BIO 407 Fall 2010
Cassie Petit
Dr. Pellock
12/15/2010
BIO 407L – Microbiology Lab
Unknown microbe report
Isolation of unknown:
Name of isolate: “Pee-Tree” (*7)
Name of researcher: Cassie Petit
Date of isolation: BIO 407 lab, 09/29/2010
Location where microbe was isolated: Handle of women’s toilet in Albertus Magnus
Most likely genus and species of unknown: Micrococcus luteus
Frozen permanent stock location: Unknown box: J2
Initial phenotypic characterization:
Color: Dark lemon yellow on outline of colony, light lemon yellow on inside
Size: Single colonies are large
Luster: Outline is dull, inside is shiny
BIO 407 Fall 2010
Form: Circular
Elevation: Raised
Margin: Undulate
Texture and consistency: Sticky
Smell of pure culture: Dirty Toilet
Notes on growth of isolate in liquid medium: Turbid culture, no excessive clumping or
sticking to sides of tube. Some sediment on bottom of tube.
Notes on growth speed: Grew into single large colonies overnight at 30oC, took four
streaks to isolate to single colonies due to rapid growth
Gram staining results:
Gram phenotype: Gram positive
Shape of individual cells: Cocci
Presence of spores: No
Growth arrangement of cells: Mostly tetrads and diplococcic, no single coccus,
irregular clusters
BIO 407 Fall 2010
Dimensions of individual cells: 1.2µm
Secondary phenotypic characterization:
Tolerance of anaerobic growth conditions/phenotype in thioglycollate broth: Obligate
Hemolytic phenotype (blood agar): Gamma
Growth at different temperatures (25°C, 30°C, 37°C, etc.): Grows at room
temperature (22°C), 30°C, and 37°C
Identification/Differential test results:
Dichotomous key: Key 2A
Differential test performed; Result: Catalase; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is in the Micrococcus or Staphylococcus group
and not the Streptococcus group
Dichotomous key: Key 2A
Differential test performed; Result: Mannitol; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is S. saprophyticus, S. epidermidis, M. luteus,
or M. varians and not S. aureus
Dichotomous key: Key 2A
Differential test performed; Result: Pigment; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is M. luteus or M. varians and not S.
epidermidis or S. saprophyticus
Dichotomous key: Key 2A
Differential test performed; Result: Glucose; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is M. luteus and not M. varians
Dichotomous key: Key 3C
Differential test performed; Result: Glucose; Negative and Colony color; Yellow
BIO 407 Fall 2010
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Micrococcus luteus and not Kocuria rosea
Dichotomous key: Key 4B
Differential test performed; Result: Arrangement; Tetrad
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is in the Micrococcus group and not the
Staphylococcus or Planococcus group
Also, this key notes that Staphylococcus are glucose positive and facultative and
Micrococcus are glucose negative and obligate aerobes, further suggesting that the
isolate is in the Micrococcus group
Dichotomous key: Key 4N
Differential test performed; Result: Pigment of Colony; Yellow and Glucose; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Micrococcus luteus and not Micrococcus
varians
Dichotomous key: Key 5E
Differential test performed; Result: Catalase; Positive, Manitol; Negative; Yellow
Pigment; Positive, and Glucose; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Micrococcus luteus and not Micrococcus
varians
---------------------------------------------------------------------------------------------------Genus and species hypothesis after using dichotomous keys: Micrococcus luteus
---------------------------------------------------------------------------------------------------Other differential tests performed after consulting Bergey’s Manual of
Determinative Bacteriology (BMoDB) and Bergey’s Manual of Systematic Bacteriology
(BMoDB):
Differential test performed; Result: Nitrate; Negative
Basis of the test: To distinguish between Micrococcus and Staphylococcus
Comparison to positive and negative controls: Yes
Conclusion: In most cases Micrococcus is negative for nitrate and Staphylococcus is
positive, so result is consistent with M. luteus hypothesis
BIO 407 Fall 2010
Differential test performed; Result: Pigment; Yellow
Basis of the test: To further confirm that the isolate is M. luteus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with M. luteus hypothesis
Differential test performed; Result: Starch; Negative
Basis of the test: To further confirm that the isolate is M. luteus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with M. luteus hypothesis
Differential test performed; Result: Glucose; Negative
Basis of the test: To further confirm that the isolate is M. luteus and not M.
varians
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with M. luteus hypothesis and
is not M. varians
Final Conclusion: Based on the dichotomous keys and confirmatory tests there is a
high probability this unknown is a strain of Micrococcus luteus. According to the
Bergey’s Manual of Systematic Bacteriology, M. luteus is very common and usually
found on mammalian skin. This makes sense since the bacteria was isolated from an
object that hands frequently come into contact with.
BIO 407 Fall 2010
Basis of differential tests
Differential test
Catalase test
Mannitol test
Glucose test
Obligate Aerobic
Growth test
Pigment test
Nitrate test
Oxidase test
Urea test
Starch test
Basis of the test
This test assays for production of the
enzyme catalase by the bacterium. If
catalase is produced, gas is generated
when the bacteria are exposed to
H2O2
Acid-induced color change of phenol
red indicator medium to yellow as a
result of production of organic acids
from fermentation of mannitol.
Acid-induced color change of phenol
red indicator medium to yellow as a
result of production of organic acids
from fermentation of glucose
This tests aerotolerance of bacteria
using thioglycollate broth
This test simply looks at whether or
not there is pigment or if it is
colorless when put on a clear slide
Nitrate broth is used to determine the
ability of an organism to reduce
nitrate to nitrite using the enzyme
nitrate reductase. It also tests the
ability of organisms to perform
nitrification on nitrate and nitrite to
produce molecular nitrogen.
Using a redox indicator, its tests for
the ability of a microorganism to
oxidize certain aromatic amines
When bacterial
cells that produce urease are grown in
this medium, urea is degraded,
ammonia is released, and
the pH become alkaline.
Tests the ability of an organism to
produce certain exoenzymes, including
a-amylase and oligo-1,6-glucosidase,
that hydrolyze starch
Positive result
Gas generation
Negative result
No gas generation
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Growth of bacteria
only forms at the
oxygen-rich top
layer
Color present
Media stays red, no
gas generation
First test: Turns
red after addition
of nitrate reagents
Second test: Does
not turn red
First test: Does not
turn red after
addition of nitrate
reagents
Second test: Turns
red
Bacteria will turn
dark purple
No color change
Media will turn
bright hot pink
Media will not turn
hot pink
Clearing around the
bacterial growth
No clearance
around growth
Media stays red, no
gas generation
Facultative or
aerotolerant
anerobes can grow
throughout medium
Colorless