Kirdahy 1

Ryan Kirdahy

Microbiology Lab

May 8, 2017

Final Lab Report

Identification of an Antibiotic Producing Bacteria

It was not until the late 1920s when antibiotics were accidentally discovered by

Alexander Flemming. One of Flemmings Petri plates were contaminated with mold, and

Flemming observed that Staphylococcus aureus was inhibited around the mold colony. It turns

out that the mold that accidentally contaminated Flemmings plate produced a substance that

inhibits other microorganisms. This inhibition was the first clue as to how antibiotics work and

how they could be used. The first clinical trials of penicillin took place in the United Kingdom

before being relocated to the United States in the early 1940s and it proved to be exceptionally

effective at killing bacteria. This was the worlds first commercially produced antibiotic, and

used heavily to treat infections during wartime (Tortora et al. 2015). Since Flemmings

discovery, much has been learned about antibiotics, how they work, and how they can be used to

kill bacteria.

Not all antibiotics work the same way or affect bacteria the same. Some antibiotics are

bacteriostatic which means they inhibit the growth of the bacteria. In comparison, others are

bactericidal which induce cell death of the bacteria (Kohanski et al. 2010). Antibiotics are also

selective to what bacteria they can affect. The antibiotics that affect the widest range of both

gram-negative and gram-positive bacteria are called broad-spectrum antibiotics. Antibiotics

that only can only affect a certain family of bacteria are called narrow-spectrum bacteria

(Tortora et al. 2015).

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Antibiotics were remarkably successful against bacteria because the bacteria had not

ever been exposed to the antibiotics before. As antibiotics were used excessively for treating

disease in humans, animals, and plants, some bacteria began to develop a resistance. Bacteria

can evolve through either horizontal or vertical gene transfer. Horizontal gene transfer allows

for bacteria to transfer their antibiotic resistant genes between another bacteria species.

Vertical gene transfer passes antibiotic resistant genes down through generations (Ventola

2015). The bacteria that develop resistance to antibiotics are no longer affected by the

antibiotics that once inhibited them. This can lead to the emergence of superbugs which pose

a serious health risk to humans due to the inability to treat the bacteria with antibiotics

(Tortora et al. 2015). The development of new antibiotics is not keeping pace with the rate in

which bacteria are becoming resistant to the current antibiotics. An example of a bacteria that

has evolved to become resistant to antibiotics is Methicillin-Resistant Staphylococcus aureus

(MRSA). This disease has spread and is a worldwide issue. MRSA is very common in hospitals

and spreads easily. In 2005 there was more than 18,000 deaths contributed to MRSA (Green

2012). The longer antibiotics are overused without the introduction of new antibiotic classes

the larger the risk of more diseases like MRSA. The most recent class of antibiotics that were

released for use was in the 1960s which may play a role in the increase in bacteria resistance.

In order to counteract the rapid increase in antibiotic resistant bacteria, it is important to

introduce new drugs that the bacteria have not yet been exposed to. An increase in research is

imperative to discover and develop these new drugs (Komolafe 2003).

A great place to look for these drugs are in fermenting foods. Many natural food

fermentations rely on the presence of bacteria. The bacteria that is found in fermenting foods
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are known to be beneficial to human health, one main benefit being the production of

antibiotics (Tamang et al. 2016). Food fermentations serve as an intermediate environment.

Many of the bacteria that go into a fermentation come from a different place such as bacteria

on vegetables that came from the soil. The largest source of microorganisms is from the soil, so

it only makes sense that when using products that originate from the soil, they will have large

source of microorganisms too. This large source helps obtain a large test sample at once

(Hibbing et al 2010).

The contribution that I specifically made to this experiment was the isolation, purification, and

identification of an unknown isolate. The first step in the process was the isolation of an

antibiotic producing bacteria found in a yogurt fermentation. This was done to separate the most

promising antibiotic producing colony from all of the bacteria isolated from a yogurt

fermentation. The next step was to purify the isolated bacteria. This was to eliminate any

contamination as well as make sure there was no other bacteria in the isolate that could influence

the results. The final step was the identification of the isolated bacteria. There were several

experiments done to obtain characteristics of the isolated bacteria that would help with

identification. The most influential identification test was the Biolog test which runs biochemical

tests and compares the results to a database. This helps produce accurate identification results.

Once the unknown was identified it was possible to compare the lab findings to current findings

and see if the results are similar (Handelsman et. al).

