4140296 pp045-076 lab13 · pdf filesystematists continue to grapple with the complex challenge...

Laboratory Objectives

After completing this lab topic, you should be able to:

1. Describe bacterial structure: colony morphology, cell shape, growthpatterns.

2. Describe the results of Gram staining and discuss the implications tocell wall chemistry.

3. Describe a scenario for succession of bacterial and fungal communitiesin aging milk, relating this to changes in environmental conditions suchas pH and nutrient availability.

4. Practice aseptic techniques producing bacterial streaks, smears, and lawns.

5. Describe the ecology and control of bacteria, applying these conceptsto life situations.

6. Describe several common bacterial relationships.

Introduction

Humans have named and categorized organisms for hundreds—perhapseven thousands—of years. Taxonomy is an important branch of biology thatdeals with naming and classifying organisms into distinct groups or cate-gories. Much of the work of early taxonomists included recording charac-teristics of organisms and grouping them based on appearance, habitat, orperhaps medicinal value. As scientists began to understand the processes ofgenetics and evolution by natural selection, they realized the value of classi-fying organisms based on phylogeny, or evolutionary history. Informationabout phylogeny was obtained from studies of development or homologousfeatures—common features resulting from common genes. In recent years,scientists have begun using biochemical evidence—studies of nucleic acidsand proteins—to investigate relationships among organisms, leading to revi-sions in the taxonomic scheme.

Systematists continue to grapple with the complex challenge of organizingthe diversity of life into categories. A three-domain system proposed in thelate 1970s is becoming widely accepted, replacing Robert Whittaker’s five-kingdom system used since 1969. The five-kingdom system places allprokaryotic organisms in the kingdom Monera, and eukaryotic organismsare in kingdoms Protista, Fungi, Plantae, and Animalia. In the three-domainsystem, the three domains—Bacteria, Archaea, and Eukarya—are essen-tially superkingdoms and include the kingdoms, historically the broadest tax-onomic category. Prokaryotes are placed in either Bacteria or Archaea, withall eukaryotes categorized in the domain Eukarya. Researchers continue to

Bacteriology

For a 2-hour lab: Omit Exercise 2,bacterial succession in milk, andpossibly the study of bacterialcolony characteristics, Exercise 1,Lab Study A. See the Teaching Plan.

L A B T O P I C 1 3

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debate the number of kingdoms to include in the domains. Some have sug-gested eight kingdoms with Bacteria and Archaea corresponding to theirrespective domains and six kingdoms in Eukarya—three protistan king-doms and Plantae, Fungi, and Animalia. Other researchers propose evenmore kingdoms. (See Figure 26.16 in Campbell and Reece, 2002.)

Although many argue that biological categories are subjective in nature andthe criteria for designating the kingdoms of life have been modified by sci-entists historically, it is nonetheless true that scientists have set definite cri-teria or guidelines that form the basis of taxonomic classification. Mostorganisms may be placed into these designated categories.

In this lab topic, you will study organisms commonly called bacteria. In thefive-kingdom scheme, bacteria were placed in the kingdom Monera. Inthe three-domain, eight-kingdom system, the common bacteria are classi-fied in the domain Bacteria, kingdom Bacteria.

Bacteria are small, relatively simple, prokaryotic, single-celled organisms.Prokaryotes, from the Greek for “prenucleus,” have existed on Earth longerand are more widely distributed than any other organismal group. They arefound in almost every imaginable habitat: air, soil, and water, in extremetemperatures and harsh chemical environments. They can be photosyn-thetic, using light, or chemosynthetic, using inorganic chemicals as thesource of energy, but most are heterotrophic, absorbing nutrients from thesurrounding environment.

Most bacteria have a cell wall, a complex layer outside the cell membrane.The most common component found in the cell wall is peptidoglycan, acomplex protein-carbohydrate polymer. There are no membrane-boundorganelles in bacteria and the genetic material is not bound by a nuclearenvelope. Bacteria do not have chromosomes; their genetic material is a sin-gle circular molecule of DNA. In addition, bacteria may have smaller ringsof DNA called plasmids, consisting of only a few genes. They reproduce bya process called binary fission, in which the cell duplicates its componentsand divides into two cells. These cells usually become independent, but theymay remain attached in linear chains or grapelike clusters. In favorable envi-ronments, individual bacterial cells rapidly proliferate, forming colonies con-sisting of millions of cells.

Differences in colony morphology and the shape of individual bacterial cells areimportant distinguishing characteristics of bacterial species. In Exercise 13.1,working independently, you will observe and describe the morphology ofcolonies and individual cells of several bacterial species. You will examine anddescribe characteristics of bacteria growing in plaque on your teeth. You andyour lab partner will compare results of all lab studies.

E X E R C I S E 1 3 . 1

Investigating Characteristics of Bacteria

Because of the small size and similarity of cell structure in bacteria, techniquesused to identify bacteria are different from those used to identify macro-scopic organisms. Staining reactions and properties of growth, nutrition,

46 Lab Topic 13: Bacteriology

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Lab Topic 13: Bacteriology 47

and physiology are usually used to make final identification of species. Thestructure and arrangement of cells and the morphology of colonies con-tribute preliminary information that can help us determine the appropriatetest necessary to make final identification. In this exercise, you will use thetools at hand, microscopes and unaided visual observations, to learn somecharacteristics of bacterial cells and colonies.

When you are working with bacteria, it is very important topractice certain aseptic techniques to make sure that the cul-tures being studied are not contaminated by organismsfrom the environment and that organisms are not releasedinto the environment.

1. Wipe the lab bench with disinfectant before and after thelab activities.

2. Wash your hands before and after performing anexperiment.

3. Using the alcohol lamp or Bunsen burner, flame all non-flammable instruments used to manipulate bacteria orfungi before and after use.

4. Place swabs and toothpicks in the disposal containerimmediately after use. Never place one of these items onthe lab bench after use!

5. Wear a lab coat, a lab apron, or a clean old shirt over yourclothes to lessen chances of staining or contaminationaccidents.

The bacteria used in these exercises are not pathogenic (disease-producing); nevertheless, use appropriate aseptictechniques and work with care! If a spill occurs, notify theinstructor. If no instructor is available, wear disposablegloves, and wipe up the spill with paper towels. Follow thisby washing the affected area with soap and water and a dis-infectant. Dispose of the gloves and soiled towels in theautoclavable plastic bag provided.

