antibiotics for growth promotion in animals being raised for food. Farmers and ranchers will require a pre~ seription from a veterinarian before using antibiotics in such animals and will have to convince the veterinarian that their animals were either sick or at risk of getting a specific illness. ‘Another controversy concerns the antimicrobial agents that are being added to toys, cutting boards, hand soaps, antibacterial kitchen sprays, and many other household products. The antimicrobial agents in these products kill any organisms that are susceptible to these drugs, but ‘what survives? Any organisms that are resistant to these gents. These drug-resistant organisms then multiply ‘and become the predominant organisms in the home. Should a member of the household become infected with these drug- and multidrug-resistant organisms, the Infection will be more difficult to treat. For many years, concerned microbiologists have attempted to eliminate or drastically reduce the practice of adding antimicrobial agents to household products. ‘Another argument against the use of antimicrobial agents in the home concerns proper development of the immune system, Many scientists believe that children must be exposed to all sorts of microbes during their growth and development so that their immune systems will de- ‘Yelop correctly and be capable of properly responding to pathogens in later years The use of household products Pontaining antimicrobial agents might be eliminating the Sery organisms that arc essential for proper maturation of the immune system. Zne scientise (Thomas McDade of Northwestern University) has sone dhat “Taflammatory networks may need the same type of sees pial exposure early in life that have been part ofthe human forall ofour evolutionary history ro function optimally Seuthood." The American Society for Microbiology (ASM) as speculated that “Ultra-clean, ultra-hygienie environments iyo life may contsibute to higher levels of inflammation (hat) Te duals experience as adults, which in turn increases their risk ae wide range of diseases.” Slonczewski JC, et al. Microbial frowdh with multiple stressors. Mere. 2010;5:98) Scanned with CamScanner