2. 1
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:
3. 2 Gram Positive
Mannitol Fermentation
Fermentation ofMannitol
Staphylcoccus aureas
s
No Fermemtnation ofMannitol
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
GlucoseandLactosefermentation
Streptococcus agalactiae
Confirmation
Glucose and Lactose
Fermentation tubes
4. 3
Gram 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
Escherichiacoli
Utilizes Citrate
Serratia marcescens
5. 4
Results:
UNKNOWNS RESULTS FORM
Student:Christine Kelly Unknown#: 3
Instructor: Dr. Paz Identification:Streptococcusagalactiae
MORPHOLOGICAL CHARACTERISTICS
Cell Shape &Arrangement:Coccusinchains ColonyGrowthon TSAYE: Small,creamandcircular
Gram’s Reaction:Gram positive reaction
Special Stains(optional): IsolationTemperature: 25C
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
IncubationTemperature/Time
25 C / 48 hours
Usedas a confirmationtest?
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
IncubationTemperature/Time
25 C/ 48 hours
Usedas a confirmationtest?
No
Media 3: Observations Interpretation
6. 5
Urea The tube remained yellow in color. There was no production of
urease.
Staphylococcus epidermidis
ruled out
IncubationTemperature/Time
25 C / 48 hours
Usedas a confirmationtest?
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.
IncubationTemperature/Time
25 C / 48 Hours
Usedas a confirmationtest?
No
Media 5:
Glucose Fermentation
Observations
The tube changed from a blue to a
more greenish color.
Interpretation
Glucose fermentation
occurred.
IncubationTemperature/Time
25 C/ 48 hours
Usedas a confirmationtest?
Yes
7. 6
UNKNOWNS RESULTS FORM
Student: Christine Kelly Unknown#: 3
Instructor: Dr. Paz Identification:Enterobacteraerogenes
MORPHOLOGICAL CHARACTERISTICS
Cell Shape &Arrangement:bacilli,chainedtogether ColonyGrowthon TSAYE: small,circular,cream
Gram’s Reaction:Gram Negative
Special Stains(optional): IsolationTemperature:25C
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
IncubationTemperature/Time
25 C / 48 Hours
Usedas a confirmationtest?
No
Media 2: Oxidative
Fermentation metabolism
Observations
Both tubes yellowed
Interpretation
Fermentationtookplace inboth
aerobic and anaerobic tubes.
Pseudomonas aeruginosa and
Alcaligenes faecalis ruled out.
IncubationTemperature/Time
25 C / 48 hours
Usedas a confirmationtest?
No
Media 3:Phenylalanine Slant Observations
The tube showed growth, and
greening.
Interpretation
The presence of green mean
Phenylalanine was catabolized
by Phenylalanine deaminase.
IncubationTemperature/Time
25 C / 48 hours
8. 7
Usedas a confirmationtest?
Yes or No
Indicates
Enterobacter aerogenes as the
gram negative bacteria
Media4: Glucose Fermentation Observations
The tube turned very yellow and
had gas.
Interpretation
Glucose was fermented
confirming Enterobacter
aerogenes
IncubationTemperature/Time
25 C / 48 hours
Usedas a confirmationtest?
Yes
Media5: Lactose Fermentation Observations
This tube was yellow and had the
presence of gas
Interpretation
Lactose was fermented
confirming Enterobacter
aerogenes
IncubationTemperature/Time
24 C / 48 hours
Usedas a confirmationtest?
Yes
Discussion:
Isolatingthe organismsthere wasaproblemgettingthe gramnegative organismtoseparate
appropriately.There were noproblemsisolatingthe grampositive organism. Bothorganismswere
viewedunderthe microscope.The grampositive organismwasobserved tobe coccus and inchains
indicatingitwouldbe aStreptococcusspecies. The flow chart for the gram positive organismwasmade
priorto the gram staining.
The gram positive flowchartwaschosento gaina direct way to identificationwithoutusing
manytests. There were no issueswiththe tests. Aftergram-stainingitwasknowntobe a
Streptococcusspecies,sothe testsperformedasexpectedgiventhe resultof Streptococcusagalactiae.
9. 8
The gram negative bacteriawere hardertoisolate.Thismayhave beenbecause the
morphologiesonboththe gram positive andgramnegative bacteriawere veryclose inappearance.
Once separatedthe gram negative bacteriabecame muchmore cooperative. The testsproceededas
expected.Againthe shortestpathtoidentificationwasused. The gramnegative flow chartstartedwith
eliminatingone possibilitywiththe BairdParkerplate.The oxidativefermentationmetabolismwould
have eliminatedtwoasthe bacteria.There wasno needtogo past the phenylalanineslantdue tothe
positive resultof the bacteriumcatabolizingphenylalaninebyphenylalaninedeaminase. The catabolism
of phenylalanine identifiedthe bacteriumas Enterobacteraerogenes.
The knowledge gainedinperforming thesetestscanbe carriedon to manydifferentfields
involvingmicrobiologytoincludethe medical professionaswellasresearchbasedprofessions. The
understandingof gramstainingandbiochemicaltestinginisolationandidentificationare reallyrequired
knowledge foranyone workinginthe microbiologyfield.
10. 9
Works Cited
Kerr,T. J., & McHale, B. B. (2003). Applicationsin General Microbiology. Winston-Salem:Hunter
TextbooksInc..