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SC246 Microbiology
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Lab: Unknown Identification
Microorganisms are the most common and the most
numerous group of organisms on Earth. The world of
Microbiology revolves around the ability to categorize and
identify these microorganisms. The identification is
especially important in the medical community to
determine the cause of disease and effectively treat
patients.
To identify unknown bacteria, Microbiologists use a
diverse collection of tests including stains, biochemical
tests, and selective and differential media. In general, the
identification begins with a Gram stain and continues with
a scientific identification process, sometimes dependent
upon which identification is suspected. For example, if a
child goes to the pediatrician with a sore throat, the doctor
might look specifically for Streptococci in the child’s
throat.
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Given the massive number of bacterial species on
Earth, you can imagine that all of this information
can pile up. For decades, Microbiologists have
relied upon a collection of books known as
Bergey's Manual. This actually refers to a
collection of books that provide detailed
information on all recognized species of
prokaryotes. Each chapter in Bergey's Manual is
written by an expert, contains tables, biochemical
test results, and further systematic information that
is useful for identification purposes.
Bergey’s Manual
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The bible for
Microbiology
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Resource that
provides
characteristics
of the genus
and species of
many bacteria
Q1
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The Gram stain is typically the
first test performed in a
microbiology laboratory in the
process of identifying an
unknown. WHY? Give some
examples of instances when a
Gram stain would NOT be a
useful first step.
Q2
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What are biochemical tests
and why are they performed?
Q3
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Give 2 examples of
biochemical tests and
what the tests are
used for
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Chapter 5 lab Manual
Indole
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SIM medium
Tests for: presence
of the enzyme
tryptophanase
Can this enzyme
break down
tryptophan into NH3
+ pyruvic acid +
indole
Kovacs reagent
MR-VP test
Methyl Red (MR)
 Voges-Proskauer test (VP)
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Same media is used for both
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Different reagents are added to do each
test
Methyl Red
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Used to identify enteric bacteria
based on glucose metabolism
Checks for mixed acid fermentation
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Reagent- methyl red
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Voges-Proskauer
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Used to identify
enteric bacteria
based on glucose
metabolism
Checks for the
production of
acetoin
Reagent- alphanapthol and KOH
Urea
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Tests for the
presence of urease
If the bacteria have
urease, they can
break down urea
into NH3 and CO2
Carbohydrate Utilization
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Tests for fermentation of a sugar
and gas production
Durham tube inside tube measures
gas production
Red/purple= negative for
fermentation
yellow = fermentation
pH indicator
Results should indicate
fermentation and gas production
Citrate
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Simmons citrate
agar
Tests for the enzyme
citrase
Green = positive
Blue = negative
Q4
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Imagine that you are working in a
microbiology lab and you are given a
sample of bacteria on an agar plate. You
are told that it is either Staphylococcus
aureus or Streptococcus pyogenes. How
would you determine which one the
sample contains? Are there any tests
you could do to differentiate them?
Staphylococcus aureus or
Streptococcus pyogenes??
Catalase
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Tests for the enzyme catalase
Catalase breaks down hydrogen
peroxide
Most aerobic and facultatively
anaerobic bacteria make catalase
Some lack this enzyme
(Streptococcus and Micrococcus)
Reagent- hydrogen peroxide
Bubbles = positive
Q5
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WHY must these steps be
followed? Wouldn’t it be
easier to just observe the
organism under a microscope
and make an identification
based on observation?
Case #1
Following a round of water testing in rural Minnesota,
contaminated water was discovered in a stream. The source
of the contamination is most likely fecal contamination
caused by run-off from a 600-acre dairy farm close to the
stream. Heavy rains in the area caused localized flooding
and the stream measured water levels well above normal.
After isolating a species of bacteria from the water, the following
laboratory tests were completed. The lab notes are listed
under laboratory observations. You should record in the
results column a positive or negative for each test. To
complete the Gram stain results, simply interpret whether it is
Gram positive or Gram negative and list the gram reaction
and shape. For example: Gram + rods in singles, Gram
negative cocci in chains.
Laboratory observations
Gram stain
Pink rods
Glucose
Yellow media, gas bubble in durham tube
Indole
Red layer of reagents at the top of the test
tube
Methyl Red
Red
Urea
Pinkish-red color
Catalase
Bubbles
Table 1.1
Results
References and photo credits
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Tortora, G., Funke, B., & Case, C.
(2007). Microbiology: An Introduction
(8th ed.). San Francisco, CA: Pearson.
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Alexander, S., & Strete, D. (2001).
Microbiology: A Photographic Atlas for
the Laboratory . San Francisco, CA:
Benjamin-Cummings.