Methods and Materials:

In order for the fermentation of milk into yogurt to take place, a type of milk and starter

culture must be selected. The combination of almond milk and vegan thermophilic starter culture

were chosen for this experiment. Two cups of almond milk were heated in a pot on a hot plate to
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140 C. Once this temperature was reached the milk was removed from the hot plate and allowed

to cool to 110 C. At this point, the vegan thermophilic starter culture was added to the almond

milk and mixed using a whisk. About one cup of the mixture was poured into a glass jar, and the

rest was given to another group. The cap of the jar was screwed on loosely, and the jar was then

placed in a yogurt maker at about 43 C for two days (Handelsman et. al).

The next step was to obtain a viable colony count from the product of the yogurt

fermentation. The jar of yogurt was obtained along with four All Culture Agar plates. A small

amount of the yogurt sample was poured into a sterile conical tube which was then briefly

vortexed. A serial dilution was performed. The 100 microliters of the dilutions, 10-1, 10-2, 10-3,

and 10-4 were plated to All Culture Agar plates. Sterile glass beads were used to thoroughly

spread the culture around the plate. The plates were placed in an incubator at 30 C for five days.

The colony morphology on the plates were observed. The colonies were counted and the cfu/mL

was solved for (Handelsman et. al).

Performing the viable colony count experiment was extremely useful for solving for the

cfu/mL, but also showed the colony morphologies present on the plates. Identifying the colony

morphology allowed for the creation of a master plate. The master plate was created by

executing the pick and patch method. Colonies with different morphology from the viable

colony count plate were picked with a toothpick and patched onto a master plate of All Culture

Agar. This was done on two All Culture Agar plate. The plates were placed in the incubator at

30 C for two days (Handelsman et. al).

Once a master plate was created, it was possible to screen the different bacteria found for

antibiotic production. All Culture Agar plates and MRS plates were used for the screening. In

order to create a lawn of bacteria, 150 microliters of Escherichia coli were micropipetted onto
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the All Culture Agar plate and spread using glass beads. This step was repeated using 150

microliters of Bacillus subtillis onto an All Culture Agar plate. In addition, one MRS Agar plate

received 150 microliters of E. coli and the other received 150 microliters of B. subtillis. The

plates were given five minutes to dry while the master plates were obtained. Using sterile

toothpicks, the pick and patch method was implemented to transfer the isolated bacteria from

the master plate to the four plates that were prepared. Once completed, all of the plates were

placed into the incubator at 30 C for five days. After five days, the plates were obtained and the

results were observed and recorded (Handelsman et. al).

Once the results from the antibiotic production screening were observed, it was possible

to pick which of the colonies seemed most promising to isolate as an antibiotic producer.

Obtaining a pure culture was possible by making streak plates. To obtain a pure culture at least

three rounds of streaking must be performed. A colony from the master plate was touched with a

sterile wooden stick and used to perform a T-streak onto an MRS agar plate. This was repeated

two more times. This completed Round 1 of the streak plates. The MRS agar plates were placed

in the incubator for 2 days. Due to lack of results it was required to attempt Round 1 again. This

time the streaking was attempted on three All Culture Agar plates. After five days of incubation

at 30 C, the plates were observed. There were sufficient results to be able to perform a Round 2

Streak Plate. A single colony from the Round 1 Streak Plate was picked using a sterile wooden

stick and it used to perform a T-streak on the All Culture Agar Round 2 Streak Plate. This was

repeated onto the other All Culture Agar Round 2 Streak Plates. The Round 2 Streak Plates were

incubated for two days at 30 C. After the two days, the Round 2 Streak Plates were observed.

The results were sufficient enough to be able perform Round 3 Streak Plates. A single colony

from the Round 2 Streak Plate was picked using a sterile wooden stick and it used to perform a
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T-streak on the All Culture Agar Round 3 Streak Plate. This was repeated onto the other All

Culture Agar Round 3 Streak Plates. The Round 3 Streak Plates were incubated for five days at

30 C (Handelsman et. al).

Once a pure isolate was obtained, further tests could be run to start identifying

characteristics of the unknown bacterial isolate. The first was a gram stain. A gram stain was

done on the bacterial isolate. On the same glass microscope slide, a gram stain of two control

bacteria was done as well. The two control bacteria were Escherichia coli as the gram-negative

control and Staphylococcus simulans as the gram-positive control. A negative stain was also

performed to help identify characteristics about the unknown isolated bacteria. The last type of

stain test that was performed was a capsule stain. The capsule stain was performed in order to

determine whether unknown bacteria had a capsule or not (Handelsman et. al).

