1. Marine Microbiology and Molecular
Biology Spring 2015
Final Lab Report
Jessica Peck
April 27, 2015

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Introduction
The purpose throughout the course of this lab is to identify bacterial communities that are
present in our samples collected from the local environment and identified using various
applications common in microbiological studies. The initial samples were collected by the
teaching assistant, Gabby Barbarite and came in numerous forms to ensure that each student was
able to cultivate various communities of bacterial content. Samples ranged from sediment
samples and shrimps to include tunicates and sponges from different locations to also include a
wider spectrum of microbiomes present on each individual. The data collected during the course
of this class seeks to give a general understanding of methods that we can use in examining a
sample found in vitro environments and approaches to understanding its microbial profile. This
particular research aims to examine the microbiome of two different types of sponges: 13-I-15-4-
1 and 13-I-15-7-4. Observations made through the course of the semester are conducted weekly
through various testing approaches and general physical observations of cell morphology, cell
structure and gram staining. Understanding preferential or ideal growth conditions such as
metabolic and enzyme activity as well as salt obligation, cellulose and xylanase usage and testing
for antimicrobial activity. The nucleotide sequences were obtained to compare in the BLAST
database of 16S rDNA that helped identify the closest match of bacterial composition which in
association with the other data collected over the semester can provide support in the bacterial
composition of both samples.
Methods and Required Materials
Sample Sources and Initial Plating
To carry out this experiment the specimens that were to be tested throughout the semester
were chosen from a variety that had been collected in vitro earlier in the day. Each of us students
were to choose two different specimens that represented aquatic marine organisms. For a
majority of the experiment these specimen were referred to as their sample ID number that
included the collection date, site location and dive number. The two samples in this study were
sponges identified as 13-I-15-4-1 from HBOI Aquaculture System (in a filter) and 13-I-15-7-4
from Little Jim in Fort Pierce. Once the samples were chosen, morphological characteristics
were recorded as well as ecological and taxonomic characteristics that help to identify the
specimens more specifically. After we observed the samples we were to grind them up using a
sterilized virtis grinder to allow bacterial colonies to be suspended in the mixture before using
them to plate the bacteria and the remaining material was frozen for future molecular work in
order to obtain DNA. Some of the excess was also fixed in in 4% formaldehyde and diluted to
plating to be examined in the future labs.
Cultivation of Microbes, Microscopy and Staining
A major portion of this lab required sub-culturing bacterial samples each week to allow
continuous growth while simultaneously testing for the best type of growth media and nutrients
required for growth. The ground sample was used to create a 10-fold dilution onto three different
agars. Marine Agar 2216 (MA), maltose-amended seawater (MAS) and oligotrophic (O) were
the three types of agar used and then incubated at room temperature to observe at a later time.

