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Fungi, Protozoa,and MulticellularParasites

he microbial world includes a broad variety of eukaryotic organ-isms having considerably more detail and structural complex-ity than bacteria. Among these organisms are the fungi, protozoa,

and multicellular parasites. Many of the organisms are harmless saprobes,but several species are human pathogens. This exercise will give you anopportunity to work with the eukaryotic microorganisms and increaseyour familiarity with them.

Fungi (Molds and Yeasts)

The fungi are a diverse group of over 100,000 species of molds and yeasts.Molds grow as long, tangled filaments of cells, each filament known as ahypha (pl. hyphae). The mass of filaments called a mycelium (pl.mycelia) soon becomes visible to the unaided eye as it spreads across thesurface of its growth medium. Spores eventually form on specializedhyphae that form the reproductive structures called fruiting bodies, andthe mold takes on the color of the spore pigments.

Yeasts, by contrast, are unicellular microorganisms whose colonies onagar resemble bacterial colonies. Like the mold cells, yeast cells are eukary-otic with a distinct nucleus, nuclear membrane, and other organelles. Thecolonies usually appear cream-colored or another light color. The primarymode of reproduction in the yeasts is budding, a process in which a newcell forms at the periphery of the parent cell, then grows and breaks free toassume an independent existence.

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PURPOSE: to identify thestructures and features thatare characteristic of themolds and yeasts.

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pecial Materials

• Selected mold cultures and/or prepared slides• Yeast cultures (Saccharomyces cerevisiae)• Plates of Sabouraud dextrose agar or potato dextrose agar• Deep tubes of Sabouraud dextrose agar or potato dextrose agar• Dropper pipettes, straws, applicator sticks• Cotton balls, glass rods or pipe cleaners, petroleum jelly• Hydrogen peroxide• Petri dishes

rocedure

I. The Study of Molds1. Mold Cultivation: Sabouraud dextrose agar (or potato dextrose agar) con-

tains the acidity and added carbohydrate favored by molds. It is a preferredmedium for cultivating fungi. A plate of the medium should be preparedaccording to the instructor’s directions. Label the plate with your name, thedate, the name of the medium, and the designation “mold isolation.”

2. Mold Isolation: To collect molds from the laboratory environment, a num-ber of methods are available. One method is to expose a plate of Sabourauddextrose agar (or potato dextrose agar) to the air for several minutes.Another method is to sprinkle dust on the surface of the medium. A thirdis to allow the exhaust from a vacuum cleaner to spray on the agar surface.(This can be particularly interesting for revealing how fungal spores arebroadcast.) A piece of blue cheese also can be rubbed on the agar surfaceto deposit fungal spores for cultivation.

3. The Agar Plug Technique: Still another way to inoculate a plate with asample of fungus is to use the agar plug technique as illustrated in Figure 9.1. To perform this technique begin with a plate of medium that sup-ports the growth of molds. Use a plastic straw to remove a plug of agarmedium from the plate (Figure 9.1A and B). The plug of agar can be pushedout of the straw with a long applicator stick placed inside the straw. Nowuse a straw to obtain a plug of fungus or a plug of blue cheese and insert theplug into the empty space, again using an applicator stick inside the straw(Figure 9.1C). The plate should be incubated at room temperature forabout one week.

4. Viewing the Mycelium: Different molds may be examined by viewing themycelium on the plate directly with the low power (10x) objective of the microscope. The underside of the plate should be used. The edge of thecolony may be observed as long as the medium is not too thick. Repre-sentations of the molds in culture should be presented in the appropriatespace in the Results section. Wet mount preparations may be made from thecolonies as described next.

5. Wet Mounts: Microscopic observations of molds may be made by prepar-ing wet mounts. To prepare a wet mount, place a large drop of water ona clean slide. Then, using an inoculating needle, pick off a small amount ofmycelium from the mold culture and deposit it in the drop of water. Next,

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!Do not open inoculatedplates for too long a periodof time because spores are easily aerated.

