CHAPTER

8 Use of Colony Morphology

for the Presumptive Identification

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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
Size ik
■ 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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■ 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
of colony was large, gray, and mucoid similar to the second
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

• Enhance the quality of patient care through rapid report-

Key Terms ing 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

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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

Escherichia/Citrobacter-like
organisms
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pathogens are presumptively identified by colonial character-
Transillumination
istics, and preliminary reporting initiates immediate therapy.
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
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
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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

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
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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

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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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
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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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laboratory. The ability to provide a presumptive identification by occurs.

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.
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.
 CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms
✓ 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.
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
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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
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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.
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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.
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
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gram-negative bacillus or coccus.
Gram-negative rods are better described on MAC agar because
these organisms produce similar-looking colonies on BAP and
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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
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on CHOC. MAC is best used to characterize gram-negative rods
because lactose fermenters can be differentiated from nonlac-
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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
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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.
✓ Case Check 8-2
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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.
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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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
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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
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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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cated guess regarding the identification of the isolate. The fol-

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
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α-Hemolysis is partial lysing of erythrocytes in a BAP around

Hemolysis and under the colony that results in a green discoloration of the
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On 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
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-Hemolysis
β-Hemolysis is complete clearing of erythrocytes in BAP around
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.

FIGURE 8-5 The use of transillumination to determine whether

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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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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.
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Certain organisms, such as group A β-hemolytic streptococci Rough

(Streptococcus pyogenes), produce a wide, deep, clear zone of
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β-hemolysis, whereas others, such as group B β-hemolytic strep-

tococci (Streptococcus agalactiae) and Listeria monocytogenes
FIGURE 8-8 Illustration of form or margin to describe colonial
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(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 identification of certain species of bacteria. (For a com-
parison of the colonial characteristics of group A and group B measures a colony. Size is generally a visual comparison between
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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
usually show a green coloration around the colony on CHOC
(Figure 8-6). However, this coloration should not be mistaken for
a hemolytic characteristic.
Size
Colonies are described as large, medium, small, or pinpoint.
However, a microbiologist rarely takes a ruler and actually
coccus species are usually larger than Streptococcus species.
Figure 8-7 shows colonies of gram-negative rods compared with
gram-positive cocci.
Form or Margin
The 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
174 PART I Introduction to Clinical Microbiology
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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FIGURE 8-10 “Diphtheroid” colonies with rough edges, dry
appearance, and umbonate center growing on blood agar plate.
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examination are described as “Medusa heads” because of the
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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
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hazy blanket of growth on the surface that extends well beyond
the streak lines. Figure 8-9 shows swarming colonies of Proteus
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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
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Figure 8-26.)
Elevation
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The 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
Flat
Raised
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Convex or dome
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Umbilicate
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Umbonate
FIGURE 8-11 Illustration of elevations to describe colonial
morphology.
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convex colonies. In comparison, β-hemolytic streptococci gener-
ally produce flat 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.
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).
Color
In 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.
Consistency
Consistency 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
 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.

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

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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
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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
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.
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• Prevotella melaninogenica—brown-black (anaerobic)
Pigment production for these organisms is variable.
Odor

Odor should be determined when the lid of the culture plate

Pigment is removed and the odor dissipates into the surrounding environ-
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Pigment 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
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
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

✓ Case Check 8-3

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

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
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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.
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Growth of Organisms in Liquid Media

Important clues to identification of an organism can also be
detected by observing the growth of the organism in liquid media
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such as thioglycollate. Streamers or vines and puffballs are

associated with certain species of streptococci (Figure 8-19).
Colonies with Multiple Characteristics
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Turbidity, which refers to cloudiness of the medium resulting

In 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).
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).
 CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms 177

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A B
FIGURE 8-19 A, “Vine” or “streamer” effect exhibited by
FIGURE 8-22 Illustration of Pseudomonas organisms produc-

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.
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ing surface “scum” at the sides of thioglycollate. Occasionally,
Pseudomonas aeruginosa produces a diffusible green pigment
and a metallic sheen at the surface.

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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).
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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
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(staphylococci and yeast) show the differences between various

organisms by colonial morphology.
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Microbiologists might become frustrated when changes in

FIGURE 8-21 Production of “scum” by yeast at the surface of
thioglycollate.
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.
178 PART I Introduction to Clinical Microbiology

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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

Translucent, may resemble a water droplet; Translucent, grayer, rough margin,

umbilicate, or flat with "penny" edge; entire umbonate center

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margin, wide and strong zone of α hemolysis

Umbilicate
Umbonate center

"Penny" edge

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B C
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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
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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

Streptococcus pyogenes Streptococcus agalactiae

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

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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

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B
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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
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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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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-
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lent, allowing the enhanced hemolysin (oxygen labile) to be seen.

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

D
U
A

N
U
FK
B
ik
C
FIGURE 8-26 A, Differentiation between staphylococci and Candida albicans (a yeast) by colonial
lin
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.
iK

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.
og

■ 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
ol
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

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:
bi

a. Streptococcus pyogenes (group A)

b. Staphylococcus aureus
c. Streptococcus agalactiae (group B)
Learning Assessment Questions
ro

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:
ik

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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nonfermenting gram-negative bacilli? d. Staphylococcus epidermidis

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?
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.