BIO 407 Fall 2010
Cassie Petit
Dr. Pellock
12/15/2010
BIO 407L – Microbiology Lab
Unknown microbe report
Isolation of unknown:
Name of isolate: “Thumbalina” (*1)
Name of researcher: Cassie Petit
Date of isolation: BIO 407 lab, 09/29/2010
Location where microbe was isolated: Under the thumbnail of the researcher
Most likely genus and species of unknown: Staphylococcus lentus
Frozen permanent stock location: Unknown box: J1
Initial phenotypic characterization:
Color: Pale yellow, darker yellow around outside
Size: Small
BIO 407 Fall 2010
Luster: Shiny
Form: Circular
Elevation: Convex
Margin: Went from undulate when not isolated in first streaking to entire when
isolated into single colonies
Texture and consistency: Creamy
Smell of pure culture: Wet dog, very strong
Notes on growth of isolate in liquid medium: Turbid culture, no excessive clumping or
sticking to sides of tube. No sediment on bottom of tube.
Notes on growth speed: Grew to single colonies overnight at 37oC
Gram staining results:
Gram phenotype: Gram-positive
Shape of individual cells: Cocci
Presence of spores: No
BIO 407 Fall 2010
Growth arrangement of cells: Mostly coccus, streptococci, some tetrad
Dimensions of individual cells: 1µm
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 37°C
Identification/Differential test results:
Dichotomous key: Key 1
Differential test performed; Result: Catalase test; 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 Test; Positive after a few days
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Staphylococcus aureus and is not S.
saprophyticus, S. epidermidis, M. luteus, or M. varians
Dichotomous key: Key 3B
Differential test performed; Result: Glucose test; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Micrococcus luteus or Kocuria rosea
Dichotomous key: Key 3C
Differential test performed; Result: Glucose test; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Micrococcus luteus or Kocuria rosea
Dichotomous key: Key 4B
Differential test performed; Result: Arrangement; mostly coccus but some tetrad
present
BIO 407 Fall 2010
Comparison to positive and negative controls: Yes
Conclusion: Since tetrad arrangements were not the majority, this key also notes
that Staphylococcus are glucose positive and facultative and Micrococcus are
glucose negative and obligate aerobes, suggesting that the isolate is in the
Micrococcus group
Dichotomous key: Key 4N
Differential test performed; Result: Pigment; White and Oxidase; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is M. sedenterius and not M. lylae
Dichotomous key: Key 4P
Differential test performed; Result: Anaerobic growth; No anaerobic growth and
Mannitol; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is S. lentus and not S. caseolyticus, S.
hominus, or S. auricularis
Dichotomous key: Key 5E
Differential test performed; Result: Catalase; Positive and Mannitol; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Staphylococcus aureus and not other
Staphylococcus or Micrococcus
---------------------------------------------------------------------------------------------------Genus and species hypothesis after using dichotomous keys: Staphylococcus aureus,
Staphylococcus lentus, or 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: Oxidase; Negative
Basis of the test: To distinguish between Micrococcus and Staphylococcus
Comparison to positive and negative controls: Yes
BIO 407 Fall 2010
Conclusion: In most cases Micrococcus is positive for oxidase and Staphylococcus is
negative, so result is consistent with Staphylococcus
Differential test performed; Result: Nitrate; Positive
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 Staphylococcus
Differential test performed; Result: Urea; Negative
Basis of the test: To distinguish between Micrococcus and Staphylococcus
Comparison to positive and negative controls: Yes
Conclusion: In most cases Micrococcus is positive for urease and Staphylococcus is
negative, so result is consistent with Staphylococcus. This is consistent with the
Staphylococcus aureus hypothesis.
**Differential test performed; Result: Sucrose; Postive
Basis of the test: To further see if the isolate is Staphylococcus lentus
Comparison to positive and negative controls: Yes
Conclusion: In most cases S. lentus is positive for sucrose so result is consistent
with Staphylococcus lentus
Final Conclusion: Based on the dichotomous keys and confirmatory tests there is a
high probability this unknown is a strain of Staphylococcus lentus. Among
Staphylococcus aureus and Micrococcus luteus, Staphylococcus lentus fit more of
the criteria than did the other two options.
BIO 407 Fall 2010
Basis of differential tests
Differential test
Catalase 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
Positive result
Gas generation
Negative result
No gas generation
Mannitol test
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
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Media stays red, no
gas generation
Obligate Aerobic
Growth test
This tests aerotolerance of bacteria
using thioglycollate broth
Pigment test
This test simply looks at whether or
not there is pigment or if it is colorless
when put on a clear slide
Growth of bacteria
only forms at the
oxygen-rich top
layer
Color present
Facultative or
aerotolerant
anerobes can grow
throughout medium
Colorless
Nitrate test
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
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
Glucose test
Oxidase test
Urea test
Maltose test
Sucrose test
Bacteria will turn
dark purple
Media stays red, no
gas generation
No color change
When bacterial
cells that produce urease are grown in
this medium, urea is degraded, ammonia
is released, and
the pH become alkaline.
Acid-induced color change of phenol
red indicator medium to yellow as a
result of production of organic acids
from fermentation of maltose.
Media will turn
bright hot pink
Media will not turn
hot pink
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Media stays red, no
gas generation
Acid-induced color change of phenol
red indicator medium to yellow as a
result of production of organic acids
from fermentation of sucrose.
Media turns yellow;
Possible gas
generation (trapped
by Durham tube)
Media stays red, no
gas generation
BIO 407 Fall 2010