Lab Study A. Colony Morphology

Introduction

A bacterial colony grows from a single bacterium and is composed ofmillions of cells. Each colony has a characteristic size, shape, consistency,texture, and color (colony morphology), all of which may be useful in pre-liminary species identification. Bacteriologists use specific terms to describecolony characteristics. Use Figure 13.1 to become familiar with this termi-nology and describe the bacterial species provided. Occasionally, one ormore fungal colonies will contaminate the bacterial plates. Fungi maybe distinguished from bacteria by the fuzzy appearance of the colony(Figure 13.2). The body of a fungus is a mass of filaments called hyphae ina network called a mycelium. Learn to distinguish fungi from bacteria.

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Procedure

1. Wipe the work area with disinfectant and wash your hands.

2. Set up your stereoscopic microscope.

3. Obtain one of the bacterial plates provided. Leaving the plate closed(unless otherwise instructed), place it on the stage of the microscope.

4. Examine a typical individual, separate colony. Measure the size and notethe color of the colony, and record this information in Table 13.1, inthe Results section.

5. Using the diagrams in Figure 13.1, select appropriate terms that describethe colony.


Provide at least six different species,six plates for every four students.Other species may be substituted.See the Prep Guide for suggestionsof bacteria. Seal the plates closedwith Parafilm® strips. Studentresults will vary. Do not expect“correct” answers. The objective isto note distinguishing variations.Do not require students to learnterms used to describe bacteria. Ifocular micrometers are available,have students measure colony sizes.

Figure 13.2. (a) Bacteria and (b) fungi growingon nutrient agar plates. The body of most fungi consists of filamentscalled hyphae in a network called a mycelium. The hyphae give fungalcolonies a fuzzy appearance. (SeeColor Plates 16–19.)

a. b.

a. Common colony shapes

b. Common colony margins

c. Common colony surface characteristics

Punctiform(under 1 mm diameter) Round Filamentous Irregular

Smooth(entire)

Curled Wavy Lobate Filamentous

Smooth Concentric Wrinkled Contoured

Figure 13.1. Terminology used in describing bacterial colonies. (a) Commonshapes, (b) margins, and (c) surfacecharacteristics are illustrated.

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6. Record your observations in Table 13.1.

7. Sketch one colony in the margin of your lab manual, illustrating thecharacteristics observed.

8. Repeat steps 2 to 6 with two additional species. Your lab partner shouldexamine three different species.

Results

1. Complete Table 13.1 at the bottom of the page using terms fromFigure 13.1 to describe the three bacterial cultures you observed.

2. Compare your observations with those of your lab partner.

Discussion

1. What are the most common colony shapes, colony margins, and colonysurface characteristics found in the species observed by you and yourlab partner?

2. Based on your observations, comment on the reliability of colony mor-phology in the identification of a given bacterial species.

Students may conclude that these criteria are not very reliablebecause they may find it difficult to be certain which features apply.Trained professionals and students, after practice, may be able to makeinitial identification based on colony morphology. However, incon-trovertible identification usually depends on results of additionaltests.


Table 13.1Characteristics of Bacterial Colonies

Name ofBacteria Size Shape Margin Surface Color

1.

2.

3.

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Lab Study B. Morphology of Individual Cells

Introduction

Microscopic examination of bacterial cells reveals that most bacteria can beclassified according to three basic shapes: bacilli (rods), cocci (spheres),and spirilla (spirals, or corkscrews). In many species, cells tend to adhereto each other and form aggregates, with each cell maintaining its indepen-dence. In this lab study, you will examine prepared slides of bacteria that illus-trate the three basic cell shapes, and then you will examine and describebacteria growing in your mouth.

Procedure

1. To become familiar with the basic shapes of bacterial cells, using thecompound microscope, examine prepared slides of the three types of bacteria, and make a sketch of each shape in the space provided.

2. Protein and carbohydrate materials from food particles accumulate at thegum line in your mouth and create an ideal environment for bacteria togrow. This mixture of materials and bacteria is called plaque. To inves-tigate the forms of bacteria found on your teeth, prepare a stained slideof plaque.

a. Set out a clean slide.

b. Place a drop of water on the slide. This must air-dry, so make thedrop of water small.

c. Using a fresh toothpick, scrape your teeth near the gum line and mixthe scraping in the drop of water.

d. Spread this plaque-water mixture into a thin film and allow it to air-dry.

e. Allow plenty of time to air dry.

Keep long hair and loose clothing away from the flame.Extinguish the flame immediately after use.

f. Place the slide on the support of a staining pan or tray and apply1–2 drops of crystal violet stain to the smear (Figure 13.3).

Crystal violet will permanently stain your clothes, and it maylast several days on your hands as well. Work carefully!


Figure 13.3. Apply several drops of crystal violetstain to the slide supported in astaining pan or tray.

To save time, set this up as ademonstration or show the threetypes of bacteria using a video-microscopy system.

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g. Leave the stain on the smear for 1 minute.

h. Wash the stain off with a gentle stream of water from a squirt bottleso that the stain goes into the staining pan (Figure 13.4).

i. Blot the stained slide gently with a paper towel. Do not rub hard oryou will remove the bacteria.

3. Examine the bacteria growing in the plaque on your teeth and deter-mine bacterial forms. Use the highest magnification on your compoundmicroscope.

Results

1. Record the individual cell shapes of bacteria present in plaque.

Cocci and bacilli are most common. Species present within a few hoursafter brushing include Streptococcus mutans (the leading causeof dental caries), S. salivarius, S. sanguis, and lactobacilli. Speciesin older plaque include Corynebacterium species (a bacillus),Actinomyces species (a filamentous form), and spirochetes. Studentsmay also see yeast and large epithelial cells.

2. What shapes are absent?

Spirilli are generally less common.

3. Estimate the relative abundance of each shape.

Usually the cocci will be in greater abundance (75%) compared tobacilli (25% or less). Proportions will vary, depending on personaloral hygiene. Older plaque has more bacilli and spirilli. We once hada student with good oral hygiene who had an abundance of spirilli.He was told by his dental hygienist that this might be an indica-tion of susceptibility to gum disease.

Discussion

1. Discuss with your lab partner information you have learned from yourdentist or health class about the relationship among plaque, dental caries(cavities), and gum disease.

