Ch_08

194

INTRODUCTIONFACTORS THAT AFFECT

MICROBIAL GROWTHAvailability of NutrientsMoistureTemperaturepHOsmotic Pressure and SalinityBarometric PressureGaseous AtmosphereENCOURAGING THE

GROWTH OF MICROORGANISMS IN VITRO

IntroductionCulturing Bacteria in the Laboratory

Bacterial GrowthCulture MediaInoculation of Culture MediaImportance of Using “Sterile

Technique”

IncubationBacterial Population CountsBacterial Population Growth Curve

Culturing Obligate Intracellular Pathogens in the Laboratory

Culturing Fungi in the LaboratoryCulturing Protozoa in the

LaboratoryINHIBITING THE GROWTH OF

MICROORGANISMS IN VITRODefinition of Terms

SterilizationDisinfection, Pasteurization,

Disinfectants, Antiseptics, andSanitization

Microbicidal AgentsMicrobistatic AgentsSepsis, Asepsis, Aseptic Technique,

Antisepsis, and AntisepticTechnique

Sterile TechniqueUsing Physical Methods to Inhibit

Microbial GrowthHeatColdDesiccationRadiationUltrasonic WavesFiltrationGaseous Atmosphere

Using Chemical Agents to Inhibit Microbial GrowthDisinfectantsAntiseptics

Inhibiting the Growth of Pathogens in Our Kitchens

Controversies Relating to the Use ofAntimicrobial Agents in Animal Feed and Household Products

AFTER STUDYING THIS CHAPTER, YOU SHOULD

BE ABLE TO:

■ List several factors that affect the growth of mi-croorganisms

■ Describe the following types of microorganisms:psychrophilic, mesophilic, thermophilic,halophilic, haloduric, alkaliphilic, acidophilic,and barophilic

■ List three in vitro sites where microbial growth isencouraged

■ Differentiate among enriched, selective, and dif-ferential media and cite two examples of each

■ Explain the importance of using “sterile tech-nique” in the microbiology laboratory

■ Describe the three types of incubators that areused in the microbiology laboratory

LEARNING OBJECTIVES

Controlling MicrobialGrowth In Vitro

Controlling the Growth of MicroorganismsIV

88

INTRODUCTION

In certain locations—such as within microbiology laboratories—the growth of mi-croorganisms is encouraged; in other words, scientists want them to grow. In otherlocations—such as on hospital wards, in intensive care units, and in operatingrooms—it is necessary to inhibit the growth of microorganisms. Both concepts—encouraging and inhibiting the in vitro growth of microorganisms—are discussedin this chapter. (Recall that in vitro refers to events outside the body, whereas invivo refers to events inside the body.) Before discussing these concepts, however,various factors that affect the growth of microorganisms are examined.

FACTORS THAT AFFECT MICROBIAL GROWTH

Microbial growth is affected by many different environmental factors, includingthe availability of nutrients and moisture, temperature, pH, osmotic pressure,barometric pressure, and composition of the atmosphere. These environmentalfactors affect microorganisms in our daily lives and play important roles in thecontrol of microorganisms in laboratory, industrial, and hospital settings.Whether scientists wish to encourage or inhibit the growth of microorganisms,they must first understand the fundamental needs of microbes.

Availability of Nutrients

As discussed in Chapter 7, all living organisms require nutrients—the variouschemical compounds that organisms use to sustain life. Therefore, to survive ina particular environment, appropriate nutrients must be available. Many nutri-ents are energy sources; organisms will obtain energy from these chemicals bybreaking chemical bonds. Nutrients also serve as sources of carbon, oxygen, hy-drogen, nitrogen, phosphorus, and sulfur as well as other elements (e.g., sodium,potassium, chlorine, magnesium, calcium, and trace elements such as iron,

Controlling Microbial Growth In Vitro 195

■ Draw a bacterial growth curve and label its fourphases

■ Cite two reasons why bacteria die during thedeath phase

■ Name three ways in which obligate intracellularpathogens can be cultured in the laboratory

■ List three in vitro sites where microbial growthmust be inhibited

■ Differentiate among sterilization, disinfection,and sanitization

■ Differentiate between bactericidal and bacterio-static agents

■ Explain the processes of pasteurization andlyophilization

■ List several physical methods used to inhibit thegrowth of microorganisms

■ Cite three ways in which disinfectants kill mi-croorganisms

■ Identify several factors that can influence the ef-fectiveness of disinfectants

■ Explain briefly why the use of antibiotics in ani-mal feed and household products is controversial

iodine, zinc, etc.) that are usually required in lesser amounts. About 25 of the 92naturally occurring elements are essential to life.

Moisture

On earth, water is essential for life. Cells consist of anywhere between 70% and95% water. All living organisms require water to carry out their normal meta-bolic processes, and most will die in environments containing too little moisture.There are certain microbial stages (e.g., bacterial endospores and protozoancysts), however, that can survive the complete drying process (desiccation). Theorganisms contained within the spores and cysts are in a dormant or restingstate; if they are placed in a moist nutrient-rich environment, they will grow andreproduce normally.

Temperature

Every microorganism has an optimum growth temperature—the temperature atwhich the organism grows best. Every microorganism also has a minimum growthtemperature, below which it ceases to grow, and a maximum growth temperature,above which it dies. The temperature range (i.e., the range of temperatures fromthe minimum growth temperature to the maximum growth temperature) at whichan organism grows can differ greatly from one microbe to another. To a large ex-tent, the temperature and pH ranges over which an organism grows best are de-termined by the enzymes present within the organism. As discussed in Chapter 7,enzymes have optimum temperature and pH ranges where they operate at peakefficiency. If an organism’s enzymes are operating at peak efficiency, the organ-ism will be metabolizing and growing at its maximum rate.

Microorganisms that grow best at high temperatures are called thermophiles(meaning 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 bottomof the ocean (check this book’s web site for “A Closer Look at HydrothermalVents”). Thermophilic cyanobacteria, certain other types of bacteria, and algaecause many of the colors observed in the near-boiling hot springs found inYellowstone National Park. Organisms that favor temperatures above 100�C arereferred to as hyperthermophiles (or extreme thermophiles). The highest tem-perature at which a bacterium has been found living is around 113�C; it was anarchaean named Pyrolobus fumarii.

Microbes that grow best at moderate temperatures are called mesophiles.This group includes most of the species that grow on plants and animals and inwarm soil and water. Most pathogens and members of the indigenous microfloraare mesophilic, because they grow best at normal body temperature (37�C).

Psychrophiles prefer cold temperatures. They thrive in cold ocean water. Athigh altitudes, algae (often pink) can be seen living on snow. Ironically, the op-timum growth temperature of one group of psychrophiles (called psychrotrophs)is refrigerator temperature (4�C); perhaps you encountered some of these mi-crobes (bread molds, for example) the last time you cleaned out your refrigera-tor. Microorganisms that prefer warmer temperatures, but can tolerate or en-dure very cold temperatures and can be preserved in the frozen state, are known

196 CHAPTER 8

as psychroduric organisms. Fecal material left by early Arctic explorers con-tained psychroduric Escherichia coli that survived the Arctic temperatures.Refer to Table 8–1 for the temperature ranges of psychrophilic, mesophilic, andthermophilic bacteria.

pH

The term “pH” refers to the acidity or alkalinity of a solution (see WebAppendix 2: Basic Chemistry Concepts). Most microorganisms prefer a neutralor slightly alkaline growth medium (pH 7.0 to 7.4), but acidophilic microbes(acidophiles), such as those that can live in the stomach and in pickled foods,prefer a pH of 2 to 5. Fungi prefer acidic environments. Acidophiles thrive inhighly acidic environments, such as those created by the production of sulfurousgases in hydrothermal vents and hot springs as well as in the debris producedfrom coal mining. Alkaliphiles prefer an alkaline environment (pH greater than8.5), such as is found inside the intestine (pH of approximately 9), in soils ladenwith carbonate, and in so-called soda lakes. Vibrio cholerae—the etiologic agentof cholera—is the only human pathogen that grows well above pH 8.

Osmotic Pressure and Salinity

Osmotic pressure is the pressure that is exerted on a cell membrane by solutionsboth inside and outside the cell. When cells are suspended in a solution, the idealsituation is that the pressure inside the cell is equal to the pressure of the solutionoutside the cell. Substances dissolved in liquids are referred to as solutes. Whenthe concentration of solutes in the environment outside of a cell is greater than theconcentration of solutes inside the cell, the solution in which the cell is suspendedis said to hypertonic. In such a situation, whenever possible, water leaves the cellby osmosis in an attempt to equalize the two concentrations. Osmosis is defined asthe movement of a solvent (e.g., water), through a permeable membrane, from asolution having a lower concentration of solute to a solution having a higher con-centration of solute. If the cell is a human cell, such as a red blood cell (erythro-cyte), the loss of water causes the cell to shrink; this shrinkage is called crenationand the cell is said to be crenated. If the cell is a bacterial cell, having a rigid cell


Minimum Growth Optimum Growth Maximum Growth Category Temperature Temperature Temperature

Thermophiles 25�C 50–60�C 113�C

Mesophiles 10�C 20–40�C 45�C

Psychrophiles �5�C 10–20�C 30�C

T A B L E 8 - 1 Categories of Bacteria on the Basis of GrowthTemperature.

wall, the cell does not shrink. Instead, the cell membrane and cytoplasm shrinkaway from the cell wall. This condition, known as plasmolysis, inhibits bacterialcell growth and multiplication. Salts and sugars are added to certain foods as a wayof preserving them. Bacteria that enter such hypertonic environments will die as aresult of desiccation.

