Haley Janowitz
Microbiology 202
Dr. Ken Keiler
April 19, 2015
What’s My Pet? : Analyzing and Defining the Characteristics of an Unknown Microbe
Description and Results from Various Tests:
When analyzing the morphology of my pet’s colonies I found that it grew in
milky white, perfectly round, smooth, almost translucent colonies. After analyzing the
Gram stain I could see extremely tiny, round cells that were purple in color and stuck
together in pairs usually.
Figure 1: Originally plate streaked with my pet
Figure 2: Gram-stain of my pet at 100x magnification
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From the Gram-stain I could conclude that my pet was Gram-positive, but I
conducted a few other tests to confirm this conclusion using McConkey and EMB agar
plates. These plates selective for Gram-negative bacteria because only microbes with a
cell wall can survive in the presence of the bile salts and crystal violet in McConkey Agar
and eosine and methylene blue dyes in EMB Agar. (1) They are also differential
mediums meaning that they can show the ability of the microbe to ferment lactose. On
McConkey agar a fermenter will grow hot pink, while a non-fermenter will grow white,
possibly light pink. On EMB agar a fermenter will grow shiny green and opaque, while a
non-fermenter will be uncolored and translucent. (1)
Table 1: Results of test for Gram-negative bacteria
McConkey Agar
No growth
Hot pink and
opaque
Negative control
No growth
B. subtilis
*This test was repeated twice just for precision
Pet
E. coli
experiment
Positive control
EMB Agar
No growth
Shiny green and
opaque
No growth
Considering my pet did not grow on either McConkey or EMB agar, as well as
the fact that the Gram stain showed it to be purple, I can conclude that my pet is in fact a
Gram-positive species.
An assay was completed to determine whether my pet could grow on salt
concentrations of 7.5% as well as its ability to ferment Mannitol. A Mannitol salt agar
was used that was selective for growth on salt and differential for identifying fermenters
of Mannitol. A fermenter would lower the pH by releasing organic acids turning the
phenol red in the agar yellow. A non-fermenter would not change the pH leaving the
surrounding area red. (1)
Table 2: Data ffrom Mannitol Salt Agar assay
Pet
S. epidermidis
S. aureus (only looked at;
did not actually test)
E. coli
Experiment
Positive control/nonfermenter
Positive control/Fermenter
Negative control
No growth
Thin, filmy white growth;
red agar
Creamy white growth;
bright yellow agar
No growth
It seems from this assay that my pet could not tolerate the salt conditions of 7.5%
so it is inconclusive on whether it can ferment mannitol sugar or not. In order to further
test the inability to grow on salt, I tested my pet on agar plates of salt concentrations
0.85%, 3.5%, 7.5%, and 15%.
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Table 3: Results from salt concentration assay
Salt Concentration
NA (no salt)
Growth
0.85%
Control for growth without
salt
Control for growth without
salt
Experiment
3.5%
Experiment
7.5%
Experiment
15%
Experiment
T-soy (no salt)
Normal growth; colonies similar
sized to original plate
Normal growth; colonies slightly
larger than on NA
Less growth than T-soy and NA;
smaller colonies
Less growth than 0.85%; very
small colonies
No growth except where streaked
on too thick
No growth
This test revealed that my pet could not tolerate high salt conditions, which is
likely the reason the mannitol salt plates had no growth. Also my pet is not really very
picky as far as whether it grows better on Nutrient Agar (NA) or Tryptic-Soy Agar (Tsoy), but just because the colonies are bigger on the T-soy that is the one with which I
continued to streak.
In order to determine which sugars my pet could ferment, as well as whether it
has the ability to grow anaerobically, a few assays were run including Phenol red tubes,
Oxidation/Fermentation Agar, and streaking a plate for growth in an anaerobic chamber.
