Unknowns Project
Christine Kelly
4/26/2015
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Introduction:
Isolating and identifying microorganisms is very important. There are many ways to
determine the differences in microorganisms. They can be determined by their shape and staining
properties, as well as the way they are grouped. They can also be determined by their reactions to
different biochemical testing. Using these methods can be very important in discovering the type
of organism present.
Understanding the procedure in isolating and identifying bacteria can help in many
different fields to include medical and biological research fields. The medical field can use
identification to assure proper treatments are given to patients. Also “many of these tests are
designed to identify Gram-negative organisms, since there is a larger percentage of pathogen”
(Kerr & McHale, 2003) that are Gram-negative. The rise in antibiotic resistance makes this even
more important. In biological research, soil and water microbiology play important parts in our
everyday lives and in many discoveries. Some of these discoveries have led to new antibiotics in
our constant battle to keep bacteria at bay within our bodies.
Morphologies of bacteria play an important role in isolation and identification. The
ability to determine different morphologies is the first step in isolation. This is followed by gram
staining to determine if you have a gram positive or gram negative bacterium. The ability to
determine these things help lead to biochemical tests which will further identify the bacteria
present.
Methods and Materials:
2 Gram Positive
Mannitol Fermentation
Fermentation of Mannitol
Staphylcoccus aureas
No Fermemtnation of Mannitol
Staphylococcus epidermidis
Streptococcus agalactiae
Streptococcus salivarius
Micrococcus luteus
Bacillus subtilis
Starch Agar
Amalayse production
Streptococcus salivarius
Bacillus subtilis
No Amylase Production
Staphylococcus epidermidis
Streptococcus agalactiae
Micrococcus luteus
Nitrate Reduction
No Nitrate Reduction
Streptococcus salivarius
NO3 to NO2
Bacillus subtilis
Urea
Urease Production
Staphylococcus Epidermidis
No Urease Production
Streptococcus agalactiae
Micrococcus luteus
TSI
Glucose Fermentation only
Micrococcus luteus
Glucose and Lactose fermentation
Streptococcus agalactiae
Confirmation
Glucose and Lactose Fermentation tubes
3Gram Negative
Baird Parker
Growth, brown colonies, no clearing
Proteus vulgaris
No growth
Alcaligenes faecalis
Enterobacter aerogenes
Escherichia coli
Psuedomonas aeruginosa
Salmonella typhimurium
Serratia marcescens
Shigella flexneri
Oxidative-Fermentation Metabolism
Fermentation
Enterobacter aerogenes
Escherichia coli
Salmonella typhimurium
Serratia marcescens
Shigella flexneri
Oxidative
Psuedomonas aeruginosa
Non saccharolytic
Alcaligenes faecalis
Phenylalanine Slant
Phenylalanine catabolism by phenylalanine deaminase
Enterobacter aerogenes
Confirmation
Glucose fermentation and Lactose fermentation
No catabolism
Escherichia coli
Salmonella typhimurium
Serratia marcescens
Shigella flexneri
Sucrose fermentation Ferments Sucrose
Escherichia coli
Serratia marcescens
No fermentation
Salmonella typhimurium
Shigella flexneri
Citrate
Citrate
Utilizes Citrate
Salmonella typhimurium
Does not utilize citrate
Shigella flexneri
Does not utilize Citrate
Escherichia coli
Utilizes Citrate
Serratia marcescens
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Results:
UNKNOWNS RESULTS FORM
Student: Christine Kelly Unknown #: 3
Instructor: Dr. Paz Identification: Streptococcus agalactiae
MORPHOLOGICAL CHARACTERISTICS
Cell Shape & Arrangement: Coccus in chains Colony Growth on TSAYE: Small, cream and circular
Gram’s Reaction: Gram positive reaction
Special Stains (optional): Isolation Temperature: 25 C
PHYSIOLOGICAL CHARACTERISTICS
Media 1:
Mannitol Fermentation
Observations
There was no change in the color of the tube and no gas present.
Interpretation
There was no fermentation of mannitol sugar.
Staphylococcus aureas ruled out
Incubation Temperature/Time
25 C / 48 hours
Used as a confirmation test?
No
Media 2:
Amylase
Observations
There was no growth on the plate and no clearing.
Interpretation
No Amylase was produced.