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A B CA plastic straw is pushed into the agar medium.

To remove the plug from the agar, a finger is placed over the end ofthe straw, and the straw is lifted. An applicator stick is then used to push the agar plug out of the straw.

For inoculation purposes, a straw is used to obtain a plug of cheese, mold, or other material. The straw is placed into the empty space in the agar, and an applicator stick is used to push the plug into place.

Straw

Agar medium

Applicatorstick

Agar pluginside straw

Plug forinoculation

tease apart the hyphae of the mycelium, and cover the preparation with acoverslip. Two or three preparations may be made next to one anotheron a slide, but they should not be allowed to dry out.

6. Observations: Observe the molds under the low (10x) and high power(40x) objectives, and choose the lens that gives the most detail and clarity.Note the size, shape, and unique characteristics of the hyphae. Locatecellular features such as nuclei, cross walls, granules, and vacuoles.Watch for various types of spores, and note the complexity or simplicity ofthe hyphae. Place labeled drawings of each mold in the appropriate spacein the Results section. Use oil immersion only at the instructor’s direction.When your work is complete, the wet mounts should be wrapped in papertoweling and saturated with disinfectant before being discarded in thedesignated container. Prepared slides also may be available for observa-tions of molds.

7. Mold Slide Culture: A slide culture is a preparation that permits the observerto view a mold microscopically while it is growing on a glass slide. Thepreparation is set up as shown in Figure 9.2 and as follows: Prepare ridgeson a slide with petroleum jelly, preferably from a syringe (Figure 9.2A).Then set a clean coverslip atop the ridges (Figure 9.2B). Now obtain a tubeof melted Sabouraud dextrose agar (or potato dextrose agar) and inoculateit with a piece of mold from a culture. Mix the contents well. Using a drop-per pipette, obtain a small amount of the liquid inoculated agar and place itinto the area under the coverslip (Figure 9.2C). The space should be half-filled.Place the slide culture on a bent glass rod or pipe cleaner within a Petri dish,and add a wet cotton ball or paper towel to provide moisture (Figure 9.2D). Setthe dish aside to incubate for several days. When growth is deemed optimal,microscopic observations of the mold may be made by placing the slideunder the microscope. You will note the complete features of the mold, andyou will be able to study all its parts without disturbing it.

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F I G U R E 9 . 1The agar plug technique.

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II. The Study of Yeasts1. Microscope Observations: To study the morphological characteristics of

yeasts, place a drop of Saccharomyces cerevisiae or commercial yeast sus-pension on a slide. Then add a coverslip, and locate the preparation underthe low (10x) and high power (40x) objectives. The yeasts will be seenfloating about. They are clear, oval organisms resembling air bubbles, butwith internal detail. Observe the nucleus, vacuoles, and other cytoplasmicdetails of the cells. Also watch for “buds” at the surface of the cells. Theseare young cells formed by mitosis and destined to break free of the parentcell. Drawings of the yeasts may be placed in the appropriate space in theResults section.

2. Staining Yeasts: Yeasts may be stained in various ways. For example, youmay add a drop of methylene blue to a drop of yeast cells to stain the cellsand increase contrast. In addition, an air-dried, heat-fixed preparation ofyeasts may be simple-stained, as outlined in Exercise 4B. This will providea permanent slide.

3. Yeast Isolation: To isolate and cultivate yeasts, a plate of Sabouraud dextroseagar (or potato dextrose agar) may be used. Yeasts may be obtained from thesurface of the tongue by swabbing the tongue and applying the swab tothe plate. The hazy surface of a piece of fresh fruit such as an apple (or asample of unpreserved apple cider) is another possible source of yeasts; stillanother is a yeast cake. After several days the yeast will form white or cream-colored colonies, and the plate will have a “yeasty” odor similar to that of beer.Slides may be made from the yeast colonies to verify the presence of yeasts.

