CHAPTER 7  Traditional Cultivation and Identification 103
important gram-negative bacteria are resistant to vanco-
mycin. Therefore when organisms with uncertain Gram
stain results are encountered, susceptibility to vancomy-
cin can be used to help establish the organism’s Gram
“status.” Any zone of inhibition around a vancomycin-
impregnated disk after overnight incubation is usually in-
dicative of a gram-positive bacterium (Figure 7-15). It is
important to understand that with the increasing use of
antibiotics to treat serious infections, some gram-positive
organisms have acquired mechanisms of resistance to
vancomycin. With few exceptions (e.g., certain Chryseo-
bacterium spp., Moraxella spp., or Acinetobacter spp. iso-
lates may be vancomycin susceptible), truly gram-negative
bacteria are resistant to vancomycin. Conversely, most
gram-negative bacteria are susceptible to the antibiotics
colistin or polymyxin, whereas gram-positive bacteria are
typically resistant to these agents.
Nutritional Requirements and Metabolic
Capabilities
Determining the nutritional and metabolic capabilities of a
bacterial isolate is the most common approach used for de-
termining the genus and species of an organism. The meth-
ods available for making these determinations share many
commonalties but also have some important differences. In
general, all methods use a combination of tests to establish
the enzymatic capabilities of a given bacterial isolate as
well as the isolate’s ability to grow or survive the presence
of certain inhibitors (e.g., salts, surfactants, toxins, and
antibiotics).
Establishing Enzymatic Capabilities
As discussed in Chapter 2, enzymes are the driving force in
bacterial metabolism. Because enzymes are genetically en-
coded, the enzymatic content of an organism is a direct re-
flection of the organism’s genetic makeup, which, in turn, is
specific for individual bacterial species.
A B
• Figure 7-15  A, Zone of growth inhibition around the 5-µg vancomycin
disk is indicative of a gram-positive bacterium. B, The gram-negative
organism is not inhibited by this antibiotic, and growth extends to the
edge of the disk.
Types of Enzyme-Based Tests
In diagnostic bacteriology, enzyme-based tests are designed
to measure the presence of a specific enzyme or a complete
metabolic pathway that may contain several different en-
zymes. Although the specific tests most useful for the iden-
tification of particular bacteria are discussed in Part III,
some examples of tests commonly used to characterize a
wide spectrum of bacteria are reviewed here.
Single Enzyme Tests
Several tests are commonly used to determine the presence
of a single enzyme. These tests usually provide rapid results
because they can be performed on organisms already grown
in culture. Of importance, these tests are easy to perform
and interpret and often play a key role in the identification
scheme. Although most single enzyme tests do not yield
sufficient information to provide species identification, they
are used extensively to determine which subsequent identi-
fication steps should be followed. For example, the catalase
test can provide pivotal information and is commonly used
in schemes for gram-positive identifications. The oxidase
test is of comparable importance in identification schemes
for gram-negative bacteria (Figure 7-13).
Catalase Test
The enzyme catalase catalyzes the release of water and oxy-
gen from hydrogen peroxide (H2O2 1 catalase d H2O 1
O2); its presence is determined by direct analysis of a bacte-
rial culture (Procedure 12-8). The rapid production of bub-
bles (effervescence) when bacterial growth is mixed with a
hydrogen peroxide solution is interpreted as a positive test
(i.e., the presence of catalase). Failure to produce efferves-
cence or weak effervescence is interpreted as negative. If the
bacterial inoculum is inadvertently contaminated with red
blood cells when the test inoculum is collected from a sheep
blood agar plate, weak production of bubbles may occur, but
this should not be interpreted as a positive test.
Because the catalase test is key to the identification
scheme of many gram-positive organisms, interpretation
must be completed carefully. For example, staphylococci
are catalase-positive, whereas streptococci and entero-
cocci are negative; similarly, the catalase reaction differ-
entiates Listeria monocytogenes and corynebacteria (catalase-
positive) from other gram-positive, non–spore-forming
bacilli (Figure 7-13).
Oxidase Test
Cytochrome oxidase participates in electron transport and
in the nitrate metabolic pathways of certain bacteria. Test-
ing for the presence of oxidase can be performed by flooding
bacterial colonies on the agar surface with 1% tetramethyl-
p-phenylenediamine dihydrochloride. Alternatively, a
sample of the bacterial colony can be rubbed onto filter
paper impregnated with the reagent (Procedure 12-33). A pos-
itive reaction is indicated by the development of a purple
color. If an iron-containing wire is used to transfer growth, a
false-positive reaction may result; therefore, platinum wire or
104 PA RT I I  General Principles in Clinical Microbiology
wooden sticks are recommended. Certain organisms may
show slight positive reactions after the initial 10 seconds
have passed; such results are not considered definitive.
The test is initially used for differentiating between groups
of gram-negative bacteria. Among the commonly encountered
gram-negative bacilli, Enterobacteriaceae, Stenotrophomonas
maltophilia, and Acinetobacter spp. are oxidase-negative,
whereas many other bacilli, such as Pseudomonas spp. and
Aeromonas spp., are positive (Figure 7-13). The oxidase test
is also a key reaction for the identification of Neisseria spp.