(You may choose to have students answer this question using ref-erences in the library.) In short, scientists have established unequiv-ocally that bacteria cause dental caries. Bacteria convert carbohy-drate to lactate, creating an acidic environment that decalcifies thetooth surface. Gum (periodontal) disease results when plaque growsunder the gum rim (gingiva). Calcium is deposited in the plaque,forming tartar. The number of bacteria increases, the percentage ofactinomycetes increases, and the gums become inflamed. Gumsbegin to bleed, they recede, and pockets form under the gums. Thebone surrounding the teeth is resorbed, and the teeth loosen.


Figure 13.4. Gently rinse the stain into the staining pan.

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2. Suggest an explanation for differences in the proportion of each type ofbacteria in the bacterial community of plaque.

Diet, time since last dental visit, even differences in genetics willbring about differences. People taking antibiotics for other bacter-ial infections usually have only bacilli in their mouth.

Lab Study C. Identifying Bacteria by the Gram Stain Procedure

Introduction

Gram stain is commonly used to assist in bacterial identification. This stain,first developed in 1884, separates bacteria into groups, depending on theirreaction to this stain. Bacteria react by testing either gram-positive, gram-negative, or gram-variable, with the first two groups being the most com-mon. Although the exact mechanisms are not completely understood, sci-entists know that the response of cells to the stain is due to differences inthe complexity and chemistry of the bacterial cell wall. Recall that bacter-ial cell walls contain a complex polymer, peptidoglycan. The cell walls ofgram-negative bacteria contain less peptidoglycan than gram-positive bac-teria. In addition, cell walls of gram-negative bacteria are more complex,containing various polysaccharides, proteins, and lipids not found in gram-positive bacteria. Studies of bacterial taxonomy have shown that these dif-ferences define major taxonomic groups.

Gram stain relies on the use of three stains: crystal violet (purple), Gramiodine, and safranin (pink/red). Gram-positive bacteria (with the thicker pepti-doglycan layer) retain the crystal violet/iodine stain and appear blue/purple.Gram-negative bacteria lose the blue/purple stain but retain the safranin andappear pink/red.

In summary:

Gram-Negative Bacteria Gram-Positive Bacteria

more complex cell wall simple cell wall

thin peptidoglycan thick peptidoglycan cell wall layer cell wall layer

outer lipopolysaccharide no outer lipopolysaccharidewall layer wall layer

retain safranin retain crystal violet/iodine

appear pink/red appear blue/purple


Bacterial colonies older than 24 hours often become gram-variable; use organisms fromyounger cultures for this study.

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Figure 13.5. Destain by dropping 95% ethyl alcohol/acetone down the slantedslide until only a faint violet color is seen in the solution.

In this lab study, you will prepare and stain slides of two different bacter-ial species. One member of the lab team should stain Micrococcus andBacillus. The other member should stain Serratia and E. coli.

Procedure

1. Prepare smears as directed for the plaque slide (Lab Study B, steps 2ato 2e), substituting the bacterial species for the plaque. If you are usinga liquid bacterial culture, do not add water to your slide (step 2b). Labelthe slide with your initials and the name of the bacterial species beinginvestigated.

2. Support the slide on the staining tray and cover the smear with 1–2 dropsof crystal violet. Wait 1 minute.

3. Rinse the stain gently into the staining pan with water from the squirtbottle.

4. Cover the smear with Gram iodine for 1 minute, setting the stain. 5. Rinse it again with water.6. Destain (remove the stain) by dropping the 95% alcohol/acetone mix-

ture down the slanted slide one drop at a time. At first a lot of violetcolor will rinse away. Continue adding drops until only a faint violetcolor is seen in the alcohol rinse. Do not overdo this step (Figure 13.5).You should be able to see some color in the smear on the slide. If not,you have destained too much. The alcohol/acetone removes the crystalviolet stain from the gram-negative bacteria. The gram-positive bacte-ria will not be destained.

7. Using the water squirt bottle, rinse immediately to prevent furtherdestaining.

8. Cover the smear with safranin for 30 to 60 seconds. This will stain thedestained gram-negative bacteria a pink/red color. The gram-positivebacteria will be unaffected by the safranin (Figure 13.6).

9. Briefly rinse the smear with water as above. Blot it lightly with a papertowel and let it dry at room temperature.

10. Examine each slide using the highest magnification on your microscope.

Gram-positivecell:

Gram-negativecell:

Purple

Purple

Step 1Crystal violet

Blue/purple

Blue/purple

Step 2Gram iodine

Losesstain

Remainsblue/purple

Step 3Alcohol/ acetone

wash

Pink/red

Remainsblue/purple

Step 4Safranin

Figure 13.6.The Gram stain. Crystal violet andGram iodine stain all cells blue/purple.Alcohol/acetone destains gram-negativecells. Safranin stains gram-negativecells pink/red.

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If you use oil immersion, remove all traces of oil from theobjective after observing the slide.

Results

Record your observations of the results of the Gram stain in Table 13.2.


Name of Bacteria Results of Gram Stain

1.

2.

Micrococcus positiveBacillus

Serratia negativeE. coli

Table 13.2Bacteria Observed and Results of Gram Stain

See the Prep Guide for suggestionsand tips about bacterial species.

For a discussion of the chemistry ofthe Gram stain, see Alcamo (1997,pp. 74–76).

Discussion

1. Which of the bacteria observed are probably more closely related taxonomically?

Micrococcus and Bacillus, being gram-positive, are probably moreclosely related. Serratia and E. coli, both being gram-negative, maybe more closely related.

2. What factors can modify the expected results of this staining procedure?

Sloppy technique, age of cells (organisms over 24 hours old areoften gram-variable, probably because the cell wall changes as cellsage). Help students reason through this question because they haveno access to the specific information.

E X E R C I S E 1 3 . 2

Ecological Succession of Bacteria in Milk

Introduction

Milk is a highly nutritious food containing carbohydrates (lactose, or milksugar), proteins (casein, or curd), and lipids (butterfat). This high level ofnutrition makes milk an excellent medium for the growth of bacteria.Pasteurizing milk does not sterilize it (sterilizing kills all bacteria) but merelydestroys pathogenic bacteria, leaving many bacteria that will multiply veryslowly at refrigerated temperatures; but at room temperature, these bacte-

✼

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ria will begin to grow and bring about milk spoilage. Biologists have discoveredthat as milk ages, changing conditions in the milk bring about a predictable,orderly succession of microorganism communities (associations of species).