Another way to test to see if the unknown bacteria is gram-positive or gram-negative is to

plate the unknown bacteria onto MacConkey Agar along with two known bacteria. The

MacConkey Agar plate was divided into thirds. On one of the thirds, the unknown bacteria

isolate was streaked. On the next third, S. simulans was plated as the gram-positive control. E.

coli was plated onto the last third of the MacConkey Agar plate. The MacConkey Agar plate was

incubated at 25 C for two days (Handelsman et. al).

A wider variety of antibiotic testing was performed during the Antibiotic Production Re-

test. Five previously prepared All Culture Agar plates were obtained. The five plates each had a

different bacterium on them. Bacillus subtillis, Escherichia coli, Enterobacter aerogenes,

Pseudomonas putida, and Enterococcus faecalis were the five bacterium that were on the All

Culture Agar plates. Using the pick and patch method, a bacteria colony from the Round 3

Streak Plate was picked and then patched onto the plate with B. subtillis. This step was repeated
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onto each of the other four plates, using a fresh toothpick and new colony from the Round 3

Streak plate each time. The Antibiotic Production Re-test plates were placed in an incubator at

25 C. After five days the Antibiotic Production Re-test plates were removed and observed for

antibiotic production (Handelsman et. al).

The next step was to perform a Biolog Identification test. This test runs 96 biochemical

reactions to identify what the unknown bacteria most likely is. Initially Protocol A was used for

the Biolog test. A sterile inoculation stick was used to touch a colony from the Round 3 Streak

Plate and then it was mixed into the inoculating fluid A. The turbidity was tested to make sure

the cell density was between 90%-98%. Once this was obtained, it was poured into a

multichannel pipette reservoir. An 8-Channel Repeating Micropipetter was used to transfer the

solution from the reservoir to the 96 well Gen III MicroPlate. The plate was incubated 25 C and

then read by the OmiLog incubator/reader. Due to an insufficient pattern, Protocol C1 had to be

performed. The same procedure was executed except inoculating fluid C was used instead of

inoculating fluid A. The second plate was incubated at 25 C and then read by the OmiLog

incubator/reader (Handelsman et. al).

In order to amplify the 16s rRNA in a sample from the isolated bacteria, a Colony PCR

test was run. The unknown isolate was run through a polymerase chain reaction (PCR). In order

to test if the PCR was successful or not, a gel electrophoresis test was performed. Due to the

results of the gel electrophoresis being lower than desired, an additional Colony PCR was

performed in order to increase the amount of DNA in the sample (Handelsman et. al).

Once the PCR reaction was complete, it was necessary to purify the PCR sample. Two

hundred microliters of CP buffer were added to the PCR sample and then the contents were

placed to a DNA mini column connected to a collection tube. The column was placed in the
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centrifuge apparatus and run for one minute. The contents of the collection tube were emptied.

Next the column received 700 microliters of wash buffer and then was placed in the centrifuge

and run for another minute. The waste from the collection tube was removed. The past two steps

were repeated once more. In order to remove any excess liquid or waste, the column was placed

in the centrifuge and run for two more minutes. A 1.5 mL capless microcentrifuge tube was

attached to the column. The column received 40 microliters of elution buffer, set for 2 minutes,

and then run in the centrifuge for one minute. The sample was moved to a capped

microcentrifuge tube and kept on ice. Two microliters of the pure sample were transferred to the

Assay Tube and was mixed with 200 microliters of working solution. This mixture was placed in

the Quibit fluorimeter. This tool helps determine the amount of DNA in the sample which can

then be calculated into the concentration of the sample (Handelsman et. al).

In order to determine if the unknown bacteria isolate was resistant to any antibiotics, an

Antibiotic Susceptibility Assay was performed. This is also known as a Kirby-Bauer test. A

colony from the Round 3 Streak Plate was touched and swirled into PBS buffer and compared to

a 0.5 McFarland Standard using a Wickerham card. Once the turbidity was matched as closely as

possible, the solution was streaked onto Muller-Hinton Agar to create a lawn. An Antibiotic Disk

Dispenser was used to dispense six antibiotic disks onto the Muller-Hinton Agar. The plate was

incubated at 25 C for five days (Handelsman et. al).