(oxidase-positive).
Indole Test
Bacteria that produce the enzyme tryptophanase are able
to degrade the amino acid tryptophan into pyruvic acid,
ammonia, and indole. Indole is detected by combining
with an indicator, aldehyde ([4-dimethylamino] benzalde-
hyde, 37% hydrochloric acid, and amyl alcohol, also re-
ferred to as Kovac’s reagent), which results in a pink to red
color formation (Procedure 12-20). Spot indole contains
p-dimethylaminocinnamaldehyde (DMACA), 37% hydro-
chloric acid, and deionized water, which results in a blue
color formation. This test is used in numerous identification
schemes, especially to presumptively identify Escherichia coli,
the gram-negative bacillus most commonly encountered in
diagnostic bacteriology.
Urease Test
Urease hydrolyzes the substrate urea into ammonia, wa-
ter, and carbon dioxide. The presence of the enzyme is
determined by inoculating an organism to broth (Stuart’s
urea broth) or agar (Christensen’s urea agar) containing
urea as the primary carbon source followed by detecting
the production of ammonia (Procedure 12-41). Ammo-
nia increases the pH of the medium so its presence is
readily detected using a pH indicator. Change in medium
pH is a common indicator of metabolic process and, be-
cause pH indicators change color with increases (alkalin-
ity) or decreases (acidity) in the medium’s pH, they
are commonly used in many identification test schemes.
Prolonged incubation of urea agar may result in false pos-
itive reactions because of the hydrolysis of proteins in the
medium. In addition, weak urease positive organisms
such as Enterobacter spp. may not demonstrate a positive
reaction in urea broth because of the buffering capacity of
the media. The urease test helps identify certain species
of Enterobacteriaceae, such as Proteus spp., and other im-
portant bacteria such as Corynebacterium urealyticum and
Helicobacter pylori.
PYR Test
The enzyme L-pyroglutamyl-aminopeptidase (PYR) hydro-
lyzes the substrate L-pyrrolidonyl-b-naphthylamide to pro-
duce a beta-naphthylamine. When the beta-naphthylamine
combines with a cinnamaldehyde reagent, a bright red color
is produced (Procedure 12-36). The PYR test is particularly
helpful in identifying gram-positive cocci such as Streptococcus
pyogenes and Enterococcus spp., which test positive, whereas
other streptococci test negative.
Hippurate Hydrolysis
Hippuricase is a constitutive enzyme that hydrolyzes the sub-
strate hippurate to produce the amino acid glycine. Glycine is
detected by oxidation with ninhydrin reagent, which results
in the production of a deep purple color (Procedure 12-19). The
hippurate test is most frequently used in the identification of
Gardnerella vaginalis, Streptococcus agalactiae, Campylobacter
jejuni, and Listeria monocytogenes.
Tests for the Presence of Metabolic Pathways
Several identification schemes are based on determining
what metabolic pathways an organism uses and the sub-
strates processed by these pathways. In contrast to single
enzyme tests, these pathways may involve several interac-
tive enzymes. The presence of an end product resulting
from these interactions is measured in the testing system.
Assays for metabolic pathways can be classified into three
general categories: carbohydrate oxidation and fermen-
tation, amino acid degradation, and single substrate
utilizations.
Oxidation and Fermentation Tests
As discussed in Chapter 2, bacteria use various metabolic
pathways to produce biochemical building blocks and en-
ergy. For most clinically relevant bacteria, this involves utili-
zation of carbohydrates (e.g., sugar or sugar derivatives) and
protein substrates. Determining whether substrate utiliza-
tion is an oxidative or fermentative process is important for
the identification of several different bacteria.
Oxidative processes require oxygen; fermentative ones
do not. The clinical laboratory determines how an organism
utilizes a substrate by observing whether acid byproducts are
produced in the presence or absence of oxygen (fermenta-
tion). In most instances, the presence of acid byproducts is
detected by a change in the pH indicator incorporated into
the medium. The color changes that occur in the presence of
acid depend on the type of pH indicator used.
Oxidation-fermentation determinations are usually ac-
complished using a special semisolid medium (oxidative-
fermentative [O-F] medium) that contains low concentra-
tions of peptone and a single carbohydrate substrate such as
glucose. The organism to be identified is inoculated into
two glucose O-F tubes, one of which is then overlaid with
mineral oil as a barrier to oxygen. Common pH indicators
used for O-F tests, and the color changes they undergo with
acidic conditions, include bromcresol purple, which changes
from purple to yellow; Andrade’s acid fuchsin indicator,
which changes from pale yellow to pink; phenol red, which
changes from red to yellow; and bromthymol blue, which
changes from green to yellow.
As shown in Figure 7-16, when acid production is de-
tected in both tubes, the organism is identified as a glucose
fermenter, because fermentation can occur with or without
oxygen. If acid is only detected in the open, aerobic tube,