Identification of Bacteria from Student I.D. Cards
Ashley Epping & Samantha Arnold
Microbiology
Dr. Stephen Baron
October 20, 2015
Purpose: In this experiment, various tests and protocols were utilized in order to isolate
unknown bacteria from student identification cards as well as identify the isolates according to
its particular genus after conducting various tests in the laboratory setting.
Introduction:
Many college students take their student ID card to various places, because it is often
needed to access most of the buildings as well as to receive meals throughout the day. It is very
likely that these cards will come into contact with a wide variety of bacteria on a daily basis.
Students tend to leave their cards on the dining hall tables, and therefore they can come into
contact with bacteria food on. Some of these bacteria could include Campylobacter and
Salmonella typhimurium. These two bacteria are commonly found in eating establishments,
especially when food is not cooked properly as well as thoroughly (9). Additionally, these cards
come into contact with several other objects found in both dorms and academic buildings. Some
of these objects include bed sheets, bathroom countertops, and toilets. Furthermore, bacteria
such as Staphylococcus aureus and Clostridium difficile have been found and identified on these
objects, which could be transferred to student ID cards as well (7).
Aside from identifying the bacteria found on these cards, the second goal of this
experiment is to isolate at least one gram-negative strain and one gram-positive strain. It is
possible that both gram-positive and gram-negative bacteria will be isolated, and that
Staphylococcus aureus will be presented as the gram-positive strain. However, it is entirely
plausible that bacteria other than the ones predicted above will be isolated.
Materials and Methods:
The beginning step in identifying the unknown bacteria includes transferring the bacteria
from the location of choice (student ID card) onto a TSA plate with a thorough swab of both
sides of the card. The TSA plate was inoculated in the first sector by the swab, and then the
streak plate technique was used to distribute the other sectors. In order to collect an appropriate
amount of bacteria, seven student ID cards were sampled and the samples were then each
transferred to an individual TSA plate. Bacterial growth was observed from the mixed culture
inoculums after incubation at 37 degrees Celsius for at least 24 hours. The next step involved
inoculating pure cultures from the mixed culture inoculums for further identification. Each
bacterium that displayed different cell morphologies were inoculated and transferred using an
isolated colony. Each TSA plate displayed pure cultures, including a total of five pure culture
isolates after the plates were incubated at 37 degrees Celsius for 24-48 hours. The success of the
growth of pure isolates was based on the similarity the morphologies of the colonies on each
culture.
In order to keep the bacteria over a period of time, the isolated pure cultures were each
transferred onto separate TSA slants. The flame loop was used to transfer a colony from each
isolated culture to the individual TSA slants. Slants were incubated at 37 degrees Celsius for 2448 hours or until sufficient growth was identified. The slants were placed in the refrigerator until
needed for further testing. When identifying the unknown bacteria, Gram staining techniques
were conducted in order to distinguish strains of Gram-positive and Gram-negative bacteria (6).
The Gram staining helps identify the morphology of the bacteria with the use of a microscope
after staining procedures are conducted. The steps involved in the process include using a
primary stain (crystal violet), mordant (iodine), decolorizer (ethanol), and counterstain (safraninbasic dye). The iodine will help to fix the stain into the cell wall. In Gram-positive bacteria as
well as Gram-negative bacteria the observation of a violet color will be present. However, the
ethanol will result in Gram-negative bacteria to turn colorless as the decolorizer will strip the
outer membrane and leach out the dye. Lastly, the safranin-basic dye will turn Gram-negative
bacteria red (6). After identification of Gram-positive and Gram-negative bacteria, additional
tests were conducted such as a catalase test, phenol red broth (glucose) test, citrate test, lactose
test, and a MRVP test (1,2). These tests have helped the process of identifying the unknown
bacteria found on student identification cards.
The DNA of both the Gram-negative and Gram-positive bacteria was extracted in order
to further verify identification through 16S rDNA (5). This was done using an extraction kit
called Quik-gDNA MiniPrep from Zymo Research. A lysozyme was used in the preparation of
the Gram-positive bacterium in order to break down the peptidoglycan present in the cell wall.
The DNA was then isolated and a PCR was run on a 1.0% agarose gel electrophoresis in order to
amplify it. The DNA was then extracted using a Zymoclean Gel DNA Recovery Kit and sent off
to a DNA sequencing company for identification (5).
Results/Discussion:
A variety of bacteria species grew into a mixed culture from the sample. Five different
isolates were successfully grown from the mixed culture inoculum in order to increase the
chance of identifying at least one Gram-positive and one Gram-negative species. Isolate #1 grew
in rounded, orange colonies that had a slimy texture and slight elevation. The Gram stain test
showed it to be Gram-negative, indicating that peptidoglycan was not heavily present in its cell
wall. The test also helped to identify the species as bacillus with a streptobacillus arrangement.
Isolate #2 grew in translucent colonies that were heavily clustered together. It showed little to no
elevation. The Gram stain test identified it as a gram positive bacillus species with a
streptobacilli and diplobacilli arrangement. The Gram-positive result of the Gram stain test
indicates that peptidoglycan is heavily present in this species’ cell wall. Isolate #3 grew in small,
yellow colonies that were also heavily clustered together. The colonies had a rough texture with
little to no elevation. The Gram stain test identified it as a Gram-positive species with a coccyx
shape and a staphylococcus arrangement. Isolate #4 grew in tough, white colonies with a slight
elevation. The gram stain test showed the bacterium to be a Gram-positive species. The test also
showed the bacterium to have a coccyx shape and a staphylococci arrangement. Isolate #5 grew
in bright white branching colonies. They were not rounded and had a fuzzy/chalky texture as the
colonies grew more spread out. The Gram stain test performed on this isolate was unsuccessful.