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Marine agar is an average agar that is considered a rich media, undefined because it possesses
the nutrients required for growth and no defined limits. MAS agar is rich is sugar and maltose
and the oligotrophic media is a minimal media that is will not necessarily grow as many colonies
since it only contains tryptone-protein. After the plates had been incubated for two weeks
observations were made on the morphology and optimal growth agar of the colonies as well as
colony forming units (CFUs). There are various forms of bacterial colonies present on the plates
and deciphering between colonies as well as what growth media works best for the organisms
helps determine the next step. The purpose of measuring the CFU of readable plates was to back-
calculate in order to determine the number of CFUs/mL of the original sample and diluted
suspension. The formula to calculate the CFU/mL of your original sample from a dilution plate is
as follows: 10 × 𝐶𝐹𝑈 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 1
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛⁄ i.e. if the sample had 7 colonies on a 10-4dilution
the math would be 10 × 7𝐶𝐹𝑈 × 1
0.0001⁄ . This helps to know the relative abundance of each
colony type. The plates were then identified as to which the colonies grew more abundantly on.
So individual colonies were separated in order to isolate onto both MA plates and the original
growth media they were found on, so long as it was different from the MA. Once the isolates
were plated the next week they were examine for macrocolony characteristics that helped to later
identify the types of bacteria present as well as ensured that only one type of bacteria was present
on the agar plate. True isolates were then sub-cultured on MA plates to continue growth and not
limit nutrients if the agar was to be overused, had the same plates been used all semester. The
sub-culture of seemingly single colony types also acted as a safety net in promoting that only one
isolated colony was actually present on the agars so as to not cause contamination in later labs
determining DNA and gram-staining. For this particular lab, of the various types of bacteriums
present on my plates I selected 11 different isolates. These isolates were then tested for further
understanding of how the bacteria, that were present on the media, grow best.
The collections that had been formaldehyde-fixed during the first week from original
suspension were also observed utilizing light microscopy for another examination of cell
morphology. These isolated samples were examined in a process known as gram staining. This
process is done by using sterilized loops and spreading 10µl of DI water (deionized) and heat
fixing bacterial isolates onto microscopic plates that are then flooded with Crystal Violet for 60
seconds. The stain remains in gram-positive cells because of the thick peptidoglycan layer
making them appear purple and the color is not removed when the sample is washed with water
and ethanol. After gram iodine (mordant) is added and another wash step then a decolorizing step
(acetone) a counterstain is added, Safranin. The Safranin is actually what cause gram negative
bacterium to appear pink in color on the microscope. This is an important factor of cellular
characteristics that is extremely beneficial in determining what bacterial colonies are present or
narrowing down possible matches prior to genomic studies.
Metagenomic DNA and LH-PCR
Another step in identifying the bacteria present in our samples we had to extract metagenomic
DNA using a FastDNA Spin Sample Kit that removed excess material from the sample. Once the
sample had been able to extract the DNA, it was retrieved from the chamber and went through
the process of thermal cycling. This process works by artificially heating the tube to denature the

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structure. Primers are then placed at particular sites, the forward primer on the 27F base pair and
355R base pair reverse primer which are fluorescently tagged allowing the ability to identify
RFUs or relatively fluorescent units of each sample. The sample then goes through the process of
annealing and once annealed the sample is able to extend and grow excessive amounts of copies
which lengthens the strands of DNA present. Since the growth of DNA is exponential, we are
cycling the samples 25 times which should result close to 33 million copies of DNA being
produced. These results were then run against positive and negative controls as well as with a
partner to allow comparison of the information we were able to obtain. Once the samples were
run through a specific system to detect fragment length we were given the percentage of lengths
of strands that each bacteria possessed. The relative abundances of each BIN that we had was
compared amongst the groups we worked in and then amongst the class.
Fermentation, Obligation, Enzyme utilization and Antimicrobial Capabilities
Although various tests are done to examine the morphology and characteristics of each
specimen, there was a continued used of agars to understand ideal or preferred conditions of
each.
To indicate whether a sample had specific saline conditions, each bacterium was plated onto
both a plate with MASW and another MANO. The MASW is a marine agar with seawater that
provides salt to the bacteria’s so if they thrived in this media it means that the bacteria has a
preference for salty environments. This result is expected for most of the organisms considering
the samples are of marine environment origins. The other plates MANO, were marine agar
without seawater to elucidate the idea if these organisms were saline obligate or required.
Another test of the samples was to determine the preferred metabolic source using both KP and
SYZ fermentation broth media. The KP media contained kelp, fish and chiton and the SYZ
solely possessed sugar. These cultures were grown at 25•C while being shaken to ensure aerobic
fluctuation for one week. Growth in these media indicate metabolic sources of the bacteria that
produce enzymes that allow them to break down the macromolecules and use them as the food
source to carry out fermentation.
Another way in which we were to examine our samples was to determine if the bacterium
could utilize cellulose or xylan in order to produce cellulose and xylanase respectively. By taking
1 µl of the broth suspension from each culture and setting it up in sections 30 mm apart from one
another with the control located in the middle. The purpose of the control was to ensure that the
cellulose could be utilized by the microbes and it was present in the agar. Digestion of either
cellulose or xylan was indicated by cellulose enzyme activity that once dyed with Congo Red
and decolorized with a NaCl wash can be observed over a fluorescent light by presence of a
yellowish to orange colored ring around the area of each isolate. The xylan was observed under a
UV light to observe xylanase activity that had a darker halo around the area of the bacteria.
Lastly, antimicrobial assays were utilized during the procedure of the lab to examine if any of
our specimen possessed the capability to fight antimicrobial growth of Candida albicans,
Pseudomonas aeruginose, and Staphylococcus aureus. Samples were taken of each bacteria
from the SYZ and KP tubes and placed onto separate plates of each of those diseases. Samples