Streptococcus salivarius and Bacillus subtilis ruled out
Incubation Temperature/Time
25 C/ 48 hours
Used as a confirmation test?
No
Media 3:
Urea
Observations
The tube remained yellow in color.
Interpretation
There was no production of
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urease.
Staphylococcus epidermidis ruled out
Incubation Temperature/Time
25 C / 48 hours
Used as a confirmation test?
No
Media 4: TSI Observations
Both the slant and the bottom of the tube turned yellow.
Interpretation
Lactose and glucose were both fermented.
Indicates
Streptococcus agalactiae is the unknown.
Incubation Temperature/Time
25 C / 48 Hours
Used as a confirmation test?
No
Media 5:
Glucose Fermentation
Observations
The tube changed from a blue to a more greenish color.
Interpretation
Glucose fermentation occurred.
Incubation Temperature/Time
25 C/ 48 hours
Used as a confirmation test?
Yes
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UNKNOWNS RESULTS FORM
Student: Christine Kelly Unknown #: 3
Instructor: Dr. Paz Identification: Enterobacter aerogenes
MORPHOLOGICAL CHARACTERISTICS
Cell Shape & Arrangement: bacilli, chained together Colony Growth on TSAYE: small, circular, cream
Gram’s Reaction: Gram Negative
Special Stains (optional): Isolation Temperature: 25 C
PHYSIOLOGICAL CHARACTERISTICS
Media 1: Baird Parker Observations
There was no growth observed on the plate
Interpretation
No coagulase, no tellurite reduction.
Proteus vulgaris ruled out
Incubation Temperature/Time
25 C / 48 Hours
Used as a confirmation test?
No
Media 2: Oxidative Fermentation metabolism
Observations
Both tubes yellowed
Interpretation
Fermentation took place in both aerobic and anaerobic tubes.
Pseudomonas aeruginosa and Alcaligenes faecalis ruled out.
Incubation Temperature/Time
25 C / 48 hours
Used as a confirmation test?
No
Media 3:Phenylalanine Slant Observations
The tube showed growth, and greening.
Interpretation
The presence of green mean Phenylalanine was catabolized by Phenylalanine deaminase.
Indicates
Incubation Temperature/Time
25 C / 48 hours
Used as a confirmation test?
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Yes or No
Enterobacter aerogenes as the gram negative bacteriaMedia 4: Glucose Fermentation Observations
The tube turned very yellow and had gas.
Interpretation
Glucose was fermented confirming Enterobacter
aerogenes
Incubation Temperature/Time
25 C / 48 hours
Used as a confirmation test?
Yes
Media 5: Lactose Fermentation Observations
This tube was yellow and had the presence of gas
Interpretation
Lactose was fermented confirming Enterobacter
aerogenes
Incubation Temperature/Time
24 C / 48 hours
Used as a confirmation test?
Yes
Discussion:
Isolating the organisms there was a problem getting the gram negative organism to separate
appropriately. There were no problems isolating the gram positive organism. Both organisms were
viewed under the microscope. The gram positive organism was observed to be coccus and in chains
indicating it would be a Streptococcus species. The flow chart for the gram positive organism was made
prior to the gram staining.
The gram positive flow chart was chosen to gain a direct way to identification without using
many tests. There were no issues with the tests. After gram-staining it was known to be a Streptococcus
species, so the tests performed as expected given the result of Streptococcus agalactiae.
The gram negative bacteria were harder to isolate. This may have been because the
morphologies on both the gram positive and gram negative bacteria were very close in appearance.
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Once separated the gram negative bacteria became much more cooperative. The tests proceeded as
expected. Again the shortest path to identification was used. The gram negative flow chart started with
eliminating one possibility with the Baird Parker plate. The oxidative fermentation metabolism would
have eliminated two as the bacteria. There was no need to go past the phenylalanine slant due to the
positive result of the bacterium catabolizing phenylalanine by phenylalanine deaminase. The catabolism
of phenylalanine identified the bacterium as Enterobacter aerogenes.
The knowledge gained in performing these tests can be carried on to many different fields
involving microbiology to include the medical profession as well as research based professions. The
understanding of gram staining and biochemical testing in isolation and identification are really required
knowledge for anyone working in the microbiology field.
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Works CitedKerr, T. J., & McHale, B. B. (2003). Applications in General Microbiology. Winston-Salem: Hunter
Textbooks Inc. .