Mikrobiologi Klinik FK UNUD

CHAPTER

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

CHAPTER OUTLINE ■ IMPORTANCE OF COLONIAL MORPHOLOGY AS A Form or Margin DIAGNOSTIC TOOL Elevation ■ INITIAL OBSERVATION AND INTERPRETATION OF Density CULTURES Color ■ GROSS COLONY CHARACTERISTICS USED TO Consistency DIFFERENTIATE AND IDENTIFY MICROORGANISMS Pigment PRESUMPTIVELY Odor Hemolysis ■ COLONIES WITH MULTIPLE CHARACTERISTICS Size ■ GROWTH OF ORGANISMS IN LIQUID MEDIA

OBJECTIVES After reading and studying this chapter, you should be able to: 1. Describe how growth on blood, chocolate, and MacConkey agars is 4. Using colonial morphology, differentiate among the following used in the preliminary identification of isolates. microorganisms: 2. Differentiate α-hemolysis from β-hemolysis on blood agar culture • Staphylococci and streptococci medium. • Streptococcus agalactiae and Streptococcus pyogenes 3. Associate the colony characteristics shown on blood, chocolate, and • Neisseria spp. and staphylococci MacConkey agars with the microscopic findings on direct smear, and • Yeast and staphylococci use the information in the presumptive identification of • “Diphtheroids” and staphylococci microorganisms. • Lactose fermenters from lactose nonfermenters • “Swarming” Proteus species from other Enterobacteriaceae

Case in Point with a surrounding pink precipitate and a few clear nonlactose fermenting colonies. Based on the Gram stain results and colo- An exudate from a sacral decubital ulcer on a 65-year-old hos- nial characteristics of the isolates, appropriate biochemical tests pital inpatient was cultured on blood agar plate (BAP), chocolate and antibiotic susceptibilities were performed to identify the (CHOC), and MacConkey (MAC) agars. Direct smear examina- causative agents of the ulcer. 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- Issues to Consider photypes were visible on the BAP. The ﬁrst was a moderate After reading the patient’s case history, consider: growth of a medium-sized β-hemolytic, which was yellowish ■ How the colony morphology of isolates is used to identify white and creamy-buttery looking. The second colony was microorganisms presumptively also β-hemolytic but larger, mucoid, and gray. The third type ■ How to correlate the direct smear examination ﬁndings Mikrobiologiof colony was large, gray, and mucoid similar to the second Klinik FK UNUD with the colony morphology of isolates on each culture but was nonhemolytic. The MAC agar showed two colony medium morphotypes—a light growth of dark pink, dry-looking colonies ■ How the colony morphology of each isolate can dif- *My comments are my own and do not represent the view of Health Resources ferentiate between pathogenic and nonpathogenic and Services Administration of the Department of Health and Human Services. microorganisms

