SECTION NINE v IDENTIFICATION OF UNKNOWNS 485

EXERCISE 9-2
Identication of
Gram-positive Cocci
v Theory
Gram-positive cocci are frequent isolates in a clinical
setting because they are common inhabitants of skin and
mucous membranes. Five main genera, briefy described
below, will be used in this lab exercise.
Staphylococcus (Figure 9-7)
Gram-positive cocci in singles, pairs, tetrads, or clusters
(especially when grown in broth)
Catalase positive.
With rare exception, facultatively anaerobic.
Most are oxidase negative.
G+C content within the genus ranges between 30
and 39%.
Grow in 6.5% NaCl.
Most produce acid from glucose.
Most are resistant to bacitracin.
Key pathogen is Staphylococcus aureus (toxic shock
syndrome and a variety of other skin and deep organ
infections, including bacterermia).
Kocuria
A small genus; most species were formerly classifed in
the genus Micrococcus.
Gram-positive cocci in pairs and tetrads
Obligately aerobic.
Do not produce acid from glucose.
Bacitracin susceptible.
Catalase positive.
Commensals or opportunistic pathogens, especially
among immunocompromised patients.
Micrococcus
Gram-positive cocci in pairs and tetrads
Obligately aerobic.
Oxidase positive.
Do not produce acid from glucose.
Bacitracin susceptible.
Catalase positive.
G+C content within the genus ranges between 66
and 75%.
Grow in 6.5% NaCl.
Commensals or opportunistic pathogens. M. luteus is a
common skin commensal.
Streptococcus (Figure 9-8)
Gram-positive cocci to ovoid cocci in singles, pairs, or
short chains (especially when grown in broth)
Gray to white, moist colonies are frequently observed.
Catalase negative.
Oxidase negative.
Facultatively anaerobic.
Nutritionally fastidious.
Some require 5% CO
2
for growth.
Ferment glucose and other carbohydrates, mostly to
lactic acid.
9-7 Staphylococcus aureus u This specimen grown in broth
illustrates the grape-like clusters of cells characteristic of the genus.
Specimens grown on solid media may not show the clusters as clearly.
9-8 Streptococcus agalactiae u This specimen grown in broth
illustrates the streptococcal arrangement of cells characteristic of the
genus. Specimens grown on solid media may not show the chains as
clearly.
Kocuria rosea (= Micrococcus roseus)
Micrococcus luteus
Staphylococcus aureus (BSL-2)
Staphylococcus epidermidis (BSL-2)
Staphylococcus saprophyticus
Gram-Positive Cocci and Identification Tests
Catalase-Positive
(Figure 9-9)
Catalase-Negative
(Figure 9-10)
Coagulase
Bacitracin Susceptibility (0.04 U)
Novobiocin Susceptibility (0.5 g)
Optochin Susceptibility (5 g)
>16
mm
16
mm
>14
mm
<14
mm
10
mm
< 9
mm
S R
+
A+ A
Mannitol Fermentation
Gelatin Hydrolysis (Gelatinase)
Enterococcus faecalis (BSL-2)
Streptococcus agalactiae (BSL-2)
Streptococcus dysigalactae subsp. equisimilis (BSL-2)
Streptococcus mutans
Streptrococcus salivarius
Streptococcus sanguinis (BSL-2)
Streptococcus pneumoniae (BSL-2)
Streptococcus pyogenes (BSL-2)
(= Streptococcus equisimilis)
Hemolysis

Arginine Decarboxylase
(Dihydrolase)
PYR Test
+
CAMP Test
NO
3
reduced to NO
2
Catalase
Starch Hydrolysis (Amylase)
Voges-Proskauer

O F
Glucose O-F Test
S
S
L
L
MICROBIOLOGY: LABORATORY THEORY AND APPLICATION, BRIEF 486
G+C content within the genus ranges between 34 and
46%.
Many species produce hemolysins that either completely
(b-hemolysins) or partially (a-hemolysins) destroy
erythrocytes. Some species are nonhemolytic.
Beta-hemolytic streptococci have traditionally been
grouped by antigens frst described by Rebecca
Lancefeld. Important Lancefeld groups include Group
A (S. pyogenes) and Group B (S. agalactiae).
Key pathogens are Streptococcus pyogenes (strep throat,
necrotizing fasciitis, scarlet fever) and S. pneumoniae
(bacterial pneumonia, otitis media, and bacteremia).
Enterococcus
Formerly members of the genus Streptococcus.
Gram-positive cocci to ovoid cocci in singles, pairs, or
short chains (especially when grown in broth); may be
more rod-shaped if grown on solid media.
Catalase negative
Oxidase negative.
Facultatively anaerobic.
Lactic acid, but no gas, is the sole end product of
fermentation.
Grow in 6.5% NaCl broth.
Grow in bile esculin.
Most are PYR positive.
G+C content within the genus ranges between 34 and
42%.
Most express the Lancefeld Group D antigen.
