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CHAPTER

8 Use of Colony Morphology for the Presumptive Identification of MicroorganismsGeorge Manuselis, Connie R. Mahon*

■ IMPORTANCE OF COLONIAL MORPHOLOGY AS A DIAGNOSTIC TOOL

■ INITIAL OBSERVATION AND INTERPRETATION OF CULTURES

■ GROSS COLONY CHARACTERISTICS USED TO DIFFERENTIATE AND IDENTIFY MICROORGANISMS PRESUMPTIVELYHemolysisSize

Form or MarginElevationDensityColorConsistencyPigmentOdor

■ COLONIES WITH MULTIPLE CHARACTERISTICS■ GROWTH OF ORGANISMS IN LIQUID MEDIA

CHAPTER OUTLINE

OBJECTIVESAfter reading and studying this chapter, you should be able to:1. Describe how growth on blood, chocolate, and MacConkey agars is

used in the preliminary identification of isolates.2. Differentiate α-hemolysis from β-hemolysis on blood agar culture

medium.3. Associate the colony characteristics shown on blood, chocolate, and

MacConkey agars with the microscopic findings on direct smear, and use the information in the presumptive identification of microorganisms.

4. Using colonial morphology, differentiate among the following microorganisms:• Staphylococci and streptococci• Streptococcus agalactiae and Streptococcus pyogenes• Neisseria spp. and staphylococci• Yeast and staphylococci• “Diphtheroids” and staphylococci• Lactose fermenters from lactose nonfermenters• “Swarming” Proteus species from other Enterobacteriaceae

Case in PointAn exudate from a sacral decubital ulcer on a 65-year-old hos-pital inpatient was cultured on blood agar plate (BAP), chocolate (CHOC), and MacConkey (MAC) agars. Direct smear examina-tion showed many white blood cells, a moderate number of gram-positive cocci in pairs and clusters, and a few gram-negative bacilli. After overnight incubation, three colony mor-photypes were visible on the BAP. The first was a moderate growth of a medium-sized β-hemolytic, which was yellowish white and creamy-buttery looking. The second colony was also β-hemolytic but larger, mucoid, and gray. The third type of colony was large, gray, and mucoid similar to the second but was nonhemolytic. The MAC agar showed two colony morphotypes—a light growth of dark pink, dry-looking colonies

with a surrounding pink precipitate and a few clear nonlactose fermenting colonies. Based on the Gram stain results and colo-nial characteristics of the isolates, appropriate biochemical tests and antibiotic susceptibilities were performed to identify the causative agents of the ulcer.

Issues to ConsiderAfter reading the patient’s case history, consider:■ How the colony morphology of isolates is used to identify

microorganisms presumptively■ How to correlate the direct smear examination findings

with the colony morphology of isolates on each culture medium

■ How the colony morphology of each isolate can dif-ferentiate between pathogenic and nonpathogenic microorganisms

*My comments are my own and do not represent the view of Health Resources and Services Administration of the Department of Health and Human Services.

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170 PART I Introduction to Clinical Microbiology

• Enhance the quality of patient care through rapid report-ing of results and by increasing the cost-effectiveness of laboratory testing. This may best be illustrated by using sputum cultures as an example. The upper respiratory tract contains many indigenous organisms, and to identify every organism in culture would be a time-consuming, cost-prohibitive, and insurmountable task. Microbiologists must be able to differentiate potential pathogens from the “usual” inhabitants of the upper respiratory tract and direct the diag-nostic workup toward only potential pathogens. Potential pathogens are presumptively identified by colonial character-istics, and preliminary reporting initiates immediate therapy.

• Play a significant role in quality control, especially of auto-mated procedures and other commercially available iden-tification systems. When commercial and automated systems are used, a mixed inoculum (polymicrobic/containing more than one genus and species or both) produces a biochemical test result or erroneous interpretation of reactions that signifi-cantly alters the identification (see Chapter 9). The ability of the microbiologist to determine whether the inoculum is mixed and to ascertain whether the results generated by a commercial or automated system correlate with the suspected identification of the organism is an important component of quality control that is accomplished by recognizing organisms by their colonial characteristics.