169 170 PART I Introduction to Clinical Microbiology

Key Terms • Enhance the quality of patient care through rapid reporting of results and by increasing the cost-effectiveness of α-Hemolysis Lactose fermenter laboratory testing. This may best be illustrated by using β-Hemolysis Margin sputum cultures as an example. The upper respiratory tract Brittle Nonlactose fermenter contains many indigenous organisms, and to identify every Butyrous Opaque organism in culture would be a time-consuming, cost- Colonial morphology Pigment prohibitive, and insurmountable task. Microbiologists must be Consistency Rhizoid able to differentiate potential pathogens from the “usual” Creamy Smooth inhabitants of the upper respiratory tract and direct the diag- Density Streamers nostic workup toward only potential pathogens. Potential Elevation Swarming pathogens are presumptively identifed by colonial character- Escherichia/Citrobacter-like Transillumination istics, and preliminary reporting initiates immediate therapy. organisms Translucent Fastidious Transparent • Play a signifcant role in quality control, especially of auto- Filamentous Turbidity mated procedures and other commercially available iden- Form Umbilicate tifcation systems. When commercial and automated systems Hemolysis Umbonate are used, a mixed inoculum (polymicrobic/containing more Klebsiella/Enterobacter-like than one genus and species or both) produces a biochemical organisms test result or erroneous interpretation of reactions that signifcantly alters the identifcation (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 he importance of mastery of colonial morphology (colony identifcation of the organism is an important component of characteristics and form) and interpretation of Gram- quality control that is accomplished by recognizing organisms Tstained smears from clinical specimens and microbial by their colonial characteristics. colonies cannot be overemphasized. Although Gram-stained smears provide initial identifcation of microorganisms by microscopic characterization, description of the physical growth char- Initial Observation and Interpretation acteristics of microorganisms on laboratory media facilitates the initial identifcation processes. of Cultures Close your eyes and imagine the physical characteristics of a Generally, microbiologists observe the colonial morphology person you know well. The person’s height, weight, shape, color of organisms isolated on primary culture after 18 to 24 hours and style of hair, eyes, freckles, and color of skin as well as voice of incubation. Incubation time may vary according to when or laugh may make that person distinctive in a crowd or when the specimen is received and processed in the laboratory, his or her back is facing you. In the same manner, many micro- which may affect the “typical” morphology of a certain isolate. organisms have specifc characteristics that distinguish them in a For example, young cultures of Staphylococcus aureus may crowd of other genera or species. appear smaller and may not show the distinct β-hemolysis This chapter explains how the characterization of colonies on that older cultures produce. In addition, the microbiologist culture media and the fndings on stained direct smears facilitate must be aware of factors that may signifcantly alter the colo- presumptive identifcation of commonly isolated organisms. The nial morphology of growing microorganisms. These factors characteristics that are used to describe colony morphology of include the ingredients present in the medium, the inhibitory certain groups of organisms and how these characteristics are nature of these ingredients, and antibiotics that may be present used to differentiate one species from a closely related species in the medium. and one genus from another are discussed. 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 Importance of Colonial Morphology culture media. Many specimens, such as sputum and wounds that arrive in the clinical laboratory, are plated on various culture as a Diagnostic Tool media such as BAP, CHOC, and MAC. Each type of agar plate In many ways, the usefulness of colonial morphology extends is examined in relationship to the other. As a set of culture media, the capabilities of the microbiologist and, ultimately, the clinical comparative colonial examination of growth from a specimen laboratory. The ability to provide a presumptive identifcation by occurs. colonial morphology may include the following: The ability to determine which organisms grow on selective Mikrobiologi• Provide a presumptive identifcation to the physician. Klinik and nonselective media FK aids the microbiologist UNUD in making an Even in this age of rapid identifcation systems, incubation initial distinction between gram-positive and gram-negative iso- times and procedures can be protracted. In a critical situation, lates. BAP and CHOC support the growth of various fastidious the microbiologist makes an educated judgment about the (hard to grow, requires additional growth factors) and nonfas- presumptive identity before performing diagnostic identifca- tidious organisms, gram-positive bacteria, and gram-negative tion procedures. bacteria. CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 171

presumptive identifcation and determine how to proceed in iden- ✓ Case Check 8-1 tifying the isolated organisms. As illustrated in the Case in Point at the beginning of the chapter, three While MAC supports most gram-negative rods, especially the colony morphotypes were observed on BAP. Because the Gram-stained Enterobacteriaceae, it inhibits growth of gram-positive organ- smear showed both gram-positive and gram-negative bacteria, three isms and some fastidious gram-negative organisms, such as Hae- types of organisms should be observed on a nonselective medium such mophilus and Neisseria spp. Growth on BAP and CHOC but not as BAP. on MAC is indicative of a gram-positive isolate or of a fastidious gram-negative bacillus or coccus. Generally, organisms that grow on BAP also grow on CHOC, Gram-negative rods are better described on MAC agar because but not all organisms that grow on CHOC grow on BAP. Although these organisms produce similar-looking colonies on BAP and BAP supports fastidious organisms, highly fastidious species, CHOC media. On BAP and CHOC, gram-negative rods produce such as Haemophilus spp. and Neisseria gonorrhoeae, do not large colonies that appear gray and sometimes mucoid, and if grow on it. CHOC provides nutritional growth requirements to hemolytic, hemolysis is seen on BAP. True hemolysis is not seen support highly fastidious organisms such as Haemophilus spp. on CHOC. MAC is best used to characterize gram-negative rods and N. gonorrhoeae. Therefore, a gram-negative bacillus that because lactose fermenters can be differentiated from nonlac- grows on CHOC but not on BAP or MAC would be suspected tose fermenters. Lactose fermenters are easily detected by the to be Haemophilus spp., whereas gram-negative diplococci with color change they produce on the medium; as the pH changes the same growth pattern would be suspected to be N. gonor- when lactose is fermented, the organisms produce pink, dark rhoeae (Figure 8-1). The microbiologist is able to provide a 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. ✓ Case Check 8-2 Certain enteric pathogens, such as Escherichia/Citrobacter-like organisms, 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.