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were placed on the antimicrobial assays by soaking 6.35mm paper discs in the bacterium and
placing in sections of the various plates that had been inoculated with bacteria.
Genomic Comparisons
Another portion of this experiment of molecular analysis was carried out by extracting
genomic DNA from each isolate. This was done by using a sterilized loop or needle to transfer
cells into 125 µl of 5% Chelex Solution. The chelex is the cheapest option and only regenerates
copies of DNA not a specific amount. So once the samples had been placed in the thermal cycler
for 10 minutes at 95 degrees Celsius, then resuspended, and centrifuged it had pressured the
genomic DNA into the supernatant which was extracted into a new sterile tube. The DNA can
then be quantified using the Nanodrop, in our case the 2000c version. The appropriate amounts
were then sent off and we were able to receive the nucleotide base pair sequence in return.
Electrophoresis
The products we received back from PCR were examined using electrophoresis, which
exemplifies the length of base pairs from the samples. The Gel electrophoresis was run slightly
above the recommended 0.02-0.03 voltage to allow for lesser time constraints in the lab. The
samples were fluorescently stained to allow tracking through the gel using SYBR Safe DNA
stain, which after the gel had been run allowed us to see the lanes in through ultraviolet lights.
The amplicons we cleaned of PCR products had been sent out and compared to registered
sequences in the GenBank through 16S sequencing since this is the most hypervariable but is the
most conservative with a little variability to allow better identification of microbial communities.
The results were then run through the BLAST nucleotide sequence database to compare with the
large system the most valid bacterium that fit our samples. Ultimately we could make a general
identification of each isolate and created our own miniature database of microbial communities
identified by their general morphology and characteristics as well as their molecular make up and
genomic comparisons.

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Results
Table 1: Origin of Samples
Figure 1: Comparative Analysis of Microbial Communities RFU

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Figure 2: Class Analysis of Bacterial Communities RFUs
Table 2: Results of potential bacterial compositions in two sponge samples

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Table 3: Overview of Potential Bacteria from each sample and their respective growth characteristics
throughout various media and antimicrobial assays
+ signifiesgrowth
- signifiesnogrowth

