Blue crab hemolymph report, Dec 2014

Quantification and Identification of Bacteria in the Hemolymph of
the Puerto Rican Blue Crab, Callinectes sapidus, Collected from the
Natural Reserve, Caño Tiburones, and the Waterfront of Islote in
Arecibo.
Research paper submitted for the project class BIOL 3108
Date submitted: 9th
of December, 2014
Students:
Barnes, Joseph A.
Garrastegui, Emmanuel
Pagán, Lillianette
Rodriguez, Génesis N.
Project supervisor:
Arbelo, Jose, PhD
University of Puerto Rico at Arecibo: Department of Microbiology
Abstract
During this research project we searched for a correlation between the density and
composition of the bacterial flora within the hemolymph of the blue crab, Callinectes
sapidus, and the elevated levels of pollution in its habitat. Specimens for the experimental
group were collected from the polluted wetlands of Caño Tiburones, while the specimens
for the control group were gathered from the waterfront of Islote. Aseptic techniques for
hemolymph extraction were designed and implemented, followed by serial dilutions for
an estimated cell count. We successfully established the presence of bacteria and, judging
by morphological and physiological characteristics, we identified a halotolerant Bacillus
spp. and a Micrococcus ssp. within the microflora isolated from the hemolymph. We
worked with standard lab techniques for bacterial isolation and biochemical tests and
staining for bacterial identification.

1 Bacteria in the Hemolymph of the Callinectes sapidus; UPRA Dec, 2014
Introduction
Caño Tiburones is the largest estuarine
wetlands in northern Puerto Rico, and a
portion of it as been designated as a natural
reserve within the municipality of Arecibo
[5]. This reserve lies adjacent to an active
landfill, which constitutes a point source of
pollution to the nearby areas. The wetland
serves as a natural habitat for various types
of plant and animal flora, including the blue
crab, Callinectes sapidus. The dominance of
the C. sapidus has made it a common food
source for the indigent who live in the
vicinity. The contamination of this wetland
by the landfill has brought to our attention
the question of how the flora which inhabits
the area may have been affected by the new
conditions. This experiment was conducted
with the purpose of assessing the microbial
flora within the C. sapidus in order to
determine any correlation that might exist
between the microbial density and
composition within the hemolymph of the
blue crab and the contamination of the
host’s environment.
The hypothesis in question is the following:
Natural habitats with elevated levels of
contamination will stimulate the
development of higher concentrations of
bacteria within the hemolymph of the C.
sapidus.
The independent variable is the level of
contamination in the habitat of the host. The
dependent variables are the concentration of
bacteria and their composition (foreign
versus natural). The null hypothesis is that
contamination does not affect bacterial cell
concentrations. The alternative hypothesis
holds that heavy contamination does alter
the levels of bacteria. For the control group
we used blue crabs fished from an area with
less contamination, while the experimental
group consisted of crabs collected from the
contaminated wetland. We selected the
dockyards at the waterfront of Islote in
Arecibo as the site for the control group.
Assessing the bacteria within the
hemolymph of crabs of the genus
Callinectes is not uncommon. Given the
commercial importance of C. sapidus as a
widely popular food source in the eastern
coastal regions of the U.S., and thus a
significant concern for organizations of
public health and safety, various studies
have been conducted to the end of
identifying the natural microflora of the C.
sapidus, as well as detecting significant
concentrations of pathogenic bacteria and
thus prospective health threats [2, 3, and 4].
Research of the microflora of a Callinectes
ssp. has also been realized here in Puerto
Rico, with a focus of quantifying total
bacterial densities and identifying species of
bacteria in the hemolymph of C. bocourti
collected from eutrophic waters as well as
coastal lagoons and estuaries [1]. This
earlier study served as the principle basis for
the following project, as it demonstrated that
crabs in eutrophic waters can sustain
bacterial concentrations up to 108
cells mL-1
within the hemolymph [1].
Materials
Sterile syringes, fishing crates, plate count
agar (PCA), tryptic soy agar (TSA), tryptic
soy broth (TSB), mannitol salt agar (MSA),
eosin methylene blue agar, crystal violet
agar, bile esculin agar, hydrogen peroxide,
lactose broth, 70% ethanol, Gram stain
reactants (crystal violet, Gram’s iodine,
safranin, 95% ethanol), toluidine blue,
citrate/EDTA buffer.
Methods
Phase 1
Collecting crabs; extracting hemolymph;
dilution in series:
We collected 5 blue crabs (4 males and 1
female) from the wetlands of Caño
Tiburones [image 5], and 3 blue crabs (2
males and 1 female) at the waterfront of
Islote [image 6]. The specimens were placed
in water igloos containing water collected
from the respective sites. The specimens
were promptly taken to the laboratory of
microbiology at UPRA, where we carefully
secured their chelas (claws) by wrapping a
rubber band around the claw, holding tightly
Students: Barnes, J.A., Garrastegui, E., Supervisor: Arbelo, J.
Pagán, L., Rodriguez, G. N.

