How-To-Do-It
Isolating
"Unknown"
Bacteria
in
the
Introductory
Microbiology
LaboratoryI
A New Selective
Medium
for Gram-Positives
John L.
McKillip MaryAnne Drake
Approximately one week after the
midterm exam in our Introductory
Microbiology lab, the time always
comes to begin the big "unknown"
assignment. This project allows the
students a chance to apply the proce-
dures presented up to this point in
the term, as well as an opportunity
to
practice
their
techniques
and reasoning
when presented with a problem. For
many of the students,
selecting
a broth
tube containing a mixture of three
unknown bacteria,
and implementing
the array of biochemical
and morpho-
logical
tests that allow them to separate
and identify
each,
is a welcome change
from working in small groups, where
perhaps only one or two members
"get their
hands dirty."
These students
enjoy the relative freedom of working
on their own problem, learning from
mistakes they make, and benefitting
from logic and common sense as they
proceed. The assignment also pro-
motes the students' critical thinking
skills.
Having completed the typical bat-
tery of biooxidation and hydrolysis
reactions a few weeks earlier, and
armed with notes on the "how" and
"why" of each of these tests, the stu-
dents are allowed to use a variety of
selective and differential
media to first
isolate each of their three microorgan-
isms. Only after a confirmatory
Gram
stain to verify purity of each isolate
are they allowed to proceed
with their
John L.
McKiIlip,
formerly
at Mississippi
State University,
is currently an Assis-
tant Professor
of Biology at Louisiana
Technical University,
Ruston,
LA
71272;
e-mail: McKillip@latech.edu. Mary-
Anne Drake is an Assistant Professor
of Food Science and Technology at
Mississippi
State University,
Mississippi
State, MS
39762.
biochemical tests and morphological
observations
to make their final deter-
mination as to the identity of each of
the three unknowns to the species
level.
Although theoretically a properly
performed
streak plate should be ade-
quate to isolate a mixture of two, or
even three different bacterial species
on a generic
medium
such as trypticase
soy agar, the students are encouraged
to use selective media (a medium that
allows certain
microorganisms
to grow,
but not others)
to assist them in getting
the Gram-negative
bacteria
away from
the Gram-positives,
and vice versa. For
Gram-negative
isolation,
a good streak
on MacConkey's
agar usually works
nicely, and also allows the student to
observe possible lactose fermentation
by the color of the isolated colonies
(red = positive lactose fermentation)
(Leboffe & Pierce 1995). The high
degree of selectivity makes this a
dependable medium for obtaining
purity of the Gram-negative
microor-
ganisms from a mixed culture. How-
ever, for Gram-positive
isolation, our
regularly used phenylethanol agar
(PEA)
plates were not nearly as effec-
tive. A number of our Gram-negative
lab stocks grew quite well on PEA,
including E. coli. Moreover, some of
our Bacillus spp. were completely
inhibited on this medium,
which made
for some frustrated
students who felt
as if they were wasting valuable time
and resources. Mannitol Salt Agar
(MSA)
works very well for staphylo-
cocci, but is too selective for most
other Gram-positive
bacteria,
such as
Bacillus spp.
Early on, we made the decision to
develop a medium that would select
against as many Gram-negatives as
possible, while still allowing growth
and differentiation
of a wide range of
Gram-positives.
Thus the medium is
not only selective,
but also differential,
based on the utilization of a particular
substrate
in the medium, imparting a
visible color change or distinctive
col-
ony appearance.
We describe in this
paper a medium that we developed
which does this better than any other
we have come across, and one which
has been successfully implemented
in
the Introductory
Microbiology lab to
replace phenylethanol
agar.
Materials
& Components
This selective and differential
medium, which we have termed GP
agar (GP for "Gram-positive")
or GP
broth, is prepared by the recipe in
Table 1.
To prepare, dissolve above compo-
nents in 1 liter of deionized water
while stirring. Add agar last while
over heat, boil gently while stirring,
then remove from
heat, and autoclave.
Analysis
To put this new medium to the test,
we inoculated
it with a wide array
of
bacteria,
most of which we routinely
use in our undergraduate
Microbiol-
ogy laboratory.
Results are scored in
Table 2 as a "+ +" for significant
Table
1. Gram-positive
medium
components,
per liter.
Sodium chloride 32 g
Sodium glycerophosphate 5 g
Lithium chloride 10 g
Phenylethanol 1.5 mL
Mannitol 15 g
Tryptone 10 g
Yeast
extract 10 g
Bromcresol purple 0.02 g
Agar 15 g
610 THE
AMERICAN
BIOLOGY
TEACHER,
VOLUME 61, NO. 8, OCTOBER
1999
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