Download Unknown Bi 324 lab T / R 8pm Evaluation and Conclusion The

Unknown
Bi 324 lab
T / R 8pm
Evaluation and Conclusion
The unknown bacteria was grown in various media, observed, and tested. Comparison to
controls identified the bacteria as serratia marcescens. The unknown, now identified as serratia
marcescens, belongs to the family enterobacteriaceae.
Triptic soy Agar (TSA-used for plating unless otherwise noted) was streaked with unknown
resulting in small to moderately sized colonies (dependant on days of growth) with a striking burnished
red pigment when grown at room temperature. It is interesting to note that there was little to no
pigmentation when the plated bacteria were incubated at 37C. Varying pigmentation depending on
growth temperatures for serratia marcescens is supported by the literature.
While testing to determine which family the unknown bacteria belonged to, we found that
it could be referred to as a facultatively anaerobic gram negative rod-shaped bacteria (see the exclusion
worksheet and template in the appendix for identifying the unknown). The rod-shape of the bacteria
was determined by a simple stain of methylene blue (all staining was observed under microscopic
examination). Another staining method was used to determine whether or not the bacteria contained
endospores. The bacteria tested negative for endospores using malachite green as the main stain and
Safranin as the counter stain. A third stain, a Gram stain, resulted in pink-colored rods, indicating the
bacteria was gram negative. Lastly, a thioglycollate medium was use to determine the aerotolerance of
the bacteria. The bacteria were more prolific towards the top of the media and the concentration visibly
decreased down the media. Such bacterial growth indicates the bacteria’s metabolic efficiency is
decreased in the absence of oxygen, but it can maintain life via fermentation or aerobic respiration.
Thus the unknown bacteria is a facultative anaerobe.
Tests used to confirm our bacterial species was a facultatively anaerobic gram negative rodshaped bacteria included a wet mount, UV light exposure, a potassium hydroxide(KOH) test, and a deep
stab test. The wet mount, using india ink as a stain, revealed clear rod shaped bacteria with growth in
singlets. A different method was used to confirm the absence of endospores. The endospore stain was
confirmed with a UV light experiment. A plated lawn of the unknown bacteria failed to grow under UV
light exposure for both 30 second and one minute intervals (indicating a absence of endospores). The
Gram negative characteristic of the unknown bacteria was confirmed with the KOH test where the
bacterial smear turned noticeably stringy when exposed to the reagent. To solidify our identification of
the unknown as facultatively anaerobic, the deep stab of the bacteria in agar showed a similar growth
pattern to the test using the thioglycollate medium.
After viewing the first flow chart (see appendix), one can see that identification of the unknown
as a facultatively anaerobic gram negative rod-shaped bacteria reveals the bacterial family. The
unknown bacterial species belongs to the family enterobacteriaceae. The second flow chart indicates the
next tests that were primarily used to determine that the bacterial species was serratia marcescens.
The main tests referenced to identify serratia marcescens as unknown were: the Indole Sim
test, gelatin hydrolysis and sugar fermentation tests. The Indole Sim media showed the bacteria as
slightly motile due to slight growth outside of the inoculation tracking. A liquefied media in the gelatin
hydrolysis test showed that the bacteria can produce gelatinases. The positive result of the gelatin
hydrolysis test is typical of serratia marcescens, but still other enterobacteriaceae as well. The last tests
used to reveal serratia marcescens as the bacterial species were the sugar fermentation tests. A red pH
indicator revealed fermentation of carbohydrates to acids. Broths containing glucose, sucrose, mannitol
and lactose were tested. The unknown yielded positive results for all but lactose. Inability of lactose
would eliminate all enterobacteriaceae but serratia marcescens and proteus rettgeri (see flowchart).
Bi 324 lab
T / R 8pm
However, the ability to ferment mannitol is not seen in proteus rettgeri. Therefore the unknown was
determined to be serratia marcescens.
