BIO 407 Fall 2010
Cassie Petit
Dr. Pellock
12/15/2010
BIO 407L – Microbiology Lab
Unknown microbe report
Isolation of unknown:
Name of isolate: MM1 (*5)
Name of researcher(s): Cassie Petit
Date of isolation: BIO 407 Lab, 9/29/2010
Location where microbe was isolated: Microbes and Man class
Most likely genus and species of unknown: Bacillus laterosporus
Frozen permanent stock location: Unknown box: J5
Initial phenotypic characterization:
Color: Strawberry red
Size: Single colonies are small, punctiform
Luster: Shiny
BIO 407 Fall 2010
Form: Circular
Elevation: Raised
Margin: Undulate
Texture and consistency: Sticky
Smell of pure culture: Stale breath
Notes on growth of isolate in liquid medium: Sediment throughout medium. Some
clumping and sticking to sides of tube. Some sediment on bottom of tube
Notes on growth speed: Grew to single small colonies at 30oC after two weeks, had
doubts that it would even grow at all, very slow growth
Gram staining results:
Gram phenotype: Gram positive
Shape of individual cells: Rods
Presence of spores: Yes
Growth arrangement of cells: Bacillus
Dimensions of individual cells: Width: 1µm, Length: 2.2µm
BIO 407 Fall 2010
Secondary phenotypic characterization:
Tolerance of anaerobic growth conditions/phenotype in thioglycollate broth: Facultative
Hemolytic phenotype (blood agar): Beta
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 1
Differential test performed; Result: Spore staining; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is either Bacillus or Clostridium groups and not
Mycobacterium, Corynebacterium, or Lactobacillus groups
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 either Bacillus megaterium or Bacillus
subtilis and not Bacillus cereus
Dichotomous key: Key 2A and Key 3B
Differential test performed; Result: Voges-Proskauer; Negative
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is Bacillus megaterium and not Bacillus subtilis
Dichotomous key: Key 4A
Differential test performed; Result: Catalase test; Positive
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is in the Bacillus group and not the Clostridium
group
Dichotomous key: Key 4D
Differential test performed; Result: Starch; Negative, Gelatin; Positive and Growth
on 5% NaCl
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is B. pasteurii or B. laterosporus and not B.
larvae, B. lentimorbus, or B. popilliae
BIO 407 Fall 2010
Dichotomous key: Key 5D
Differential test performed; Result: Starch; Negative, Catalase; Positive, Swollen
Cell; Positive, Spore shape; oval
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the isolate is B. laterosporus and not B. sphaericus or B.
pasteurii
---------------------------------------------------------------------------------------------------Genus and species hypothesis after using dichotomous keys: B. laterosporus
---------------------------------------------------------------------------------------------------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: Mannitol; Positive
Basis of the test: To further confirm the isolate is B. laterosporus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with B. laterosporus hypothesis
and not B. pasteurii or B. sphaericus
Differential test performed; Result: Spore shape; oval
Basis of the test: To further confirm the isolate is B. laterosporus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with B. laterosporus hypothesis
and not B. pasteurii or B. sphaericus
Differential test performed; Result: Anaerobic growth; Positive
Basis of the test: To further confirm the isolate is B. laterosporus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with B. laterosporus hypothesis
Differential test performed; Result: Voges-Proskauer test; Negative
Basis of the test: To further confirm the isolate is B. laterosporus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with B. laterosporus hypothesis
BIO 407 Fall 2010
Differential test performed; Result: Urea; Negative
Basis of the test: To further confirm the isolate is B. laterosporus
Comparison to positive and negative controls: Yes
Conclusion: Suggests that the results are consistent with B. laterosporus hypothesis
Final Conclusion: Based on the dichotomous keys and confirmatory tests there is a
high probability this unknown is a strain of Bacillus laterosporus. This conclusion is
consistent with the Bergey’s Manual of Systematic Bacteriology which describes
this bacteria being isolated from water, soil, and some food samples. B.
laterosporus has also been found in larvae of diseased bees. The student who
isolated this bacterium could have picked it up anywhere.
BIO 407 Fall 2010
Basis of differential tests
Differential test
Catalase test
Mannitol test
Glucose test
Obligate Aerobic
Growth test
Nitrate test
Urea test
Voges-Proskauer
test
Citrate test
Gelatin test
5% and 2% NaCl
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
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.
When bacterial
cells that produce urease are grown in
this medium, urea is degraded,
ammonia is released, and
the pH become alkaline.
Detects organisms that utilize the
butylene glycol pathway and produce
acetoin
To determine the ability to use citrate
as its sole carbon source
Gelatinase catalyzes the hydrolysis of
the protein gelatin (collagen). Gelatin
in solution liquefies above 25 degrees
C. At room temperature or below it
becomes a solid (gel).
Tests if the bacterium will grow in
NaCl of different concentrations
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
First test: Turns
red after addition
of nitrate reagents
Second test: Does
not turn red
Media stays red, no
gas generation
Media stays red, no
gas generation
Facultative or
aerotolerant
anerobes can grow
throughout medium
First test: Does not
turn red after
addition of nitrate
reagents
Second test: Turns
red
Media will turn
bright hot pink
Media will not turn
hot pink
Media will turn red
Media will not turn
red
Media will turn blue
Media will stay
green
Gelatin solidifies
Gelatin cannot
solidify
Bacteria will grow
Clearing around the
bacterial growth
Bacteria will not
grow
No clearance
around growth
BIO 407 Fall 2010