Microbiology 205
Major Unknown Report
Suzanne Ricca - Lab #22
Gram (+) Unknown #13 – Bacillus subtilis
Gram (-) Unknown #13 – Proteus mirabilis
Isolation and Identification of Gram (+) Organism #13 – Bacillus subtilis
4.16.2012 – Unknown organism was streaked from a mixed broth onto both TSA and BHA plates in an
attempt to isolate pure colonies for testing.
4.18.2012 – Although growth was best on the TSA plate, the Gram(+) organism was unable to be
isolated because of overgrowth of the Gram(-) organism. The plate showed areas of tiny pinpoint
colonies surrounded by cloudy areas, indicating the presence of the Gram(-) organism throughout the
plate. This was confirmed with gram staining of the pinpoint colonies which showed the presence of
both organisms. Another streak was done on a PEA plate in an attempt to inhibit the growth of the
Gram(-) organism enough to isolate the Gram(+).
4.23.2012 – One large circular colony grew on the PEA plate. Gram staining indicated the presence of
long pinkish rods with circular purple spots inside the cells. The presence of a faint smaller shape
throughout the slide was also noticed, and thought to be spores. Spore staining was inconclusive and
the PEA plate was returned to the incubator for further aging. Two TSA slants were inoculated to
produce working samples, as well as a motility deep and FTM broth.
4.25.2012 – Gram staining of the TSA working slant showed the same long pinkish rods with purple dots
as well as faint shapes in the background. The FTM showed a facultative anaerobe and the motility test
showed the organism to be motile. Another spore stain of the PEA plate remained inconclusive and the
plate was returned to the incubator. In the likelihood the organism was in fact a spore former and
therefore in the Bacillus genus, a mannitol broth was inoculated to begin to differentiate among the
Bacillus species.
4.30.2012 – A final spore stain was done on samples taken from the PEA plate as well as the working
slant. Although there was no change to the stain from the PEA plate, fortunately the sample from the
working slant produced the appearance of spores and confirmed the genus Bacillus. Unfortunately the
faint background shapes were not the actual spores but short rod-shaped cells and indicated the
continuing presence of the Gram(-) organism. Since the motility test and FTM test were done with the
organisms still mixed, they are not able to be used. The mannitol test (read on 4.26) appeared negative,
indicating neither organism is a mannitol fermenter, so that test is still good. A sample from the working
slant was streaked onto another PEA plate to try to further inhibit the Gram(-) growth and isolate the
Bacillus.
5.02.2012 - There was heavy growth throughout the new PEA plate with a few tiny pinpoint colonies but
they were not isolated form the rest of the growth. It did not appear as if the second organism was still
present however and this was confirmed by doing a gram stain from the growth at the end of the streak.
The long pinkish rods with purple spots inside the cells were still present, but no smaller rods were
present and the Bacillus seemed to finally be isolated. Two new slants were inoculated to produce new
working samples. Since the mannitol test was already done and negative, the species B. subtilis is
eliminated. A MRVP broth was inoculated to differentiate between B. megatarium (VP-), B. sphaericus
(VP-), B. cereus (VP+) and B. thuringiensis (VP+).
5.04.2012 – VP test was positive. To differentiate between B. cereus and B. thuringiensis a blood agar
plate was streaked to determine strength of hemolysis, since the species are similar in almost every
other way. They are both hemolytic but B. cereus is stronger. Samples of known B. cereus and B.
thuringiensis were also streaked on the plate to compare with the unknown.
5.07.2012 – The hemolysis result was not consistent enough with either known species to be definitive.
It looked most like B. thuringiensis but the unknown growth had raised wrinkled areas and appeared
unique enough to be a separate species. A problem with the mannitol test is suspected and another
mannitol broth was inoculated to retest for B. subtilis.
5.09.2012 – The mannitol test was not a strong positive, but still clearly positive. The broth turned
yellow-orange at the top although remained red toward the bottom and there were a few bubbles in
the tube. Most of the growth was at the top indicating the possibility of an aerobe rather than
facultative anaerobe. Since the VP test is already done and positive, Bacillus subtilis is confirmed.