A litmus milk test was used as an overall confirmation that the unknown was serratia
marcescens because it is the only enteric bacteria that results in casein coagulation. Tests used to
confirm motility, glucose fermentation, and the absence of lactose fermentation included a wet mount,
other carbohydrate fermentation and Maconkey agar respectively. A wet mount showed clear bacterial
movement or motility under microscopic examination. Glucose fermentation was naturally confirmed
with fermentation of mannitol. It is important to note that the fermentation of mannitol could not be
confirmed with growth on mannitol salt agar as serratia marcescens is not a halophile nor salt tolerant.
Maconkey agar is used to reveal lactose fermenting bacteria and confirmed that the unknown did not
ferment lactose. There was growth on the Maconkey agar but not the characteristic purple or pink
appearance indicating lactose fermentation.
Other tests detailed in the test template (appendix) supported the identification of unknown
as serratia marcescens. As with many other enterobacteriaceae, unknown tested positive for the
presence of catalase but negative for the presence of oxidase and starch hydrolysis. In addition,
unknown showed complete resistance to the antibiotics penicillin and bacitracin. As is more
common specifically to serratia marcescens, the unknown showed a zone of inhibition less than 22mm
for amoxicillin and tetracycline, indicating resistance to the aforementioned antibiotics as well. Along
the same lines, our unknown yielded a blue color in the Simmons Citrate Media Test indicating the
unknown bacteria utilitzes citrate and ammonium ion. Also more typical to serratia marcescens was
testing positive for the Vogue-Proskauer test and negative for the methyl red test. Such results show
that the bacteria does produce acetoin from fermenting glucose but does not perform mixed acid
fermentation of glucose.
Based on the series of tests used, comparison to controls, and confirmed results in the
literature, unknown is the bacterial species serratia marcescens belonging to the family
enterobacteriaceae. As to be expected, tests confirmed that the facultatively anaerobic gram negative
rod-shaped bacteria was of the family enterobacteriaceae. The gelatin hydrolysis, carbohydrate
fermentation and litmus milk tests were most helpful in determining unknown as serratia
marcescens. However, it is important not to forget that something as simple as the characteristic red
pigmentation of serratia marcescens differentiates it from many other bacterial species. Such
pigmentation makes it easily identifiable in restrooms where it commonly proliferates outside the
human body.
T / R 8pm
APPENDIX
Exclusion Worksheet
Full Name:
Unknown bacteria
Selected Sections of Bergey’s Manual of Systematic Bacteriology.
Fill in the third column using your lab notes on your unknown. Your goal is to exclude each section except for one, the section
your unknown belongs to. Indicate which section you cannot exclude. Provide a reason for excluding each section, and the
reason must exclude all members of the section. The first section has been filled in as an example to help you.
Section
Spirochetes (filled in as an example
on how to do this)
Aerobic, Motile, Helical Gramnegative bacteria
Gram Negative Aerobic
Rods and Cocci
Gram Negative Cocci and
Coccobacilli
Facultatively Anaerobic
Gram Negative Rods
Rickettsiae & Chlamydiae
Mycoplasmas
Gram Positive Cocci
Endospore-forming Gram
Positive Rods and Cocci
Regular Nonsporing
Gram Positive Rods
Irregular Nonsporing
Gram Positive Rods
Actinomycetes
(Gram Positive Filamentous cells)
Key Characteristics of Members of the
Section
Gram Negative, helical cells, move by axial
filaments
move by flagella, either helical or comma
shaped cells
Some are pigmented or motile, some have
oxidase, some require special nutrients, and
some fix nitrogen. Except for the nitrogen
fixers, cell shape is distinct.
Nonmotile and nonspore forming aerobes.
Cell shape is difficult to classify, as cocci
are flattened and rods are very short, often
being pleomorphic.
Some have flagella, many ferment some
or all sugars
intracellular pathogens
(live within cells)
extremely small cells, lacking cell walls,
require special media
non-spore forming cells that grow in
aerobic to anaerobic conditions
some are motile, grow in aerobic to
anaerobic conditions
growth is facultative or strictly anaerobic,
nonmotile
club-shaped, pleomorphic, and filamentous
cells, grow in aerobic to anaerobic
Streptomycetes
(Gram Positive Aerobes)
Filamentous cells that branch and divide in
more than one plane, some reproduce by
reproductive spores
Filamentous cells that resemble fungal
hyphae, reproductive spores
Mycobacteria
(Gram Positive Rods)
Acid Fast staining, occasional filaments and
rare branching
Characteristics of your Unknown that
exclude it from the Section.