The Phenol red tubes consisted of either glucose, sucrose, or lactose media with little
tubes upside down in the liquid. Each solution had phenol red indicator in it. If the
bacteria fermented that particular sugar they would release organic acids that turn the
solution yellow, or orange if they slightly ferment it. They could also ferment it and
release gas, which would be collected in the upside down tubes. If they were nonfermenters, the solution would remain red. The Oxidation/Fermentation tubes test for the
bacteria’s ability to ferment and/or oxidize glucose, as well as grow anaerobically. They
have bromothymol blue as an indicator so they start green, but when the bacteria process
glucose the tubes turn yellow in the presence of organic acids. Also one tube has a layer
of mineral oil on top meaning that if the bacteria grow in that tube they can survive
without oxygen. (2)
Table 4: Data from Phenol Red tube assays
Lactose
Pet
Some Acid
1 bubble of gas
Orange
E. coli
B. subtilis
M. lateus
Acid
Gas
Yellow
No acid
No gas
Red
No acid
No gas
Red
Sucrose
Acid
No gas
Yellow
No acid
No gas
Red
Some acid
No gas
Orange
No acid
No gas
Red
Glucose
Acid
No gas
Yellow
Acid
Gas
Yellow
Acid
No gas
Yellow
No acid
No gas
Red/Orange
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Table 5: Results from Oxidation/Fermentation tube assay
Presence of Acid
Pet
No color change, but it
grew in both tubes
E. coli
P. fluorescens
Yellow throughout
Yellow top only
Anaerobic/Aerobic
Both
Motility
No
Both
Aerobic only
Yes
Yes
Table 6: Results for anaerobic growth chamber
Growth; smaller colonies
Growth
No growth
Pet
E. coli
P. fluorescens
My pet is a strong fermenter of sucrose and glucose and a moderate to weak
fermenter of lactose. It also was able to grow in both Oxidation/Fermentation tubes, even
though it neither fermented nor oxidized glucose, and it grew in the anaerobic chamber,
although the colonies were smaller. This most likely means that my pet is a facultative
anaerobe, which allows it to switch its metabolism between aerobic and anaerobic
respiration. (3) The Oxidation/ Fermentation tubes also test another characteristic of
microbes known as motility. The agar was semi-solid meaning that if the bacteria are
motile then they can disperse and/or swim to the top near the oxygen if that is their
preferred/necessary method of respiration. My pet did not seem to be motile at all;
however, another two assay was conducted to determine motility.
Both a double well plate (one side water agar, the other side GYE agar) with filter
strips bridging the wells, and semi-solid agar plates were used to test motility. The
microbe was either inoculated on the water agar side of the double well plate or in the
center of the semi-solid agar. If it grew on the nutrient side (GYE) or in a large ring
around the point of inoculation, then the microbe is motile.
Table 7: Data on bridge plate and semi-solid agar assays
Bridge Plates
Pet
Experiment
Growth on water agar
P. fluorescens
Positive control
Growth on GYE agar
M. luteus
Negative control
Growth on water agar
Semi-Solid Agar
No movement from
point of inoculation
Large ring of growth
away from center
No movement from
point of inoculation
*Repeated the bridge plate assay with my pet because the first time it didn’t grow on
either side
Since my pet only grew on the water agar plate and at the point of inoculation, it
is a most likely a non-motile species of bacteria.
A few other tests were conducted to understand other qualities of my pet. A plate
was streaked and incubated at 50OC producing much tinier colonies that were closer
together. Its optimal growing conditions seemed to be a 28OC on T-soy agar. Another test
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was to see if my pet would grow and/or infect a pepper and an onion. After streaking
these vegetables with the microbe, they appeared dried out, but not particularly “sick.”
When I streaked from the pepper onto another T-soy plate I got colonies with the same
morphology and color as my pet so I concluded that it can grow on vegetables, but for the
short amount of time that it existed on them it isn’t pathogenic. It is possible that the
vegetables could have developed a disease later, but initially the vegetables were not
harmed. A dilution was also done on a liquid culture of my pet to see how many colony
forming units per milliliter there are. Using the seventh dilution, I calculated 1.9 x 109
CFU/mL.
The last set of assays conducted was to understand what bacterial resistance my
pet has. Because my pet is Gram-positive I tested a variety of antibiotics including ones
that inhibit peptidoglycan synthesis, protein synthesis, and enzymatic pathways. If the
bacteria are not resistant it will form a large ring of clearing around the antibiotic disc. If
it they are resistant then they will grow closer, or ever right up to the disc.
Table 8: Data for Antibiotic Resistance assay
Antibiotic/Treatment
Plain (negative control)
Penicillin
Cephalothin
Ampicillin
Tetracycline
Minocycline
Doxycycline
Erythromycin
Nalidixic Acid
Sulfamethoxazole Trimethoprim
Cloramphenicol
Clindamycin
Radius of Clearing
0cm
0.75cm (outer)/ 0.47cm (inner)
0.40cm
0.80cm (outer)/ 0.49cm (inner)
0.75cm
1.60cm
1.32cm
1.38cm (outer)/ 0.45 (inner)
0.98cm
1.20cm
1.05cm
0.88cm
My pet was most susceptible to the inhibitors of protein synthesis, specifically
Minocycline, because they had rings with the largest radii. The inhibitors of
peptidoglycan synthesis were the most resisted antibiotics by my pet, especially
Cephalothin. They even had double rings; the inner ring clear and the outer ring more
turbid, meaning that some bacteria were more resistant than others. This is possibly
because the bacteria don’t have a cell wall so it is easier for the protein synthesis
inhibitors to get into the cell and disrupt normal functions.