4. Spore Staining: In their sexual mode of reproduction, yeasts form ascosporesand are thus classified as members of the phylum Ascomycota. Ascosporesmay be stained in yeast preparations by using the spore stain techniquedescribed in Exercise 7A. Malachite green and safranin are used in thisprocedure. The ascospores will appear green, and the vegetative yeast cellswill appear orange to red. Representations of the ascospores and vegeta-tive cells should be placed in the appropriate space of the Results section.


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Place two lines of petroleum jelly on a glass slide.

Add a coverslip and cover the edges with the petroleum jelly.

Place agar inoculated with fungal spores under the coverslip andincubate.

Mold growth occurs on the slide.

Petroleum jelly

Inoculated agar

Mold growth

Moistened cotton ball

F I G U R E 9 . 2Preparation of a slide culture for studying molds.

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5. Catalase Production: Yeasts are known producers of catalase. Catalase isan enzyme that breaks down hydrogen peroxide to form water and oxygen:

catalase2 H2O2 2 H2O � O2

Yeast catalase may be demonstrated as follows: Obtain a long pipette (itneed not be sterile). Also obtain a sample of yeast either as a dried powderor as a colony on a plate. Using a mechanical pipetter or bulb, fill thepipette with hydrogen peroxide and hold it there. Now touch the end of thepipette to the yeast, thereby adding some of the cells to the hydrogen per-oxide. Almost immediately, the hydrogen peroxide will start to break downand oxygen bubbles will rise in the tube.

6. Yeast Enzymes: Yeasts contain many of the enzymes used in the metab-olism of glucose and other carbohydrates. The presence of these enzymesis demonstrated by the following procedure: Obtain a small 10-ml test tubeand place in it 9 ml of 5% glucose solution and 1 ml of yeast suspension.This should fill the tube to the brim (add water if necessary). Now obtaina larger 25-ml test tube and invert it over the small tube. Using a pencil,push the small tube high up into the larger tube until the surface of the smalltube touches the bottom of the large tube. Quickly invert the large tube. Atthis point the large tube will be upright and the small tube will be upsidedown. Set the tube aside in a warm environment, and observe the contentsof the small tube at regular intervals. Refer to step 7 to complete thisexercise.

As the minutes pass, you will see the bubble in the small tube gradu-ally increase in size. Through the chemical process of respiration, yeastenzymes are breaking down the glucose and releasing gas, mainly carbondioxide gas. The process is identical to that happening in the early stagesof fermentation where yeasts metabolize the glucose in grape juice. Theexercise may be modified to study the effects of yeasts on different carbo-hydrates (for example, sucrose, lactose, maltose) or on different concen-trations of the same carbohydrate, (for example, 1%, 5%, 10%). It also ispossible to study the effects of an inhibitor substance on the chemicalprocess. The effects of temperature, pH or other factors may also be mea-sured by altering the procedure to suit your needs.

7. Lactose Metabolism: It is interesting to note that many humans lack theenzyme lactase and are unable to digest lactose. These individuals suf-fer from a condition called lactose intolerance. Fortunately, they canadd a commercially prepared lactase product such as Lactaid® or DairyEase® to dairy foods and digest the lactose this way. Can yeasts metab-olize lactose?

The procedure explained in Steps 6 and 7 will be used in this exercise.Obtain a small 10-ml test tube and fill it with 9 ml of 5% lactose solution and 1 ml of yeast suspension. This should fill the tube up to the brim (addwater if necessary). Now prepare a second tube of lactose and yeast, but thistime add two drops of commercial Lactaid® or Dairy Ease®. Invert twolarger 25-ml test tubes over the two smaller tubes and push the smalltubes high up into the larger tubes until they touch the bottoms of the

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large tubes. Quickly invert the large tubes so the small tubes are upsidedown. Set the tubes aside in a warm environment, and observe the contentsat regular intervals.

As the minutes pass, you will note the development of gas bubbles inone tube but not the other. As described on the previous page, gas bubblesmean that carbohydrate metabolism is taking place. In this instance, the lac-tose is being broken down and the glucose resulting from its breakdown isbeing metabolized. In which tube is this metabolism taking place? Why?Enter your observations in the Results section and suggest ways of modi-fying the exercise to discover other properties of lactase activity.