The final step of the procedure was to perform an Antibiotic Re-screen. The purpose was

to test if the isolated bacteria produced antibiotics against five bacteria, and to see if the results

were consistent with previous tests. Bacillus subtillis, Escherichia coli, Enterobacter aerogenes,

Pseudomonas putida, and Enterococcus faecalis were the five bacterium that were on the All

Culture Agar plates that were being tested against. The pick and patch method was used to
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transfer colonies of the isolated bacteria from the Round 3 Streak Plate to the All Culture Agar

plates with bacteria on them. The Antibiotic Re-Screen plates were incubated at 25 C for five

days (Handelsman et. al).

Results:

Figure 1: Pure Culture Agar Plate of bacteria isolated from yogurt on All Culture Agar

The isolate produced whole, off white, circular, smooth colonies with an entire edge

when incubated at 30 C for two days.

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Figure 2a: Initial Antibiotic Production test on MRS Agar

During the initial antibiotic production test on MRS Agar it was observed that the

isolated bacteria inhibited the growth of both E. coli and B. subtillis when incubated at 30 C for

five days.

Figure 2b: Initial Antibiotic Production test on All Culture Agar

During the initial antibiotic production test on All Culture Agar it was observed that the

isolated bacteria did not inhibit the growth of E. coli or B. subtillis when incubated at 30 C for

five days.
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Figure 3a: Antibiotic production re-screen (Section labeled RK) against B. subtillis, E. coli, E.

aerogenes, P. putida, and E. faecalis on All Culture Agar

During the antibiotic production re-screen on All Culture Agar it was observed that the

isolated bacteria did not inhibit the growth of B. subtillis, E. coli, E. aerogenes, P. putida, or E.

faecalis in the section labeled RK when incubated at 25 C for five days.

Figure 3b: Second antibiotic production re-screen against B.s subtillis, E. coli, E. aerogenes, P.

putida, and E. faecalis on All Culture Agar.

During the second antibiotic production re-screen on All Culture Agar it was observed

that the isolated bacteria did not inhibit the growth of B. subtillis, E. coli, E. aerogenes, P.

putida, or E. faecalis when incubated at 25 C for five days.

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Figure 4: Bacteria isolate being tested on MacConkey Agar along with the tester strains of E. coli

and S. stimulans

The MacConkey test revealed that the bacteria isolate is a gram-positive because it did

not grow on the MacConkey Agar. The gram-negative tester strain, E. coli, did successfully grow

while the gram-positive tester strain, S. stimulans, did not grow. This plate was incubated at 25

C for two days.

Figure 5: Test results in lane 9 (indicated by the red stars) from the 16s rRNA gel.

The results of the 16s rRNA were not as strong as expected so an additional PCR was run

to amplify the 16s rRNA more for better results. Band runs near the 2.0kb ladder.
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Figure 6: Antibiotic Resistance Assay Plate on Muller-Hinton Agar with six antibiotic disks

There was no growth of the bacteria isolate on the Muller-Hinton Agar. The six antibiotic

disks were streptomycin, tetracycline, novobiocin, chloromycetin, erythromycin, and penicillin.

The antibiotics completely inhibited all growth of the bacteria isolate which means the bacteria

isolate was completely susceptible to all of the antibiotics present.

Table 1: MacConkey Agar Test and Gram Stain

Test Results Conclusion

MacConkey Agar No growth Gram-positive

Gram Stain Purple rods Gram-positive bacillus

Negative Stain Rods Bacillus

Capsule stain No halo No capsule

The gram stain results showed that the bacteria stained purple and was rod shaped. These

results are standard of a gram-positive bacillus bacterium. The negative stain confirmed the

results of the rod shape of the bacteria. A capsule stain was also performed in order to test to see

if the bacteria produced a capsule which it did not. The results from the MacConkey Test showed

that the isolated bacteria did not grow on the MacConkey Agar. No growth on MacConkey Agar

corresponds with the gram stain that the bacteria is gram-positive.

Table 2a: Biolog Plate Results-Protocol A

Bacteria Species PROB SIM DIST Organism Type

No ID: insufficient data N/A N/A N/A N/A

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The results from the Biolog Plate when using Protocol A came back with insufficient data

and was unable to identify the unknown isolate. The results suggested rerunning the test using

Protocol C1.

Table 2b: Biolog Plate Results-Protocol C1

Bacteria Species PROB SIM DIST Organism Type

Lactobacillus rhamnosus 0.961 0.669 4.307 GP-Rod

Lactobacillus plantarum 0.016 0.008 6.941 GP-Rod

Lactobacillus casei 0.014 0.007 7.041 GP-Rod

Lactobacillus mali 0.010 0.005 7.240 GP-Rod

After rerunning the Biolog test again using Protocol C1, an identification was made on

the unknown isolate. The unknown was identified to be a 96.1% match with Lactobacillus

rhamnosus. This is the most likely match considering the other results are only about a 1%

match.