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Classification:
Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Streptococcaceae
Genus: Lactococcus
Species: lactis (4)
L. lactis is classified as a Gram-positive bacterium that typically is used in the
production of cheese and other dairy products. (4) It is a non-motile cocci that has
colonies with a medium sized, white, translucent morphology. (5) L. lactis is capable of
growing anaerobically, although it mostly grows aerobically, and it is able to ferment
glucose, sucrose, and lactose. (4) L. lactis can also ferment mannitol sugar, but it does not
grow as quickly. (4) Most strains show variable resistance to antibiotics, but for the most
part are susceptible equally susceptible. (5) The optimum growth range is between 20 and
35oC, and growth is typically inhibited beyond 39oC with an upper limit of 53oC in some
species. (6) Using this information, I was able to identify my pet as L. lactis. I was able to
rule out all the Gram-negative bacteria on the list (E. coli, X. campestris, P. ananatis, and
P. fluorescens) as well as K. rosea because of the obvious colony morphology and color.
I also ruled out B. subtilis and M. luteus based on the observation that my pet is has cocci
cell morphology and it can ferment a variety of sugars. (7) That left just S. epidermidis
and L. lactis. My pet failed to grow on Mannitol salt agar as well as a variety of salt
concentrations, where growth is a key characteristic for several varieties of
Staphylococcus. Despite the fact that my pet only marginally fermented lactose, a key
characteristic of L. lactis, I determined that it must be L. lactis. The selectivity of salt is a
much more reliable test than the Phenol tubes because other conditions can effect the pH
of the medium and therefore the color of the phenol indicator. Aside from these
characteristics, my pet matched the colony and cell morphology descriptions, the lack of
motility, optimum growth conditions, and the classification as a facultative anaerobe of L.
lactis bacterium. My pet was not highly resistant to any one antibiotic, and any
differences may not be statistically significant. L. lactis is also capable of growing on
plants, but it is non-pathogenic, which seemed to be the same as my pet. (4) Due to the
acute similarities of the characteristics of my pet, and the characteristic of L. lactis, I can
confidently classify my pet to be Lactococcus lactis.
Others who have L. lactis:
Nobody else in the class has identified as having L. lactis as their pet.
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Works Cited
1. Microbiology 202. (2015). Pet Microbe 1: Lab Guide. In Piazza. Retrieved from
https://s3.amazonaws.com/piazzaresources/i49z7te1tfc1zq/i58cvrh46113pd/PetMicrobeGuide.pdf?AWSAccessKey
Id=AKIAJKOQYKAYOBKKVTKQ&Expires=1429485446&Signature=CTIb5h
SXvK8ys7vQMgJMz8TA%2B%2FI%3D
2. Microbiology 202. (2015). Pet Investigations. In Piazza. Retrieved from
https://s3.amazonaws.com/piazzaresources/i49z7te1tfc1zq/i6fpwsfxlq2lt/PetInvestigations2.pdf?AWSAccessKeyId
=AKIAJKOQYKAYOBKKVTKQ&Expires=1429491442&Signature=EFUIMN
LchFBc%2FYqvczJ8gmp1FRY%3D
3. "facultative anaerobes." A Dictionary of Biology. 2004. Retrieved April 19, 2015
from Encyclopedia.com: http://www.encyclopedia.com/doc/1O6facultativeanaerobes.html
4. Lactococcus lactis. (2012, January 13). Retrieved April 21, 2015, from
http://microbewiki.kenyon.edu/index.php/Lactococcus_lactis
5. Elliot, J., & Facklam, R. (1996). Antimicrobial Susceptibilities of Lactococcus
lactis and Lactococcus garvieae and a Proposed Method To Discriminate between
Them. Journal of Clinical Microbiology, 34(5), 1296–1298-1296–1298.
Retrieved April 20, 2015, from http://jcm.asm.org/content/34/5/1296.full.pdf html
6. Toqeer Ahmed, Rashida Kanwal and Najma Ayub. (2006). Influence of
Temperature on Growth Pattern of Lactococcus lactis, Streptococcus cremoris
and Lactobacillus acidophilus Isolated from Camel Milk. Biotechnology, 5: 481488.
7. Bacillus subtilis. (2013, March 5). Retrieved April 21, 2015, from
https://microbewiki.kenyon.edu/index.php/Bacillus_subtilis