Protozoa and Multicellular Parasites

The protozoa include over 65,000 species of eukaryotic animal-like organisms.The organisms are unicellular and microscopic, and most are free-livingspecies, although a few protozoal species cause serious human diseases suchas malaria, sleeping sickness, and trichomoniasis.

Multicellular parasites are the flatworms and roundworms that com-monly inhabit the natural environment and infect millions of people on aglobal basis. In the strict sense, they are animals, but they are studied inmicrobiology because of their small size and ability to cause infectiousdisease. The study of pathogenic protozoa and multicellular parasites con-stitutes the discipline of parasitology.

pecial Materials

• Samples of pond and aquarium water• Fresh hay infusions• 10% methylcellulose• Prepared slides of protozoa and multicellular parasites• Dilute acid, India ink, yeasts• Salt and sugar solutions

rocedure

1. Microscope Observations: Protozoa may be observed from cultures, sam-ples of pond or aquarium water, or a fresh hay infusion. Plant materialfrom a pond or aquarium is another good source of protozoa. When usingplant material, rub a piece of the plant on the slide to dislodge the proto-zoa. A drop of 10% methylcellulose may be added to provide a viscous envi-ronment and slow the movement of protozoa. Alternately, a thin fragmentof lens paper will provide threads to trap the protozoa for observation.Observe the organisms for size, shape, cellular features, locomotororganelles (e.g., flagella, cilia, pseudopods) and other distinctive charac-teristics. Drawings should be prepared in the appropriate spaces, withlabels pointing to the important characteristics. Prepared slides may beused for this exercise.

2. Estimating Size: To estimate the size of protozoa, follow the procedure out-lined in exercise 3B.

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PURPOSE: to become famil-iar with some features char-acteristic of the protozoaand multicellular parasites.

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3. Protozoal Behavior: Various types of behavior may be observed in the pro-tozoa. For example, a drop of dilute acid may be placed at the edge of theslide to see how the protozoa respond. India ink may be used in a seconddemonstration. It also is possible to mix a sample of yeasts with the protozoato observe the feeding mechanism. Still another way to study protozoalbehavior is to dip a piece of thread in a salt or sugar solution and place thethread in the drop of protozoa before adding the coverslip. Thread dippedin sugar solution and ammonia solution should elicit different behaviors.

4. Multicellular Parasites: To study the multicellular parasites, preparedslides will be available. Using whichever magnification is best for thespecimen, observe their shape, size, and distinctive features. Anatomicalstructures should be visible in the parasite, noting the salient featuresused in its identification. Representations of the parasites may be placed inthe appropriate space of the Results section.

Multicellular parasites also may be sought in fresh feces from a dog, cat,or other animal. Another source is an animal specimen used in anatomy lab-oratories. For example, the intestinal contents of preserved rats and cats maycontain parasites suitable for study. The instructor will give directions onlocating the parasites. Parasitology manuals will assist in their identification.

uestions

1. How do the fungi, protozoa, and multicellular parasites compare with thebacteria in size and morphological features?

2. Why is Sabouraud dextrose agar of greater value than nutrient agar for theisolation of molds from the environment?

3. What conditions might prevent a successful slide culture of a mold?

4. Explain the advantages of a wet mount preparation over a prepared slide.What disadvantages are there?

5. It is noted that certain molds come close but do not overgrow one anotheron Sabouraud dextrose agar. How might this be explained?

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Organism: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

Organism: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________


Name

Date Section

Exercise Results

Fungi, Protozoa, and Multicellular Parasites

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Organism: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

Organism: ______________________ ______________________ ______________________

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Organism: ______________________ ______________________ ______________________

Magnif.: ______________________ ______________________ ______________________

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Organism: ______________________ ______________________ ______________________

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Organism: ______________________ ______________________ ______________________

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43038 ch09 0079

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