FIGURE 8-1 Clockwise from the top: chocolate (CHOC), blood This is a comparative analysis of the growth on the three types agar plate (BAP), and MacConkey (MAC). The large colonies of culture media. Microorganisms grow on culture media in the growing on all three plates are gram-negative rods (enterics). same proportion or concentration in which they are present in the These gram-negative rods grow larger, gray, and mucoid on BAP and CHOC. Notice the smaller, grayish brown fastidious clinical specimen. Because many specimens are polymicrobic, colonies of Haemophilus organisms growing on CHOC (arrow), this feature can be benefcial in identifying different colony which are not growing on BAP or MAC. types.

Mikrobiologi Klinik FK UNUD

A B 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. 172 PART I Introduction to Clinical Microbiology

A B 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.

discoloration of the medium is the result of growth of the Gross Colony Characteristics Used organism on the plate. Often the colony has to be removed to Differentiate and Identify with a loop to visualize the hemolytic pattern. Proper technique Microorganisms Presumptively requires the passing of bright light through the bottom of the plate (transillumination) to determine whether the organism By observing the colonial characteristics of the organisms that is hemolytic (Figure 8-5). Many organisms have no lytic effect have been isolated, the microbiologist is able to make an edu- on the RBCs in BAP and are referred to as nonhemolytic. cated guess regarding the identifcation of the isolate. The fol- Although there are many types of hemolysis, only α-hemolysis lowing descriptions are routinely used to examine colony and β-hemolysis are illustrated in this chapter. characteristics. Many of the following colony characteristics may vary among species and strains of the same genus. α-Hemolysis α-Hemolysis is partial lysing of erythrocytes in a BAP around Hemolysis and under the colony that results in a green discoloration of the On BAP, hemolysis (Greek hemo: pertaining to red blood cells medium. Examples of organisms that produce α-hemolysis [RBCs]; lysis: dissolution or break apart) observed in the media include Streptococcus pneumoniae and certain viridans strepto- Mikrobiologiimmediately surrounding or underneath the colony is a reaction Klinik cocci. (For a comparison FK of the colonial UNUD morphology of these two caused by enzymatic or toxin activity of bacteria. Hemolysis organisms, see Figure 8-24.) (e.g., α, β, or no hemolysis with other colony characteristics) on BAP is helpful in presumptive identifcation, particularly of β-Hemolysis streptococci and enterococci (see Chapter 15). It is important β-Hemolysis is complete clearing of erythrocytes in BAP around to determine whether true hemolysis is present or whether or under the colonies because of the complete lysis of RBCs. CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 173

Colonies

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

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. Filamentous

Irregular

Smooth

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.

