Pet Microbe Final Report

Emily Cribas
Introduction An unidentified microbe was assigned to each individual and several experiments were designed to determine the overall classification of the bacteria.

Figure 1. Original Assigned Microbe on TSA Medium

Treatment and Controls
Preferred Medium and Gram Stain The purpose of this first lab was to determine
what the morphology of the microbe was as well as what the optimal growth
medium was: NA (Nutrient Agar) or TSA (Tripticase Soy Agar). The bacteria
was plated onto each medium using the pure culture, three-streak, and gram staining methods, where a purple color indicates a gram-positive bacteria, while pink
indicates the presence of gram-negative bacteria.
Salt Effects and Lactose Fermentation The purpose of this experiment was to
determine optimal salt concentrations of the microbe, as well as whether the microbe is a weak, strong, or non-fermenter of lactose. The microbe was streaked
onto plates with salt concentrations of 0, 3.5, and 7.5%. As for determining fermentation ability, MacConkey agar (supports growth of gram-negative bacteria)
was used and the plate was divided into 5 parts: my pet, E. coli (a known strong
lactose fermenter), P. fluorescens (negative control, non fermenter), B. subtilis
(gram positive bacteria), and E. aerogenes (known weak fermenter).
Protease Secretion and Temperature Effects This experiment will determine whether
my microbe can secrete proteases by comparing it to two other species: E. coli,
a negative control, and B. subtilis on a skim milk agar. If the bacteria secretes
protease, it should show clearing of casein around the bacteria, and if not, it should
1

show no clearing of the agar. Separately, to determine optimal temperature conditions, the microbe was also plated onto 4, 37, and 50 plates.
Motility and Chemotaxis Ability This design will determine whether my pet is
mobile and/or chemotactic. For the first motility experiment, P. fluorescens was
used as a positive, mobile control, and M. luteus was the negative non-motile
control. These bacteria along with my pet were placed on a sterile strip, where
half of it was placed over water agar, and the other half was placed over GYE agar.
Motile bacteria are expected to move towards the GYE agar, while nonmotile
bacteria are expected to stay stationary. For the chemotaxis experiment, I used
a semi-solid tryptone agar plate, and depending on whether it formed large rings
will determine whether it is motile and chemotactic or motile and non-chemotactic.
This agar was compared with the non-motile, non-chemotactic M. luteus.
Sugar Fermentation and Aerobic Growth Ability This experiment tested glucose,
lactose, and sucrose fermentation abilities of my microbe, as well as whether it is
aerobic and/or anaerobic. Specifically, the phenol red broth tested for fermentation
of these sugars, as well as if the end products of fermentation (if it does ferment)
are organic acids or gases. If acids are produced, there will be a pH change that
is accompanied by a change in color ranging from yellow as acidic (<6.3) to red
as basic (>8.3). Gas production is detected through gas collection tubes inverted
into the bigger tubes.
Lactose

Sucrose

Glucose

Acid Gas Acid Gas

Acid
Gas
Yes
No
Yes
E. coli
Yes
No
Yes
Yellow
Red
Yellow
No
+/Yes
B. subtilis
No
No
No
Red
Orange
Yellow
No
No
No
M. luteus
No
No
No
Red
Red
Red/Orange
Table 1. Phenol Red Broth Controls
The oxidation/fermentation (O/F) tubes tested for: metabolism of glucose to
produce acids oxidatively or by fermentation, as well as motility and anaerobic
growth. These tubes are also pH indicators (blue is basic, green is neutral, and
yellow is acidic). Finally, the microbe was placed in an anaerobic chamber.

Acid

Anaerobic/
Motility
Aerobic

E. coli

Yes
Both
Yellow
P. fluorescens
Yellow
Aerobic
Oxidative
Table 2. O/F Tube Controls

Yes
Yes

Antibiotic Resistance My microbe was grown in liquid BHIB culture for a week.
A cotton swab was used to extract the liquid culture and spread it evenly on
3 plates which contained 4 equidistant antibiotic disks each, with only 3 on the
third plate. This tested for antibiotic resistance, and a small or no clearing area
indicates resistance, because the bacterial growth was not affected or deterred by
the antibiotic.
Results
Preferred Medium and Gram Staining [ht]
TSA

NA

Thick, creamy texture

Lighter color
A lot of growth
Less growth than TSA
Yellowish white colonies
Easily identifiable
Lumped colonies
individual colonies
Table 3. Bacterial Growth Observations

Figure 2. Gram Stain and Morphology

Salt Effects and Lactose Fermentation The microbe grew equally well under 0 and
3.5% salt concentrations, but showed no growth under salt concentrations at or
above 7.5%. In Figure 3, E. coli appeared pink, as did E. aueogenes, while B.
subtilis, showed no growth as expected, and P. fluorescens and my pet both had
a creamy yellow color.