Mannitol retest (+)
VP (+)
Hemolysis: Unknown in Middle
Purpose
Result
Determine morphology and
arrangement
Differentiate gram positive bacilli
between spore formers and nonspore formers
Determine oxygen requirements
and differentiate Bacillus species
Gram positive, long pinkish rods
(bacilli) with purple dots
Spore former, indicating Bacillus,
also showed gram negative
organism still present
Facultative anaerobe (growth
throughout), may not be
accurate due to gram negative
Motile, consistent with all
possible Bacillus species
Gram positive isolated,
confirmed with gram srain
Negative (red) indicates B.
cereus, B. megatarium, B.
sphaericus or B. thuringiensis
VP positive (red) indicates B.
cereus or B. thuringiensis
Media Table
Test
Gram stain
Spore stain
FTM broth
Motility Deep
Mannitol broth
(Mannitol Fermentation)
Determine motility and
differentiate Bacillus species
Inhibit gram negative growth
and isolate gram positive
Differentiate Bacillus between
fermenters and non-fermenters
MRVP
(Acetoin production)
Use VP test to differentiate
mannitol non-fermenters
PEA Plate
Blood Agar Hemolysis
Streak unknown between given
samples of B. cereus and B.
thuringiensis to compare
strength of hemolysis
Mannitol broth
(Mannitol fermentation)
Retest mannitol fermentation to
differentiate VP positive species
B. subtilis and B. megatarium
Growth inconsistent with either
species as unknown exhibited
unique contoured wrinkles,
contamination of mannitol test
suspected
Weak Positive (yellow-orange
with some red remaining),
confirms Bacillus subtilis
Habitat & Lifestyle Bacillus subtilis
Generally considered a ubiquitous organism, B. subtilis is found diversely in the soil, plant roots and
even the GI tract of animals. It is gram positive and motile by peritrichous flagella. Since it is a sporeformer it is able to assume a vegetative state under harsh conditions such as temperature variations and
lack of nutrients, and is most often found in this state. It can appear to be an obligate aerobe but more
recently it has been observed to grow anaerobically in the presence of nitrate, making it a facultative
anaerobe. This supports the idea that B. subtilis can actually germinate within the GI tract of animals,
where it has a beneficial probiotic effect that was previously attributed to an unknown property of the
spore.
Special Characteristics of Bacillus subtilis
When biologically active, B. subtilis also produces a variety of enzymes which make it an important
contributor to nutrient cycling in the soil. It is a promoter of plant growth as well, because it grows in
close association with plant roots where it forms beneficial biofilms. It seems B. subtilis has a mutually
beneficial symbiotic relationship with the plant rhizosphere; the presence of the microbe prevents the
plant from being colonized by other bacteria that would be harmful to it, and the microbe is able to have
a place to grow its biofilm and access nutrients.
Clinical Significance of Bacillus subtilis
Although it can be associated with food contamination and occasionally food poisoning, B. subtilis is
otherwise non-pathogenic to humans. It exhibits minimal if any virulence, confirmed by genome
sequencing of a strain which showed no genes encoding for virulence factors. It is generally not an
animal or plant pathogen either, but is actually hazardous to other microorganisms. Genome
sequencing has also revealed B. subtilis has a large portion of its genes dedicated to producing the many
compounds which inhibit other bacteria and fungi. This feature is what probably enables it to compete
in nature and allows it to promote plant growth and act as a probiotic. B. subtilis is used to produce
many antibiotics including the commonly known bacitracin, and its enzymes are used for industrial
purposes such as protease for detergents. It is also used to produce antifungal agents for plants in
agricultural settings.
