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Part V MedicalMicrobiology

evolutions, be they political, social, or scientific, are events thattransform the world in which we live. Certainly, this applies to theClassical Golden Age of microbiology (1854–1914) and the break-

through in identifying bacteria as a cause of infectious disease. Pasteur,Koch, and their contemporaries were among the first to identify a specific bac-terium with a specific disease. Since that time, medical microbiology hasmade great advances in laboratory diagnosis of disease, and in under-standing pathogenesis and the role of immunity in the infectious cycle. Yet,many of the diseases common in the late 1800s remain with us today.

Tuberculosis (TB) was called the white plague when Koch was study-ing the organism because of the white granules present in the lungs of itsvictims. TB is caused by just one of more than 80 species of the genusMycobacterium. Most species though are not pathogenic. Identificationincludes its mold-like morphology and thick, waxy cell walls that can beidentified with an acid-fast staining technique (Exercise 16). The genusStreptococcus consists of nearly 40 species of gram-positive cocci thattypically grow in chains or pairs. Certain groups can be identified by theirhemolytic activity in culture and others by their growth on selective and dif-ferential media (Exercise 17).

The genus Neisseria consists of gram-negative diplococci. The patho-genic members are difficult to distinguish using morphological and culturalcharacteristics, although they are recognizable by the diseases they cause—a form of meningitis and gonorrhea. Many species are commensals of theupper respiratory tract (Exercise 18). Alexander Ogston, a Scottish sur-geon, was the first to describe the cluster-forming bacterial cells causingpyogenic (pus-forming) diseases in humans in the 1880s. He named thegenus Staphylococcus. The genus consists of three major species thatcan be identified from other bacteria and from one another by using selec-tive and differential growth media (Exercise 19).

The enteric bacteria consist of a large number of genera that livewithin or are responsible for infections in the intestinal tract. Species can beseparated and identified on differential and selective growth media (includ-ing a rapid Enterotube II system), by the presence or absence of gas and acidproduction, and with other special tests (Exercise 20).

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The anthrax bacillus, the subject of some of Koch’s most elegant work,represents the most pathogenic species of the genus Bacillus. However,many species are common soil inhabitants and can be identified by severaltypical characteristics (Exercise 21). The genus Clostridium also consistsof sporeforming species but it can be separated from Bacillus by its neces-sity for growth under anaerobic conditions. Most of the clostridia are sapro-bic species that naturally grow in the soil and can be isolated and studiedusing anaerobic techniques such as the GasPak system (Exercise 22).

Unlike some of the other species in this discussion, the genus Lacto-bacillus is not a significant pathogen, although it may be associated withdental caries (Exercise 23). Still, it is of medical significance because it is partof the human microbiota (normal flora). Lactobacillus species normallyresiding in the vagina can protect against fomite-transmitted gonorrheaand produce a low pH that is inhibitory to other potential disease-causingmicroorganisms. The ability of Lactobacillus species to produce acid is ofgreat significance in dairy microbiology and the genus can be easily isolatedfrom dairy products like yogurt (Exercise 23).

Many of the exercises in Part V also can be used to demonstrate the use-fulness of special media formulations to select for or differentiate betweenbacterial species. Selective media are used to isolate (select) specificgroups of bacteria growing on agar (Exercises 17, 23), while differentialmedia facilitate a visual differentiation of different species of bacterialcolonies growing on an agar plate (Exercises 19, 20). Enriched media,where additional growth factors are added to a medium, also can act as aselective or differential medium (Exercises 17, 18).

earning Objectives

When you have completed the exercises in Part V, you should be capable of:• Performing an acid-fast stain to identify Mycobacterium species. • Isolating and culturing streptococci from the upper respiratory tract and oral cavity.• Identifying Neisseria species isolated from the throat.• Using selective and differential media, and employing biochemical tests, to

isolate and differentiate staphylococci.• Using selective and differential media to isolate enteric bacteria, and employing

TSI agar and the IMViC series to identify enteric species.• Identifying enteric bacteria using the Enterotube II system. • Performing the appropriate biochemical tests and staining procedures to isolate

and identify members of the genus Bacillus.• Employing anaerobic techniques to isolate and identify Clostridium species.• Isolating lactobacilli from a mixed population and performing a caries

susceptibility test.• Distinguishing between selective and differential media.