together the dactyl and propodus [image 1].
We then randomly selected one male from
each site, weighed it, and measured the
width of the carapace, which we defined as
the distance between the lateral spikes
located on either side of the carapace. We
did the same for the female from each site
[table 2]. Using either a sterile 21 or 22 ½
gauge needle, we extracted 1.0 mL of
hemolymph by inserting the needle into the
body at the base of one of the posterior
pereopods (legs), preferably the swimming
leg [image 2]. Before inserting the needle,
we applied 70% ethanol over the area so as
to disinfect the insertion point. Each sample
of hemolymph was immediately diluted in
series following the procedure delineated in
the lab manual [6] [image 3]. The diluted
samples were used to prepare pour plates
using Plate Count Agar (PCA) as the
nutrient medium. After 48 hours of
incubation, we counted for colonies using a
dark-field colony counter.
Preparing bacterial cultures; isolating
bacteria:
Following the serial dilutions and incubation,
we prepared 20 bacterial cultures from the
agar plates, each from a distinctive colony.
Each bacterial culture was grown in a tryptic
soy broth (TSB), incubated for 24 hours. We
then prepared a streak plate, using tryptic
soy agar (TSA), for each culture and
incubated them for 22 hours; from the streak
plates we observed 10 distinct types of
colonies, and from each we prepared a pure
culture using TSA slants. Following 21 hr
incubation, we prepared Gram stains for
each of the 10 slants to confirm whether or
not they were pure cultures, as well as to
observe morphology and gram-staining. The
10 bacterial cultures were sub-cultured
periodically to maintain a fresh stock culture.
Differential media; biochemical tests;
identification:
We attempted to grow selected bacterial
cultures on differential media on the basis of
observed morphology and gram-staining.
The differential media we used are the
following: mannitol salt agar, bile esculin,
crystal violet, and eosin methylene blue.
We performed the following biochemical
tests: catalase test and carbohydrate (lactose)
fermentation (standard test for detecting
coliform bacteria).
We also tested for endospore-formation in
the case of the gram (+) rod-shaped bacteria
we isolated.
Phase 2
Collecting crabs; extracting hemolymph;
dilution in series:
We fished 5 blue crabs from the waterside of
Islote. We collected the specimens in a
water igloo containing water gathered from
the same site. Securing their claws in the
same manner of phase 1, we selected three
crabs, all females, and extracted 1.0 ml of
hemolymph from each. We used sterile 21
gauge needles with 3ml-syringes; prior to
extraction we aseptically transferred 0.5 ml
of sterile citrate/EDTA buffer into each
syringe. The citrate/EDTA buffer was
prepared in the lab using the substances
published by an earlier experiment with
Callinectes ssp. crabs [1] [table 4]. The
needle insertion point was also disinfected
with 70% ethanol before extraction. Each
sample of hemolymph/buffer was promptly
diluted in series following the procedure
delineated in the lab manual [1]. PCA was
used to prepare the pour plates and after 45
hours of incubation, we counted for colonies
using a dark-field colony counter.
Results and discussion
Phase 1
We were unable to determine the cell
concentration for any of the 4 hemolymph
samples extracted and diluted. The samples
from the female crabs generated only 6
colonies, numbers far below the permissible
counting range. The samples from the male
crabs also generated colony counts outside
the acceptable counting range, however the
male from Caño Tiburones (specimen 1)
produced colony numbers that exceeded the
counting range in the plates with dilutions of
10-6
and 10-7
[table 3]. The serial dilutions