Initial Observation and Interpretation of CulturesGenerally, microbiologists observe the colonial morphology of organisms isolated on primary culture after 18 to 24 hours of incubation. Incubation time may vary according to when the specimen is received and processed in the laboratory, which may affect the “typical” morphology of a certain isolate. For example, young cultures of Staphylococcus aureus may appear smaller and may not show the distinct β-hemolysis that older cultures produce. In addition, the microbiologist must be aware of factors that may significantly alter the colo-nial morphology of growing microorganisms. These factors include the ingredients present in the medium, the inhibitory nature of these ingredients, and antibiotics that may be present in the medium.

The interpretation of primary cultures, commonly referred to as plate reading, is actually a comparative examination of the colony morphology of microorganisms growing on various culture media. Many specimens, such as sputum and wounds that arrive in the clinical laboratory, are plated on various culture media such as BAP, CHOC, and MAC. Each type of agar plate is examined in relationship to the other. As a set of culture media, comparative colonial examination of growth from a specimen occurs.

The ability to determine which organisms grow on selective and nonselective media aids the microbiologist in making an initial distinction between gram-positive and gram-negative iso-lates. BAP and CHOC support the growth of various fastidious (hard to grow, requires additional growth factors) and nonfas-tidious organisms, gram-positive bacteria, and gram-negative bacteria.

The importance of mastery of colonial morphology (colony characteristics and form) and interpretation of Gram-stained smears from clinical specimens and microbial

colonies cannot be overemphasized. Although Gram-stained smears provide initial identification of microorganisms by micro-scopic characterization, description of the physical growth char-acteristics of microorganisms on laboratory media facilitates the initial identification processes.

Close your eyes and imagine the physical characteristics of a person you know well. The person’s height, weight, shape, color and style of hair, eyes, freckles, and color of skin as well as voice or laugh may make that person distinctive in a crowd or when his or her back is facing you. In the same manner, many micro-organisms have specific characteristics that distinguish them in a crowd of other genera or species.

This chapter explains how the characterization of colonies on culture media and the findings on stained direct smears facilitate presumptive identification of commonly isolated organisms. The characteristics that are used to describe colony morphology of certain groups of organisms and how these characteristics are used to differentiate one species from a closely related species and one genus from another are discussed.

Importance of Colonial Morphology as a Diagnostic ToolIn many ways, the usefulness of colonial morphology extends the capabilities of the microbiologist and, ultimately, the clinical laboratory. The ability to provide a presumptive identification by colonial morphology may include the following:• Provide a presumptive identification to the physician.

Even in this age of rapid identification systems, incubation times and procedures can be protracted. In a critical situation, the microbiologist makes an educated judgment about the presumptive identity before performing diagnostic identifica-tion procedures.

Key Termsα-Hemolysisβ-HemolysisBrittleButyrousColonial morphologyConsistencyCreamyDensityElevationEscherichia/Citrobacter-like

organismsFastidiousFilamentousFormHemolysisKlebsiella/Enterobacter-like

organisms

Lactose fermenterMarginNonlactose fermenterOpaquePigmentRhizoidSmoothStreamersSwarmingTransilluminationTranslucentTransparentTurbidityUmbilicateUmbonate

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CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 171

presumptive identification and determine how to proceed in iden-tifying the isolated organisms.

While MAC supports most gram-negative rods, especially the Enterobacteriaceae, it inhibits growth of gram-positive organ-isms and some fastidious gram-negative organisms, such as Hae-mophilus and Neisseria spp. Growth on BAP and CHOC but not on MAC is indicative of a gram-positive isolate or of a fastidious gram-negative bacillus or coccus.

Gram-negative rods are better described on MAC agar because these organisms produce similar-looking colonies on BAP and CHOC media. On BAP and CHOC, gram-negative rods produce large colonies that appear gray and sometimes mucoid, and if hemolytic, hemolysis is seen on BAP. True hemolysis is not seen on CHOC. MAC is best used to characterize gram-negative rods because lactose fermenters can be differentiated from nonlac-tose fermenters. Lactose fermenters are easily detected by the color change they produce on the medium; as the pH changes when lactose is fermented, the organisms produce pink, dark pink, or red colonies (Figure 8-2, A). Colonies of nonfermenters remain clear and colorless (Figure 8-2, B).

This differentiation is particularly important in screening for enteric pathogens from stool cultures. Most enteric pathogens do not ferment lactose.