Most species are commensals or opportunistic
pathogens. The key opportunistic pathogen is E. faecalis
(urinary tract infections, wound infections, and
bacteremia in seriously ill elderly persons).
The organisms to be used as unknowns are listed in
Table 9-2, as are the tests to be used in their identifcation.
(Note: Your instructor will choose Gram-positive coccus
unknowns appropriate to your microbiology course and
facilities.)
v Application
Identifcation of Gram-positive cocci from human specimens
requires a coordinated and integrated use of biochemical
tests and stains. Although several serological tests allow
rapid identifcation, fowcharts are still a useful way to
visualize the process of identifcation by elimination.
v In This Exercise
This exercise will span several lab
periods. You will be given an
unknown Gram-positive coccus
from the organisms listed in Table
9-2, then run biochemical tests as
directed by the fowcharts in Figure
9-9 or Figure 9-10 to identify it.
TABLE 9-2 List of Organisms
and a Key to the Icons Used in This
Exercise u The organisms listed are
Gram-positive cocci available from Wards
or Carolina Biological Supply Companies.
Icon colors are to remind you what the
results look like. They should not be used
to compare with your results. Note: For
the fermentation tests, A1 means
acid is produced; A2 means no acid is
produced.
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Kocuria rosea (= Micrococcus roseus)
Micrococcus luteus
+
Coagulase Tube Test
Micrococcus luteus
Staphylococcus saprophyticus Staphylococcus epidermidis
NO
3
reduced to NO
2
+
Kocuria rosea
NO
3
reduced to NO
2

+
Gelatin Hydrolysis +
Novobiocin (5 g) S R
>16
mm
16
mm
S R Bacitracin (0.04 U)
10
mm
< 9
mm
Glucose Oxidation-Fermentation Test
O F
K. rosea (= M. roseus)
M. luteus
S. aureus
S. epidermidis
S. saprophyticus
Staphylococcus aureus S. epidermidis
S. saprophyticus
Gram-Positive, Catalase-Positive Cocci
S L
S L
SECTION NINE v IDENTIFICATION OF UNKNOWNS 487
v Materials
Per Class
media listed in Table 9-2 for biochemical testing
pure cultures of organisms listed in Table 9-2 to be used
for positive controls and unknowns (BSL2 organisms
should not be chosen by schools or classes not
adequately prepared to handle them.)
Todd-Hewitt Broth or BrainHeart Infusion Broth
candle jar set-up
Per Student
two PEA, CNA, or 5% Sheep Blood Agar plates
Gram-stain kit
gloves
microscope slides
3% H
2
O
2
one unknown organism
1
from the list in Table 9-2
compound microscope with oil objective lens and
ocular micrometer
lens paper
immersion oil
1
Note to instructor: This exercise can be combined with Exercise 9-1 by
passing out a mixture of a Gram-negative enteric and a Gram-positive
coccus as unknowns. The student then must isolate the two organisms
from a mixed culture, and then proceed to identify the two independently.
If this option is chosen, grow the two unknowns separately, and then mix
them immediately prior to handing them out in lab.
9-9 Identifcation Flowchart for Gram-positive, Catalase-Positive Cocci (Usually in Tetrads or Clusters) u See the appropriate exercises
for instructions on how to run each test. Novobiocin is covered in this exercise. In some cases, fnal identifcation to species is diffcult. Try to get
that far, but you may reach a point where your lab strains cant be differentiated any further. (Compiled from sources listed in the References.)
Enterococcus faecalis
Streptococcus agalactiae
Streptococcus equisimilis
Streptococcus mutans
Hemolysis Reaction
or
Streptococcus equisimilis
PYR +
S. agalactiae
S. equisimilis
S. pyogenes
E. faecalis
Streptococcus agalactiae
S R Bacitracin (0.04 U)
10
mm
<9
mm
Streptococcus pyogenes Enterococcus faecalis
R
S Optochin (5 g)
<14
mm
>14
mm
Streptrococcus salivarius
Streptococcus sanguinis
Streptococcus pneumoniae
Streptococcus pyogenes
CAMP Test +
Gram-Positive, Catalase-Negative Cocci
E. faecalis
S. agalactiae
S. equisimilis
S. pyogenes
E. faecalis
S. mutans
S. pneumoniae
S. salivarius
S. sanguinis
A A+ Mannitol Fermentation
R S Optochin (5 g )
<14
mm
>14
mm
E. faecalis
S. mutans
S. salivarius
S. pneumoniae
S. sanguinis
+ Voges-Proskauer
Streptococcus pneumoniae
+ Arginine Decarboxylase
Streptococcus sanguinis
Streptococcus mutans
A A+ Mannitol Fermentation
Streptococcus salivarius E. faecalis
S. mutans
Enterococcus faecalis
Starch Hydrolysis
+
MICROBIOLOGY: LABORATORY THEORY AND APPLICATION, BRIEF 488
Procedure
1
Obtain an unknown. Record its number on the Data
Sheet.