for specimen 1 generated higher colony
counts in the pour plates with higher dilution
factors, an outcome contrary to the natural
assumption that colony count on the agar
plate should be inversely proportional to the
dilution factor. The coagulation of the
hemolymph outside of the crab body might
account for this unexpected result. It is
possible that due to coagulation, the
bacterial cells were mostly clumped together,
thus the first dilution would generated sparse
but very large growths, each arising from an
agglomeration of bacterial cells rather than
from a single cell. After the second dilution
and vigorous shaking, however, the cell
clumps were loosened and distributed more
evenly across the solution, and thus
produced numerous, smaller colonies, each
arising from a single cell.
Of the 10 pure cultures which we isolated, 4
of them (cultures 1, 2, 3 and 10) appear to
belong to a strain of a Bacillus ssp. in
accordance to Bergey’s Manual of
Determinative Bacteriology flow chart [7],
wherein large, gram (+), rod-shape, spore-
forming, not strictly anaerobic bacteria are
classified as members of the genus Bacillus
[image 8]. This strain has demonstrated the
unique ability of growing on media with a
high salt concentration. All four cultures
grew on mannitol salt agar (MSA) with a
salt concentration of 7.5% [table 1], and
were able to ferment the mannitol sugar as
well. It had been possible that the culture
was a mixed culture, with a strain of
staphylococcus hidden among the more
prominent bacillus rods; however, we
removed these doubts by carrying out a
simple stain for the cultures that grew on the
MSA. The observed morphology of the
bacteria was no different from what we had
seen earlier, they remained bacillus rods,
thus we ruled out the possibility of
contamination by staphylococcus. The
presence of a halotolerant Bacillus ssp. can
be attributed to the fact that the natural
habitat of the host blue crab is in salt water
from the ocean. The bacteria which live
naturally in its host, or invaded it, are
adapted to the marine environment, having
halotolerance as a requisite characteristic
necessary for survival.
2 of the 10 pure cultures isolated (cultures 4
and 8) were gram (+), catalase positive,
coccus-shaped bacteria capable of growth on
MSA [image 7]. They did not ferment
mannitol, thus ruling out S. aureus as a
possible identity. However, culture 4 had a
yellow pigment, which is characteristic of
Micrococcus ssp., according to Bergey’s
Manual of Determinative Bacteriology [7].
Culture 7 displayed streptococcus
morphology and was negative for catalase.
However, it failed to grow on bile esculin
medium, and no blood agar was available to
carry out further tests.
Culture 5, 6, and 9 demonstrated small-rod
shape cells, and their Gram stains were
inconclusive despite repeated attempts.
Their growth on crystal violet suggests that
they are gram (--), however, only culture 6
grew on eosin methylene blue agar, which
differential medium is inhibitory towards
gram (+); thus, although culture 6 may be
classified as gram (--), cultures 5 and 9
remain inconclusive. All three cultures were
tested for lactose fermentation and were
ruled out as possible coliform bacteria;
culture 6 fermented lactose without
production of gas, while cultures 5 and 9
were unable to ferment lactose [table 1].
Phase 2
As table 5 demonstrates, the pour plates
generated hardly any growth, far too little
for any viable cell count. To account for this
absence of colony growth, it is important to
note significant differences between phase 1
and 2. Whereas in phase 1, we tested the
hemolymph of 2 males and 2 females from 2
different sites, in phase 2 we only tested
females from one site. It is particularly
evident that the males in phase 1 produced
the greatest number of colonies, particularly
the male from Caño Tiburones [table 3],
whereas the two females generating only a
few colonies more than those tested in phase
2. A final distinction is that the specimens
tested in phase 2 were conspicuously smaller
in size compared to those in phase 1.

Tables and Images
Table 1
Morphological and Physiological Properties
Culture
Sex/Site
Cellular
morpho-
logy
Gram
Catalase
Endo-
spore
forma-
tion
Mannitol
Salt Agar
7.5% NaCl
Eosin
methy-
lene
blue
Lac-
tose
Ferm
enta-
tion
gr/gas
Cryst
al
violet
Bile
Escu-
lin
1
M – CT
Strepto-
bacillus
+ +
Growth;
Mannitol
fermentation
2
M – CT
Strepto-
bacillus
+ +
Growth;
Mannitol
fermentation
3
M – CT
Strepto-
bacillus
+ +
Growth;
Mannitol
fermentation
4
F – I
Coccus + +
Growth;
No
fermentation
5
F – I
Small-
short
bacillus
N/A No
growth
--/-- Pale
pink
6
F – CT
Small-
short
bacillus
N/A Tan
color
--/+ Tan
color
7
F – CT
Strepto-
coccus
+ -- --
8
F – I
Staphylo
coccus
+ +
Growth;
No
fermentation
9
M – I
Small-
short
bacillus
N/A
No
growth --/--
Soft
violet
10
M – I
Strepto-
bacillus
+ +
Growth;
Mannitol
fermentation
M / male; F / female; CT / Caño Tiburones; I / Islote; + / positive; -- / negative
N/A – non applicable: Gram stains for cultures 5, 6, and 9 were inconclusive despite
repeated attempts.