Generally, organisms that grow on BAP also grow on CHOC, but not all organisms that grow on CHOC grow on BAP. Although BAP supports fastidious organisms, highly fastidious species, such as Haemophilus spp. and Neisseria gonorrhoeae, do not grow on it. CHOC provides nutritional growth requirements to support highly fastidious organisms such as Haemophilus spp. and N. gonorrhoeae. Therefore, a gram-negative bacillus that grows on CHOC but not on BAP or MAC would be suspected to be Haemophilus spp., whereas gram-negative diplococci with the same growth pattern would be suspected to be N. gonor-rhoeae (Figure 8-1). The microbiologist is able to provide a

FIGURE 8-1 Clockwise from the top: chocolate (CHOC), blood agar plate (BAP), and MacConkey (MAC). The large colonies growing on all three plates are gram-negative rods (enterics). These gram-negative rods grow larger, gray, and mucoid on BAP and CHOC. Notice the smaller, grayish brown fastidious colonies of Haemophilus organisms growing on CHOC (arrow), which are not growing on BAP or MAC.

FIGURE 8-2 A, Lactose-fermenting, gram-negative rods producing pink colonies on MacConkey (MAC). B, Nonlactose-fermenting, gram-negative rods producing colorless colonies on MAC.

A B

✓ Case Check 8-1 As illustrated in the Case in Point at the beginning of the chapter, three colony morphotypes were observed on BAP. Because the Gram-stained smear showed both gram-positive and gram-negative bacteria, three types of organisms should be observed on a nonselective medium such as BAP.

✓ Case Check 8-2 Certain enteric pathogens, such as Escherichia/Citrobacter-like organ-isms, produce dry, pink colonies with a surrounding “halo” of pink, precipitated bile salts (Figure 8-3), whereas Klebsiella/Enterobacter-like organisms produce large, mucoid pink colonies that occasionally have cream-colored centers (Figure 8-4). These characteristics on MAC are helpful in presumptive identification. In the Case in Point, dry, dark pink colonies were observed on MAC, indicating the presence of a lactose-fermenting, gram-negative rod. Colonies of nonfermenters that were clear and colorless (see Figure 8-2, B) were also recovered from this patient’s sample.

This is a comparative analysis of the growth on the three types of culture media. Microorganisms grow on culture media in the same proportion or concentration in which they are present in the clinical specimen. Because many specimens are polymicrobic, this feature can be beneficial in identifying different colony types.

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discoloration of the medium is the result of growth of the organism on the plate. Often the colony has to be removed with a loop to visualize the hemolytic pattern. Proper technique requires the passing of bright light through the bottom of the plate (transillumination) to determine whether the organism is hemolytic (Figure 8-5). Many organisms have no lytic effect on the RBCs in BAP and are referred to as nonhemolytic. Although there are many types of hemolysis, only α-hemolysis and β-hemolysis are illustrated in this chapter.

α-Hemolysisα-Hemolysis is partial lysing of erythrocytes in a BAP around and under the colony that results in a green discoloration of the medium. Examples of organisms that produce α-hemolysis include Streptococcus pneumoniae and certain viridans strepto-cocci. (For a comparison of the colonial morphology of these two organisms, see Figure 8-24.)

β-Hemolysisβ-Hemolysis is complete clearing of erythrocytes in BAP around or under the colonies because of the complete lysis of RBCs.

Gross Colony Characteristics Used to Differentiate and Identify Microorganisms PresumptivelyBy observing the colonial characteristics of the organisms that have been isolated, the microbiologist is able to make an edu-cated guess regarding the identification of the isolate. The fol-lowing descriptions are routinely used to examine colony characteristics. Many of the following colony characteristics may vary among species and strains of the same genus.

HemolysisOn BAP, hemolysis (Greek hemo: pertaining to red blood cells [RBCs]; lysis: dissolution or break apart) observed in the media immediately surrounding or underneath the colony is a reaction caused by enzymatic or toxin activity of bacteria. Hemolysis (e.g., α, β, or no hemolysis with other colony characteristics) on BAP is helpful in presumptive identification, particularly of streptococci and enterococci (see Chapter 15). It is important to determine whether true hemolysis is present or whether

FIGURE 8-3 A, Lactose-fermenting Escherichia/Citrobacter-like organisms growing on MacConkey (MAC). Notice the dry appearance of the colony and the pink precipitate of bile salts extending beyond the periphery of the colonies. B, Close-up of dry, flat Escherichia/Citrobacter-like lactose fermenters growing on MAC. Compare with Figure 8-4, B.