Community succession is a phenomenon observed in the organizationalhierarchy of all living organisms, from bacterial communities in milk to ani-mal and plant communities in a maturing deciduous forest. In each exam-ple, as one community grows, it modifies the environment, and a differentcommunity develops as a result.

In this laboratory exercise, you will work in pairs and observe successionalpatterns in two types of milk, plain whole milk and milk with sucrose andchocolate added. You will record changes in the environmental conditionsof the two types of milk as they age. Note certain observations scientistshave made about milk bacteria and their environment.1. Lactobacillus (gram-positive rod) and Streptococcus (gram-positive coc-

cus) survive pasteurization.2. Lactobacillus and Streptococcus ferment lactose to lactate and acetic acid.3. An acidic environment causes casein to solidify, or curd.4. Two bacteria commonly found in soil and water, Pseudomonas and

Achromobacter (both gram-negative rods), digest butterfats and give milka putrid smell.

5. Yeasts and molds (both fungi) grow well in acidic environments.

Scenario

Propose a scenario (the hypothesis) for bacterial succession in each typeof milk.

After rea

ding the information given, students might propose that succes-sion in the cultures will begin with a greater proportion of gram-positive coccus bacteria that survive pasteurization with somegram-positive rods. As the milk ages and the pH becomes moreacidic, the proportion of gram-negative rods will increase and thegram-positive coccus will decrease. In later stages of succession,yeasts and molds, previously absent, will begin to grow. The addi-tional sucrose in chocolate milk may accelerate the successionprocess and the growth of yeast. Accept any reasonable scenario.

On each lab bench are four flasks of plain whole milk, four flasks of soy milk,and four flasks of chocolate milk. One flask of each has been kept underrefrigeration. One flask of each has been at room temperature for 24 hours,one for 4 days, and one for 8 days. On each bench there are also TGY (tryp-tone, glucose, yeast) agar plate cultures of each of the types of milk.

One team of two students should work with plain milk, another with choco-late milk. Teams will then exchange observations and results.


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Procedure

1. Using the pH paper provided, take the pH of each flask. Record yourresults in Table 13.3, in the Results section.

2. Record the odor (sour, putrid), color, and consistency (coagulationslight, moderate, chunky) for the milk in each flask.

3. Using the TGY agar plates, observe and describe bacterial/fungal coloniesin each age and type of milk. Use the vocabulary you developed whiledoing Exercise 13.1.

4. Prepare Gram stains of each different bacterial type on each plate usingthe staining instructions in Exercise 13.1, Lab Study C.

5. Record the results of the Gram stains in Table 13.3, in the Results section.

Results

Complete Table 13.3, in the Results section, describing the characteristicsof each milk culture and the bacteria present in each.

Discussion

1. Describe the changing sequence of organisms and corresponding envi-ronmental changes during succession in plain milk. Do the results of yourinvestigation match your hypothesis?

As milk ages, the predominant coccus forms feed on milk sugar(lactose), converting it to lactate and lowering the pH. A low pH isunfavorable to the coccus species and favors the bacillus species.Bacillus species multiply as the pH continues to fall, creating anenvironment that favors the growth of filamentous fungi and yeasts.

2. Describe the changing sequence of organisms and corresponding envi-ronmental changes during succession in chocolate milk. Do the resultsof your investigation match your hypothesis?

The pH rapidly becomes more acidic, which favors the growth of rods,yeasts, and filamentous fungi. Pseudomonas and Achromobactermust be increasing as the putrid smell increases.

3. Compare succession in plain and chocolate milk. Propose reasons fordifferences.

Additional sugar in chocolate milk encourages more rapid bacter-ial growth than in plain milk and a more drastic pH change earlyin the experiment. This seems to favor the rapid growth and ulti-mate dominance of fungi and the putrid-smelling bacteria.

4. Propose an experiment to test the environmental factors and/or organ-isms changing in your proposed scenario for milk succession.

Pure cultures of bacteria or fungi could be grown under controlledpH conditions or on media that lack certain nutrients, such as sugar(lactose).


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filamentous fungi andyeasts dominate; rodsmore common; cocci

declining

pH 4; heavy coagulationseparating solid chunks;

strong odor

pH 4; increased coagulation; strong

sour odor


Table 13.3Physical Features and Bacterial/Fungal Communities of Aged Plain and Chocolate Milk

Environmental Organisms PresentCharacteristics (Bacteria: Gram +/–,

(pH, Consistency, Shapes; Yeasts Age/Type Milk Odor, Color) or Fungi)

Refrigerated plain

24-hr plain

48-hr plain

4-day plain

8-day plain

Refrigerated soy

24-hr soy

48-hr soy

4-day soy

pH 4; heavy coagulationseparating solid chunksfrom liquid; strong odor

pH 5; coagulation beginning; no odor

cocci continue to domi-nate, but rods are pro-portionally increasing;yeasts very common

cocci dominant; rods present

cocci and rods present;yeasts more common;

filamentous fungi appear

pH 5; no coagulation; no odor

pH 4; slight coagulation;strong sour odor

cocci dominant; rodspresent; occasional

yeasts

cocci dominant; rods present

Results will vary depending on the variety of milk. Let students’ observations stand. Do not correct them.


cocci dominant; since bacteria are dilute, few, if any, grow on plates


cocci dominant; since bacteria are dilute, few, if any, grow on plates

Make an overhead acetate of thisblank table from the student man-ual. Have a representative fromeach group fill in results so that all students have a completed table.

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Environmental Organisms PresentCharacteristics (Bacteria: Gram +/–,

(pH, Consistency, Shapes; Yeasts Age/Type Milk Odor, Color) or Fungi)

8-day soy

Refrigeratedchocolate

24-hr chocolate

48-hr chocolate

4-day chocolate

8-day chocolate

E X E R C I S E 1 3 . 3

Bacteria in the Environment

In these experiments, you will sample different environments, testing forthe presence of bacteria and fungi. In Experiment A, pairs of students willinvestigate one of five different environments. Each pair will report resultsto the entire class. In Experiment B, your team will investigate an environ-ment of your choice.

Table 13.3Continued

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Experiment A. Investigating SpecificEnvironments

Introduction

The instructor will assign team numbers to each pair of students. Each pair(team) of students will sample bacteria and fungi from one of five environ-ments: food supply, soil, air, stream water, and hands. Read the instructionsfor all investigations. Think about the following questions, and before youbegin your investigation, hypothesize about the relative growth of bacteriaand fungi in the different environments.

