Lydia Jordache

Keiler

MICRB 203 sec 001

31 March 2018

Pet Lab Report: Bacteria #28

Description of Microbe

The bacteria formed small, shiny, yellow colonies with a domed texture, and appeared

rod shaped through microscopy. Under two separate trials of gram staining, the bacteria

appeared as Gram positive, due to its appearance of retaining the crystal violet stain that was

used, however it should be noted that in all of the Gram-identifying test performed, it acted as

Gram negative. Those tests included EMB and McConkey plates. On the EMB and McConkey

plates, the streaking was divided into fifths in order to support four control groups and my pet

microbe, bacteria #28. The control groups for both tests consisted of Bacillus subtilis,

Escherichia coli, Enterobacter aerogenes, and Pseudomonas fluorescens. If, after incubation,

the pet bacteria does not grow on either plate, it can be concluded as Gram positive. However,

if the bacteria grows on either the EMB or McConkey plate, then it is Gram negative. That

distinction makes B.subtilis the negative control for both tests, as it is Gram positive and not

expected to grow. After incubation, the pet bacteria grew on both plates and created a

noticeable color gradient of red/purple on the outside of the growth, and orange/yellow on the

inside on McConkey agar and pink on EMB. For the EMB plate, this experiment was performed

twice with the same results, leaving the appearance that this bacteria is actually Gram negative,

unlike the earlier Gram stain tests. Because Gram staining results can be more easily

misinterpreted than growth-or-no growth tests like EMB and McConkey, the bacteria is probably

Gram negative.
 The preferred growth medium for bacteria #28 is TSA, and grows best at 28 degrees

celsius, with an ideal salt concentration range of 0.85% to 3.5% NaCl. To determine the

preferred growth medium, the bacteria was streaked on a TSA plate and an NA plate, and

incubated at 28 degrees celsius. There was more growth on the TSA plate after incubation, and

therefore seemed to prefer that medium over NA. To determine optimal temperature settings,

five individual plates of the bacteria were prepared, with one incubated at 4 degrees celsius, the

second plate incubated at 28 degrees celsius, the third plate at 30 degrees celsius, the fourth

plate at 37 degrees celsius, and the fifth plate at 55 degrees celsius. If one of the plates

produced more growth than another after incubation, then it can be said that that is the optimal

temperature for this bacteria out of the temperatures tested. It was determined that the largest

amount of growth occurred on the plates that sat at 28 degrees celsius, due to the appearances

of the plates after incubation. The following were the results of each temperature:

Temperature 4 28 30 37 55
(°C):

Bacteria Some Growth Significant Some Growth Some Growth No Growth

Appearance: Growth

To determine the ideal level of salt for the survival of bacteria #28, and therefore how salty the

bacteria’s natural environment would be, four plates of varying salt concentrations were

streaked with the microbe and allowed to incubate at 28 degrees celsius. If one plate held more

bacterial growth than another, that salt concentration would be the preferred concentration of

the bacteria. These concentrations and their results were as follows:

 NaCl 0.85% 3.5% 7.5% 15%
Concentration:

Bacterial growth: Significant Significant Some growth No growth

growth growth
Due to the resulting appearances of the bacteria on each plate after incubation, it can be

concluded that the pet bacteria prefers a less salty environment, somewhere between or

including 0.85% to 3.5% NaCl concentration. In addition to the plates of varying salt content, the

salt tolerance of this bacteria was also tested in part by a mannitol salt agar test. In this test, the

mannitol salt agar used contained 7.5% NaCl, and growth determined the bacteria’s ability to

tolerate 7.5% salt concentration. The bacteria did grow some, though not significantly, meaning

that it does not seem to prefer 7.5% salt environments even if it can survive them.

This bacteria was able to grow anaerobically. This was determined by streaking a TSA

plate with the bacteria and placing the plate into an anaerobic chamber. If, after incubation, the

plate has successfully grown the bacteria, then it can be concluded that the microbe can survive

anaerobically. The plate tested did have growth.