A B

FIGURE 8-4 A, Klebsiella/Enterobacter-like lactose fermenters growing on MacConkey (MAC). Notice the pink, heaped, mucoid appearance. B, Close-up of Klebsiella/Enterobacter-like colonies on MAC. Notice the mucoid, heaped appearance and the slightly cream-colored center after 48 hours’ growth.

A B

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measures a colony. Size is generally a visual comparison between genera or species. For example, gram-positive bacteria generally produce smaller colonies than gram-negative bacteria. Staphylo-coccus species are usually larger than Streptococcus species. Figure 8-7 shows colonies of gram-negative rods compared with gram-positive cocci.

Form or MarginThe edge of the colonies should be observed and the form, or margin, described as smooth, filamentous, rough or rhizoid, or irregular (Figure 8-8). Colonies of Bacillus anthracis on visual

FIGURE 8-5 The use of transillumination to determine whether colonies are hemolytic. The technique can be used for MacConkey also to see slight color differences in nonlactose fermenters.

Light sourceBlood agar plate

Colonies

FIGURE 8-6 Chocolate (CHOC) does not display true hemolysis because the red cells in the medium have already been lysed. Bacteria that are hemolytic on blood agar plate usually are green around the colony on CHOC.

FIGURE 8-7 Left, blood agar plate (BAP): small, white colonies are gram-positive cocci. Right, BAP: large, gray, mucoid colo-nies are enteric gram-negative rods.

Certain organisms, such as group A β-hemolytic streptococci (Streptococcus pyogenes), produce a wide, deep, clear zone of β-hemolysis, whereas others, such as group B β-hemolytic strep-tococci (Streptococcus agalactiae) and Listeria monocytogenes (a short, gram-positive rod) produce a narrow, diffuse zone of β-hemolysis close to the colony. These features are helpful hints in the identification of certain species of bacteria. (For a com-parison of the colonial characteristics of group A and group B streptococci, see Figure 8-25.) CHOC does not display true hemolysis because the RBCs in the medium have already been lysed. Organisms that are α-hemolytic or β-hemolytic on BAP usually show a green coloration around the colony on CHOC (Figure 8-6). However, this coloration should not be mistaken for a hemolytic characteristic.

SizeColonies are described as large, medium, small, or pinpoint. However, a microbiologist rarely takes a ruler and actually

FIGURE 8-8 Illustration of form or margin to describe colonial morphology.

Filamentous

Irregular

Smooth

Rough

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convex colonies. In comparison, β-hemolytic streptococci gener-ally produce flat colonies.

DensityThe density of the colony can be transparent, translucent, or opaque. To see the differences in the density of colonies, it is useful to look through the colony while using transillumination. Translucent colonies allow some light to pass through the colony, and opaque colonies do not (Figure 8-12). β-Hemolytic strep-tococci except group B (S. agalactiae) are described as trans-lucent. S. agalactiae produces colonies that are semiopaque, with the organisms concentrated at the center of the colony, sometimes described as a bull’s-eye colony. Staphylococci and other gram-positive bacteria are usually opaque. Most gram-negative rods are also opaque. Bordetella pertussis is described as shiny, similar to a half-pearl, on blood-containing media (see Chapter 19).

ColorIn contrast to pigmentation, color is a term used to describe a particular genus in general. Colonies may be white, gray, yellow, or buff. Coagulase-negative staphylococci are white (Figure 8-13), whereas Enterococcus spp. may appear gray. Certain Micrococcus spp. and Neisseria (nonpathogenic) spp. are yellow or off-white (Figure 8-14). “Diphtheroids” are buff. Most gram-negative rods are gray on BAP.

ConsistencyConsistency is determined by touching the colony with a sterile loop. Colony consistency may be brittle (splinters), creamy (butyrous), dry, or waxy; occasionally, the entire colony adheres (sticks) to the loop. S. aureus is creamy, whereas certain

examination are described as “Medusa heads” because of the filamentous appearance. Certain genera such as Proteus spp. (especially Proteus mirabilis and Proteus vulgaris) may swarm on nonselective agar such as blood or chocolate. Swarming is a hazy blanket of growth on the surface that extends well beyond the streak lines. Figure 8-9 shows swarming colonies of Proteus spp. Diphtheroids produce colonies that have rough edges (Figure 8-10), whereas certain yeast produce colonies that are described as stars or colonies with feet or pedicles. (For a comparison of the colonial morphology of yeast and staphylococci, see Figure 8-26.)