Table 3: Isolates Antibiotic Spectrum of Activity

Bacteria Plate Initial Initial Antibiotic Antibiotic

Screening Screening All Production First Re- Production Second
MRS: Culture Agar: test- All Culture Re-test- All Culture
Agar: Agar:
Bacillus Growth No zone No zone clearing No zone clearing
subtillis inhibition clearing

Escherichia Growth No zone No zone clearing No zone clearing

coli inhibition clearing
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Enterobacter N/A N/A No zone clearing No zone clearing

aerogenes

Pseudomonas N/A N/A No zone clearing No zone clearing

putida
Enterococcus N/A N/A No zone clearing No zone clearing
faecalis

During the initial screening of antibiotics, it was found that the isolate produced an

antibiotic against both gram-positive and gram-negative tester strains on MRS Agar due to the

zone clearings found around the isolate. In comparison, the isolate did not create any zone

clearings on the All Culture Agar plate against either tester strain during the initial screening.

The Antibiotic Production First Re-test showed no zone clearings which indicates that the isolate

did not produce antibiotics against the five tester strains. The results from the Antibiotic

Production Second Re-test corresponded with the first re-test. There were no zone clearings on

any of the plate. This indicates a loss in antibiotic production

Table 3: Isolates Antibiotic Resistance Profile

Antibiotic Disk: Zone of Inhibition: Conclusion:

Streptomycin >30 Completely susceptible

Tetracycline >30 Completely susceptible

Novobiocin >30 Completely susceptible

Chloromycetin >30 Completely susceptible

Erythromycin >30 Completely susceptible

Penicillin >30 Completely susceptible

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The Kirby-Bauer test reveals the isolates resistance to different antibiotics. The isolate

was plated onto Muller-Hinton Agar and six antibiotic disks were placed onto the plate. The

antibiotic disks completely inhibited the growth of the isolate preventing the isolate from any

growth on the plate. These results indicate that the isolate was completely susceptible to the

antibiotic disks on the plate.

Discussion:

The isolated bacteria initially produced a broad-spectrum antibiotic because it inhibited

the growth against both the gram-negative tester strain of E. coli and the gram-positive tester

strain of B. subtillis on MRS Agar. However, the bacteria isolate was not able to inhibit growth

of these same tester strains on All Culture Agar. After further purification, the isolated bacteria

did lose its antibiotic producing capabilities. In the first antibiotic re-test there was no antibiotic

production against B. subtillis, E. coli, E. aerogenes, P. putida, or E. faecalis when tested on All

Culture Agar at 25 C for five days. The results remained consistent on the second antibiotic re-

test when there was no antibiotic production against B. subtillis, E. coli, E. aerogenes, P. putida,

or E. faecalis when tested on All Culture Agar at 25 C for five days. The bacteria were tested on

All Culture Agar instead of MRS Agar because the bacteria were not growing well on MRS Agar

plates, and grew much better on All Culture Plates. The original antibiotic production was

observed on MRS Agar plates, so had the isolated bacteria been retested on MRS Agar there may

have been different results. However, after the initial antibiotic screening, the isolated bacteria

struggled to grow on MRS Agar which is why the switch to All Culture was made. Other studies

have shown that L. bacillus does not grow very well on MRS Agar either

When dealing with natural isolates in the lab it is possible for them to adapt to the lab

conditions. If the bacteria have optimal growing conditions like temperature, nutrients and do not
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have to compete like it would naturally, the bacteria tend to adapt to the lab conditions. This may

be the reason that the bacteria stopped producing antibiotics. There is likelihood that the isolated

bacteria may not have all of the same characteristics as it did when it was first isolated (Palkova

2004).

The isolated bacteria were gram-positive and this was shown through multiple test

results. During a gram stain, the bacteria showed up as purple on multiple glass slides. The

purple is indicative of gram-positive bacteria because it has a thick layer of peptidoglycan that

helps protect against decolonization of the cell after the crystal violet stain is set using iodine.

Gram-negative bacteria are known for staining pink due to their thin layer of peptidoglycan

which is easily affected by the decolorizer. The Safranin stain follows the decolorizer to give it

the pink color (Beveridge 2001).