Table 2
Specimen Characteristics
Specimen
(Callinectes
sapidus)
Site Sex Carapace width Mass
1 Caño Tiburones Male 13.0 cm 168.0 g
2 Caño Tiburones Female 14.5 cm 180.0 g
3 Islote Female 13.8 cm 156.0 g
4 Islote Male 13.6 cm 232.0 g
Table 3
Phase 1: Colony count for series dilution with dilution factors of 4 through 7
Specimen 10-4
10-5
10-6
10-7
1 11 8 TNTC TNTC
2 1 0 0 1
3 1 1 0 2
4 1 0 9 3
Table 4
Ingredients for citrate/EDTA buffer
Substances Molar concentration Calculated mass Measured mass
NaCl ………………...
D-glucose …………...
Trisodium citrate ........
Citric acid ……….…..
EDTA ……………….
(Ethylenediaminetetra
acetic acid)
……... 0.14 M
……... 0.10 M
……... 0.030 M
……... 0.026 M
……... 0.010 M
……… 3.2726 g
……… 7.2072 g
……… 3.0967 g
……… 1.9983 g
……… 1.4890 g
……. 3.27262 g
……. 7.19840 g
……. 3.09650 g
……. 1.99146 g
……. 1.48931 g
All substances were dissolved in 400 ml of distilled water; the solution was sterilized in
the autoclave at 121 °C at 15 psi.
Table 5
Phase 2 colony counts for serial dilutions with dilution factors of 4 through 7
Specimen 10-4
10-5
10-6
10-7
1 1 0 0 0
2 1 0 1 0
3 0 0 0 0
All specimens were collected from the Islote site; all were female; all were noticeably
younger and smaller than the earlier specimens collected during phase 1.

Images
Photos taken during phase 1 and 2
Image 1 Image 2
Image 3 Image 4
Image 5 Image 6
Image 7 Image 8

It should also be brought to attention the fact
that the nutrient media (i.e. PCA, TSA) we
used in growing the bacteria may not have
all the requisite nutrients necessary for
sustaining growth for the bacteria associated
with the blue crab hemolymph. In the work
by Corwell, Wicks and Tubiash [2],
bacterial counts for the hemolymph of
collected specimens were higher when using
MSYE growth media, which contains the
major ionic constituents of seawater, than
when using SMA, the Standard Methods
Agar which is comparable to the TSA and
PCA commonly used here. In other words,
our choice of growth media may be
inadequate to maintain the growth of
bacteria collected from the hemolympth, and
thus our plate counts may be biased against
more fastidious marine microorganisms.
Conclusions
The tests and results here are not enough to
prove or disprove our hypothesis. Sample
numbers were too small and furthermore the
testing procedure itself is experimental and
under development. We have, however,
demonstrated that the hemolympth of the
blue crab is not aseptic and can hold a
notable density of bacterial cells [table 3],
and that the bacterial flora can be diverse
[table 1]. Furthermore, the specimen with
the highest density of bacteria in the
hemolymph was isolated from Caño
Tiburones.
It is recommended that we perfect the
procedure for counting cells in the
hemolymph with further testing. For
instance, we can attempt to grow E. coli on
growth media supplemented with the
citrate/EDTA buffer, and compare its
growth to a control group, E. coli on growth
media not supplemented with the buffer.
This simple test would determine whether or
not the buffer in any way inhibits cell
growth. Another recommended step would
be to more carefully control any
confounding factors, such as differences in
the weight, age, sex, size and health of the
specimens.
Future research on this project bares the
promise of demonstrating to others the
harmful impact that pollution can have on
the native flora of the wetlands of Caño
Tiburones, and the potential health risks it
signifies towards those humans who fish and
consume blue crabs which inhabit the same
contaminated waters.
References
[1] Rivera, A., Santiago, K., Torres, J.,
Sastre M.P., Fuentes Rivera, F., 1999.
Bacteria associated with hemolymph in the
crab Callinectes bocourti in Puerto Rico.
Bulletin Marine Science, Vol. 64, No. 3, p.
543 – 548.
[2] Colwell, R.R., Sizemore, R.K., Tubiash,
H.S., Lovelace, T.E., 1974. Bacterial flora of
the hemolymph of the blue crab, Callinectes
sapidus: numerical taxonomy. Applied
Microbiology, Vol. 29, No. 3, p. 393-399.
[3] Colwell, R.R., Tubiash, H.S., Wicks,
T.C., 1975. A comparative study of the
bacterial flora of the hemolymph of
Callinectes sapidus. Marine Fisheries
Review, Vol. 37, Nos. 5-6
[4] Givens, C.E., Burnett, K.G., Burnett,
L.E., Hollibaugh, J.T., 2013. Microbial
communities of the carapace, gut, and
hemolympth of the Atlantic blue crab,
Callinectes sapidus. Spinger-Verlag Berlin
Heidelberg, DOI 10.1007/s00227-013-2275-
8.
[5] Estado libre asociado de Puerto Rico,
Departamento de recursos naturales y
ambientales (2007). Hojas de nuestro
ambiente: reserva natural caño tiburones.
San Juan, Puerto Rico.
[6] Cappuccino, J.G., Sherman, N., (2011).
Microbiology: a laboratory manual. Pearson
Benjamin Cummings, San Francisco, United
States of America.
[7] Bergey’s manual of determinative
bacteriology: identification flow charts.
Accessed 30, Nov 2014.
http://www.uiweb.uidaho.edu/micro_biolog
y/250/IDFlowcharts.pdf

Blue crab hemolymph report, Dec 2014

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