ElevationThe elevation should be determined by tilting the culture plate and looking at the side of the colony (Figure 8-11). Elevation may be raised, convex, flat, umbilicate (depressed center, concave—an “innie”), or umbonate (raised or bulging center, convex—an “outie”). S. pneumoniae typically produces umbili-cate colonies, unless the colonies are mucoid because of the presence of polysaccharide capsule. S. aureus typically produces

FIGURE 8-9 Swarming colonies of Proteus spp. The organism was inoculated in the middle of the blood agar plate (arrow).

FIGURE 8-10 “Diphtheroid” colonies with rough edges, dry appearance, and umbonate center growing on blood agar plate.

FIGURE 8-11 Illustration of elevations to describe colonial morphology.

Flat

Raised

Convex or dome

Umbilicate

Umbonate

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• Chromobacterium violaceum—purple• Prevotella melaninogenica—brown-black (anaerobic)

Pigment production for these organisms is variable.

OdorOdor should be determined when the lid of the culture plate is removed and the odor dissipates into the surrounding environ-ment. The microbiologist should never inhale directly from the plate. Examples of microorganisms that produce distinctive odors are as follows:• S. aureus—old sock (stocking that has been worn continu-

ously for a few days without washing); this odor is evident when growing on mannitol salt agar

• P. aeruginosa—fruity or grapelike• P. mirabilis—putrid

Neisseria spp. are sticky. Nocardia spp. produce colonies that are brittle, crumbly, and wrinkled, resembling bread crumbs on a plate. Diphtheroid colonies are usually dry and waxy. Most β-hemolytic streptococci are dry (except for mucoid types), and when pushed by a loop, the whole colony remains intact.

PigmentPigment production is an inherent characteristic of a specific organism confined generally to the colony. Examples of organ-isms that produce pigment include the following:• P. aeruginosa—green, sometimes a metallic sheen (Figure

8-15)• Serratia marcescens—brick-red (Figure 8-16), especially at

room temperature• Kluyvera spp.—blue

FIGURE 8-12 Density.

transparent transparent translucent translucentcolony colony

opaqu paque

FIGURE 8-13 Example of white colonies of coagulase-negative staphylococci on blood agar plate.

FIGURE 8-14 Example of the yellow colonies characteristic of certain nonpathogenic species of Neisseria organisms on blood agar plate.

FIGURE 8-15 A, Pseudomonas aeruginosa illustrating the metallic sheen and green pigmentation of colonies on blood agar plate (BAP). B, Not all strains of the same organism have the same colonial appearance. This is a mucoid strain of P. aeruginosa on BAP.

A

B

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• Haemophilus spp.—musty basement, “mousy” or “mouse nest” smell

• Nocardia spp.—freshly plowed field

FIGURE 8-17 Large, rough, hemolytic colonies of Bacillus cereus on blood agar plate.

FIGURE 8-18 Small, “fuzzy-edged,” umbonate center–appearing colony of Eikenella corrodens on chocolate. This organism has the tendency to “pit” the agar.

✓ Case Check 8-3 The Case in Point illustrates the sequential deductive reasoning that occurs during “plate reading” of the culture, the direct smear of the clinical specimen, and colony morphology. Both techniques have an important role in the presumptive identification required in plate reading. The first step is to examine the direct smear of the specimen for impor-tant clues, for example, the presence of white blood cells (an inflamma-tory process) and specific Gram stain morphology. Gram-positive cocci in pairs and clusters in the direct smear are suggestive of staphylococci (see Chapter 7); it is difficult to distinguish between enteric gram-negative bacilli. The β-hemolytic, white with a light yellow tinge, creamy-butter–looking, medium colonies on BAP are highly suggestive of S. aureus (see Figure 8-26, B). S. aureus would be inhibited by MAC and would not grow, leaving the other two colony types to identify. The lactose fermenter (pink) on MAC with a halo of pink precipitate sur-rounding the colonies is indicative of Escherichia/Citrobacter-like organ-isms. Of these two, Escherichia coli can be β-hemolytic on BAP (see Chapter 20). The nonlactose fermenter is the third type of colony present in the clinical specimen. Both lactose fermenters and nonlactose fermen-ters are growing on BAP because this medium is noninhibitory but are best differentiated on MAC.

