Part IIIViruses andEukaryoticMicro-organisms

ertainly the “menagerie” of microorganisms is immense. Besidesthe unicellular bacteria, which are prokaryotic organisms,there are the viruses, which are acellular entities many of which

consist of nothing more than genetic information wrapped in a proteincoat. The eukaryotic organisms, including the unicellular protists (pro-tozoa and algae), and the multicellular fungi, plants, and animals are all sim-ilar in having a nuclear envelope that surrounds the DNA and contains avariety of membrane-enclosed cellular structures that comprise most of thecellular organelles (Table A).

The viruses are truly different from any of the prokaryotic and eukary-otic microorganisms. Most have very definite geometric shapes (Exercise 8).When bacteriophages (phages), which are viruses that infect bacteria, findsuitable bacteria, the viruses enter the cells and replicate, eventually releas-ing a substantial number of progeny phage and killing the host cells. In culture, waves of successive infections and cell lysis produce clear areas

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TABLE

Basic Characteristics of Microorganisms

CHARACTERISTIC VIRUSES PROKARYOTES EUKARYOTES

Contain genetic information Yes Yes Yes

Presence of a cell nucleus No No Yes

Presence of membrane-enclosed No No Yesorganelles

Capable of independent No Yes (most) Yesreproduction or replication

A

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called plaques that are produced in the bacterial lawn. The detection ofphages in such materials such as sewage can be detected by such plaque for-mation, which also can be used to estimate bacteriophage numbers (Exer-cise 8).

The fungi include the molds and yeasts. These eukaryotic organismshave a unique growth form and produce many products that are beneficialin industrial and commercial industries. The study of the molds and yeasts(Exercise 9) provides a strong contrast to laboratory studies of the bacteria.Some of the unicellular protozoa and multicellular flatworms androundworms (animals) are considered parasites because they are patho-genic or parasitic on other organisms, including humans. Most protozoa arenot parasitic and survive on dead and decaying matter in ponds and verydamp soil (Exercise 9). The multicellular parasites mentioned aboveare uncommon in developed countries, so they often are studied in themicrobiology lab through microscopic observation of prepared slides or per-haps from feces from other animals (Exercise 9).

earning Objectives

When you have completed the exercises in Part 3, you should be capable of:• Distinguishing the structure of viruses from other microorganisms. • Detecting the presence of replicating bacteriophages through cell lysis and

plaque formation, and enumerating phage numbers.• Contrasting mold and yeast growth and reproduction. • Testing for the presence of yeast metabolism through enzyme activity. • Comparing the characteristics of protozoa and multicellular parasites.

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Viruses

iruses are obligate intracellular parasites that multiply within thecytoplasm of a host organism. Certain viruses replicate withinanimal cells, others within plant cells, and still others within

bacterial cells. Viruses multiplying within bacteria are called bacterio-phages, or phages.

Viruses are ultramicroscopic entities, and an electron microscope isneeded to see them clearly. Many viruses appear as 20-sided geometrical fig-ures, while others are helical, and others have complex shape. The shapeis determined by the viral genes and is an important characteristic in viralclassification.

When viruses multiply within their host cells, they rely upon the meta-bolic machinery of the cell for the necessary functions of replication. Bac-teriophages fit this pattern and multiply within bacterial cells. The mostdramatic type of phage infection is the lytic cycle of infection in which thehost bacterium disintegrates when new viral particles are released. Shouldthe viruses multiply within and be released from bacteria growing on thesurface of a nutrient agar plate, clear areas of bacterial destruction willresult.

The interaction between a bacteriophage and its host bacterium can bean important tool in the identification of bacterial strains because bacte-riophages are highly specific for their bacterial hosts. In this exercise, var-ious activities and experiments will be performed with phages and bacteria.

Constructing a Virus

The capsids of certain viruses have the shape of the geometric figure knownas an icosahedron. Among the notable human icosahedral viruses arethose causing chickenpox, polio, and hepatitis A. The icosahedron is com-posed of 20 triangular faces, with 12 edges and 12 points. This activity willhelp you construct and visualize an icosahedron in three dimensions.

pecial Materials

• Scissors, tape, glue

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PURPOSE: to visualize inthree dimensions the shapeof an icosahedral virus.

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rocedure

1. Refer to the template of an icosahedron in Figure 8.1.

2. Make a copy of the icosahedron onto a sheet of heavy cardboard or a filefolder. You may reduce the size or enlarge it according to your needs. Alter-nately, you may draw an icosahedron on heavy stock paper using the illus-tration as a guide. The illustration itself may be used, but the paper will bedifficult to work with because it is thin and difficult to fold.

3. Once you have prepared an outline of the icosahedron, fold the trianglestogether by matching the numbers. The tabs containing the numbers shouldbe folded down as you proceed.