Where in the environment would bacteria be more common, and wherewould fungi be more common? Would any of these environments be freeof bacteria or fungi?

Seal all plates with Parafilm after preparation! Discard allused swabs in the designated receptacle!

Hypothesis

Hypothesize about the presence of bacteria and fungi in the differentenvironments.

All environments will have some bacteria and fungi present.Bacteria will be more common than fungi on food and hands, andfungi will be more common in soil and water. (Accept any testablehypothesis.)

Prediction

Predict the results of the experiment based on your hypothesis (if/then).

If bacteria and fungi are present in all environments, then afterincubation, all plates will show colonies of each. There will bemore bacteria on the food and hands cultures and more fungi onthe soil and water cultures.


Have trays labeled for each lab sec-tion to store students’ cultures.

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Procedure

Team 1

1. Holding the lid in place, invert an agar plate and label the bottom“chicken.”

2. Open the dish containing the piece of chicken, and swab the chickensurface using a sterile cotton swab.

Avoid touching the chicken. Use the swab. Always wash your hands thoroughly after touching raw chicken, owing to the potential presence of Salmonella, bacteria that cause diarrhea.

3. Isolate bacteria by the streak plate method.

a. Carefully lift the lid of the agar plate to 45° and lightly streak theswab back and forth across the top quarter of the agar (Figure 13.7a).Close the lid and discard the swab in the receptacle provided. Minimizeexposing the agar plate to the air.

b. Flame the bacterial inoculating loop using the alcohol lamp or Bunsenburner. Allow the loop to cool; starting at one end of the swab streak,lightly streak the microorganism in the pattern shown in Figure 13.7b.Do not gouge the medium.

c. Reflame the loop and continue to streak as shown in Figure 13.7c anddescribed in the figure legend.

By the end of the last streak, the bacteria should be separated andreduced in density so that only isolated bacteria remain. These shouldgrow into isolated, characteristic colonies.

4. Write the initials of your team members, the lab room, and the date onthe petri dish.

5. Seal the dish with Parafilm and place it in the area indicated by theinstructor.

6. Incubate the culture 1 week and observe results during the next labo-ratory period.


1 1

2

a. b.

1

23

c.

Swab

Transferloop

Use pieces of fresh chicken. Placethe pieces in the appropriate num-ber of petri dishes and refrigeratethem until needed for the experi-ment. Return the dishes to therefrigerator after the experiment.

Figure 13.7. Isolating bacterial colonies using the streak technique. (a) Streak the swabover the top quarter of the agar plate, region 1. (b) Using the newly flamed loop,pick up organisms from region 1 and streak them into region 2. (c) Reflame theloop and pick up organisms from region 2 and streak them into region 3.

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The following week, to avoid exposure to potentially patho-genic bacteria, do not open the petri dish containing thechicken bacteria. Wash hands after handling cultures.

Team 2

1. Holding the lid in place, invert an agar plate and label the bottom “soil.”

2. Using a cotton swab, pick up a small amount of soil from the sample.

3. Prepare a streak culture by following step 3 in the procedure for Team 1.




Team 3

1. Holding the lid in place, invert an agar plate and label the bottom “air.”

2. Collect a sample of bacteria by leaving the agar plate exposed (lidremoved) to the air in some interesting area of the room for 10 to 15 min-utes. Possible areas might be near a heat duct or an animal storage bin.

3. If additional agar plates are available, you may choose to sample severalsites.

4. Write the initials of your team members, the lab room, and the date oneach petri dish.


6. Incubate the culture(s) 1 week and observe results during the next lab-oratory period.

Team 4

1. Holding the lid in place, invert an agar plate and label the bottom “streamwater.”

2. Using a sterile cotton swab, take a sample from the stream water.

3. Prepare a streak culture by following step 3 in the procedure for Team 1.




Team 5

1. Draw a line across the center of the bottom of an agar plate. Write“unwashed” on the dish bottom on one side of the line and “washed”on the other side of the line.


For a class of 24 students, you willhave six teams. Choose an interest-ing environment for team 6. Youmight choose compost, pond water,sand, hands washed with hand san-itizer, etc.

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2. Select one person who has not recently washed his or her hands to bethe test subject. The subject should open the petri dish and lightly pressthree fingers on the agar surface in the half of the dish marked “unwashed.”Do not break the agar. Close the petri dish.

3. The subject should wash his or her hands for 1 minute and repeat theprocedure, touching the agar with the same three fingers on the side ofthe dish marked “washed.”




Results

Include results from the entire class.

1. During the following laboratory period, observe your agar cultures ofbacteria and fungi from the environment and record your observationsin Table 13.4.

2. Place your agar culture on the demonstration table and make a label ofthe environment being investigated. All students should observe everyculture.

3. Observe the agar plates prepared by your classmates. Record observa-tions in Table 13.4.


Place large labels on a demonstra-tion table and have students placetheir cultures in the appropriatearea.

Table 13.4Abundance and Types of Colonies Associated with Food (Raw Chicken),Soil, Air, Water, and Hands

Environment Colony Type(s) and Abundance

Chicken

Soil

Air

Stream water

Hands before washing

Hands after washing

Other

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Discussion

1. How did the plates differ in the number and diversity of bacterial andfungal colonies?

Chicken gives an extensive bacterial growth but has less diversity.The results of cultures from soil, air, and water will vary, with soilusually giving the greatest diversity. What happens with the air cul-ture will depend on your particular facilities.

2. Did your predictions match your observations? Describe any discrepancies.

Students might predict that washing would reduce numbers of bac-terial colonies. However, the results may show that after washing,new bacterial species are present and the new species are more com-mon than the original. Washing removes outer cell layers and exposesbacterial species that live deep in underlying dead cells. Point outthat washing removes pathogenic bacteria picked up from surfaces.Standard infection control is washing for 1 full minute. Nurses andsurgeons scrub and use brushes for 5 minutes before surgery.

3. What factors might be responsible for your results?

Such things as source of substances tested, ventilation systems inroom; for stream water, results will depend on degree of pollution.

4. Based on the results of your experiments, suggest health guidelines forworkers in the food industry, as well as for schoolchildren or otherswho might be concerned with sanitary conditions.

Students should conclude that the presence of bacteria and fungi inmost environmental areas, including human hands, compels us towash hands before eating and before and after handling food beingprepared for eating. Food preparation areas should be washedfrequently.