When the concentration of solutes outside a cell is less than the concentra-tion of solutes inside the cell, the solution in which the cell is suspended is saidto hypotonic. In such a situation, whenever possible, water enters the cell in anattempt to equalize the two concentrations. If the cell is a human cell, such as anerythrocyte, the increased water within the cell causes the cell to swell. If suffi-cient water enters, the cell will burst (lyse). In the case of erythrocytes, thisbursting is called hemolysis. If a bacterial cell is placed in a hypotonic solution(such as distilled water), the cell may not burst (due to the rigid cell wall), butthe fluid pressure within the cell increases greatly. This increased pressure oc-curs in cells having rigid cell walls such as plant cells and bacteria. If the pressurebecomes so great that the cell ruptures, the escape of cytoplasm from the cell isreferred to as plasmoptysis.

When the concentration of solutes outside a cell equals the concentration ofsolutes inside the cell, the solution is said to be isotonic. In an isotonic environ-ment, excess water neither leaves nor enters the cell and, thus, no plasmolysis orplasmoptysis occurs; the cell has normal turgor (fullness). Refer to Figure 8–1for a comparison of the effects of various solution concentrations on bacteriaand red blood cells.

Sugar solutions for jellies and pickling brines (salt solutions) for meats preservethese foods by inhibiting the growth of most microorganisms. However, some typesof molds and bacteria can survive and even grow in a salty environment.

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Isotonic solution Hypotonic solution

Plasmoptysisof bacteria

Hemolysis(red blood cell)

Hypertonic solution

Plasmolysisof bacteria

Crenation ofred blood cell

Figure 8-1. Changes in osmotic pressure. No change in pressure occurs inside the cell in anisotonic solution. Internal pressure is increased in a hypotonic solution, resulting in swellingof the cell. Internal pressure is decreased in a hypertonic solution, resulting in shrinking ofthe cell. (Arrows indicate the direction of water flow. The larger the arrow, the greater theamount of water flowing in that direction.)

Those microbes that actually prefer salty environments (such as the concen-trated salt water found in the Great Salt Lake and solar salt evaporation ponds)are called halophilic; halo referring to “salt” and philic meaning “to love.”Microbes that live in the ocean, such as Vibrio cholerae (mentioned earlier) andother Vibrio species, are halophilic. Organisms that do not prefer to live in saltyenvironments but are capable of surviving there (such as Staphylococcus aureus)are referred to as haloduric organisms.

Barometric Pressure

Most bacteria are not affected by minor changes in barometric pressure. Somethrive at normal atmospheric pressure (about 14.7 pounds per square inch orpsi). Others, known as barophiles (baro, referring to “pressure”), thrive deepin the ocean and in oil wells, where the atmospheric pressure is very high. Somearchaeans, for example, are barophiles, capable of living in the deepest parts ofthe ocean. Check this book’s web site for “A Closer Look at BarometricPressure.”

Gaseous Atmosphere

As discussed in Chapter 4, microorganisms vary with respect to the type ofgaseous atmosphere that they require. For example, some microbes (obligateaerobes) prefer the same atmosphere that humans do (i.e., about 20% to 21%O2 and 78% to 79% N2, with all other atmospheric gases combined representingless than 1%). Although microaerophiles also require oxygen, they require re-duced concentrations of oxygen (around 5% O2). Obligate anaerobes are killedby the presence of oxygen. Thus, in nature, the types and concentrations of gasespresent in a particular environment determine which species of microbes areable to live there. To grow a particular microorganism in the laboratory, it wouldbe necessary to provide the atmosphere that it requires. For example, to obtainmaximum growth in the laboratory, capnophiles require increased concentra-tions of carbon dioxide (usually from 5% to 10% CO2).


The suffix “-phile” means to love something. For example, acidophiles are organismsthat love acidic conditions; therefore, they live in acidic environments. Alkaliphileslive in alkaline environments. Halophiles live in salty environments. Barophiles livein environments where there is high barometric pressure, such as at the bottom ofthe ocean. Thermophiles prefer hot temperatures. Mesophiles prefer moderatetemperatures. Psychrophiles prefer cold temperatures. Microaerophiles live in envi-ronments containing reduced concentrations of oxygen (around 5% O2).Capnophiles grow best in environments rich in carbon dioxide.

“-Phile”

ENCOURAGING THE GROWTH OFMICROORGANISMS IN VITRO

Introduction

There are many reasons why the growth of microorganisms is encouraged in mi-crobiology laboratories. For example, technologists and technicians who work inclinical microbiology laboratories must be able to isolate microorganisms fromclinical specimens and grow them on culture media so they can then gather in-formation that will enable identification of any pathogens that are present. Inmicrobiology research laboratories, scientists must culture microbes so that theycan learn more about them, harvest antibiotics and other microbial products,test new antimicrobial agents, and produce vaccines. Microbes must also be cul-tured in genetic engineering laboratories and in the laboratories of certain foodand beverage companies as well as other industries.

Many different types of microorganisms can be cultured (grown) in vitro, in-cluding viruses, bacteria, fungi, and protozoa. In this chapter, emphasis is placedon culturing bacteria. Culturing other types of microorganisms will be men-tioned only briefly.

Culturing Bacteria in the Laboratory

In many ways, modern microbiology laboratories resemble those of 50, 100, oreven 150 years ago. Today’s laboratories still use many of the same basic “tools”that were used in the past. For example, microbiologists still use compound lightmicroscopes, Petri dishes containing solid culture media, tubes containing liquidculture media, Bunsen burners, wire inoculating loops, bottles of stainingreagents, and incubators. However, a closer inspection will reveal many modern,commercially available products and instruments that would have been incon-ceivable in the days of Louis Pasteur and Robert Koch.

Bacterial Growth“Human growth” refers to an increase in size; going from a tiny newborn babyto a large adult. Although bacteria do increase in size before cell division, “bac-terial growth” refers to an increase in the number of organisms rather than anincrease in their size. When each bacterial cell reaches its optimum size, it di-vides by binary fission (bi meaning “two”) into two daughter cells (i.e., each bac-terium simply splits in half to become two identical cells). )Recall from Chapter3, that DNA replication must occur before binary fission occurs, so that eachdaughter cell has exactly the same genetic makeup as the parent cell.) On solidmedium, binary fission continues through many generations until a colony isproduced. A bacterial colony is a mound or pile of bacteria containing millionsof cells. Binary fission continues for as long as the nutrient supply, water, andspace allow and ends when the nutrients are depleted or the concentration ofcellular waste products reaches a toxic level. The division of staphylococci by bi-nary fission is shown in Figure 8–2.

The time it takes for one cell to become two cells by binary fission is calledthe generation time. The generation time varies from one bacterial species to

200 CHAPTER 8

another. In the laboratory, under ideal growth conditions, E. coli, Vibriocholerae, Staphylococcus, and Streptococcus all have a generation time ofabout 20 minutes, whereas some Pseudomonas and Clostridium species maydivide every 10 minutes, and Mycobacterium tuberculosis may divide onlyevery 18 to 24 hours. Bacteria with short generation times are referred to asrapid growers, whereas those with long generation times are referred to as slowgrowers.

The growth of microorganisms in the body, in nature, or in the laboratory isgreatly influenced by temperature, pH, moisture content, available nutrients,and the characteristics of other organisms present. Therefore, the number ofbacteria in nature fluctuates unpredictably because these factors vary with theseasons, rainfall, temperature, and time of day.


Figure 8-2. Binary fission of staphylococci. (Original magnification, �30,000.) (Photographcourtesy of Ray Rupel.)

In the laboratory, however, a pure culture of a single species of bacteria canusually be maintained if the appropriate growth medium and environmental con-ditions are provided. The temperature, pH, and proper atmosphere are quite eas-ily controlled to provide optimum conditions for growth. Appropriate nutrientsmust be provided in the growth medium, including an appropriate energy and car-bon source. Some bacteria, described as being fastidious, have complex nutritionalrequirements. Often, special mixtures of vitamins and amino acids must be addedto the medium to culture these fastidious organisms. Some organisms will notgrow at all on artificial culture; these include obligate intracellular pathogens, suchas viruses, rickettsias, and chlamydias. To propagate obligate intracellularpathogens in the laboratory, they must be inoculated into live animals, embry-onated chicken eggs, or cell cultures. Other microorganisms that will not grow onartificial media include Treponema pallidum (the etiologic agent of syphilis) andMycobacterium leprae (the etiologic agent of leprosy).