Bacillus subtilis Identification Flow Chart
Gram (+)
Cocci
Bacilli
Spore
Former
Non-Spore
Former
Bacillus
Mannitol Fermentation (-)
B. sphaericus, B. cereus, B.
thuringiensis,
B. megatarium (V)
VP (+)
B. cereus
B. thuringiensis
Hemolytic
Activity
VP (-)
B. megatarium
B. sphaericus
Mannitol
Fermentation (+)
B. subtilus
B. megatarium (V)
VP (+)
B. subtilis
Bacillus subtilis
VP (-)
B. megatarium
Isolation and Identification of Gram (-) Organism #13 – Proteus mirabilis
4.16.2012 – Unknown organism was streaked from a mixed broth onto both TSA and BHA plates in an
attempt to isolate pure colonies for testing.
4.18.2012 – Although growth was best on the TSA plate, the organism was not isolated into pure
colonies because of its swarming growth throughout the entire plate. It grew over and around the
colonies of the other organism and appeared as a cloudy area where the other organism did not grow. A
sample was taken from the cloudy area, gram stained, and determined to be mostly short and some
longer red rods, indicating isolation of the Gram(-) organism. Two slants were inoculated to produce
working samples as well as inoculation of a motility deep and incubated.
4.23.2012 – The motility test was positive and an FTM broth was inoculated to determine respiration.
4.25.2012 – The FTM test indicated a facultative anaerobe. Combining this result with the motility test
determines the organism is an enteric. A lactose broth was inoculated to differentiate between the
lactose fermenting and non-lactose fermenting genera. Chromobacterium is already eliminated as a
possibility because the organism did not have the characteristic violet pigment on the growth media. A
Simmon’s citrate slant was also inoculated to further differentiate, as the only genus with citrase
variability is Proteus.
4.30.2012 – The lactose test (read on 4.26) was negative indicating a non-lactose fermenter and
eliminating Escherichia, Enterobacter and Citrobacter. The citrate test was positive indicating the
organism produces the citrase enzyme to use citrate as its carbon source. This eliminates Morganella
and Edwardsiella and narrows the potential genus to Salmonella, Serratia or Proteus. An MRVP broth
was inoculated to differentiate Serratia (VP+) from Salmonella (VP-) and Proteus (VP-, although one
species can be variable). A urea broth was inoculated to further differentiate Salmonella (urease-) and
Proteus (urease+, although one species can be variable).
5.02.2012 – As suspected the organism was VP(-) and urease(+), confirming Proteus as the genus. A
tryptone broth to test for indole production and an Orthinine decarboxylase broth were both inoculated
to differentiate between P. vulgaris and P. mirabilis.
5.04.2012 - Ornithine decarboxylase test was positive and indole test was negative, both consistent for
Proteus mirabilis.
Left: Urease (+), Right: VP (-)
Left: Tryptone (-), Right: Ornithine (+)
Media Table
Test
Gram stain
FTM broth
Motility Deep
Lactose broth
Simmon’s Citrate
(Citrase production)
MRVP
Urease
Ornithine Decarboxylase
Tryptone
Purpose
Determine morphology and
arrangement
Determine oxygen requirements
to differentiate bacilli between
aerobes and facultative
anaerobes
Differentiate facultative
anaerobes between motile and
non-motile
Determine lactose fermentation
to differentiate enterics between
fermenters and non-fermenters
Determine ability to use citrate
as carbon source to differentiate
non-lactose fermenting enterics
Use VP test to determine acetoin
production and differentiate
between Salmonella, Serratia
and Proteus
Determine ability to metabolize
urea and differentiate between
Salmonella and Proteus
Determine ability to
decarboxylate ornithine to
putrescine and differentiate
between P. mirabilis and P.
vulgaris
Determine ability to produce
indole and confirm
differentiation between P.
mirabilis and P. vulgaris
Result
Gram negative, short red rods
(bacilli)
Facultative anaerobe (growth
throughout)
Motile (growth spread
throughout) indicates organism
is an enteric
Negative (red) indicates nonlactose fermenter
Positive (blue) indicates
Salmonella, Serratia or Proteus
VP negative (amber/green)
indicates Salmonella or Proteus
Positive (bright pink) indicates
Proteus mirabilis or vulgaris
Positive (purple) indicates P.
mirabilis
Negative (no color change)
confirms Proteus mirabilis
Habitat & Lifestyle of Proteus mirabilis
As a member of the Enterobacteriaceae Family, P. mirabilis is a gram-negative facultative anaerobe and
motile by peritrichous flagella which contributes to its swarming ability. It is among the normal flora of
the human intestine and also found in the urinary tract. In nature it can be found freely living in water
and moist soil. P. mirabilis survives best in an alkaline environment, with a pH between 8-9.