FIGURE 8-16 Brick-red pigment of Serratia marcescens, which is evident on MacConkey (right). This brick-red pigment should not be confused with lactose fermentation. The pigment is slightly visible on chocolate (left). Additional incubation at room temperature enhances the brick-red pigmentation.

Colonies with Multiple CharacteristicsIn addition to the organisms already mentioned, other bacteria fit in multiple descriptive categories of colonial morphology. Bacil-lus cereus forms large, rough, greenish, hemolytic colonies on BAP (Figure 8-17). Eikenella corrodens forms a small, “fuzzy-edged” colony with an umbonate center on BAP or CHOC (Figure 8-18).

Growth of Organisms in Liquid MediaImportant clues to identification of an organism can also be detected by observing the growth of the organism in liquid media such as thioglycollate. Streamers or vines and puffballs are associated with certain species of streptococci (Figure 8-19). Turbidity, which refers to cloudiness of the medium resulting from growth (and usually gas if the medium contains glucose), is produced by many Enterobacteriaceae (Figure 8-20). Yeast and Pseudomonas species produce scum at the sides of the tube (Figures 8-21 and 8-22). In addition, yeast occasionally grows below the surface, in the microaerophilic area of the media (Figure 8-23).

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FIGURE 8-20 Turbidity produced by enterics when growing in thioglycollate. Notice the gas bubbles at the surface of and in the middle of the medium (arrow).

FIGURE 8-21 Production of “scum” by yeast at the surface of thioglycollate.

FIGURE 8-22 Illustration of Pseudomonas organisms produc-ing surface “scum” at the sides of thioglycollate. Occasionally, Pseudomonas aeruginosa produces a diffusible green pigment and a metallic sheen at the surface.

FIGURE 8-23 Yeast growing in the microaerophilic area of thioglycollate.

Figure 8-24, A and B (S. pneumoniae and α-hemolytic strep-tococci), Figure 8-24, C (Enterococcus spp. [see Chapter 15]), Figure 8-25 (S. pyogenes and S. agalactiae), and Figure 8-26 (staphylococci and yeast) show the differences between various organisms by colonial morphology.

Microbiologists might become frustrated when changes in colony morphology, Gram staining, and biochemical reactions occur in microorganisms that produce characteristic features. Organisms frequently exhibit characteristics far different from those previously described for them. The ability to recognize these differences and changes in characteristics makes this dis-cipline a challenge.

FIGURE 8-19 A, “Vine” or “streamer” effect exhibited by certain species of streptococci when growing in thioglycollate. The effect is more prevalent toward the bottom of the tube. B, “Puffed balls” effect exhibited by certain streptococcal species when growing in thioglycollate.

A B

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FIGURE 8-24 A, Differentiation of Streptococcus pneumoniae and α-hemolytic viridans strepto-cocci by colonial morphology. B, S. pneumoniae growing on blood agar plate (BAP). Notice the strong zone of α-hemolysis, umbilicate center, and wet (mucoid) appearance of the colonies. C, Enterococcus growing on BAP. It does not have an umbilicate or umbonate center, but it is more heaped and gray-appearing than S. pneumoniae. Enterococci have larger colonies and a smooth, darker margin, in contrast to many strains of α-hemolytic streptococci. The green color on the plate is not hemolysis but is a characteristic of growth.

Streptococcus pneumoniae α-Hemolytic viridans streptococci

Translucent, may resemble a water droplet;umbilicate, or flat with "penny" edge; entiremargin, wide and strong zone of α hemolysis

Translucent, grayer, rough margin,umbonate center

Umbilicate

Differentiation of streptococcus pneumoniae, α-hemolytic viridans streptococci, and Enterococcus by colonial morphology

Umbonate center

"Penny" edge

A

B C

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FIGURE 8-25 A, Differentiation of Streptococcus pyogenes and Streptococcus agalactiae by colo-nial morphology. B, Pinpoint colony of S. pyogenes exhibiting large, deep zone of β-hemolysis on blood agar plate (BAP). C, Colonies of S. agalactiae growing on BAP. This organism produces a larger colony and a smaller, more diffuse zone of hemolysis than S. pyogenes. The hemolysis is not evident in this photograph. Compare with B. D, Colonies of S. agalactiae growing on BAP. Through the use of transillumination, the hemolytic pattern is now evident; hemolysis is diffuse, and it remains close to the periphery of the colony. The same colonial morphology is produced by Listeria monocytogenes, a gram-positive rod. Compare with B. S. pyogenes (arrow) produces two hemolysins; one is oxygen stabile, and the other is oxygen labile. Stabbing the medium with an inoculating loop carries the organism into areas where anaerobic conditions are more preva-lent, allowing the enhanced hemolysin (oxygen labile) to be seen.