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Culture MediaThe media (sing., medium) that are used in microbiology laboratories to culturebacteria are referred to as artificial media or synthetic media, because they donot occur naturally; rather, they are prepared in the laboratory. There are anumber of ways of categorizing the media that are used to culture bacteria.

One way to classify culture media is based on whether the exact contents ofthe media are known. A chemically defined medium is one in which all the in-

The earliest successful attempts to culture microorganisms in a laboratory settingwere made by Ferdinand Cohn (1872), Joseph Schroeter (1875), and Oscar Brefeld(1875). Robert Koch described his culture techniques in 1881. Initially, Koch usedslices of boiled potatoes on which to culture bacteria, but he later developed bothliquid and solid forms of artificial media. Gelatin was initially used as a solidifying agentin Koch’s culture media, but in 1882, Fanny Hess, the wife of Dr. Walther Hesse—one of Koch’s assistants—suggested the use of agar. Frau Hesse (as she is most com-monly called) had been using agar in her kitchen for many years as a solidifying agentin fruit and vegetable jellies. Another of Koch’s assistants, Richard Julius Petri, in-vented glass Petri dishes in 1887 for use as containers for solid culture media andbacterial cultures. The Petri dishes in use today are virtually unchanged from the orig-inal design, except that most of today’s laboratories use plastic, presterilized, dispos-able Petri dishes. In 1878, Joseph Lister became the first person to obtain a pure cul-ture of a bacterium (Streptococcus lactis) in a liquid medium. As a result of their abilityto obtain pure cultures of bacteria in their laboratories, Louis Pasteur and RobertKoch made significant contributions to the germ theory of disease.

Culturing Bacteria in the Laboratory

gredients are known; this is because the medium was prepared in the laboratoryby adding a certain number of grams of each of the components (e.g., carbohy-drates, amino acids, salts). A complex medium is one in which the exact contentsare not known. Complex media contain ground up or digested extracts from an-imal organs (e.g., hearts, livers, brains), fish, yeasts, and plants, which providethe necessary nutrients, vitamins, and minerals.

Culture media can also be categorized as liquid or solid. Liquid media (alsoknown as broths) are contained in tubes and are thus often referred to as tubedmedia. Solid media are prepared by adding agar to liquid media and then pour-ing the media into tubes or Petri dishes, where the media solidifies. Bacteria arethen grown on the surface of the agar-containing solid media. Agar is a complexpolysaccharide that is obtained from a red marine alga; it is used as a solidifyingagent, 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 ofspecial nutrients that promotes the growth of fastidious organisms. It is usuallyprepared by adding extra nutrients to a medium called nutrient agar. Blood agar(nutrient agar plus 5% sheep red blood cells) and chocolate agar (nutrient agarplus powdered hemoglobin) are examples of solid enriched media that are usedroutinely in the clinical bacteriology laboratory. Blood agar is bright red,whereas chocolate agar is brown (the color of chocolate). Although both ofthese media contain hemoglobin, chocolate agar is considered to be more en-riched than blood agar, because the hemoglobin is more readily accessible inchocolate agar. Chocolate agar is used to culture important, fastidious, bacterialpathogens, like Neisseria gonorrhoeae and Haemophilus influenzae, that will notgrow on blood agar.

A selective medium has added inhibitors that discourage the growth of cer-tain organisms without inhibiting growth of the organism being sought. For ex-ample, MacConkey agar inhibits growth of Gram-positive bacteria and thus isselective for Gram-negative bacteria. Phenylethyl alcohol (PEA) agar and colistin-nalidixic acid (CNA) agar are selective for Gram-positive bacteria. Thayer-Martin agar, Martin-Lewis agar, and New York City agar (chocolate agars con-taining extra nutrients plus several antimicrobial agents) are selective forNeisseria gonorrhoeae. Only salt-tolerant (haloduric) bacteria can grow on man-nitol salt agar (MSA).

A differential medium permits the differentiation of organisms that grow onthe medium. For example, MacConkey agar is frequently used to differentiate be-tween various Gram-negative bacilli that are isolated from fecal specimens.Gram-negative bacteria able to ferment lactose (an ingredient of MacConkeyagar) produce pink colonies, whereas those unable to ferment lactose produce col-orless colonies. Thus, MacConkey agar differentiates between lactose fermenting(LF) and nonlactose fermenting (NLF) Gram-negative bacteria. Mannitol saltagar is used to screen for Staphylococcus aureus; not only will S. aureus grow onMSA, but it turns the originally pink medium to yellow due to its ability to fer-ment mannitol. In a sense, blood agar is also a differential medium because it isused to determine the type of hemolysis (alteration or destruction of red bloodcells) that the bacterial isolate produces (see Color Figures 14, 15, 16, and 24).


The various categories of media (enriched, selective, differential) are notmutually exclusive. For example, as just seen, blood agar is enriched and differ-ential. MacConkey agar and MSA are selective and differential. PEA and CNAare enriched and selective: they are blood agars to which selective inhibitorysubstances have been added. Thayer-Martin agar is highly enriched and highlyselective; Martin-Lewis agar, and New York City agar are also.

Thioglycollate broth (THIO) is a very popular liquid medium for use in thebacteriology laboratory. THIO supports the growth of all categories of bacteriafrom obligate aerobes to obligate anaerobes. How is this possible? Within thetube of THIO there is a concentration gradient of dissolved oxygen. The con-centration of oxygen decreases with depth. The concentration of oxygen in thebroth at the top of the tube is about 20% to 21%. At the bottom of the tube,there is no oxygen in the broth. Organisms will grow only in that part of thebroth where the oxygen concentration meets their needs (Fig. 8–3). For exam-ple, microaerophiles will grow where there is around 5% oxygen, and obligateanaerobes will only grow at the very bottom of the tube where there is no oxy-gen. Facultative anaerobes can grow anywhere in the tube. (Recall that faculta-tive anaerobes can live in the presence or absence of oxygen.)

Inoculation of Culture MediaIn clinical microbiology laboratories, culture media are routinely inoculated withclinical specimens (i.e., specimens that have been collected from patients sus-

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Obligate aerobes will grow wherethere is 20–21% oxygen.

20–21%

15%

10%

5%

0%

Dissolvedoxygen

Obligate anaerobes will growwhere there is 0% oxygen.

Microaerophiles will grow wherethere is about 5% oxygen.

Figure 8-3. Thioglycollate broth contains a concentration gradient of dissolved oxygen, rang-ing from 20–21% O2 at the top of the tube to 0% O2 at the bottom of the tube. A particularbacterium will grow only in that part of the broth containing the concentration of oxygenthat it requires.

pected of having infectious diseases). Inoculation of a liquid medium involvesadding a portion of the specimen to the medium. Inoculation of a solid or platedmedium involves the use of a sterile inoculating loop to apply a portion of thespecimen to the surface of the medium; a process commonly referred to as“streaking” (Fig. 8–4). The proper method of inoculating plated media to obtainwell-isolated colonies is described in Web Appendix 4: Clinical MicrobiologyLaboratory Procedures.

Importance of Using “Sterile Technique”Individuals working in a microbiology laboratory must practice what is known assterile technique, and must understand its importance. For example, while inoc-ulating plated media, it is important to keep the Petri dish lid in place at alltimes, except for the few seconds that it takes to inoculate the specimen to thesurface of the culture medium. Every additional second that the lid is off pro-vides an opportunity for airborne organisms (e.g., bacterial and fungal spores) toland on the surface of the medium, where they will then grow. Such unwantedorganisms are referred to as contaminants, and the plate is said to be contami-nated. Of equal importance is to maintain the sterility of the media before inoc-ulation and to avoid touching the agar surface with fingertips or other non-sterile objects. Inoculating media within a biological safety cabinet (BSC)minimizes the possibility of contamination and protects the laboratory workerfrom becoming infected with the organism(s) that he or she is working with.BSCs are further discussed in Web Appendix 3 on the web site.

IncubationAfter media are inoculated, they must be incubated (i.e., they must be placedinto a chamber [called an incubator] that contains the appropriate atmosphereand moisture level and is set to maintain the appropriate temperature). This iscalled incubation. To culture most human pathogens, the incubator is set at 35�to 37�C. Three types of incubators are used in a clinical microbiology laboratory:


Figure 8-4. The propermethod of inoculating anagar plate. The plate isheld in the palm of onehand. The other hand isused to lightly drag the in-oculating loop over thesurface of the solid cul-ture medium. The inocu-lating loop is held in muchthe same manner as asmall camel-hair paintbrush is held by an artistwhile applying paint to thesurface of a canvas.