Certain organisms, such as group A β-hemolytic streptococci Rough (Streptococcus pyogenes), produce a wide, deep, clear zone of β-hemolysis, whereas others, such as group B β-hemolytic streptococci (Streptococcus agalactiae) and Listeria monocytogenes FIGURE 8-8 Illustration of form or margin to describe colonial (a short, gram-positive rod) produce a narrow, diffuse zone of morphology. β-hemolysis close to the colony. These features are helpful hints in the identifcation of certain species of bacteria. (For a comparison of the colonial characteristics of group A and group B measures a colony. Size is generally a visual comparison between streptococci, see Figure 8-25.) CHOC does not display true genera or species. For example, gram-positive bacteria generally hemolysis because the RBCs in the medium have already been produce smaller colonies than gram-negative bacteria. Staphylo- lysed. Organisms that are α-hemolytic or β-hemolytic on BAP coccus species are usually larger than Streptococcus species. usually show a green coloration around the colony on CHOC Figure 8-7 shows colonies of gram-negative rods compared with Mikrobiologi(Figure 8-6). However, this coloration should not be mistaken Klinikfor gram-positive cocci. FK UNUD a hemolytic characteristic. Form or Margin Size The edge of the colonies should be observed and the form, or Colonies are described as large, medium, small, or pinpoint. margin, described as smooth, flamentous, rough or rhizoid, or However, a microbiologist rarely takes a ruler and actually irregular (Figure 8-8). Colonies of Bacillus anthracis on visual 174 PART I Introduction to Clinical Microbiology

Flat

Raised

Convex or dome

Umbilicate

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

Umbonate

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

convex colonies. In comparison, β-hemolytic streptococci generally produce fat colonies. Density The 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. FIGURE 8-10 “Diphtheroid” colonies with rough edges, dry Translucent colonies allow some light to pass through the colony, appearance, and umbonate center growing on blood agar plate. and opaque colonies do not (Figure 8-12). β-Hemolytic streptococci except group B (S. agalactiae) are described as translucent. S. agalactiae produces colonies that are semiopaque, with the organisms concentrated at the center of the colony, examination are described as “Medusa heads” because of the sometimes described as a bull’s-eye colony. Staphylococci and flamentous appearance. Certain genera such as Proteus spp. other gram-positive bacteria are usually opaque. Most gram- (especially Proteus mirabilis and Proteus vulgaris) may swarm negative rods are also opaque. Bordetella pertussis is described on nonselective agar such as blood or chocolate. Swarming is a as shiny, similar to a half-pearl, on blood-containing media (see hazy blanket of growth on the surface that extends well beyond Chapter 19). the streak lines. Figure 8-9 shows swarming colonies of Proteus spp. Diphtheroids produce colonies that have rough edges (Figure Color 8-10), whereas certain yeast produce colonies that are described In contrast to pigmentation, color is a term used to describe as stars or colonies with feet or pedicles. (For a comparison a particular genus in general. Colonies may be white, gray, of the colonial morphology of yeast and staphylococci, see yellow, or buff. Coagulase-negative staphylococci are white Figure 8-26.) (Figure 8-13), whereas Enterococcus spp. may appear gray. Certain Micrococcus spp. and Neisseria (nonpathogenic) spp. Elevation are yellow or off-white (Figure 8-14). “Diphtheroids” are buff. The elevation should be determined by tilting the culture plate Most gram-negative rods are gray on BAP. Mikrobiologiand looking at the side of the colony (Figure 8-11). Elevation Klinik FK UNUD may be raised, convex, fat, umbilicate (depressed center, Consistency concave—an “innie”), or umbonate (raised or bulging center, Consistency is determined by touching the colony with a sterile convex—an “outie”). S. pneumoniae typically produces umbili- loop. Colony consistency may be brittle (splinters), creamy cate colonies, unless the colonies are mucoid because of the (butyrous), dry, or waxy; occasionally, the entire colony adheres presence of polysaccharide capsule. S. aureus typically produces (sticks) to the loop. S. aureus is creamy, whereas certain CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 175

transparent transparent translucent translucent opaqu paque colony colony

FIGURE 8-12 Density.

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

B FIGURE 8-14 Example of the yellow colonies characteristic of FIGURE 8-15 A, Pseudomonas aeruginosa illustrating the certain nonpathogenic species of Neisseria organisms on blood metallic sheen and green pigmentation of colonies on blood agar plate. 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.