Figure 3. MacConkey Agar with 5 Samples

Protease Secretion and Temperature Effects In Figure 4, E. coli and my pet do

not have a clearing area, while B. subtilis has a very pronounced clearing area.

Figure 4. Protease Secretion on Skim Milk Agar

On the temperature plates (Figure 5), the 4 plate minimal growth, as did 50 ,
while the 37 plate showed the most growth.

Figure 5. Temperature Effects

Chemotaxis and Motility Figure 6 shows clear growth and movement of my microbe from the water to the GYE agar. On the left side, the bacteria moved from
the water to the GYE agar, while, on the right, it grew on the GYE agar and did
not move to the water side.

Figure 6. Motility Experiment

Figure 7 shows the entire plate covered in bacteria from my pet, while the initial
deposit of M. luteus is visible under the handwriting.

Figure 7. Chemotaxis Experiment

Sugar Fermentation and Aerobic Growth For the red phenol broth experiment,
the microbe turned yellow in glucose, and stayed red for sucrose and lactose. As
for the O/F tubes, my pet turned yellow without mineral water and had no color
change with mineral water. Finally, there was no growth in the anaerobic chamber.

Figure 8. O/F Tube without Mineral Water

Antibiotic Resistance The first plate contained: Penicillin (P), Doxycycline (D),
Ampicillin (Am), and Cephalothin (CF). Three of these plates were noticeably
covered in bacteria with no clearing area, while the area around the Doxycycline
contained a clearing space with a diameter of 1.8 cm. The second plate was completely covered in bacteria and contained: Chloramphenicol (C), Tetracyclin (TE),
Monocyclin (MI), and Clinadomycin (CC). Finally, the third plate showed absolutely no bacterial growth and contained: Nalidixic Acid (NA), Sulfamethaxozoic
Trimethoprin (SXT), and Erythrorycin (E).

Figure 9. First Plate with Antibiotic Disks

Individual Experimental Conclusions
Preferred Medium and Gram Stain Based on the plating results, my microbe
clearly grew best on the TSA medium. Notably, the streaks could have been
diffused more clearly to show individual colonies on both the TSA and NA plates.
From the gram staining, it can be deduced that the microbe is also gram-negative
and rod-shaped.
Salt Effects and Lactose Fermentation My microbe grows well with no salt, and
cannot ferment lactose because it is most similar in appearance to P. fluorescens,
a known non-lactose fermenter. The results also support the conclusion from the
gram stain: my microbe is indeed a gram-negative bacteria.
Protease Secretion and Temperature Effects Similar to B. subtilis, my pet microbe showed no clearing of the agar, indicating that it does not secrete proteases.
According to the temperature experiment, it cannot survive under extreme conditions, but can tolerate a higher temperature (37 ) decently.
Chemotaxis and Motility My microbe did grow and move on the GYE/water plate,
indicating motility, and covered the entire semi-solid agar plate with rings, indicating that it is chemotaxic as well.
Sugar Fermentation and Aerobic Growth My bacteria can ferment and oxidize
glucose, but cannot ferment sucrose or lactose, does not produce gas, and is strictly
aerobic.
Antibiotic Resistance My bacteria is resistant against P, CF, Am, NA, E, SXT,
but is not resistant against D, TE, CC, MI, C.

Overall Conclusions and Classification Based on the results from each experiment, I have concluded that my microbe is Pseudomonas fluorescens. My microbe
is a rod-shaped gram-negative bacteria, that can oxidize glucose, is chemotactic
and motile, cannot ferment lactose, grows optimally at room temperature with no
salt, does not secrete protease, is fairly resistant, and exhibits tan-colored colonies:
all characteristics of P. fluorescens. Two other classmates that have the same bacteria are: (1) Nhi Hoang and (2) Rebecca Beacham. The classification of my
microbe is as follows:
Domain: Bacteria
Phylum: Proteobacteria
Class: Gamma Proteobacteria
Order: Pseudomonadales
Family: Pseudomonadaceae
Genus: Pseudomonas
Species: P. fluorescens
References Pseudomonas Fluorescens. MicrobeWiki. Web. 20 Apr. 2015.

The genome portal of the Department of Energy Joint Genome Institute:2014 updates.
Nordberg H, Cantor M, Dusheyko S, Hua S, Poliakov A, Shabalov I, Smirnova T,
Grigoriev IV, Dubchak I. Nucleic Acids Res. 2014,42(1):D26-31.