Bacteria #28 can ferment glucose, lactose, and sucrose, but only produces gas as a

result of glucose fermentation, and not for the fermentation of lactose or sucrose. To establish

this, three durham tubes of liquid broth of this bacteria were prepared as a test group, all

containing Phenol red to determine organic acid-producing fermentation , and all containing a

small glass chamber to determine the production of gas as a result of the fermentation. One

tube incorporated glucose in its broth, one incorporated lactose, and the third contained

sucrose. As a control group, durham tubes of E.coli, B.subtilis, and Micrococcus luteus were

similarly prepared, three nutrient tests for each bacteria. E.coli is expected to strongly ferment

two nutrients (glucose and lactose), and used as a positive control for strong fermentation.
B.subtilis is expected to weakly ferment glucose and sucrose, and used as a negative control for

lactose. M.luteus is expected to ferment glucose and lactose weakly, and used as a negative

control for sucrose. If a bacteria ferments any of the three nutrients tested, then the broth will

turn yellow (from its original phenol-red color) after incubation, and if it produces gas a result, a

gas bubble will appear in the glass chamber in the tube. Weak fermentation creates an orange

color. This color change is due to the indicator sensitivity towards the organic acids, and

subsequent change in pH, that occurs from the fermentation. The control group bacteria acted

as expected:

Nutrient: Sucrose Lactose Glucose

Phenol Broth result: B.subtilis-red B.subtilis-red B.subtilis-orange

E.coli- red E.coli- yellow,gas E.coli- yellow,gas
M.luteus- red M.luteus- red/orange M.luteus- orange

and for the test bacteria, the results were as follows:

Nutrient Sucrose Lactose Glucose

Color of Broth/Gas Orange-yellow, no Yellow, no gas Yellow, gas present

Present gas

It can be concluded from these results that bacteria #28 can ferment glucose, lactose, and

(weakly) sucrose, but a gas-byproduct is only present for glucose fermentation. In the second

test for lactose fermentation, the results for the EMB agar test mentioned previously were

interpreted to show that this bacteria ferments lactose. In addition to the durham tube tests, the

EMB plate also supported the idea that this bacteria ferments lactose., as a result of bacteria

#28 having a very colorful appearance on the EMB plate, similar to E.coli, which is a known
lactose fermenter. If the colonies that grew appeared clear or weak, then it would not be

considered a lactose fermenter, but that was not the case.

While the pet ​can​ ferment glucose, sucrose, and lactose, it cannot ferment the sugar

mannitol. This was found to be true after a test on a mannitol salt agar plate. In this test, the

bacteria was streaked onto a mannitol salt agar plate and incubated at 28 degrees celsius. The

positive control was Staphylococcus auerus, which is expected to grow and turn the agar

yellow. The negative control was Staphylococcus epidermis, which should grow but leave the

agar in its original red color. If the bacteria ferments the mannitol in the agar, then the organic

acids produced from the fermentation will the turn the agar from red to yellow, like S.aureus. If

there is no color change, then the bacteria does not ferment mannitol. The result of this

experiment was no change in the color of the agar, and therefore this bacteria does not ferment

mannitol.

This microbe is motile. Two tests were performed in order to determine this, the first of

which was a bridge plate assay. This involved two plates, each composed to of two divided

sections, one side being nutrient agar and the other being non-nutritive agar (just water and

agar). Two strips of paper were placed on each plate in a manner that bridged the divider

between agars. On the first plate, my pet and the positive control, P.Fluorescens, were placed

on the side of the papers that touched the non-nutritive agar. On the second plate, my partner,

Erin W.’s, pet bacteria and the negative control, M.luteus, were also placed on the papers that

touched the non-nutritive agar. After incubation, if a bacteria on any of the paper strips moved

from the water agar to the nutrient agar side of the plate, that bacteria is considered motile.