■ A CO2 (carbon dioxide) incubator is an incubator to which a cylinder ofCO2 is attached. CO2 is periodically introduced into the incubator tomaintain a CO2 concentration of about 5% to 10%. Such an incubatoris used to isolate capnophiles (organisms that grow best in atmospherescontaining increased CO2). It is important to keep in mind that a CO2incubator contains oxygen (about 15% to 20%) in addition to CO2.Thus, a CO2 incubator is not an anaerobic incubator.

■ A non-CO2 incubator is an incubator containing room air; thus, it con-tains about 20% to 21% O2.

■ An anaerobic incubator is an incubator containing an atmosphere de-void of oxygen.

Once a particular species of bacteria has been isolated from a clinical speci-men, it can be separated from any other organisms that were present in the spec-imen and can be grown as a pure culture. The term pure culture refers to the factthat there is only one bacterial species present. The changes in a bacterial pop-ulation over an extended period follows a definite predictable pattern that canbe shown by plotting the population growth curve on a graph (discussed later inthis chapter).

Bacterial Population CountsMicrobiologists sometimes need to know how many bacteria are present in aparticular liquid at any given time (e.g., to determine the degree of bacterial con-tamination in drinking water, milk, and other foods). The microbiologist may (1)determine the total number of bacterial cells in the liquid (the total numberwould include both viable and dead cells) or (2) determine the number of viable(living) cells.

Various types of instruments are available to determine the total number ofcells (e.g., a spectrophotometer could be used). In a spectrophotometer, a beamof light is passed through the liquid. When no bacteria are present in the liquid,the liquid is clear, and a large amount of light passes through. As bacteria in-crease in number, the liquid becomes turbid (cloudy), and less light passesthrough. Turbidity increases (i.e., the solution becomes more cloudy) as the num-ber of organisms increases; therefore, the amount of transmitted light decreasesas the bacteria increase in number. Formulas are available to equate the amountof transmitted light to the concentration of organisms in the liquid, which is usu-ally expressed as the number of organisms per milliliter (mL) of suspension.

The viable plate count is used to determine the number of viable bacteria ina liquid sample, such as milk, water, ground food diluted in water, or a broth cul-ture. In this procedure, serial dilutions of the sample are prepared, and then 0.1mL or 1.0 mL aliquots (portions) are inoculated onto plates of nutrient agar.Following overnight incubation, the number of colonies are counted. (Usually, aplate containing 30 to 300 colonies is used.) To determine the concentration ofbacteria in the original sample, the number of colonies must be multiplied by thedilution factor(s). For example, if 220 colonies were counted on an agar platethat had been inoculated with a 1.0 mL sample of a 1:10,000 dilution, there were220 � 10,000 � 2,200,000 bacteria/ mL of the original material at the time the di-

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lutions were made and cultured. If, however, 220 colonies were counted on anagar plate that had been inoculated with a 0.1 mL sample of a 1:10,000 dilution,there were 220 � 10 � 10,000 � 22,000,000 bacteria/mL of the original materialat the time the dilutions were made and cultured.

In the clinical microbiology laboratory, a viable cell count is an importantpart of a urine culture. (The technique is described in Chapter 13.) The numberof viable bacteria per milliliter of a urine specimen is used as an indicator of aurinary tract infection (UTI). As explained in Chapter 13, high colony countsmay also be due to contamination of the urine specimen with indigenous mi-croflora during specimen collection or failure to refrigerate the specimen be-tween collection and transport to the laboratory.

Bacterial Population Growth CurveA population growth curve for any particular species of bacterium may be de-termined by growing a pure culture of the organism in a liquid medium at a con-stant temperature. Samples of the culture are collected at fixed intervals (e.g.,every 30 minutes), and the number of viable organisms in each sample is deter-mined. The data are then plotted on logarithmic graph paper. The graph inFigure 8–5 was obtained by plotting the logarithm (log10) of the number of vi-able bacteria (on the vertical or Y axis) against the incubation time (on the hor-izontal or X axis). (If you are not familiar with logarithms, refer to a math book.)

The growth curve consists of the following four phases.

1. The first phase of the growth curve is the lag phase (A in Fig. 8–5), duringwhich the bacteria absorb nutrients, synthesize enzymes, and prepare forcell division. The bacteria do not increase in number during the lag phase.


Log of thenumber oforganismsper mL

Time (hours)

A

B D

C

Figure 8-5. A population growth curve of living organisms. The logarithm of the number ofbacteria per milliliter of medium is plotted against time. (A) Lag phase. (B) Logarithmic growthphase. (C) Stationary phase. (D) Death phase.

2. The second phase of the growth curve is the logarithmic growth phase(also known as the log phase or exponential growth phase; B in Fig.8–5). In the log phase, the bacteria multiply so rapidly that the numberof organisms doubles with each generation time (i.e., the number of bac-teria increases exponentially). Growth rate is the greatest during the logphase. The log phase is always brief, unless the rapidly dividing cultureis maintained by constant addition of nutrients and frequent removal ofwaste products. When plotted on logarithmic graph paper, the log phaseappears as a steeply sloped straight line.

3. As the nutrients in the liquid medium are used up and the concentrationof toxic waste products from the metabolizing bacteria build up, the rateof division slows, such that the number of bacteria that are dividingequals the number that are dying. The result is the stationary phase (Cin Fig. 8–5). It is during this phase that the culture is at its greatest pop-ulation density.

4. As overcrowding occurs, the concentration of toxic waste products con-tinues to increase and the nutrient supply decreases. The microorgan-isms then die at a rapid rate; this is the death phase or decline phase (Din Fig. 8–5). The culture may die completely, or a few microorganismsmay continue to survive for months. If the bacterial species is a spore-former, it will produce spores to survive beyond this phase. When cellsare observed in old cultures of bacteria in the death phase, some of themlook different from healthy organisms seen in the log phase. As a resultof unfavorable conditions, morphological changes in the cells may ap-pear. Some cells undergo involution and assume a variety of shapes, be-coming long, filamentous rods or branching or globular forms that aredifficult to identify. Some develop without a cell wall and are referred toas protoplasts, spheroplasts, or L-phase variants (L-forms). When theseinvoluted forms are inoculated into a fresh nutrient medium, they usu-ally revert to the original shape of the healthy bacteria.

Many industrial and research procedures depend on the maintenance of anessential species of microorganism. These are continuously cultured in a con-trolled environment called a chemostat (Fig. 8–6), which regulates the supplyof nutrients and the removal of waste products and excess microorganisms.Chemostats are used in industries where yeast is grown to produce beer andwine, 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 con-stant source of microorganisms.

Culturing Obligate Intracellular Pathogens in the Laboratory

Recall from Chapter 4 that obligate intracellular pathogens are microorganismsthat can only survive and multiply within living cells (called host cells). Obligate in-tracellular pathogens include viruses and two groups of Gram-negative bacteria—rickettsias and chlamydias. Because obligate intracellular pathogens will not growon artificial (synthetic) media, they present a challenge to laboratorians when large

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numbers of the organisms are required for diagnostic or research purposes (e.g.,development of vaccines and new drugs). To grow such organisms in the labora-tory, they must be inoculated into embryonated chicken eggs, laboratory animals,or cell cultures.

Culturing Fungi in the Laboratory

Fungi (including yeasts, molds, and dimorphic fungi) will grow on and in a vari-ety of solid and liquid culture media. There is no one medium that is best for allmedically important fungi. Examples of solid culture media used to grow fungiinclude brain heart infusion (BHI) agar, BHI agar with blood, and Sabourauddextrose agar (SDA). Antibacterial agents are often added to the media to sup-press the growth of bacteria. The low pH of SDA (pH 5.6) inhibits the growth ofmost bacteria; thus, SDA is selective for fungi. Laboratory personnel must exer-cise caution when culturing fungi, because the spores of certain fungi are highlyinfectious. A Class II biological safety cabinet must be used.

Culturing Protozoa in the Laboratory

Most clinical microbiology laboratories do not culture protozoa, but techniquesare available for culturing protozoa in reference and research laboratories.Examples of protozoa that can be cultured in vitro are amebae (e.g.,Acanthamoeba spp., Balamuthia spp., Entamoeba histolytica, Naegleria fowleri,Giardia lamblia, Leishmania spp., Toxoplasma gondii, Trichomonas vaginalis,and Trypanosoma cruzi). Of these protozoa, it is of greatest importance to cul-ture Acanthamoeba, Balamuthia, and Naegleria fowleri in a clinical microbiology


Freshmedium

Stopcock tocontrol rate

Fritted glass discto break air

into tiny bubbles Growthchamber

Collection vessel

Forcedsterileair

Figure 8-6. Chemostat used forcontinuous cultures. Rates ofgrowth can be controlled eitherby controlling the rate at whichnew medium enters the growthchamber or by limiting a requiredgrowth factor in the medium.

laboratory. These amebae can cause serious (often fatal) infections of the cen-tral nervous system—infections that are difficult to diagnose by other methods.Parasitic protozoa are further discussed in Chapter 18.

