Mikrobiologi Klinik FK UNUD: Use of Colony Morphology For The Presumptive Identification of Microorganisms
Mikrobiologi Klinik FK UNUD: Use of Colony Morphology For The Presumptive Identification of Microorganisms
Mikrobiologi Klinik FK UNUD: Use of Colony Morphology For The Presumptive Identification of Microorganisms
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of Microorganisms
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George Manuselis, Connie R. Mahon*
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CHAPTER OUTLINE
■ IMPORTANCE OF COLONIAL MORPHOLOGY AS A Form or Margin
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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
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■ COLONIES WITH MULTIPLE CHARACTERISTICS
■ GROWTH OF ORGANISMS IN LIQUID MEDIA
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OBJECTIVES
After reading and studying this chapter, you should be able to:
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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
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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
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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
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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-
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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
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*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
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170 PART I Introduction to Clinical Microbiology
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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
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pathogens are presumptively identified by colonial character-
Escherichia/Citrobacter-like Transillumination
istics, and preliminary reporting initiates immediate therapy.
organisms Translucent
• Play a significant role in quality control, especially of auto-
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Fastidious Transparent
Filamentous Turbidity mated procedures and other commercially available iden-
Form Umbilicate tification systems. When commercial and automated systems
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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 signifi-
cantly alters the identification (see Chapter 9). The ability of
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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
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he importance of mastery of colonial morphology (colony identification of the organism is an important component of
characteristics and form) and interpretation of Gram- quality control that is accomplished by recognizing organisms
stained smears from clinical specimens and microbial by their colonial characteristics.
colonies cannot be overemphasized. Although Gram-stained
smears provide initial identification of microorganisms by micro-
scopic characterization, description of the physical growth char-
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Initial Observation and Interpretation
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acteristics of microorganisms on laboratory media facilitates the
initial identification processes.
of Cultures
Close your eyes and imagine the physical characteristics of a Generally, microbiologists observe the colonial morphology
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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 specific characteristics that distinguish them in a For example, young cultures of Staphylococcus aureus may
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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 findings on stained direct smears facilitate must be aware of factors that may significantly alter the colo-
presumptive identification of commonly isolated organisms. The nial morphology of growing microorganisms. These factors
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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.
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and one genus from another are discussed. The interpretation of primary cultures, commonly referred
to as plate reading, is actually a comparative examination of
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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
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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
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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
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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-
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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
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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.
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).
These gram-negative rods grow larger, gray, and mucoid on
same proportion or concentration in which they are present in the
clinical specimen. Because many specimens are polymicrobic,
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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
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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
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fermenters growing on MAC. Compare with Figure 8-4, B.
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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
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on MAC. Notice the mucoid, heaped appearance and the slightly cream-colored center after 48
hours’ growth.
to Differentiate and Identify with a loop to visualize the hemolytic pattern. Proper technique
requires the passing of bright light through the bottom of the
Microorganisms Presumptively plate (transillumination) to determine whether the organism
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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.
Although there are many types of hemolysis, only α-hemolysis
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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-
immediately surrounding or underneath the colony is a reaction cocci. (For a comparison of the colonial 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 identification, 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
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Colonies
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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 colo-
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nies are enteric gram-negative rods.
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colonies are hemolytic. The technique can be used for MacConkey
also to see slight color differences in nonlactose fermenters.
Filamentous
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Irregular
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Smooth
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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-
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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
(Figure 8-6). However, this coloration should not be mistaken for gram-positive cocci.
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, filamentous, 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
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Convex or dome
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Umbilicate
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FIGURE 8-9 Swarming colonies of Proteus spp. The organism
was inoculated in the middle of the blood agar plate (arrow).
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Umbonate
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convex colonies. In comparison, β-hemolytic streptococci gener-
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ally produce flat colonies.
Density
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filamentous 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
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hazy blanket of growth on the surface that extends well beyond Chapter 19).
the streak lines. Figure 8-9 shows swarming colonies of Proteus
Color
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Figure 8-26.) (Figure 8-13), whereas Enterococcus spp. may appear gray.
Certain Micrococcus spp. and Neisseria (nonpathogenic) spp.
Elevation
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FIGURE 8-13 Example of white colonies of coagulase-negative A
staphylococci on blood agar plate.
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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
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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.
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Neisseria spp. are sticky. Nocardia spp. produce colonies that are • Chromobacterium violaceum—purple
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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
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Pigment production is an inherent characteristic of a specific ment. The microbiologist should never inhale directly from the
organism confined generally to the colony. Examples of organ- plate. Examples of microorganisms that produce distinctive
isms that produce pigment include the following: odors are as follows:
• 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
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FIGURE 8-16 Brick-red pigment of Serratia marcescens, which
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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
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room temperature enhances the brick-red pigmentation.
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• Haemophilus spp.—musty basement, “mousy” or “mouse
nest” smell
• Nocardia spp.—freshly plowed field
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-
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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
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A B
FIGURE 8-19 A, “Vine” or “streamer” effect exhibited by
FIGURE 8-22 Illustration of Pseudomonas organisms produc-
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certain species of streptococci when growing in thioglycollate.
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.
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Differentiation of streptococcus pneumoniae, α-hemolytic viridans streptococci, and Enterococcus by colonial morphology
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Streptococcus pneumoniae α-Hemolytic viridans streptococci
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margin, wide and strong zone of α hemolysis
Umbilicate
Umbonate center
"Penny" edge
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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
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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.
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CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 179
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
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of organisms concentrated in center
Colony Colony
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Zone of β-hemolysis Zone of β-hemolysis
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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
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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
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FIGURE 8-26 A, Differentiation between staphylococci and Candida albicans (a yeast) by colonial
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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.
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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.
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■ 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
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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:
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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:
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3. What genus of bacteria would you suspect if you were to find a. Streptococcus agalactiae (group B)
α-hemolytic colonies from a respiratory sample? b. Listeria monocytogenes
4. How would you describe the colonies produced on MAC by c. Streptococcus pneumoniae
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