Experiment B. Investigating the Environment of Your Choice

Introduction

In the previous experiment, you tested specific environments for the pres-ence of bacteria and fungi. In this lab study, you will study an environmentof your choice. If extra agar plates are available, you may choose to inves-tigate bacteria in an environment before and after some treatment, such asbacteria on the water fountain before and after cleaning.

Seal all plates with Parafilm after preparation!


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Hypothesis

Hypothesize about the growth of bacteria in an environment of your choice.

Prediction

Predict the results of the experiment based on your hypothesis.

Procedure

1. Decide what environment you will investigate. It might be some envi-ronment in the lab room or somewhere in the biology building. Carrythe sterile cotton swab and agar plate to the environment, and use theswab to collect the sample. If you are collecting from a dry surface, youshould first dip the cotton swab in the sterile water and then swab thesurface. If you apply any treatment to the surface, describe the treat-ment in the margin of your lab manual.

Do not do throat or ear swabs! Pathogenic bacteria may bepresent.

2. Open the agar plate and lightly streak the swab back and forth acrossthe agar. Discard the swab in the receptacle provided.

3. Label the bottom of the agar plate to indicate the environment tested.Record the environment tested in the Results section.



6. Incubate the culture 1 week and then observe and describe results dur-ing the next laboratory period.

Results

1. What environment did you investigate? Indicate any treatment youapplied.


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2. Characterize the bacterial and fungal colonies from your experiment.

Discussion

1. Do your results match your predictions for the presence of bacteria andfungi in this environment?

2. What factors might be responsible for your results?

E X E R C I S E 1 3 . 4

Controlling the Growth of Bacteria

Bacteria are found almost everywhere on Earth, and most species aredirectly or indirectly beneficial to other organisms. Bacteria are necessaryto maintain optimum environments in animal and plant bodies and inenvironmental systems. However, even beneficial species, if they are repro-ducing at an uncontrolled rate, are potentially harmful or destructive totheir environment. In addition, several species of bacteria and fungi are knownto be pathogenic, that is, to cause disease in animals and plants. Theirgrowth must be controlled. Agents have been developed that control bac-terial and fungal growth. In this exercise, you will investigate the efficacyof three of these growth-controlling agents: antibiotics, antiseptics, anddisinfectants.

Lab Study A. Using Antibiotics to Control Bacterial Growth

Introduction

An antibiotic is a chemical produced by a bacterium or fungus that has thepotential to control the growth of another bacterium or fungus. Many antibi-otics are selective, however, having their inhibiting effect on only certainspecies of bacteria or fungi. In this lab study, you will apply an assortmentof antibiotics to a lawn culture of a bacterial species. Working in pairs, youwill determine which antibiotics are able to control the growth of the bac-teria. Each pair of students in a group of eight should culture a differentbacterium. All four species should be cultured.

A lawn of bacteria is like a lawn of grass—a uniform, even layer of organ-isms covering an entire surface. Prepare the lawn of bacteria carefully. Thesuccess of this experiment will largely depend on the quality of your lawn.


If you have bacteria spreaders, theycan be used to produce lawns in thisexercise. (One kind commerciallyavailable is called Bacti-Spreader.)

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Hypothesis

Hypothesize about the effect of different antibiotics on the growth of bacteria.

All antibiotics control the growth of all bacterial species.

Prediction


If all antibiotics control the growth of all bacterial species, then thezone of inhibition will be the same around each type of antibioticdisk on all bacterial lawns, regardless of species.

Procedure

1. Label the bottom of an agar plate with your initials, the lab room, thedate, and a word to indicate the experiment (such as “antibiotic”).

2. Prepare a bacterial lawn.

a. Insert a sterile swab into the bacterial culture in liquid nutrient broth.

b. Allow the swab to drip for a moment before taking it out of the cul-ture tube, but do not squeeze out the tip. The swab should be soakedbut not dripping.

c. Carefully lift the lid of the agar plate to about 45° and swab the entiresurface of the agar, taking care to swab the bacteria to the edges ofthe dish (Figure 13.8a).

d. Rotate the plate 45° and swab the agar again at right angles to thefirst swab (Figure 13.8b). Close the lid.

3. Carry the agar plate swabbed with bacteria to the demonstration table.

4. Remove the plate lid, place the antibiotic disk dispenser over the plate,and push down on the handle to dispense the disks (Figure 13.9). (Eachdisk has been saturated with a particular antibiotic. The symbol on thedisk indicates the antibiotic name. Your instructor will provide a keyto the symbols.)

5. Replace the lid, seal the plate with Parafilm, and place the plate in thearea indicated by the instructor. Incubate the dishes at 37°C for 24–48hours and then refrigerate them.

6. Next week, examine cultures to determine bacterial sensitivity to antibi-otics. Measure the diameter of the zone of inhibition (area around diskwhere bacteria growth has been inhibited) for each antibiotic.

7. Record the measurement for your bacterial species and each antibioticin Table 13.5. If the antibiotic had no effect on bacterial growth, recordthe size of the zone as 0.

Results

1. Using your results and the results from other teams, complete Table 13.5.Record the sizes of the zone of inhibition for all species of bacteria andall antibiotics.


a.

b.

Figure 13.8. Preparation of a bacterial lawn.(a) Apply the bacteria evenly over theentire agar surface. (b) Rotate the plate and swab at right angles to thefirst application.

Petri dish

Antibioticdisk

Figure 13.9. The antibiotic dispenser. Press downthe dispenser handle to dispense the disks.

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2. Use the following arbitrary criteria to rank relative bacterial sensitivityto antibiotics:

NS = not sensitive = no zone of inhibition

S = sensitive = zone size above 0 but less than 1 cm

VS = very sensitive = zone size greater than 1 cm

Write the designation in each blank in the table.

Discussion

1. Did your results support your hypothesis? Was the zone of inhibitionthe same for all bacteria?

2. Were any bacteria very sensitive (greater than 1 cm) to all antibiotics?If so, which bacteria?

3. Based on your results, which antibiotic would you prescribe for each micro-organism?

Depending on the antibiotics and bacteria chosen, neomycin,kanamycin, and erythromycin are usually more effective thanpenicillin.

Table 13.5Results of Antibiotic Sensitivity Tests (Size of inhibition zone for each antibiotic is given in centimeters.)

Bacteria(Name, Antibiotic

Gram +or –)

1.

2.

3.

4.