INHIBITING THE GROWTH OFMICROORGANISMS IN VITRO

In certain environments, it is necessary and/or desirable to inhibit the growth ofmicrobes. In hospitals, nursing homes, and other healthcare institutions, for ex-ample, it is necessary to inhibit the growth of pathogens so that they will not in-fect patients, staff members, or visitors. Other environments where it is neces-sary and/or desirable to inhibit microbial growth include food and beverageprocessing plants, restaurants, kitchens, and bathrooms.

Definition of Terms

Before discussing the various methods used to destroy or inhibit the growth ofmicrobes, a number of terms should be understood as they apply to microbiology.

SterilizationSterilization is the complete destruction of all living organisms, including cells,spores, and viruses. When something is sterile, it is devoid of microbial life.Sterilization of objects can be accomplished by dry heat, autoclaving (steam un-der pressure), gas (ethylene oxide), various chemicals (such as formaldehyde),and certain types of radiation (e.g., ultraviolet light and gamma rays). Thesetechniques are discussed later in this chapter.

Disinfection, Pasteurization, Disinfectants, Antiseptics, and SanitizationDisinfection is the destruction or removal of pathogens from nonliving objectsby physical or chemical methods. The heating process developed by Pasteurto kill microbes in wine—pasteurization—is a method of disinfecting liquids.Pasteurization is used today to eliminate pathogens from milk and most otherbeverages. It should be remembered that pasteurization is not a sterilization pro-cedure, because not all microbes are destroyed.

Chemical agents are also used to eliminate pathogens. Chemicals used todisinfect inanimate objects, such as bedside equipment and operating rooms, arecalled disinfectants. Disinfectants are strong chemical substances that cannot beused on living tissue. Antiseptics are solutions used to disinfect skin and otherliving tissues. Sanitization is the reduction of microbial populations to levelsconsidered safe by public health standards, such as those applied to restaurants.

Microbicidal AgentsThe suffix “-cide” or “-cidal” refers to “killing,” as in the words homicide andsuicide. General terms like germicidal agents (germicides), biocidal agents (bio-cides), and microbicidal agents (microbicides) are disinfectants that kill mi-crobes. Bactericidal agents (bactericides) are disinfectants that specifically killbacteria but not necessarily bacterial endospores. Because spore coats are thick

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and resistant to the effects of many disinfectants, sporicidal agents are requiredto kill bacterial endospores. Fungicidal agents (fungicides) kill fungi, includingfungal spores. Algicidal agents (algicides) are used to kill algae in swimmingpools and hot tubs. Viricidal agents (or virucidal agents) destroy viruses.Pseudomonicidal agents kill Pseudomonas species, and tuberculocidal agentskill Mycobacterium tuberculosis.

Microbistatic AgentsA microbistatic agent is a drug or chemical that inhibits growth and reproduc-tion of microorganisms. A bacteriostatic agent is one that specifically inhibitsthe metabolism and reproduction of bacteria. Some of the drugs used to treatbacterial diseases are bacteriostatic, whereas others are bactericidal. Freeze-drying (lyophilization) and rapid freezing (using liquid nitrogen) are microbista-tic techniques that are used to preserve microbes for future use or study.

Lyophilization is a process that combines dehydration (drying) and freez-ing. Lyophilized materials are frozen in a vacuum; the container is then sealed tomaintain the inactive state. This freeze-drying method is widely used in industryto preserve foods, antibiotics, antisera, microorganisms, and other biological ma-terials. It should be remembered that lyophilization cannot be used to kill mi-croorganisms, but rather, is used to prevent them from reproducing and to storethem for future use.

Sepsis, Asepsis, Aseptic Technique, Antisepsis, and Antiseptic TechniqueSepsis refers to the presence of pathogens in blood or tissues, whereas asepsismeans the absence of pathogens. Various techniques, collectively referred to asaseptic techniques, are employed to eliminate and exclude pathogens. Aseptictechniques include hand washing; the use of sterile gloves, masks, and gowns;sterilization of surgical instruments and other equipment; and the use of dis-infectants, including antiseptics. Antisepsis is the prevention of infection.Antiseptic technique, developed by Joseph Lister in 1867, refers to the use of an-tiseptics. Antiseptic technique is a type of aseptic technique. Lister used dilutecarbolic acid (phenol) to cleanse surgical wounds and equipment and a carbolicacid aerosol to prevent harmful microorganisms from entering the surgical fieldor contaminating the patient.

Sterile TechniqueSterile technique is practiced when it is necessary to exclude all microorganismsfrom a particular area, so that the area will be sterile. Earlier in this chapter, youlearned of the importance of using sterile technique in the microbiology labora-tory when inoculating culture media. In Chapter 12, you will learn how steriletechnique is employed in other areas of the hospital (e.g., in the operating room).

Using Physical Methods to Inhibit Microbial Growth

The methods used to destroy or inhibit microbial life are either physical orchemical, and sometimes both types are used. The physical methods commonlyused in hospitals, clinics, and laboratories to destroy or control pathogens


include heat, the combination of heat and pressure, desiccation, radiation, sonicdisruption, and filtration.

HeatHeat is the most practical, efficient, and inexpensive method of sterilization ofthose inanimate objects and materials that can withstand high temperatures.Because of these advantages, it is the means most frequently employed.

Two factors, temperature and time, determine the effectiveness of heat forsterilization. There is considerable variation from organism to organism in sus-ceptibility to heat; pathogens usually are more susceptible than nonpathogens.Also, the higher the temperature, the shorter the time required to kill the or-ganisms. The thermal death point (TDP) of any particular species of microor-ganism is the lowest temperature that will kill all the organisms in a standardizedpure culture within a specified period. The thermal death time (TDT) is thelength of time necessary to sterilize a pure culture at a specified temperature.

In practical applications of heat for sterilization, one must consider the ma-terial in which a mixture of microorganisms and/or their spores may be found.Pus, feces, vomitus, mucus, and blood contain proteins that serve as a protectivecoating to insulate the pathogens; when these substances are present on bedding,bandages, surgical instruments, and syringes, very high temperatures are requiredto destroy vegetative (growing) microorganisms and spores. In practice, the mosteffective procedure is to wash away the protein debris with strong soap, hot wa-ter, and a disinfectant, and then sterilize the equipment or materials with heat.

Dry Heat. Dry heat baking in a thermostatically controlled oven provideseffective sterilization of metals, glassware, some powders, oils, and waxes. Theseitems must be baked at 160� to 165�C for 2 hours or at 170� to 180�C for 1 hour.An ordinary oven of the type found in most homes may be used if the tempera-ture remains constant. The effectiveness of dry heat sterilization depends onhow deeply the heat penetrates throughout the material, and the items to bebaked must be positioned so that the hot air circulates freely among them.

Incineration (burning) is an effective means of destroying contaminated dis-posable materials. An incinerator must never be overloaded with moist or protein-laden materials, such as feces, vomitus, or pus, because the contaminating micro-organisms within these moist substances may not be destroyed if the heat doesnot readily penetrate and burn them. Flaming the surface of metal forceps andwire bacteriological loops is an effective way to kill microorganisms and, formany years, was a common laboratory procedure. Flaming is accomplished bybriefly holding the end of the loop or forceps in the yellow portion of a gas flame(Fig. 8–7). Open flames are dangerous, however, and, for this reason, are rarelyused in modern laboratories, where sterile, disposable, plastic inoculating loopsare primarily used. Today, whenever wire inoculating loops are used, heat steril-ization is usually accomplished using electrical heating devices (Fig. 8–7).

Moist Heat. Heat applied in the presence of moisture, as in boiling or steam-ing, is faster and more effective than dry heat, and can be accomplished at a

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lower temperature; thus, it is less destructive to many materials that otherwisewould be damaged at higher temperatures. Moist heat causes proteins to coagu-late (as occurs when eggs are hard boiled). Because cellular enzymes are pro-teins, they are inactivated by moist heat, leading to cell death.

The vegetative forms of most pathogens are quite easily destroyed by boil-ing for 30 minutes. Thus, clean articles made of metal and glass, such as syringes,needles, and simple instruments, may be disinfected by boiling for 30 minutes.Since the temperature at which water boils is lower at higher altitudes, watershould always be boiled for longer times at high altitudes. Boiling is not alwayseffective, however, because heat-resistant bacterial endospores, mycobacteria,and viruses may be present. The endospores of the bacteria that cause anthrax,tetanus, gas gangrene, and botulism, as well as hepatitis viruses, are especiallyheat resistant and often survive boiling. Also, because thermophiles thrive athigh temperatures, boiling is not an effective means of killing them.