Streptococcus pyogenes Streptococcus agalactiae

Pinpoint, brittle, translucent, gray that mayturn brownish on continued incubation, largeand deep zone of β-hemolysis in comparisonto colony size

Medium-size colony compared with Streptococcuspyogenes, creamy texture, gray, small and diffusezone of β-hemolysis compared with colony size; often need to remove colony with a loop to see β-hemolysis; "bull's eye"–appearing colony because of organisms concentrated in center

Colony

Zone of β-hemolysis

Colony

Zone of β-hemolysis

A

B C

D

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BIBLIOGRAPHYLeBeau LJ: Effective lighting systems for photography of microbial

colonies, J Biol Photogr Assoc 44:4, 1976.

FIGURE 8-26 A, Differentiation between staphylococci and Candida albicans (a yeast) by colonial morphology. B, Large, white, convex, shiny, moist, β-hemolytic colonies of Staphylococcus aureus growing on blood agar plate (BAP). C, “Heaped” or convex, white, dull appearance and butyrous texture of C. albicans on BAP. Notice the tiny projections or “feet” at the edge of the colonies.

Staphylococcus organisms

Large, flat, or convex or possesses an umbonatecenter after 24 hours of incubation; shiny, moist,creamy, white to yellowish; S. aureus—usuallyβ-hemolytic

Smaller than staphylococci; convex, grows upwardmore than outward; creamy, white, dull surface;usually displays tiny projections at the base of thecolony after 24 hours of incubation

Candida albicans (yeast)

A

B C

Points to Remember■ The colonial morphology described in this chapter is not infallible.

Variations occur quite frequently. The morphologies described are general characteristics for any given organism.

■ The identification process must include Gram stain and biochemi-cal reactions in addition to colonial morphology.

■ Gram stain of the colony from the culture plate may look different from the direct smear from the specimen itself. Competition, crowding, and metabolic by-products may alter the Gram stain microscopic morphology. For example, in contrast to the direct smear or liquid media, streptococci may not appear as positive cocci in chains from the colony.

Learning Assessment Questions1. What do the dark pink colonies on MAC agar indicate?2. Why are there three colony types that grow on the BAP but only

two on MAC agar?3. What genus of bacteria would you suspect if you were to find

α-hemolytic colonies from a respiratory sample?4. How would you describe the colonies produced on MAC by

nonfermenting gram-negative bacilli?5. How would you differentiate β-hemolysis from α-hemolysis?6. What would you suspect if you noticed “puffballs” growing in

the broth medium?7. “Swarming” colonies is a characteristic of which genus of

bacteria?8. A moderate growth of a heaped, dry-appearing, white organism

is isolated from a patient with “thrush.” The colony has tiny

projections or “feet” projecting out along the edge of its margin. A presumptive identification of this organism would be:a. Staphylococcus aureusb. Staphylococcus epidermidisc. Neisseria spp.d. Candida albicans

9. Moderate growth of a β-hemolytic, gray colony is seen on a vaginal culture from a 25-year-old pregnant woman. The colonies are growing on the BAP and CHOC, but the MAC is negative for growth. The colonies are described as large with small, diffuse zones of β-hemolysis. This type of hemolysis is noticed when a colony is removed with a loop. A presumptive identification of this organism would be:a. Streptococcus pyogenes (group A)b. Staphylococcus aureusc. Streptococcus agalactiae (group B)d. Streptococcus pneumoniae

10. If a smear of an individual colony from the BAP (in Question 9) indicated a regular, short, gram-positive bacilli, the organism would be presumptively identified as a:a. Streptococcus agalactiae (group B)b. Listeria monocytogenesc. Streptococcus pneumoniaed. Staphylococcus epidermidisM

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