MacConkey Agar holds both selective and differential properties. It is selective against

gram-positive bacteria because of the bile salts and crystal violet present in the agar. It is

differential because it helps determine whether or not the bacteria are fermenting (Handelsman

et. al). After incubation at 25 C for two days, it was observed that the isolated bacteria were not

able to grow on the MacConkey Agar plate because it was gram-positive. When compared the

tester strains the results were accurate. E. coli was the gram-negative tester strain and it was able

to grow effectively on the MacConkey Agar plate. S. stimulans was the gram-positive tester

strain and it was not able to grow on the MacConkey Agar as expected.

The Biolog plates lead to an identification because it ran 96 biochemical tests and

compared the results to the Biolog Gen III database to get a match. This produces very accurate

results in a short amount of time. Originally, the isolated bacteria were not able to be identified
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by Biolog Protocol A. The results read insufficient pattern and the bacterial isolate was not

identified. Protocol C1 was recommended for the next test. Protocol C1 initially showed no

results as well, but after extended incubation at 25 C the results came through. The bacterial

isolate was a 96.1% match to Lactobacillus rhamnosus. The other three matches, Lactobacillus

plantarum, Lactobacillus casei, Lactobacillus mali were only about a 1% match. This allows for

a more confident identification of the isolate to be L. rhamnosus. All four possible bacteria

identifications were gram-positive rods, so the results from gram staining, negative staining, and

the MacConkey tests would not have helped confirm the results. It is possible that the initial

protocol either did not have L. rhamnosus in the database, or it did not have long enough to

incubate, and that is why it was not identified.

The 16s rRNA PCR test was not able to be performed because the sample was not

concentrated enough. Therefore, there was no result that could confirm the Biolog results. The

results of the electrophoresis test showed that the bands ran near the 2.0kb ladder, but not in a

very clear, concentrated result. An additional Colony PCR was performed and added to the

sample, but the results still were not concentrated enough to send for testing. Had the results

been concentrated enough it is possible that a very accurate result would have been obtained. The

16s rRNA is very accurate and tests a very large database of known bacteria (Janda and Abbot

2007). This could have confirmed the Biolog results and made a more accurate identification of

the bacteria.

Lactobacillus rhamnosus belongs the genus Lactobacillus. This genus is known for either

being microaerophilic or facultative anaerobic. They typically are rod shaped and gram-positive

bacteria. L. rhamnosus belongs to the lactic acid bacteria (LAB) group (Fijan 2014). L.

rhamnosus is very diverse in the places it exists. A few popular places it can be located is in the
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mammalian gastrointestinal tract and plant and dairy products. A very popular commercial use of

L. rhamnosus is using it as a probiotic for intestinal health (Ceapa et al. 2015). Due to the

location that L. rhamnosus is usually found, it grows best around 37 C in low oxygen

environments. These conditions replicate environmental conditions such as the gastrointestinal

tract where L. rhamnosus exists (Soukoulis 2014). L. rhamnosus is a known antibiotic producer.

It is known for helping reducing severity of some gastrointestinal diseases with its production of

broad-spectrum antibiotics (Herbel et al. 2013).

The largest problem during the experiment was identification. The initial Biolog Protocol

A was unsuccessful in identifying the isolated bacteria. Even Protocol C1 struggled to identify

the unknown at first. Also, the PCR sample that was prepared to be sent for identification was

not concentrated enough to provide results. This left the isolate unidentified for a while, but this

was eventually resolved.

An additional problem that was encountered was obtaining a pure culture. The most

challenging part of this was that the bacteria was not growing well on the MRS Agar. This is the

agar plate that the bacteria produced an antibiotic on, but would no longer grow on it. Once the

MRS Agar was swapped for All Culture Agar, the bacteria isolate was successfully grown and a

pure culture was obtained.

The Kirby-Bauer test revealed that the isolate was completely susceptible to all antibiotic

disks. There was no growth on the Muller-Hinton Agar. Had there been growth with zones of

inhibition, it could have been calculated how susceptible the bacteria were to each antibiotic,

but there was no growth. L. rhamnosus is typically susceptible to many antibiotics (Korhonen
 Kirdahy 20

et. al 2009).

A future test that could be performed would be antibiotic production retests on MRS

Agar. Also, it may be beneficial to replicate the antibiotic production screenings under more

optimal conditions now that they are known. Another step that could be taken would be

obtaining results from the 16s rRNA PCR test. The results obtained from the PCR amplification

were not concentrated enough to send a sample, so it may have been beneficial to obtain a more

concentrated sample to send for testing. This would have helped solidify the identification results

obtained from the Biolog test.

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