4. Use pieces of tape to hold the sides together as the icosahedron develops.For a more permanent figure, glue the sides together.

5. Once the icosahedron has been constructed, its area may be calculated.Assuming that, in rough terms, about 500 viruses may fit within a cell, therelative areas of virus and cell may be calculated and compared. Otherexercises may also be developed to give an appreciation of the minute sizeof the virus.

The Effect of Bacteriophages on Bacteria

Virulent bacteriophages are those that destroy their bacterial hosts at theconclusion of the replication cycle. The viral nucleic acid enters the bacterialcytoplasm and after about 15 minutes, the viral genes have encoded enzymesand other proteins for the construction of new viruses. Several hundred bac-teriophages are produced per bacterium, and as they are released, the bac-teria undergo lysis and die. The new bacteriophages infect other bacterialcells and the cycle is repeated until all the bacteria have been destroyed. Ina broth culture, a cloudy suspension of bacteria would become relativelyclear.

pecial Materials

• Suspension of bacteriophage T4• Young culture of Escherichia coli strain B• Young cultures of another strain of Escherichia coli

• Tubes of nutrient broth• Tubes of phenol red lactose broth

rocedure

1. Obtain three tubes of nutrient broth, a tube of the phage suspension, andbroth cultures of E. coli B and a second strain of E. coli. Also obtain threetubes of phenol red lactose broth. This medium contains a pH indicatorcalled phenol red and an inverted Durham tube. The indicator in the tubeturns yellow when acid is produced, and a bubble appears in the invertedtube when gas is produced.

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PURPOSE: to detect thepresence of replicatingphage in susceptiblebacteria.

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F I G U R E 8 . 1Template of an icosahedron.

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2. Inoculate one of the nutrient broth tubes with a loopful of E. coli B and a second broth tube with the other strain of E. coli. Leave the third tube as anuninoculated control. (At the instructor’s suggestion, a different species of bac-terium may also be included using a fourth tube of nutrient broth.) Inoculatetwo tubes of phenol red lactose broth in the same way and leave the third tubeas a control. Label each tube with your name, the date, the name of themedium, and the contents. (Another control tube of each medium inoculat-ed with E. coli B but no phage may be included at the instructor’s suggestion.)

3. Inoculate all six tubes with a loopful of a suspension of bacteriophage T4.

4. Incubate all the tubes at 37ºC for 24 to 48 hours. If the tubes cannot beexamined immediately, they may be refrigerated until the next laboratorysession.

5. Examine the three nutrient broth tubes. Look for evidence of growth in thetubes indicated by cloudiness in the tube. It may be necessary to vigorouslytap the bottom of the tube to suspend any bacteria that have sedimented.The control tube should be clear with no evidence of bacterial growth.Note the tubes in which growth has or has not occurred, and draw your con-clusions on the effect of the bacteriophage T4 on its host bacterium. Notewhether the phage had any effect on the other strain of E. coli tested, andplace your conclusions in the Results section.

6. Examine the three tubes of phenol red lactose broth. Normally, Escherichiacoli will grow in this medium and produce both acid and gas. As noted pre-viously, acid is displayed by a yellowing of the medium, and gas is indicatedby a gas bubble trapped in the inverted vial. As you examine the tubes, notein the Results section whether Escherichia coli has multiplied and grownin the tube. If no acid or gas is present and the tube remains the original col-or, then E. coli has not grown. Alternately, if acid and gas have been produced,the E. coli has multiplied. From your observations, conclude whether the bac-teriophage has destroyed the host organism.

Plaque Formation

When bacteriophages lyse their bacterial hosts on an agar surface, a moth-eaten clear area is seen amid the lawn of bacterial growth. This moth-eatenarea is called a plaque. It indicates that a bacteriophage has destroyed itsspecific bacterial host.

pecial Materials

• Suspension of bacteriophage T4• Young culture of Escherichia coli strain B• Young cultures of another strain of E. coli

• Plates of nutrient agar, or materials for their preparation• Tubes of 9.9 ml sterile saline• Mechanical pipetters and pipettes

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PURPOSE: to detect andvisualize areas of phage lysisin a bacterial lawn.

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rocedure

1. Obtain two plates of nutrient agar, or pour two plates at the direction of theinstructor.

2. After the agar has hardened, inoculate one plate with E. coli B, and oneplate with another strain of E. coli. To prepare an even lawn of bacteria,streaking should be performed with a sterile swab. Dip a sterile swab intoa broth culture of E. coli, and prepare a lawn of bacteria by swabbingthe surface of the agar in three directions, as shown in Figure 8.2. Be sureto cover all parts. Then make a final swab around the perimeter. A sep-arate swab should be used for each plate, and precautions should betaken to avoid airborne contamination by lifting the lid only enough topermit access. When the swabbing is complete, the swab should bedeposited in the beaker of disinfectant as directed by the instructor.After inoculation, the plates should be labeled with your name, the date,the medium, and the name of the bacteria used. They may be dividedinto sectors if the instructor recommends.