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The GenusMycobacterium

embers of the genus Mycobacterium include several sapro-bic commensals of the body as well as Mycobacterium tuber-culosis, the agent of tuberculosis, and Mycobacterium leprae, the

cause of leprosy. The cells are straight or slightly curved rods without flagella,spores, or capsules. They often display a filamentous, funguslike growth onagar media but quickly fragment to rods when disturbed. The prefix myco-refers to the funguslike property. Mycobacterium species grow slowly inagar media and may require special supplements such as egg, potato, orserum. Lowenstein-Jensen medium commonly is used to cultivate them.

In this exercise, laboratory cultures of Mycobacterium will be examinedfor typical colony and cellular appearance, and the acid-fast technique willbe performed to demonstrate an important property used for detectingMycobacterium species.

Colony and Cellular Morphology

The colony and cellular morphology of an organism are inherited traitsderived from biochemical information stored in the chromosomal DNA.They are distinctive for each species and are passed from generation togeneration in the genes. In this section, the colony and cellular morphologyof Mycobacterium species will be studied by noting the characteristics ofcolonies isolated on agar media and by preparing simple stains.

pecial Materials

• Agar cultures of Mycobacterium smegmatis

• Plates of Lowenstein-Jensen medium

rocedure

1. Examine the cultures of Mycobacterium for unique features of this organ-ism, including pigmentation and consistency of the growth. Information inExercise 2 (Figure 2.3) may be used as a guide. With a sterile inoculatingneedle, touch the growth to determine its texture. Note your observationsin the Results section.

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PURPOSE: to recognize thecolony and cellularcharacteristics of themycobacteria.

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2. Isolate colonies of Mycobacterium by streaking a plate of Lowenstein-Jensen medium using the streak plate technique described in Exercise 2A.Incubation should be at 37° C for at least 7 days since Mycobacteriumgrows slowly. Examine the isolated colonies with reference to Exercise 2,and enter your observations in the Results section together with a dia-gram of individual colonies.

3. Cellular morphology may be examined by preparing an air-dried, heat-fixedsmear of the organism and staining by the simple stain procedure out-lined in Exercise 4. Since heat is necessary to assist stain penetration, theslide should be thoroughly heat-fixed and the stain should be appliedimmediately. In the Results section, enter a representation of typicalMycobacterium cells.

Acid-Fast Stain Technique

Members of the genus Mycobacterium contain an abundance of fats andwaxes in their cell walls, including mycolic acid. Excessive physical treat-ment such as heat is necessary to penetrate this layer. However, once stainhas penetrated and combined with the mycolic acid, the cells resist decol-orization even when a dilute acid-alcohol solution is applied. The organismstherefore are said to be acid-resistant or acid-fast.

The acid-fast technique is important in the diagnosis of such diseasesas tuberculosis and leprosy. The procedure is essentially similar to thatdevised by Ziehl and Neelsen in the 1880s. In a mixed culture, the primarystain colors all cells, but only the acid-fast organisms resist the subsequenttreatment with acid-alcohol. A counterstain is then applied for bacteria thathave been decolorized. These bacteria are said to be nonacid-fast.

Commercially-available prepared slides can be used to replace the stain-ing procedure.

pecial Materials

• Agar cultures of Mycobacterium smegmatis• Selected nonacid-fast organisms as controls• Ziehl-Neelsen carbolfuchsin• Acid alcohol• Methylene blue• Steaming apparatus

rocedure

1. Set up a steaming apparatus as shown in Figure 16.1A and explained inExercise 7 (for spore staining). Bring the water to a rolling boil.

2. On a clean slide, prepare three air-dried, heat-fixed smears: a Myco-bacterium species, a nonacid-fast bacterium, and a mixture of the two.The mixture is prepared by adding a loopful of one bacterium to a loop-

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154 16 T H E G E N U S M Y C O B A C T E R I U M

!The acid-fast staining shouldonly be performed in a wellventilated area, preferably afume hood.

PURPOSE: to identifyMycobacterium species bythe acid-fast procedure.

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A

B C

D E F

Saturate paper with carbolfuchsin;steam for 5 minutes.

Remove paper, cool, and rinse with water.