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4. Were the results different for gram-positive and gram-negative bacteria?

This will vary depending on which antibiotics and bacteria you use.

5. Can you think of alternative explanations for the differential effective-ness of some antibiotics?

Students should discuss the resistance of bacteria to widely usedantibiotics as well as the different modes of action by antibiotics.

Lab Study B. Using Antiseptics and Disinfectantsto Control Bacterial Growth

Introduction

Other agents besides antibiotics are often used to control bacterial growth.Those used to control bacteria on living tissues such as skin are called anti-septics. Those used on inanimate objects are called disinfectants. Antisepticsand disinfectants do not kill all bacteria, as would occur in sterilization, butthey reduce the number of bacteria on surfaces.

Hypothesis

Hypothesize about the effect of antiseptics and disinfectants on the growthof bacteria.

Disinfectants are more effective than antiseptics in controlling bac-terial growth.

Prediction


If disinfectants are more effective than antiseptics in controlling bac-terial growth, then zones of inhibition around disinfectant disks willbe larger than those around antiseptic disks.

Procedure

1. Holding the lid in place, invert a sterile agar plate and label the bottomwith your initials, the lab room, and the date. Draw four circles on thebottom. Number the circles.

2. Using the same bacterial culture as you used in Lab Study A, prepare alawn culture as instructed in Lab Study A.

3. Carry the closed agar plate swabbed with bacteria to the demonstrationtable.

4. Open the agar plate; using forceps soaking in alcohol, pick up a disk soakedin one of the antiseptics or disinfectants, shake off the excess liquid,and place the disk on the agar above one of the circles. Repeat this pro-


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cedure with two more antiseptics and/or disinfectants. Place a disksoaked in sterile water above the fourth circle to serve as a control.

5. Record the name of the agent placed above each numbered circle inTable 13.6 below. (Example: 1 = Lysol, 2 = Listerine, and so on.) Sealthe plate with Parafilm.

6. Place the agar plate in the area indicated by the instructor. Incubate theagar plates at 37°C for 24–48 hours and then refrigerate them.

7. Next week, examine the cultures to determine the bacterial sensitivityto disinfectants and antiseptics. Measure the diameter of the zone ofinhibition for each agent.

8. Record the measurement for your bacterial species and each inhibitingagent in Table 13.6. If the agent had no effect on bacterial growth, recordthe size of the zone as 0.


Table 13.6Results of Sensitivity Tests of Antiseptics and Disinfectants (Size of inhibition zones given in centimeters.)

Antiseptic/Disinfectant/Control

Bacteria 1. 2. 3. 4. Control

1.

2.

3.

4.

Check the plates after 24–48 hoursand refrigerate them if there is ade-quate growth.

Results

1. Using your results and the results from other teams, complete Table 13.6.Record sizes of the zone of inhibition for all species of bacteria and allantiseptics and disinfectants.

2. Use the following arbitrary criteria to rank relative bacterial sensitivityto antiseptics and disinfectants:

NS = not sensitive = no zone of inhibition

S = sensitive = zone size above 0 but less than 1 cm

VS = very sensitive = zone size greater than 1 cm

Write the designation in each blank in the table.

Discussion

1. Did your results support your hypothesis? Explain.

Yes. Zones of inhibition are larger around disinfectant disks thanaround antiseptic disks.

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2. Based on your results, which disinfectant is most effective in controllingthe growth of bacteria?

Household bleach works best.

3. Which antiseptic is most effective?

Rubbing alcohol usually works best.

4. In which situations is it appropriate to use a disinfectant?

An antiseptic?

E X E R C I S E 1 3 . 5

Bacterial Relationships

As mentioned in Exercise 13.4, most species of bacteria are directly or indi-rectly beneficial to other organisms. A relationship that is beneficial to thehost as well as the bacteria is called a mutalistic relationship. One veryimportant form of mutalisim is nitrogen fixation in which bacteria convertunusable nitrogen into a form that plants can utilize.

Lab Study A: Nitrogen-Fixing Bacteria—Symbiosis

Rhizobium

Legumes, the plant family containing peas, beans, clover and alfalfa, are thelargest group of plants that form symbiotic associations with bacteria of thegenus Rhizobium. Rhizobium enter the legume root tissue, inducing it to formnodules, pinkish bulbous structures where the bacteria live. These bacteriasupply the plant with a usable source of nitrogen by nitrogen fixation—converting atmospheric N

2into ammonia (NH

3), which plants can then use

in manufacturing protein. The plant supplies Rhizobium with sugar, theproduct of photosynthesis.

When legumes are planted as a crop, the Rhizobium existing in symbiosis withthem may add hundreds of pounds of ammonia or nitrate per acre to thesoil.

Examine the plants which have been inoculated with Rhizobium.


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How are the nodules arranged on the roots?

Examine a demonstration slide of a nodule cross-section.

Why might farmers rotate leguminous crops with other plantings?

Lab Study B: Cyanobacteria: ModernPhotoautotrophic Bacteria

Cyanobacteria (formerly called blue-green algae) contain chlorophyll a, asdo the photosynthetic eukaryotes. They also possess additional photosyn-thetic pigments, including the blue pigment, phycobilin.

Cyanobacteria occupy wide geographical ranges and extremely variablehabitats, including fresh water and oceans. Representatives of the groupmay be found in the hottest deserts, in arctic waters, in hot springs, and inextremely polluted ecosystems.

Although sometimes found as single cells, more often they form cell clus-ters or filaments.

Make a wet mount of Oscillatoria. Sketch some Oscillatoria cells.

Is a gelatinous sheath present?

Watch Oscillatoria under the light for a few minutes. Explain how it got itsname.

Lab Study C: Nitrogen-Fixing Cyanobacteria—Symbiosis

Anabaena

Another important mutualistic symbiotic relationship is formed betweenAzolla, a small, floating water fern, and Anabaena azollae, a nitrogen-fixingcyanobacterium. Anabaena lives within the pores of the Azolla fronds. Incertain parts of the world, Azolla-Anabaena are allowed to grow heavily inrice paddies. The rice crop shades out the Azolla late in the growing season,


Boxes of milk seen on grocery storeshelves are prepared by ultrahightemperatures (UHT), 285°F (about140°C) for 1–2 seconds. At thistemperature, the milk is sterilizedrather than pasteurized.

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and as the fern plants die, as much as 50 kilograms of nitrogen may bereleased per hectare. This nitrogen is then used by the maturing rice plants.