Bacteria #28 moved across the paper bridge, and therefore appears to be motile. The positive

control moved across the paper bridge, as expected, and the negative control stayed on the

water agar side, as expected. The second motility test involved inoculating a tryptone-infused
semi-soft agar plate with the pet bacteria. If the bacteria grew over a wide expanse of the plate,

the bacteria can be considered not only motile but also chemotactic. If the bacteria grows, but

only in a tight, limited space around the inoculation site, then the bacteria is motile but not

chemotactic. If the bacteria does not grow away from the inoculation site, it is neither

chemotactic nor motile. From this test, my bacteria actually covered the entirety of the plate, and

therefore seems to be both chemotactic and motile.

The pet microbe appeared to be sensitive to the following antibiotics: Sulfamethoxazole-

Trimethoprim (SXT), Nalidixic Acid (NA), Ampicillin (AM), Tetracycline (TE), and Erythromycin

(E). The approach used for testing this antibiotic resistance involved two plates that used a disc

diffusion method. Both plates were coated in the liquid broth of bacteria #28. On one plate, three

small paper discs were placed, one was erythromycin, another was tetracycline, and the third

was a plain disc. On the second plate, a paper disc of SXT, a disc of ampicillin, and a disc of

nalidixic acid were placed. The plain disk served as a negative control. If, after incubation, the

bacterial growth was halted around any of the disks, resulting in a clear ring of agar around it,

then the bacteria is sensitive to the antibiotic present on that disc. However, if there is no

clearing of bacteria and the growth goes right up to the paper disc, then the bacteria is resistant

to that antibiotic. The pet bacteria had a ring of clearing around every antibiotic tested, though in

varying degrees. The results were as follows:

Antibiotic SXT NA AM TE E

Diameter of 3.2 3.7 1.7 3.0 2.1

Zone of
Clearing (cm)
Due to the fact that all antibiotic discs had a zone of clearing around them, it can be concluded

that this bacteria is not resistant to Sulfamethoxazole- Trimethoprim, Nalidixic Acid, Ampicillin,

Textracycline, or Erythromycin.

This bacteria also does not secrete the enzyme protease. In a test with skim milk agar,

two control groups were used. The positive control was B.subtilis, which produces a lot of

protease. The negative control was E.coli, which does not produce protease. If the bacteria

does produce protease, a ring of clearing should appear in the skim milk agar to show that the

milk proteins are being degraded. If it does not produce the enzyme, then there would be no

such zone of clearing. There was no clearing around the pet bacteria, and therefore does not

produce protease.

Classification of Pet Microbe

This microbe appears to be ​Pantoea ananatis, a species of bacteria that identifies with the

following:

[Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Pantoea

Species: Pantoea ananatis]1

This seems to be the correct identification of this pet microbe because P.ananatis can grow

both aerobically and anaerobically, is gram-negative, rod-shaped, and is susceptible to all antibiotics

that were tested in this lab report2. Pantoea bacteria are almost always motile3. Likewise, the colony

1
Mergaert, Et al. (1993). Pantoea ananatis. Retrieved March 31, 2018, from
https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=964578#null
2
​Baere, T. D., Verhelst, R., Labit, C., Verschraegen, G., Wauters, G., Claeys, G., & Vaneechoutte, M. (2004, September).
Bacteremic Infection with Pantoea ananatis. Retrieved March 31, 2018, from ​https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516322/
3
​https://onlinelibrary.wiley.com/doi/pdf/10.1002/9781118960608.gbm01157
morphology of P.ananatis appears nearly identical to bacteria #28, as yellow, round, and shiny

domes4:

The following students seem to have the same pet:

Eric Kraus and Adam Laudenslager

4
​Gegenhuber, K. (2013, July 29). First Report of Pantoea ananatis (Syn. Erwinia uredovora) Being Associated with Peanut Rust in
Georgia. Retrieved March 31, 2018, from ​https://www.plantmanagementnetwork.org/pub/php/brief/2013/peanut/