3. Obtain a loopful of phage suspension from the phage culture and make onestreak across the center of the inoculated plate being careful not to diginto the surface of the agar. Inoculate both plates in this manner. At theinstructor’s direction, various dilutions of the phage suspensions may bemade and samples of various dilutions may be inoculated to different sec-tors of the plate.

4. Incubate all plates at 37°C for 24 to 48 hours. Observe the plates for clearlines in an otherwise uniform lawn of bacterial growth. These lines indi-cate regions where bacteriophages have multiplied within the bacteriaand destroyed them. Note the plate in which a clearing has occurred andcompare this plate with the other containing a nonspecific bacterial host.Consider and explain the specificity of the bacteriophage with its hostorganism. Results may be expressed in the appropriate diagrams in theResults section, and conclusions concerning virus–host specificity shouldbe drawn.

5. It is valuable to make dilutions of the phage suspension to determine thenumber of phage particles in a sample (see Part E).

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(a) (b) (c) (d)F I G U R E 8 . 2The “3+1” procedure for making a lawn of bacteria on a plate of medium.

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Identification of Bacteriophages from Sewage

Because Escherichia coli is commonly found in sewage, its bacteriophageis also located here. Identifying E. coli bacteriophages from sewage is arelatively easy exercise requiring all bacteria be removed from the sewageand the liquid tested. Bacterial cell lysis and/or plaque formation describedin the previous sections are used for the identification.

pecial Materials

• Sample of raw sewage from a local sewage treatment plant• Filtering apparatus with 0.45 �m filter• Nutrient agar plates, or materials for their preparation• Young culture of Escherichia coli strain B

rocedure

1. A sewage treatment plant should be visited to obtain a small sample ofraw sewage. Approximately 100 ml is needed.

2. The sewage fluid should be filtered through a membrane filter apparatussuch as that described in Exercise 33D (Figure 33.1). A filter pore size of 0.45μm is used to trap the very small bacteria. The material emerging from thefilter (the filtrate) should contain only bacteriophages.

3. To confirm the presence of bacteriophages, proceed as in Parts B and C ofthis exercise. For example, loopfuls of the phage filtrate may be inoculatedto tubes containing E. coli strain B and incubated to see whether the bacteriaare destroyed. Alternately, loopfuls of the filtrate can be streaked onto platescontaining a lawn of E. coli strain B. The filtrate can also be diluted, and sam-ples used for streaking.

4. Record your results in the appropriate portion of the Results section andindicate whether the identification of bacteriophages was successful.

Estimating the Number of Bacteriophages

The ability of bacteriophages to replicate in and lyse susceptible host bac-teria to form plaques can be used to enumerate (count) the number ofphage particles in a solution or sample.

The enumeration procedure uses a double-layer of agar. Over a “hardagar” base is poured a “soft agar,” consisting of a mixture of phage and sus-ceptible bacterial cells in molten agar. This forms an upper layer of softeragar on solidification. During the incubation period at 37ºC, susceptibleEscherichia coli cells will divide rapidly and form an even lawn of bacterialcells. After a phage particle attaches to, penetrates, and replicates in thehost cell, phage release lyses the host cell. The released phages then infectsurrounding cells and quickly produce a single plaque in the bacterial lawn.

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!Handle raw sewage carefully,since it may contain micro-bial pathogens.

PURPOSE: to estimate thenumber of phage particles ina sample.

PURPOSE: to detect thepresence of phage particlesin sewage.

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Each plaque formed is referred to as a plaque-forming unit (PFU) andis assumedto be the result of one initial phage particle. Each PFU then repre-sents a phage particle in the original mixture. Therefore, the number of PFUsformed can be used to estimate the number of infective phages in the dilutedmixture and extrapolated back to estimate the numbers in original sample.

pecial Materials

• Young broth cultures of E. coli strain B• Suspensions of bacteriophage T4 • Plates of nutrient hard agar• Tubes containing 1.8 ml sterile, nutrient soft agar (kept at 45ºC)• Tubes containing 9.9 ml sterile, nutrient broth• Mechanical pipetters• 1 ml sterile pipettes

rocedure

Enumeration requires that there be a sufficient number of phages present to get astatistically accurate count, yet not too many phage particles that counting plaquesbecomes impossible. Therefore, a series of dilutions of the phage should be made.

1. To prepare dilutions, first label all dilution tubes and media with your nameand the dilution designations as described below.a. Four nutrient broth tubes: labeled 10-2, 10-4, l0-6, 10-8.b. Four nutrient agar “soft agar” tubes: labeled 10-3, 10-5, 10-7, l0-9.c. Four nutrient agar “hard agar” plates: labeled 10-3, 10-5, l0-7, 10-9.