An autoclave is like a large metal pressure cooker that uses steam underpressure to completely destroy all microbial life (Fig. 8–8). The increased pres-sure raises the temperature above the temperature of boiling water (i.e., above100�C), and forces the steam into the materials being sterilized. Autoclaving ata pressure of 15 pounds per square inch (psi), at a temperature of 121.5�C, for 20minutes, kills vegetative microorganisms, bacterial endospores, and viruses, aslong as they are not protected by pus, feces, vomitus, blood, or other proteina-ceous substances. Some types of equipment and certain materials, such as rub-ber, which may be damaged by high temperatures, can be autoclaved at lowertemperatures for longer periods. The timing must be carefully determined basedon the contents and compactness of the load. All articles must be properly pack-aged and arranged within the autoclave to allow steam to penetrate each package


Figure 8-7. Dry heat sterilization. (A) Flaming a wire inoculating loop in a Bunsen burnerflame. (B) Sterilizing a wire inoculating loop using an electrical heating device.

A B

completely. Cans should remain open, bottles covered loosely with foil or cot-ton, and instruments wrapped in cloth. Sealed containers should not be auto-claved. Pressure-sensitive autoclave tape (Fig. 8–9) and commercially availablesolutions containing bacterial spores can be used as quality control measures toensure that autoclaves are functioning properly.

Home canning done without the use of a pressure cooker does not destroythe endospores of bacteria—notably the anaerobe, Clostridium botulinum.Occasionally, local newspapers report cases of food poisoning resulting from theingestion of C. botulinum toxins in improperly canned fruits, vegetables, and

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Figure 8-8. A large,built-in autoclave.(Photograph courtesy ofDr. Janet Duben-Engelkirk and Scott &White MemorialHospital, Temple, TX.)

Figure 8-9. Pressure-sensitive autoclave tape showing dark stripes after sterilization. (VolkWA, et al.: Essentials of Medical Microbiology, 4th ed. Philadelphia, JB Lippincott, 1991.)

meats. Botulism food poisoning is preventable by properly washing and pressurecooking (autoclaving) food.

An effective way to disinfect clothing, bedding, and dishes is to use hot wa-ter (greater than 60�C) with detergent or soap and to agitate the solution aroundthe items. This combination of heat, mechanical action, and chemical inhibitionis deadly to most pathogens.

ColdMost microorganisms are not killed by cold temperatures and freezing, but theirmetabolic activities are slowed, greatly inhibiting their growth. Refrigerationmerely slows the growth of most microorganisms; it does not completely inhibitgrowth. Slow freezing causes ice crystals to form within cells and may rupturethe cell membranes and cell walls of some bacteria; hence, slow freezing shouldnot be used as a way to preserve or store bacteria. Rapid freezing, using liquidnitrogen, is a good way to preserve foods, biologic specimens, and bacterial cul-tures. It puts bacteria into a state of suspended animation. Then, when the tem-perature is raised above the freezing point, the organisms’ metabolic reactionsspeed up and the organisms begin to reproduce again.

Persons who are involved in the preparation and preservation of foods mustbe aware that thawing foods allows bacterial spores in the foods to germinateand microorganisms to resume growth. Consequently, refreezing of thawedfoods is an unsafe practice, because it preserves the millions of microbes thatmight be present, leading to rapid deterioration of the food when it is rethawed.Also, if the endospores of Clostridium botulinum or C. perfringens were present,the viable bacteria would begin to produce the toxins that cause food poisoning.

DesiccationFor many centuries, foods have been preserved by drying. When moisture andnutrients are lacking, many dried microorganisms remain viable, although theycannot reproduce. Foods, antisera, toxins, antitoxins, antibiotics, and pure cul-tures of microorganisms are often preserved by lyophilization (freeze-drying;discussed previously).

In the hospital or clinical environment, healthcare professionals should keepin mind that dried viable pathogens may be lurking in dried matter, includingblood, pus, fecal material, and dust that are found on floors, in bedding, on cloth-ing, and in wound dressings. Should these dried materials be disturbed, such as bydry dusting, the microbes would be easily transmitted through the air or by con-tact. They would then grow rapidly if they settled in a suitable moist, warm nu-trient environment such as a wound or a burn. Therefore, important precautionsthat must be observed include wet mopping of floors, damp dusting of furniture,rolling bed linens and towels carefully, and proper disposal of wound dressings.

RadiationThe sun is not a particularly reliable disinfecting agent because it kills only thosemicroorganisms that are exposed to direct sunlight. The rays of the sun includethe long infrared (heat) rays, the visible light rays, and the shorter ultraviolet(UV) rays. The UV rays, which do not penetrate glass and building materials,


are effective only in the air and on surfaces. They do, however, penetrate cellsand, thus, can cause damage to DNA. When this occurs, genes may be so se-verely damaged that the cell dies (especially unicellular microorganisms) or isdrastically changed.

In practice, a UV lamp (often called a germicidal lamp) is useful for reduc-ing the number of microorganisms in the air. Its main component is a low-pressuremercury vapor tube. Such lamps are found in newborn nurseries, operatingrooms, elevators, entry ways, cafeterias, and classrooms, where they are incor-porated into louvered ceiling fixtures designed to radiate across the top of theroom without striking people in the room. Sterility may also be maintained byhaving a UV lamp placed in a hood or cabinet containing instruments, paper andcloth equipment, liquid, and other inanimate articles. Many biologic materials,such as sera, antisera, toxins, and vaccines, are sterilized with UV rays.

Those whose work involves the use of UV lamps must be particularly care-ful not to expose their eyes or skin to the rays, because they can cause seriousburns and cellular damage. Because UV rays do not penetrate cloth, metals, andglass, these materials may be used to protect persons working in a UV environ-ment. It has been shown that skin cancer can be caused by excessive exposure tothe UV rays of the sun; thus, extensive suntanning is harmful.

X-rays and gamma and beta rays of certain wavelengths from radioactivematerials may be lethal or cause mutations in microorganisms and tissue cells,because they damage DNA and proteins within those cells. Studies performed inradiation research laboratories have demonstrated that these radiations can beused for the prevention of food spoilage, sterilization of heat-sensitive surgicalequipment, preparation of vaccines, and treatment of some chronic diseasessuch as cancer, all of which are very practical applications for laboratory re-search. The Food and Drug Administration (FDA) approved the use of gammarays (from cobalt-60) to process chickens and red meat in 1992 and 1997, re-spectively. Since then, gamma rays have been used by some food processingplants to kill pathogens (like Salmonella and Campylobacter spp.) in chickens;the chickens are labeled “irradiated” and marked with the green internationalsymbol for radiation.

Ultrasonic WavesIn hospitals, medical clinics, and dental clinics, ultrasonic waves are a frequentlyused means of cleaning and sterilizing delicate equipment. Ultrasonic cleanersconsist of tanks filled with liquid solvent (usually water); the short sound wavesare then passed through the liquid. The sound waves mechanically dislodge or-ganic debris on instruments and glassware. Glassware and other articles thathave been cleansed in ultrasonic equipment must be washed to remove the dis-lodged particles and solvent and are then sterilized by another method beforethey are used.

FiltrationFilters of various pore sizes are used to filter or separate cells, larger viruses, bac-teria, and certain other microorganisms from the liquids or gases in which they

216 CHAPTER 8

are suspended. Filters with tiny pore sizes (called millipore filters) are used inlaboratories to filter bacteria and viruses out of liquids. The variety of filters islarge and includes sintered glass (in which uniform particles of glass are fused),plastic films, unglazed porcelain, asbestos, diatomaceous earth, and cellulosemembrane filters. Small quantities of liquid can be filtered through a syringe, butlarge quantities require larger apparatuses.

A cotton plug in a test tube, flask, or pipette is a good filter for preventingthe entry of microorganisms. Dry gauze and paper masks prevent the outwardpassage of microbes from the mouth and nose, at the same time protecting thewearer from inhaling airborne pathogens and foreign particles that could dam-age the lungs. Biological safety cabinets contain high-efficiency particulate air(HEPA) filters to protect workers from contamination. HEPA filters are also lo-cated in operating rooms and patient rooms to filter the air that enters or exitsthe room.

Gaseous AtmosphereIn limited situations, it is possible to inhibit growth of microorganisms by alter-ing the atmosphere in which they are located. Because aerobes and mi-croaerophiles require oxygen, they can be killed by placing them into an atmos-phere devoid of oxygen or by removing oxygen from the environment in whichthey are living. Conversely, obligate anaerobes can be killed by placing theminto an atmosphere containing oxygen or by adding oxygen to the environmentin which they are living. For instance, wounds likely to contain anaerobes arelanced (opened) to expose them to oxygen. Another example is gas gangrene, adeep wound infection that causes rapid destruction of tissues. Gas gangrene iscaused by various anaerobes in the genus Clostridium. In addition to debride-ment of the wound and the use of antibiotics, gas gangrene can be treated byplacing the patient in a hyperbaric (meaning increased pressure) oxygen cham-ber or in a room with high oxygen pressure. Because of the pressure, oxygen isforced into the wound, providing oxygen to the oxygen-starved tissue and killingthe clostridia.