Examine the Azolla cultures. Using a coverslip, crush a fragment of anAzolla frond in a drop of water. Examine the slide microscopically.

Are the numbers of Anabaena low, moderate or high?

Sketch some Anabaena cells alongside of some cells from Azolla.

Does this Anabaena exist as single cells, clusters or filaments?

Anabaena can also be free-living cyanobacteria. The cellular machinery thatenables this species to take nitrogen (N

2) from the surrounding environ-

ment and convert it into organic molecules (a useable source of nitrogenfor plants) is located in certain enlarged cells called heterocysts. Sketch aportion of Anabaena with one or more heterocysts.

Questions for Review

1. Once you have completed this lab topic, you should be able to defineand use the following terms, providing examples if appropriate: steril-ize, nutrient broth and agar, coccus, bacillus, spirillum, antibiotic, antiseptic,disinfectant, peptidoglycan, aseptic technique.

2. Compare the techniques used to prepare a lawn culture and a streakculture.

Applying Your Knowledge

1. Would you expect the community of bacteria in plaque sampled 1 weekafter you have your teeth cleaned to differ from the community of bac-teria found 1 week before you have your teeth cleaned? Explain. In youranswer, consider the results of the milk succession experiment.

Initial bacterial communities will produce an environment that willfavor the invasion of new species (such as changing pH or creatingpockets for anaerobic bacteria to grow), as students observed in themilk experiment.


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2. Bacterial species that are harmful, as well as others that are beneficial,are found living in the human body. How can the information learnedby antibiotic sensitivity testing be used by physicians who must chooseantibiotics that inhibit the growth of bacteria causing disease but thatdo not interfere with beneficial bacteria?

Samples of the bacterium could be plated (for example, throat swabs)and antibiotic disks applied. The physician should choose an antibi-otic that specifically inhibits the infectious bacterium but does notinhibit beneficial bacteria. Students will see that penicillin, for asymbiotic resident of the human intestine, but is effective in con-trolling the growth of other bacteria.

3. Scientists measure the effectiveness of antiseptics and disinfectants in con-trolling bacterial growth by a standard called the phenol coefficient(PC). PC compares a germicidal agent (antiseptic or disinfectant) withphenol, a disinfectant used since the 1860s. A PC of “1” means that thegermicide is as effective as phenol in controlling the growth of germs.A substance with a PC greater than “1” is more effective than phenol,and a substance with a PC less than “1” is less effective than phenol.

Salmonellosis (caused by ingesting Salmonella sp.) is one of the mostserious foodborne diseases of our time. Salmonella bacteria may be foundin any food substance but are particularly common on poultry and eggs.Using Table 13.7 for reference, which germicide would you recommendto control the growth of Salmonella in egg- and poultry-processingplants?

Table 13.7Phenol Coefficients of Some Common Antiseptics and Disinfectants Used to Control Staphylococcus and Salmonella Growth*

Germicide Staphylococcus Salmonella

Phenol 1.0 1.0

Iodine 6.3 5.8

Lysol 5.0 3.2

Clorox 133.0 100.0

Ethyl alcohol 6.3 6.3

Hydrogen peroxide – 0.01

Formalin 0.3 0.7

*Modified from Table 22.1 in Alcamo (1997).

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4. Search the Web for information about milk seen in boxes on grocerystore shelves. How is this milk prepared? How would you expect bac-terial succession in milk prepared in this fashion to differ from succes-sion in milk as investigated in Exercise 13.2?

5. In 1998 physicians discovered that the deadly bacterium Staphylococcusaureus had become resistant to the antibiotic vancomycin, once veryeffective in controlling this bacterium. Using information from your textor the Web, discuss how this unfortunate situation could have comeabout.

Investigative Extensions

1. Design experiments to test diffusion rates and dilution factors of anti-septics and disinfectants.

2. Investigate the efficacy of hand washing by varying the type of soap(liquid, bar, antibacterial, deodorant) or the manner of washing (scrub-bing time, use of a brush).

3. Investigate the efficacy of waterless hand sanitizers in killing bacteriaafter various activities—for example, using the rest room, touching rawchicken, shaking hands, or cracking a raw egg. Wear disposable gloveswhen performing this experiment.

4. The chicken industry and the FDA have recently been criticized for hav-ing low health and safety standards. Pursue this topic by a survey ofbrands or handling techniques. Health officials now recommend that alleggs be cooked before eating to avoid Salmonella. Determine the extentof contamination in store-bought eggs and in eggs from local sources.

5. Design an experiment to test bacterial succession in plaque.

6. Onions, garlic, green tea, and grapefruit seeds have all been suggestedas having antibiotic properties. Design an experiment to test this.


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References

Alcamo, E. I. Fundamentals of Microbiology, 5th ed. MenloPark, CA: Benjamin/Cummings, 1997. Excellent cov-erage of microbiology. Good discussion of the role ofbacteria in dental diseases.

Campbell, N., and J. Reece. Biology, 6th ed. San Francisco:Benjamin/Cummings, 2002.

Dill, B., and H. Merilles. “Microbial Ecology of the OralCavity,” in Tested Studies for Laboratory Teaching (Volume10). Proceedings of the 10th Workshop/Conference of theAssociation for Biology Laboratory Education (ABLE), CoreyGoldman, editor, 1989.

Doolittle, W. F. “Uprooting the Tree of Life” ScientificAmerican 2000, vol. 282, pp. 90–95.

Gillen, A. L., and R. P. Williams. “Pasteurized Milk as anEcological System for Bacteria” The American BiologyTeacher 1988, vol. 50, pp. 279–282.

Jarrell, K. F., D. P. Bayley, J. D. Correia, and N. A. Thomas.“Recent Excitement about the Archaea” Bioscience 1999,vol. 49, pp. 530–541.

Levy, S. B. “The Challenge of Antibiotic Resistance.”Scientific American 1998, vol. 278, pp. 46–53.

Nester, E. W., C. E. Roberts, N. N. Pearsall, and B. J.McCarthy. Microbiology, 2nd ed. Dubuque, IA:WCB/McGraw-Hill, 1998. Good discussion of ecolog-ical succession of microbes.

“The Race Against Lethal Microbes.” A Report from theHoward Hughes Medical Institute, 1996.

Website

Resource for learning about the Domain Bacteria:http://www.ucmp.berkeley.edu/bacteria/bacteria.html

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