Isolate #1 and #4 were the Gram-negative and Gram-positive chosen to identify further.
The results of the catalase test on the Gram-positive unknown showed a positive result due to the
fact that there were heavy levels of fizzing when the bacterium was placed into a few drops of
3% H2O2. A positive result for the catalase test indicated that the bacterium produces the enzyme
catalase (2). Because the colonies grown on the TSA plate were white, a mannitol test was then
performed. The medium was cherry red in color and produced no gas, indicating an alkaline, and
therefore negative result (2). Using the dichotomous key for Gram positive enteric bacteria, the
combined results of all of these tests led us to conclude that the Gram-positive unknown
bacterium species in Staphylococcus epidermidis (4).
The Gram-negative unknown bacterium tested positive for glucose fermentation due to
the fact that the medium turned yellow in color. A citrate test was then performed and the
medium remained blue-green in color, indicating a negative result. The results of this test show
that the gram negative unknown bacteria does not use citrate as its sole carbon source (1). The
results of the methyl red and Voges-Proskauer tests also proved to be negative due to the fact
that the color of the medium did not change. The negative result of the methyl red test indicates
that the Gram-negative bacterium does not ferment glucose to high acid. Likewise, the negative
result of the Voges-Proskauer test indicates that the bacterium also does not ferment glucose to
butanediol (2). The bacterium had a positive result for the oxidase test due to the fact that the
swab containing the Gram-negative species turned black after being saturated with the oxidase
reagent. The results of this test show that the bacterium contains the proton pump cytochrome
oxidase (cyt a/a3) (2). Using the dichotomous key for Gram-negative enteric bacteria, the
combined results of all of these tests led us to conclude that the Gram-negative unknown
bacterium is an Aeromonas species (3).
Unfortunately a valid result was not received from the DNA sequencing company on
both the Gram-positive and Gram-negative bacteria. This could be due to a variety of factors. For
one, the sequencing procedure only works for enteric bacteria so it will not yield a valid result on
DNA from non-enteric bacteria. However, since both the Gram-negative and Gram-positive
bacteria were identified as enteric bacteria through identification tests performed in the lab, this
explanation is unlikely. Another explanation for the invalid results is that there was not enough
DNA present in order to properly identify the bacteria. It is also possible that something went
wrong during the sequencing procedure. Very rarely is an invalid result due to human error in the
isolation and extraction procedure; however it is still a possibility.
Although the predicted bacteria were not identified, it is possible that they were, in fact
present on the student ID cards. Only one gram positive and one gram negative bacterium was
chosen to fully identify out of five isolates. Additionally, those five isolates were chosen out of
seven mixed culture inoculums. Therefore, it is entirely plausible that the predicted bacteria were
present on the sample but it is not possible to know for sure because they did were not chosen to
identify further.
A wide variety of bacteria can be present on objects that people take with them as they go
about their daily lives. The more locations that an object comes into contact with, the more likely
it is going to harbor a more diverse population of bacteria. Not all of these bacteria are
necessarily harmful in healthy adults. However, it is possible for them to act as pathogens and
cause diseases. For example, Aeromonas can cause a variety of respiratory tract infections,
including epiglottis. It can also cause diarrheal disease such as gastroenteritis but it is unclear
whether many of the strains isolated from feces are involved in diarrheal diseases (8).
Staphylococcus epidermis is one of the most important pathogens involved in nosocomial
bloodstream infections, cardiovascular infections, and infections of the eye, ear, nose and throat
(10).
Fig. 1. Gram stain of Staphylococcus epidermis.
Fig. 2. Gram stain of Aeromonas species.
Test
Result
Catalase
+
White colonies present?
+
Mannitol
-
Table 1. Identification tests performed on the Gram positive unknown species. The combined
results of this test helped to identify the bacterium as Staphylococcus epidermis, using the
dichotomous key (4).
Test
Result
Glucose
+
Citrate
-
Methyl Red
-
Voges-Proskauer
-
Oxidase
+
Table 2. Identification tests performed on the Gram negative unknown species. The combined
results of this test helped to identify the bacterium as an Aeromonas species, using the
dichotomous key (3).
References
1. Baron, S. (2015). Alternate Growth Substrates-Hydrolytic and Degradative Reactions.
2. Baron, S. (2015). Carbohydrate Fermentation and Respiration.
3. Baron, S. (2015). Dichotomous Key for Gram Negative Enteric Bacteria.
4. Baron, S. (2015). Dichotomous Key for Gram Positive Enteric Bacteria
5. Baron, S. (2015). Identification of Unknown Microorganisms by 16S rDNA.
6. Baron, S. (2015). Staining Techniques for Light Microscopy Simple Stains and the Gram
Stain.
7. Changym, Xy, et al. “Sanitary Status and Incidence of Methicillin-Resistant
Staphylococcus Aureus and Clostridium Difficile within Canadian Hotel Rooms.”
Journal of Environment Health 77.8 (2015): 8-15. Academic Search Complete. Web.
30 Aug. 2015.
8. Janda, Michael J, Abbot, Sharon L. “Evolving Concepts Regarding the Genus
Aeromonas: An Expanding Parorama of Species, Disease Presentations, and
Unanswered Questions.” Oxford Journals 27.2 (1998): 332-334. Academic
Search Complete. Web. 18 Oct. 2015.
9. Sirat, Suiata A,. et al. “Persistence of Salmonella and E. coli on the Surface of Restaurant
Menus.” Journal of Environmental Health 75.7 (2013): 8-14. Academic Search
Complete. Web. 30 Aug, 2015.
10. Vuong, Cuong, Otto, Michael. “Staphylococcus epidermis infections.” Microbes &
Infection 4.4 (2002): 481. Academic Search Complete. Web. 18 Oct. 2015.