Using Chemical Agents to Inhibit Microbial Growth

DisinfectantsChemical disinfection refers to the use of chemical agents to inhibit the growthof pathogens, either temporarily or permanently. The mechanism by which var-ious disinfectants kill cells varies from one disinfectant to another. Various fac-tors affect the efficiency or effectiveness of a disinfectant (Fig. 8–10), and thesefactors must be taken into consideration whenever a disinfectant is used. Thesefactors include the following:

■ Prior cleaning of the object or surface to be disinfected■ The organic load that is present, meaning the presence of organic mat-

ter (e.g., feces, blood, vomitus, pus) on the materials being treated■ The bioburden, meaning the type and level of microbial contamination■ The concentration of the disinfectant


■ The contact time, meaning the amount of time that the disinfectant mustremain in contact with the organisms in order to kill them (see thisbook’s web site for “A Closer Look at Contact Time”)

■ The physical nature of the object being disinfected (e.g., smooth orrough surface, crevices, hinges)

■ Temperature and pH

Directions for preparing the proper dilution of a disinfectant must be fol-lowed carefully, because too weak or too strong a concentration is usually lesseffective than the proper concentration. (Information about preparing dilu-tions can be found on this book’s web site.) The items to be disinfected mustfirst be washed to remove any proteinaceous material in which pathogens maybe hidden. Although the washed article may then be clean, it is not safe to useuntil it has been properly disinfected. Healthcare personnel need to under-stand an important limitation of chemical disinfection—that many disinfec-tants that are effective against pathogens in the controlled conditions of thelaboratory may be ineffective in the actual hospital or clinical environment.Furthermore, the stronger and more effective antimicrobial chemical agentsare of limited usefulness because of their destructiveness to human tissues andcertain other substances.

Almost all bacteria in the vegetative state, as well as fungi, protozoa, andmost viruses, are susceptible to many disinfectants, although the mycobacteriathat cause tuberculosis and leprosy, bacterial endospores, pseudomonads, fungalspores, and hepatitis viruses are notably resistant. Therefore, chemical disinfec-tion should never be attempted when it is possible to use proper physical steril-ization techniques.

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Effectiveness ofEffectiveness ofantimicrobialantimicrobialproceduresprocedures

Presence of proteinsin feces, blood,vomitus, pus

Time

Temperature

Concentration

Type of microbesNumber of microbesPresence of spores

Figure 8-10. Factors that determine the effectiveness of any antimicrobial procedure: time,temperature, concentration, the type and number of microbes present, the presence ofspores, and the presence of proteinaceous materials.

The disinfectant most effective for each situation must be chosen carefully.Chemical agents used to disinfect respiratory therapy equipment and thermome-ters must destroy all pathogenic bacteria, fungi, and viruses that may be found insputum and saliva. One must be particularly aware of the oral and respiratorypathogens, including Mycobacterium tuberculosis; species of Pseudomonas,Staphylococcus, and Streptococcus; the various fungi that cause candidiasis, blas-tomycosis, coccidioidomycosis, and histoplasmosis; and all the respiratory viruses.

Because most disinfection methods do not destroy all bacterial endosporesthat are present, any instrument or dressing used in the treatment of an infectedwound or a disease caused by sporeformers must be autoclaved or incinerated.Gas gangrene, tetanus, and anthrax are examples of diseases caused by spore-formers that require the healthcare worker to take such precautions.Formaldehyde and ethylene oxide, when properly used, are highly destructive tospores, mycobacteria, and viruses. Certain articles are heat sensitive and cannotbe autoclaved or safely washed before disinfection; such articles are soaked for24 hours in a strong detergent and disinfectant solution, washed, and then ster-ilized in an ethylene oxide autoclave. The use of disposable equipment wheneverpossible in these situations helps to protect patients and healthcare personnel.

The effectiveness of a chemical agent depends to some extent on the physi-cal characteristics of the article on which it is used. A smooth, hard surface isreadily disinfected, whereas a rough, porous, or grooved surface is not. Thoughtmust be given to selection of the most suitable germicide for cleaning patientrooms and all other areas where patients are treated.

The most effective antiseptic or disinfectant should be chosen for the spe-cific purpose, environment, and pathogen or pathogens likely to be present. Thecharacteristics of an ideal chemical antimicrobial agent include the following:

■ It should have a wide or broad antimicrobial spectrum, meaning that itshould kill a wide variety of microorganisms

■ It should be fast-acting, meaning that the contact time should be short■ It should not be affected by the presence of organic matter■ It must be nontoxic to human tissues and noncorrosive and nondestruc-

tive to materials on which it is used (for instance, if a tincture [e.g.,alcohol-water solution] is being used, evaporation of the alcohol solventcan cause a 1% solution to increase to a 10% solution, and at this con-centration, it may cause tissue damage)

■ It should leave a residual antimicrobial film on the treated surface■ It must be soluble in water and easy to apply■ It should be inexpensive and easy to prepare, with simple, specific di-

rections■ It must be stable both as a concentrate and as a working dilution, so that

it can be shipped and stored for reasonable periods■ It should be odorless

How do disinfectants kill microorganisms? Some disinfectants (e.g., surface-active soaps and detergents, alcohols, and phenolic compounds) target and de-stroy cell membranes. Others (e.g., halogens, hydrogen peroxide, salts of heavy


metals, formaldehyde, and ethylene oxide) destroy enzymes and structural pro-teins. Others attack cell walls or nucleic acids. Some of the disinfectants that arecommonly used in hospitals are discussed in Chapter 12.

The effectiveness of phenol as a disinfectant was demonstrated by JosephLister in 1867, when it was used to reduce the incidence of infections followingsurgical procedures. The effectiveness of other disinfectants is compared withthat of phenol using the phenol coefficient test. To perform this test, a series ofdilutions of phenol and the experimental disinfectant are inoculated with the testbacteria, Salmonella typhi and Staphylococcus aureus, at 37�C. The highest dilu-tions (lowest concentrations) that kill the bacteria after 10 minutes are used tocalculate the phenol coefficient.

AntisepticsMost antimicrobial chemical agents are too irritating and destructive to be ap-plied to mucous membranes and skin. Those that may be used safely on humantissues are called antiseptics. An antiseptic merely reduces the number of organ-isms on a surface; it does not penetrate pores and hair follicles to destroy mi-croorganisms residing there. To remove organisms lodged in pores and folds ofthe skin, healthcare personnel use an antiseptic soap and scrub with a brush. Toprevent resident indigenous microflora from contaminating the surgical field,surgeons wear sterile gloves on freshly scrubbed hands, and masks and hoods tocover their face and hair. Also, an antiseptic is applied at the site of the surgicalincision to destroy local microorganisms.

Inhibiting the Growth of Pathogens in Our Kitchens

Many of the foods that we bring into and work with in our kitchens are contam-inated with pathogens (see “Insight: Microbes in Our Food” on the web site).For example, gastrointestinal pathogens such as E. coli O157:H7, Salmonella,and Campylobacter are often present on poultry, ground beef, and other meatproducts. Salmonella and Campylobacter may also be present within and on thesurface of eggs. Protozoan parasites also gain access to our kitchens via contam-inated foods. Toxoplasma gondii cysts may be present in meat, especially porkor mutton, and Cyclospora outbreaks in 1996 and 1997 were associated with im-ported raspberries.

Assuming that the meat and poultry that you serve to your family are prop-erly and thoroughly cooked, the pathogens that are present on the surface of, orwithin, these foods are usually killed. They are not the problem. The real prob-lem concerns the handling of these foods before cooking them. As we handle andprepare foods in the kitchen, pathogens from the foods get on our hands,counter tops, plates, cutting boards, knives, and almost anything else that wetouch in the kitchen.

Here is a typical scenario. You place a package of chicken breasts on a plateand then unwrap it. You then place the chicken breasts, one at a time, on a cut-ting board and trim away the excess fat with a knife. By the time the chickenbreasts are placed into the oven, the plate, the cutting board, the knife, yourhands, and anything that you have touched with your hands have become con-

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taminated. Now you take a head of lettuce from the refrigerator, place it on thecutting board, and proceed to chop it for use in a salad. It is quite possible thatany pathogens that were present on your hands, the knife, or the cutting boardare now in the salad. It is not likely that you will be cooking the salad, so laterwhen you eat it, you and your family will be ingesting live pathogens.

It is very important that you remain aware of the presence of pathogens asyou prepare foods in the kitchen and take steps to eliminate contamination ofyourself, your kitchen, and other foods with those pathogens. Wash your handsoften, using hot water and antibacterial soap. Do not merely rinse them. In thekitchen, just as in the hospital setting, frequent and thorough handwashing is themost important way to prevent the transmission of pathogens.