Neisseria spp. are sticky. Nocardia spp. produce colonies that are • Chromobacterium violaceum—purple brittle, crumbly, and wrinkled, resembling bread crumbs on a • Prevotella melaninogenica—brown-black (anaerobic) plate. Diphtheroid colonies are usually dry and waxy. Most Pigment production for these organisms is variable. β-hemolytic streptococci are dry (except for mucoid types), and when pushed by a loop, the whole colony remains intact. Odor Odor should be determined when the lid of the culture plate Pigment is removed and the odor dissipates into the surrounding environ- Pigment production is an inherent characteristic of a specifc ment. The microbiologist should never inhale directly from the organism confned generally to the colony. Examples of organ- plate. Examples of microorganisms that produce distinctive Mikrobiologiisms that produce pigment include the following: Klinikodors are as follows: FK UNUD • P. aeruginosa—green, sometimes a metallic sheen (Figure • S. aureus—old sock (stocking that has been worn continu- 8-15) ously for a few days without washing); this odor is evident • Serratia marcescens—brick-red (Figure 8-16), especially at when growing on mannitol salt agar room temperature • P. aeruginosa—fruity or grapelike • Kluyvera spp.—blue • P. mirabilis—putrid 176 PART I Introduction to Clinical Microbiology

FIGURE 8-16 Brick-red pigment of Serratia marcescens, which is evident on MacConkey (right). This brick-red pigment should FIGURE 8-17 Large, rough, hemolytic colonies of Bacillus not be confused with lactose fermentation. The pigment is cereus on blood agar plate. slightly visible on chocolate (left). Additional incubation at room temperature enhances the brick-red pigmentation.

• Haemophilus spp.—musty basement, “mousy” or “mouse nest” smell • Nocardia spp.—freshly plowed feld

✓ 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 important 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- FIGURE 8-18 Small, “fuzzy-edged,” umbonate center– butter–looking, medium colonies on BAP are highly suggestive of S. appearing colony of Eikenella corrodens on chocolate. This aureus (see Figure 8-26, B). S. aureus would be inhibited by MAC and organism has the tendency to “pit” the agar. would not grow, leaving the other two colony types to identify. The lactose fermenter (pink) on MAC with a halo of pink precipitate surrounding the colonies is indicative of Escherichia/Citrobacter-like organisms. 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 fermenters are growing on BAP because this medium is noninhibitory but are best differentiated on MAC. Growth of Organisms in Liquid Media Important clues to identifcation 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). Colonies with Multiple Characteristics Turbidity, which refers to cloudiness of the medium resulting In addition to the organisms already mentioned, other bacteria ft from growth (and usually gas if the medium contains glucose), Mikrobiologiin multiple descriptive categories of colonial morphology. BacilKlinik- is produced by many FK Enterobacteriaceae UNUD (Figure 8-20). Yeast lus cereus forms large, rough, greenish, hemolytic colonies on and Pseudomonas species produce scum at the sides of the tube BAP (Figure 8-17). Eikenella corrodens forms a small, “fuzzy- (Figures 8-21 and 8-22). In addition, yeast occasionally grows edged” colony with an umbonate center on BAP or CHOC below the surface, in the microaerophilic area of the media (Figure 8-18). (Figure 8-23). CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 177

A B FIGURE 8-19 A, “Vine” or “streamer” effect exhibited by certain species of streptococci when growing in thioglycollate. FIGURE 8-22 Illustration of Pseudomonas organisms produc- The effect is more prevalent toward the bottom of the tube. ing surface “scum” at the sides of thioglycollate. Occasionally, B, “Puffed balls” effect exhibited by certain streptococcal Pseudomonas aeruginosa produces a diffusible green pigment species when growing in thioglycollate. and a metallic sheen at the surface.

FIGURE 8-23 Yeast growing in the microaerophilic area of FIGURE 8-20 Turbidity produced by enterics when growing in thioglycollate. thioglycollate. Notice the gas bubbles at the surface of and in the middle of the medium (arrow).