Special Characteristics of Proteus mirabilis
A unique feature of P. mirabilis is the ability to differentiate its morphology between swimmer cells and
swarmer cells. Swimmer cells, very small flagellated rods, are found in liquid suspension. Conversion to
swarmer cells, in which the cell becomes elongated with highly flagellated filaments, occurs on sold
surfaces and the cells line up and move in raft formation. On growth media such as agar, P. mirabilis has
the ability to produce geometric shapes as it grows, called concentric patterns. This is caused by
repeating swarming-plus-consolidation cycles in which the organism switches between its swimmer and
swarmer state, forming circular terraces. The swarming ability is important in the organism’s virulence.
P. mirabilis raft formation
P. mirabilis concentric growth on agar
Clinical Significance of Proteus mirabilis
In a normally functioning urinary tract, P. mirabilis is of little risk and is flushed out regularly before it
colonizes. It can become pathogenic and is the cause of complicated urinary tract infections in cases of
structural abnormalities of the urinary tract, in immune-compromised individuals, or most commonly in
cases of long-term catheterization. P. mirabilis is able to adhere to the catheter and form a biofilm. Due
to its motility it can travel up the urethra to the bladder. It also possesses various kinds of fimbriae
which enhances its ability to adhere to the host tissue. Once established the ability to produce urease
further enhances virulence, allowing P. mirabilis to efficiently metabolize urea into ammonia and carbon
dioxide. This increases pH of the area to the microbe’s preferred alkaline level, as well as causes bladder
and kidney stones, which it is also able to colonize for use as further protection from host defenses and
antimicrobial drugs. In addition, P. mirabilis can evade host defenses by producing protease which
degrades the IgA released by the host, as well as releasing its endotoxin hemolysin which is cytotoxic to
the host epithelial cells.
Although other Proteus species have become resistant to many antibiotics, P. mirabilis remains
susceptible to most except tetracycline. Infections caused by P. mirabilis can most likely be treated with
ampicillin and other broad spectrum penicillins, cephalosporins, imipenem, and aztreonam, but is still
difficult to clear with antibiotics alone. This is due to the stone formation which can become reservoirs
for the infection to reoccur, eventually travelling to the kidneys and causing further complications
including pyelonephritis and renal damage. Even more serious, the microbe can cause bacteremia by
invading the bloodstream where its endotoxin can induce sepsis, resulting in Systemic Inflammatory
Response Syndrome (SIRS) which can be fatal. Research is also currently in process to develop a vaccine
involving the MR/P (mannose-resistant Proteus-like) fimbriae, one of at least four types of known
fimbriae produced by P. mirabilis and known to function as a surface antigen. The vaccine would not
only have the potential to prevent complicated urinary tract infections from Proteus in high-risk
individuals but from other microbes as well which cause similar conditions.
Proteus mirabilis Identification Flow Chart
Gram (-)
Cocci
Bacilli
Facultative
Anaerobe
Aerobe
Motile
Lactose
Fermentation (+)
Lactose
Fermentation (-)
Escherichia,
Enterobacter,
Citrobacter
Proteus, Morganella,
Salmonella,
Edwardsiella, Serratia
Citrate (-)
Morganella
Edwardsiella
Citrate (+)
Salmonella,
Serratia, Proteus
VP (+)
VP (-)
Serratia
Salmonella, Proteus
Urease (+)
Proteus
Indole (+), Orthinine (-)
P. vulgaris
Urease (-)
Salmonella
Indole (-), Orthinine (+)
P. mirabilis
Proteus
mirabilis
Works Cited
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