Using hot water, thoroughly rinse poultry and meat blood from plates, and thenplace them in the dishwasher or wash them with hot, soapy water. Do not use themfor anything else until after they have been washed. Always wash knives beforereusing them. After working with poultry and meat, be sure to thoroughly washcounter tops and cutting boards with hot, soapy water. Because bacteria can get be-tween the slats in wooden cutting boards, consider replacing them with smooth plas-tic cutting boards. Use an antibacterial kitchen spray to clean counter tops, refriger-ator and oven handles, and anything else that you touched as you prepared the food.Remember to follow the manufacturer’s directions regarding the length of time toleave the disinfectant in place before wiping it off. Because wet sponges and dish-cloths are havens for pathogens, be sure to wash or replace them often. Wheneverpossible, people should place their sponges or dishcloths in the dishwasher eachtime they wash dishes and should never use dish towels to dry their hands.

Controversies Relating to the Use of AntimicrobialAgents in Animal Feed and Household Products

It has been estimated that approximately 40% of the antibiotics manufactured inthe United States are used in animal feed. The reason is obvious: to prevent in-fectious diseases in farm animals—infections that could lead to huge economicloses for farmers and ranchers. The problem is that when antibiotics are fed to ananimal, the antibiotics kill any indigenous microflora organisms that are suscep-tible to the antibiotics. But what survives? Any organisms that are resistant to theantibiotics. Having less competition now for space and nutrients, these drug-resistant organisms multiply and become the predominant organisms of the ani-mal’s indigenous microflora. These drug-resistant organisms are then transmittedin the animal’s feces or food products (e.g., eggs, milk, and meat) obtained fromthe animal. Many multi–drug-resistant Salmonella strains—strains that cause dis-ease in animals and humans—developed in this manner. The use of antibiotic-containing animal feed is quite controversial. Microbiologists concerned aboutever increasing numbers of drug-resistant bacteria are currently attempting toeliminate or drastically reduce the practice of adding antibiotics to animal feed.

Another controversy concerns the antimicrobial agents that are being addedto toys, cutting boards, hand soaps, antibacterial kitchen sprays, and many otherhousehold products. The antimicrobial agents in these products kill any organ-isms that are susceptible to these drugs, but what survives? Any organisms that


are resistant to these agents. These drug-resistant organisms then multiply andbecome the predominant organisms in the home. Should a member of the house-hold become infected with these drug-resistant and multi–drug-resistant organ-isms, the infection will be more difficult to treat. Concerned microbiologists arecurrently attempting to eliminate or drastically reduce the practice of adding an-timicrobial agents to household products.

Another argument against the use of antimicrobial agents in the home con-cerns the immune system. Many scientists believe that children must be exposedto all sorts of microorganisms during their growth and development so that theirimmune systems will develop correctly and be capable of properly responding topathogens in later years. The use of household products containing antimicro-bial agents might be eliminating the very organisms that are essential for propermaturation of the immune system.

222 CHAPTER 8

■ Microbial growth is affected by many differ-ent environmental factors, including theavailability of nutrients and moisture, tem-perature, pH, osmotic pressure, barometricpressure, and composition of the atmosphere.

■ Some microorganisms (thermophiles) preferto live in hot temperatures, others (meso-philes) prefer moderate temperatures, andothers (psychrophiles) prefer cold tempera-tures. Psychrotrophs are a group of psy-chrophiles that prefer to live at refrigeratortemperature (4�C).

■ Acidophiles live in acidic environments, al-kaliphiles live in alkaline environments,halophiles live in salty environments, andbarophiles live where there is high baromet-ric pressure.

■ The growth of microorganisms is encour-aged in microbiology laboratories, researchlaboratories, and in various industries (e.g.,antibiotic industry and certain food, bever-age, and chemical industries). To culture mi-croorganisms in the laboratory, they must beprovided with appropriate nutrients, mois-ture, and the temperature, pH, and atmos-phere that they require.

■ Enriched media are necessary to growpathogens, and especially fastidiouspathogens require highly enriched media.Selective media are used to grow specifictypes of bacteria while inhibiting the growthof unwanted bacteria. Differential mediaare used to differentiate between differentgroups of bacteria (e.g., MacConkey agar isused to differentiate between lactose fer-menters and nonlactose fermenters).

■ Cell cultures, laboratory animals, or embry-onated chicken eggs are required to growobligate intracellular pathogens. They willnot grow on artificial (synthetic) media.

■ A population growth curve consists of fourphases: lag phase, log phase, stationaryphase, and death phase. Cells are healthiest

and the growth rate is greatest (shortest gen-eration time) during the logarithmic growthphase. This phase may be perpetuated in achemostat by maintaining optimum growthconditions (i.e., by adding nutrients and re-moving toxic waste products and excess mi-croorganisms).

■ It is necessary or desirable to inhibit thegrowth of microorganisms in certain environ-ments, such as various locations in hospitals(e.g., patients’ rooms, operating rooms, inten-sive care units), food and beverage processingplants, restaurants, kitchens, and bathrooms.

■ Sterilization is the complete destruction ofall microbial life, whereas disinfection is thedestruction of pathogens. Pasteurization andthe use of antiseptics are examples of disin-fection techniques.

■ A variety of physical methods can be used toinhibit microbial growth, including dry heat,moist heat, desiccation, various types of ra-diation, ultrasonic waves, and filtration.

■ Bacterial endospores and certain viruses arevery resistant to heat and desiccation.

■ A variety of chemical agents can be used toinhibit microbial growth. Bactericidal agentskill bacteria, whereas bacteriostatic agentsstop bacteria from growing and dividing.Sporicidal agents kill bacterial endospores.Fungicidal agents kill fungi. Algicidal agentskill algae. Viricidal (or virucidal) agents de-stroy viruses. Pseudomonicidal agents killPseudomonas species, and tuberculocidalagents kill Mycobacterium tuberculosis.

■ Some disinfectants target and destroy cellwalls, whereas others attack cell mem-branes. Others destroy enzymes, structuralproteins, or nucleic acids.

■ The effectiveness of a chemical disinfectantdepends on many factors, including priorcleaning of the object or surface to be disin-fected, the presence of organic matter (e.g.,feces, blood, vomitus, pus) on the materials


Review of Key Points

being treated, the type and level of micro-bial contamination, the concentration of thedisinfectant, the contact time, the physicalnature of the object being disinfected (e.g.,smooth or rough surface, crevices, hinges),temperature, and pH.

■ Lyophilization (freeze-drying) will not killmicrobes but it does prevent microbialgrowth. It is a method of preserving mi-

crobes for future use. Rapid freezing, usingliquid nitrogen, is another method of pre-serving microbes.

■ Healthcare professionals must take specialcare not to transfer potentially pathogenicmicrobes from patient to patient, fromthemselves to patients, or from patients tothemselves, by using physical and chemicalmethods to inhibit the growth of pathogens.

224 CHAPTER 8

On the Web—h t t p : / / c o n n e c t i o n . l w w . c o m / g o / b u r t o n 7 e

■ Insight■ Microbes in Our Food

■ Increase Your Knowledge■ Critical Thinking■ Additional Self-Assessment Exercises

Self-Assessment Exercises

After you have read Chapter 8, answer the following multiple choice questions.

1. It would be necessary to use a tu-berculocidal agent to kill a partic-ular species of:

a. Clostridium.b. Mycobacterium.c. Pseudomonas.d. Staphylococcus.e. Streptococcus.

2. Pasteurization is a type of whatkind of technique?

a. antisepticb. disinfectionc. medical asepticd. sterilizatione. surgical aseptic

3. The combination of freezing anddrying is known as:

a. desiccation.b. lyophilization.c. pasteurization.d. sterilization.e. tyndallization.

4. Organisms that live in andaround hydrothermal vents at thebottom of the ocean are:

a. acidophilic, psychrophilic,and halophilic.

b. halophilic, alkaliphilic, andpsychrophilic.

c. halophilic, psychrophilic, andbarophilic.

d. halophilic, thermophilic, andbarophilic.

e. mesophilic, halophilic, andbarophilic.

5. When placed into a hypertonicsolution, a bacterial cell will:

a. become crenated.b. hemolyze.c. lyse.d. shrink.e. swell.

6. To prevent Clostridium infectionsin a hospital setting, what kind ofdisinfectant should be used?

a. fungicidalb. pseudomonicidalc. sporicidald. tuberculocidale. virucidal

7. Sterilization can be accomplishedby use of:

a. an autoclave.b. antiseptics.c. disinfectants.d. medical aseptic techniques.e. pasteurization.

8. The goal of medical asepsis is tokill __________, whereas the goalof surgical asepsis is to kill__________.

a. all microorganisms . . . . .pathogens

b. bacteria . . . . . bacteria andviruses

c. nonpathogens . . . . .pathogens

d. pathogens . . . . . all micro-organisms

e. pathogens . . . . . non-pathogens

9. Which of the following types ofculture media is selective and dif-ferential?

a. blood agarb. MacConkey agarc. phenylethyl alcohol agard. Sabouraud agare. Thayer-Martin agar

10. All the following types of culturemedia are enriched and selectiveexcept:

a. blood agar.b. colistin-nalidixic acid agar.c. phenylethyl alcohol agar.d. Thayer-Martin agar.e. New York City agar.


Ch_08

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