Excluded-unknown is not helical
Excluded-unknown is not helical or
comma shaped
Excluded-unknown was not completely
aerobic
Excluded-unknown is not nonmotile, the
cell shape is not difficult to classify, and
the rod shape is not particularly short
CANNOT EXCLUDE UNKNOWNunknown did ferment Dextrose,
Mannitol and Sucrose, no flagella
viewed but unknown was motile
Excluded-unknown grows extracellularily
Excluded-unknown did not require special
media-grows readily on nutrient agar, was
not relatively small, and no cell wall was
present
Excluded-unknown was not cocci or gram
positive
Excluded-unknown did not contain
endospores, and was not Gram positive
Excluded- unknown is not nonmotile and
unknown was not Gram positive
Excluded-unknown was not Gram positive,
no apparent pleomorphism, and there
wasn’t any filamentous growth
Excluded-unknown was not Gram positive,
was not filamentous and there was no
evidence of reproductive spores
Excluded-unknown was not Gram positive,
was not a complete aerobe, cells did not
resemble fungal hyphae and there was not
evidence of reproductive spores
Excluded-unknown was not Gram positive,
unknown was not filamentous or acid fast
T / R 8pm
Template for Identifying Unknown
Test (indentations are not in the chart of
unknowns) Fill in results as provided.
Results
Confirmation (y/n)
(if no, discuss why it is no)
Cell Shape (cocci, bacilli, etc.)
Bacilli
can wet mount confirm?
Y
Other cellular characteristics
(describe cell size, growth pattern, etc)
Gram Stain (Gram-/Gram+)
Colonial characteristics (describe
colony pigment/shape/margin/etc.)
Gelatin hydrolysis (+/-)
Motility test (+/-, if growth is it black?)
Litmus Milk (0-6, C and R)
Growth is in singlets
Gram (-)
can KOH test confirm?
Y
Pigment- burnished red pigment
Shape-circular
Margin-entire
Elevation-slightly raised
Size-Small
Texture-Smooth
Appearance-Dull
Optical property-Opaque
+
+
Y
can wet mount or broth culture confirm?
Y
(Broth culture indicated slightly and wet
mount confirmed)
C
One tube was solid and another
had coagulation at the bottom
indicating casein protein
coagulation
Glucose fermentation (-,A,*,K)
A
can another sugar confirm?
Y
Lactose (-,A,*,K)
_
can Mac/Litmus confirm?
Macokey: Y (growth but no pink appearance)
Litmus: Y (solution did not turn pink)
Sucrose (-,A,*,K)
A
can MSA confirm?
Mannitol (-,A,*,K)
Starch (+/-)
A
_
(growth but not fermentation)
N
(confirmation was not attainable because
this bacterial species is not salt tolerant and
did not grow. There was a negative result
for salt tolerance)
Bi 324 lab
T / R 8pm
Indole (+/-)
+
VP (+/-)
+
MR (+/-)
_
Hydrogen Sulfide (from TSI, +/-)
_
Simmons Citrate (–, G, or B)
B
Hemolysis (alpha, beta, or gamma)
can another test confirm?
Y
(the wet mount showed motility and the
gelatin agar yielded a positive result with
Kovac’s reagent)
gamma
Capsules (+/-)
_
Salt tolerance of MSA
(+/-, if +, does it have a yellow halo?)
_
Catalase (+i, +s, or -)
+i
Endospores (+/-, if + give position)
_
Can UV test confirm?
Y
Acid Fast Stain (+/-)
_
Can liquid culture confirm?
N/A
Oxygen requirements (a, fa, other)
Oxidase (+/-) (if time permits)
Crystal violet/bile tolerance of Mac
(+/-, if growth, is it pink?)
FA
_
(took longer than 30 sec)
+
Tetracycline sensitive
_
(15 mm)
Bacitracin sensitive
_
(1-5 mm)
Amoxicillin Sensitive
_
(10 mm)
Penicillin Sensitive
_
(1-5mm)
Can deep stab confirm?
Y