Decolorize for 15 seconds two times.

Rinse with water. Counterstain with methyleneblue for 1 minute.

Rinse with water, blot dry, and observe.

Carbolfuchsin

Water

Acidalcohol

Methyleneblue

Water

Water

ful of water, and then adding a loopful of the second bacterium before thewater dries.

3. Cut a piece of blotting paper just large enough to cover the smears. Placethe paper over the smears, and saturate it with carbolfuchsin, the primarystain. Place the slide on the rack over the boiling water.

4. Allow the slide to remain over the boiling water for 5 minutes whileadding sufficient carbolfuchsin to keep the smears wet. At the conclusionof the staining period, remove the paper and wash the slide with a gen-tle stream of distilled water (Figure 16.1B).

5. Decolorize the slide with acid-alcohol by placing several drops on thesmears and rocking them back and forth for 15 seconds as performed in theGram stain technique (Exercise 6). Allow the excess to drip off, then repeat the decolorization for another 15-second interval or until the drippings areclear (Figure 16.1C). Gently wash the slide with distilled water (Figure 16.1D).

6. Stain the slide with methylene blue, the counterstain, for 1 minute (Fig-ure 16.1E). Rinse the slide and blot it dry (Figure 16.1F).


Quick ProcedureAcid-Fast Stain

1. Place the heat-fixedslide over boiling water.

2. Stain with carbolfuchsinfor 5 min; wash.

3. Decolorize for two 15-sec periods withacid-alcohol; wash.

4. Stain with methyleneblue for 1 min; wash;dry; observe.

F I G U R E 1 6 . 1The acid-fast stain technique.

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7. Locate the stained cells using the low power (10x) lens. Then, examine thesmears under 40x and oil immersion, and note the red acid-fast rods andthe blue nonacid-fast organisms. The slide containing the bacterial mixtureshould contain both red and blue forms. Enter representations of theorganisms in the appropriate space in the Results section indicatingwhether they are acid-fast or nonacid-fast.

Cold Acid-Fast Stain Technique

The acid-fast technique may also be performed by a cold method that elim-inates the steaming step. The carbolfuchsin used in this method contains thedetergent tergitol 7. This substance dissolves the waxes in the cell walls ofMycobacterium species and thereby facilitates penetration of the dye. Thestaining reagent is called Kinyoun carbolfuchsin after its developer.

pecial Materials

• Kinyoun carbolfuchsin

rocedure

1. Perform the acid-fast technique as described in Part B, with the followingexception: Instead of saturating a piece of paper with stain and steaming itfor 5 minutes, flood the smears with Kinyoun carbolfuchsin, and permit thestain to remain for 5 minutes. Wash the stain free, and continue with theacid-alcohol decolorization step.

2. Conclude the procedure by staining with Brilliant Green for 1 minute.The colors of acid-fast bacteria will be similar to those in the hot stainingmethod; nonacid-fast bacteria will be green. Enter your drawings in theResults section.

uestions

1. In what ways is the acid-fast technique similar to the Gram stain tech-nique? How does it differ?

2. If the cells from the mixed smear were observed after each step of theacid-fast or cold acid-fast procedure, predict the colors of the cells at eachstep.

3. Why are the acid-fast procedures considered differential staining techniques?

4. Suppose alcohol instead of acid-alcohol were used in the acid-fast technique.Would decolorization take place? How would this affect the results?

5. Explain the diagnostic importance of the acid-fast technique.

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PURPOSE: to identifyMycobacterium specieswithout using steam.

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Organism:

Form:

Elevation:

Margin:


Name

Date Section

Exercise Results

The Genus Mycobacterium

A. Colony and Cellular Morphology

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Cultural characteristics:

Colony characteristics:

Simple Stained Smear

Colonial Morphology

Organism:

Magnif.:

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C. Cold Acid-Fast Stain Technique

Observations and Conclusions:

B. Acid-Fast Stain Technique

Organism: ______________________ ______________________ ______________________

Acid-fast reaction: ______________________ ______________________ ______________________

Magnif: ______________________ ______________________ ______________________

Organism: ______________________ ______________________ ______________________

Acid-fast reaction: ______________________ ______________________ ______________________

Magnif: ______________________ ______________________ ______________________

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43038 CH16 0151