Figure 8-24, A and B (S. pneumoniae and α-hemolytic streptococci), 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 Mikrobiologi Klinikoccur in microorganisms FK that produce UNUD characteristic features. Organisms frequently exhibit characteristics far different from those previously described for them. The ability to recognize FIGURE 8-21 Production of “scum” by yeast at the surface of these differences and changes in characteristics makes this dis- thioglycollate. cipline a challenge. 178 PART I Introduction to Clinical Microbiology

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

Streptococcus pneumoniae α-Hemolytic viridans streptococci

Translucent, may resemble a water droplet; Translucent, grayer, rough margin, umbilicate, or flat with "penny" edge; entire umbonate center margin, wide and strong zone of α hemolysis

Umbilicate Umbonate center

"Penny" edge A

B C

FIGURE 8-24 A, Differentiation of Streptococcus pneumoniae and α-hemolytic viridans streptococci 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.

Mikrobiologi Klinik FK UNUD CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 179

Streptococcus pyogenes Streptococcus agalactiae

Pinpoint, brittle, translucent, gray that may Medium-size colony compared with Streptococcus turn brownish on continued incubation, large pyogenes, creamy texture, gray, small and diffuse and deep zone of β-hemolysis in comparison zone of β-hemolysis compared with colony size; to 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 Colony

Zone of β-hemolysis Zone of β-hemolysis A

B C

D FIGURE 8-25 A, Differentiation of Streptococcus pyogenes and Streptococcus agalactiae by colonial 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- Mikrobiologilent, allowing the enhanced hemolysin (oxygenKlinik labile) to be seen. FK UNUD 180 PART I Introduction to Clinical Microbiology

Staphylococcus organisms Candida albicans (yeast)

Large, flat, or convex or possesses an umbonate Smaller than staphylococci; convex, grows upward center after 24 hours of incubation; shiny, moist, more than outward; creamy, white, dull surface; creamy, white to yellowish; S. aureus—usually usually displays tiny projections at the base of the β-hemolytic colony after 24 hours of incubation

B C 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.

Points to Remember projections or “feet” projecting out along the edge of its margin. A presumptive identification of this organism would be: ■ The colonial morphology described in this chapter is not infallible. a. Staphylococcus aureus Variations occur quite frequently. The morphologies described are b. Staphylococcus epidermidis general characteristics for any given organism. c. Neisseria spp. ■ The identification process must include Gram stain and biochemi- d. Candida albicans cal reactions in addition to colonial morphology. 9. Moderate growth of a β-hemolytic, gray colony is seen on a ■ Gram stain of the colony from the culture plate may look different vaginal culture from a 25-year-old pregnant woman. The colonies from the direct smear from the specimen itself. Competition, are growing on the BAP and CHOC, but the MAC is negative for crowding, and metabolic by-products may alter the Gram stain growth. The colonies are described as large with small, diffuse microscopic morphology. For example, in contrast to the direct zones of β-hemolysis. This type of hemolysis is noticed when a smear or liquid media, streptococci may not appear as positive colony is removed with a loop. A presumptive identification of cocci in chains from the colony. this organism would be: a. Streptococcus pyogenes (group A) b. Staphylococcus aureus c. Streptococcus agalactiae (group B) Learning Assessment Questions d. Streptococcus pneumoniae 1. What do the dark pink colonies on MAC agar indicate? 10. If a smear of an individual colony from the BAP (in Question 9) 2. Why are there three colony types that grow on the BAP but only indicated a regular, short, gram-positive bacilli, the organism two on MAC agar? would be presumptively identified as a: 3. What genus of bacteria would you suspect if you were to find a. Streptococcus agalactiae (group B) b. Listeria monocytogenes α-hemolytic colonies from a respiratory sample? 4. How would you describe the colonies produced on MAC by c. Streptococcus pneumoniae nonfermenting gram-negative bacilli? d. Staphylococcus epidermidis 5. How would you differentiate β-hemolysis from α-hemolysis? Mikrobiologi6. What would you suspect if you noticed “puffballs” growing Klinikin FK UNUD the broth medium? 7. “Swarming” colonies is a characteristic of which genus of bacteria? BIBLIOGRAPHY 8. A moderate growth of a heaped, dry-appearing, white organism is isolated from a patient with “thrush.” The colony has tiny LeBeau LJ: Effective lighting systems for photography of microbial colonies, J Biol Photogr Assoc 44:4, 1976.