These plates contain 2 ml of molten agar that was allowed to solidify.

2. Maintain the four nutrient agar “soft agar” tubes in a 45ºC water bath.

3. Use a mechanical pipetter and sterile pipettes to make 100-fold serial dilu-tions of the phage suspension as follows: a. Pipette 0.1 ml of the phage suspension into the nutrient broth tube

labeled 10-2. This is a 1/100 (10-2) dilution. Mix by rotating the tubebetween the palms of your hands.

b. Using another sterile pipette, transfer 0.1 ml of the phage suspensionfrom the 10-2 dilution into the nutrient broth tube labeled 10-4. This isa 1/100 (10-2) dilution. Mix.

c. With another sterile pipette, transfer 0.1 ml of the phage suspensionfrom the 10-4 dilution into the nutrient broth tube labeled 10-6. This isa 1/100 (10-2) dilution. Mix.

d. With one more sterile pipette, transfer 0.1 ml of the phage suspensionfrom the 10-6 dilution into the nutrient broth tube labeled 10-8. This isa 1/100 (10-2) dilution. Mix.

4. To each nutrient “soft agar” tube add the following:a. To the soft agar tube labeled 10-3, aseptically add two drops of the

E. coli B culture with a sterile pipette and 0.2 ml of the 10-2 nutrientbroth phage dilution. This is a 1/10 (10-1) dilution. Rapidly mix byrotating the tube between the palms of your hands and pour the con-tents over the hard nutrient agar plate labeled 10-3. Swirl the plategently to spread the agar. Allow the soft agar to harden.

b. To the soft agar tube labeled 10-5, aseptically add two drops of the E. coli B culture with a sterile pipette and 0.2 ml of the 10-4 nutrient

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!Don’t let the soft agar tubes solidify.

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broth phage dilution. This is a 1/10 (10-1) dilution. Rapidly mix byrotating the tube between the palms of your hands and pour the con-tents over the hard nutrient agar plate labeled 10-5. Swirl the plategently to spread the agar. Allow the soft agar to harden.

c. To the soft agar tube labeled 10-7, aseptically add two drops of the E. coli B culture with a sterile pipette and 0.2 ml of the 10-6 nutrientbroth phage dilution. This is a 1/10 (10-1) dilution. Rapidly mix byrotating the tube between the palms of your hands and pour the con-tents over the hard nutrient agar plate labeled 10-7. Swirl the plategently to spread the agar. Allow the soft agar to harden.

d. To the soft agar tube labeled 10-9, aseptically add two drops of the E. coli B culture with a sterile pipette and 0.2 ml of the 10-8 nutrientbroth phage dilution. This is a 1/10 (10-1) dilution. Rapidly mix byrotating the tube between the palms of your hands and pour thecontents over the hard nutrient agar plate labeled 10-9. Swirl the plategently to spread the agar. Allow the soft agar to harden.

5. Once the soft agar over-lay has solidified, incubate the four agar plates inan inverted position for 24 hours at 37°C. If the plates cannot be exam-ined after 24 hours, refrigerate them until the next laboratory session.

6. The number of phage particles contained in the original phage suspensioncan be estimated by counting the number of plaques formed in the soft agar.This number then is multiplied by the dilution factor. For a valid phagecount, the number of plaques per plate should not exceed 300 or be fewerthan 30.For example, suppose you count 100 PFUs in the 10-7 dilution plate.

(100) × (107) = 100 × 107 or 1 × 109 PFUs per ml of phage suspension.

7. Record your calculations in the Results section.

8. Other samples also could be examined for phage concentrations. The sewagesample used in Part D might be a very interesting sample to quantify.

uestions

1. Since phages only infect and destroy bacterial cells, might it be possible touse viruses to treat and cure bacterial diseases in humans?

2. Which factors might account for the specificity of certain viruses for certainbacteria?

3. Suppose a nutrient broth tube was inoculated with nothing but a loopful ofphage and it became cloudy after several hours of incubation. What mightbe a possible explanation?

4. What might provide possible alternatives if sewage could not be obtainedfor the isolation of bacteriophages specific for E. coli?

5. In the phage enumeration exercise, what is the significance of having the“hard agar” base?

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Name

Date Section

Exercise Results

Viruses

B. The Effect of Bacteriophages on Bacteria

8

Contents: ________________ ________________ ________________ ________________

Growth: ________________ ________________ ________________ ________________

Contents: ________________ ________________ ________________ ________________

Growth: ________________ ________________ ________________ ________________

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C. Plaque Formation

Observations and Conclusions:

Bacterium:

Observations and Conclusions:

Bacterium:

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D. Identification of Bacteriophages from Sewage

Observations and Conclusions:

Filtrateplus

E. coli BE. coli BAlone Plaques From

Sewage Phages

E. Estimating the Number of Bacteriophages

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