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Discussion
Now, further examination of the results of the data collected throughout this lab will allow
understanding to how the results are intertwined and allow more accreditation to the information
found. To begin with it is understood there was a variety of organisms in which samples were
extracted from as seen in Figure 1 including sponges, shrimp, anemones, sediment, algae etc.,
which is the first indication that there was going to be varying bacterial communities due to
organisms examined and their locations.
Of the organisms studied in this particular study there were two sponges, 13-I-15-4-1 located
from HBOI Aquaculture and 13-I-15-7-4 found in Little Jim in Fort Pierce. Already differing in
their locations the bacterial communities also greatly differed. Of the eleven samples collected 6
belonged to the sponge sample of 13-I-15-4-1, of which only four different types of bacterial
composition could be identified. A sponge provides a soft substrate for many microbes to readily
attach and work on the surface of the organisms. This particular sponge, found in Aquaculture,
was actually growing on the inside of a filter where the Bioflock was examined by another
student. Examining Table 2 you can get a better visualization of the similar RFUs that the two
specimens shared. However there is still high variability amongst which types of bacteria were
present. The physical morphology of an organism can greatly factor which types of bacteria are
able to grow on it’s surface or in some cases inside the organism itself. Through examination of
Table 3 it can be observed that all of the bacterial specimens studied during this experiment
possessed the characteristic of being gram-negative bacteria. Gram-negative signifies the
bacteria has a thin peptidoglycan wall allowing easier penetration and thus a lesser barricade to
entering the cell. Of the bacterial communities found by utilizing the DNA extraction of LH PCR
and then comparing the nucleotide sequences in the BLAST system database the potential
bacterial strands were examined and to identify the most accurate and properly depicted bacteria
comparisons to cell morphology, salt obligation and enzyme utilization. All of these
characteristics assist in determining the bacteria actually present. As seen from the information
depicted in Table 3, the variable growth or lack thereof helped determine the presence of Vibrio
chagasii, Alteromonas macleodii, Pseudoalteromonas piscicida and Vibrio rotiferanus as part of
the bacterial composition for the sponge found in HBOI’s Aquaculture Facility. Vibrio
rotiferanus was deemed a likely factor because it is a gram negative bacterium, shaped like small
rods, and Alteromonas macleodii was a likely candidate within the sponge because it is a marine
proteobacterium found in coastal and open ocean environments. Although the sponge was found
locally, there could be the possibility that it began growing by contamination after being
transferred from another location. Indications that these are the proper bacteria is supported
through the results found throughout the semester not just from the BLASTing database. They
are gram negative and grow mainly in marine environments, and also grew best on the MASW
agar, indicating that the bacterial community is majority of salt obligates. None of the samples
collected possessed the ability to break down xylan and cellulose however they were all able to
utilize the breakdown of KP made of the kelp, fish and chiton. Similar information can be found
for all of the samples I had processed and the second sponge appeared to house a bacterial
community composed of Spongiibacterium flavum, Vibrio brasiliensis, Endozoicomonas
numazuensis, and Ruegeria atlantica. Other factors that help in determining the presence of these

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particular bacteria include Spongiibacterium flavum being a common bacteria found in marine
sponges as well as Endozoicomonas numazuensis commonly found in marine environments
especially amongst sponges. Amongst all of the bacteria found none possessed the ability to
combat antimicrobial activity, exemplified by the negative results in the zones of inhibition of P.
aeruginosa, S. aureus, and C. albicans.
Conclusion
After examining the results of this lab it can be understood that one approach does not
provide enough sufficient evidence in identifying bacterial communities. Through the course of
this lab, an array of bacteria were expected to be found amongst various organisms but even
amongst similar individuals such as the two sponges examined for this report, a wide variety of
bacterial communities were found despite being in close proximity. Through the course of the
semester more data was collected about each individual such as salt obligation, enzyme activity,
and the ability to utilize the KP or SYZ media types to better determine and provide support
what bacteria was present in each of the samples. The process of determining the nucleotide
sequences and comparing the information to that found in the BLAST database directed the
bacterial types into a more narrow decision in what bacteria were present and in association with
the other information we collected that was useful in determining the best possible match based
upon cell morphology, enzyme activity and the obligation of salt.
So through the attempt to find the composition of bacterial communities in organisms this
semester there was a wide variety present in these two sponges. When comparing the class data
there was an immense, even wider, variety of bacterial isolates that were found but also
possessed similarities amongst the class data as a whole representing the variable association
organisms possess, even being within close proximity. To avoid contamination of any specimen
that could result in human error it was important to wear gloves throughout the course of the
semester as well as labelling the plates on the underside so we could read the plates after
incubation and ensuring they remained isolated during the lab by parafilming the plates. There
was some difficulty in reading the cellulose and xylan plates for the reason that all of the plates
for this experiment did not show any positive result even from the control, but this does not play
part in deciding the final bacterial communities present in our samples this semester. It is
important to know that there are many approaches in understanding what composes a bacterial
community and they must all be used in association to better determine the appropriate bacteria
present in each sample.