2
Streak the sample for isolation on two Sheep Blood
Agar, PEA, or CNA plates. Incubate one in the candle
jar and the other aerobically. Incubate both at 30
35C. It is best to check your plate for isolation at 24
hours. If you have isolation, continue with Step 3. If
you dont have time to continue with Step 3, refriger-
ate your plates until you do. If you dont see isolation,
ask your instructor if you should restreak for isola-
tion, let the original plates continue to incubate, or do
both. Record your isolation procedure as directed on
the Data Sheet.
9-10 Identifcation Flowchart for Gram-positive, Catalase-Negative Cocci (Usually in Pairs or Chains) u See the appropriate
exercises for instructions on how to run each test. In some cases, fnal identifcation to species is diffcult. Try to get that far, but you may reach
a point where your lab strains cant be differentiated any further. (Compiled from sources listed in References.)
SECTION NINE v IDENTIFICATION OF UNKNOWNS 489
3
After incubation, record colony morphology
(in cluding medium), optimum growth temperature,
and CO
2
requirement under Preliminary Results on
the Data Sheet. Also include the result of the differen-
tial component of blood agar as Test #1 if you used it.
Continue to record your isolation procedure on the
Data Sheet.
4
Perform a Gram stain on a portion of one well-
isolated colony. Continue testing colonies until you
fnd one that is a Gram-positive coccus.
Record its Gram reaction and cell morphology,
arrangement and size under Preliminary
Obser vations on the Data Sheet.
If the isolate is a Gram-positive coccus, transfer a
portion of the same colony to a Todd-Hewitt Broth
(or another suitable growth medium as available in
your lab) and incubate it at 3562C. This is your
pure culture to be used as a source of organisms
for further testing. Complete the record of your
isolation procedure on the Data Sheet.
5
Perform a catalase test on what remains of the isolated
colony, or wait until your pure culture has grown and
do it then. Record the result as Test #1 (or #2 if
hemolysis reaction is already recorded) under Differ-
ential Tests on the Data Sheet. (Note: Be sure to use
growth from the top of the colony if isolation was
performed on a blood agar plate. This minimizes the
possibility of a false positive from the catalase-positive
erythrocytes in the medium. Alternatively, you can
postpone the catalase test until your pure culture has
grown.)
6
Use the chart in Figure 9-9 for identifcation of
catalase-positive cocci (usually in tetrads or clusters).
Use the chart in Figure 9-10 if the isolate is catalase-
negative (usually with cocci in pairs or chains).
Re cord the Figure number of the fowchart you will
use on the Data Sheet. If your isolate is catalase-
negative, ask your instructor if you would get better
growth of your pure culture using Todd-Hewitt Broth
or BrainHeart Infusion Broth and incubating in a
candle jar or CO
2
incubator.
7
Now you will begin biochemical testing to identify
your organism. The tests in the fowcharts were chosen
because of their uniformity of results as published in
standard microbiological references, so they give the
best chance for correct identifcation. Be aware,
however, that the symbol 1 indicates that 90% or
more of the strains tested give a positive result. This
means that as many as 10% of the strains tested give
a negative result. The same is true of the symbol 2.
That is, as many as 10% of the strains give a positive
result. Bottom line: There are no guarantees that your
lab strains will behave in the majority, and if not, you
will misidentify your unknown. This issue, if relevant
to your unknown, will be addressed in Step 10.
8
Follow the tests in the appropriate fowchart to
identify your unknown.
2
Use these guidelines.
Do not run more than one test at a time unless your
instructor tells you to do so.
Where multiple tests are listed at a branch point,
run only one, and then move to the next level in the
fowchart as indicated by the result of that test.
Continue to record the relevant inoculation infor-
mation and results on the Data Sheet as you go.
Keep accurate records of what you have done. Do
not enter all your data at the end of the project.
9
When you have identifed your organism, use Bergeys
Manual or another standard reference to fnd one
more test to run for confrmation. The confrmatory
test doesnt have to separate the fnal organisms on
the fowchart. It only has to be a test for which you
know the result and that you havent run already as
part of the identifcation process. Record this test on
the Data Sheet, and identify it as the confrmatory
test. Record the expected result on the comments line.
After incubation, note whether the organisms result
matches or differs from the expected result.
10
After the confrmatory test, write the organisms name
on the back of the Data Sheet under My Unknown
is and check with your instructor to see if you are
correct. If you are, congratulations! If you arent, your
instructor will advise you as to which test(s) gave
incorrect results by writing the test name(s) in the
rerun space next to your identifcation. These
should be rerun with appropriate controls from your
schools inventory of organisms. Record the test(s)
and control(s) on your Data Sheet. Checking the
results with controls and your unknown will indicate
if the incorrect test result was truly incorrect, or if
the strain of organism your school is using doesnt
match the majority of strains for that test result.
2
These fowcharts can be used only for the organisms listed in Table 9-2.
MICROBIOLOGY: LABORATORY THEORY AND APPLICATION, BRIEF
References
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Landry, and David W. Warnock. ASM Press, Washington, DC.
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