The Isolation and Characterization of Vibrio harveyi from Oysters
Patrick Kudyba and Shaker Abdullatif Microbiology 421W, Section 002
Microbiology dept. Pennsylvania State University
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Abstract Bioluminescent bacteria can be found in marine ecosystems and can be isolated and characterized within the lab. Vibrio harveyi was the prime candidate to isolate and characterize a notable species that could be differentiated from other bacteria within marine wildlife. The purpose of the lab was to isolate and characterize bioluminescent bacteria and V. harveyi was the most likely choice because of ease of access and is one of the most common bioluminescent bacterial strains known. The results of the experiment were all negative. There was no sign of bioluminescent bacteria in any of the oyster samples that were used. In conclusion it is possible that oysters may carry V. harveyi but not ones that were bought through a fish market within a grocery store. There are better ways to carry out the experiment and possibly get better results but due to time and location restraints the results ended up negative.
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Introduction
The purpose of this experiment is to find and isolate cultures of bioluminescent bacteria. The bacteria chosen to find was Vibrio harveyi. V. harveyi is a bioluminescent bacteria that is found most notably in a marine environment. It can be found free floating or associated living in the gut micro flora of marine animals (1). Oysters were used in this experiment to look for V. harveyi living symbiotically within the oysters’ gut. After isolating colonies, they must be able to be identified. Aside from being bioluminescent and displaying a blue glow, V. harveyi are gram negative, rod shaped ranging from 0.5 microns to 2 microns long, and are motile possessing flagellum organelles (2). Additionally, they are halophilic and facultative anaerobes (3). All of these characteristics could be used to determine if the bacteria isolated is V. harveyi or not. The hypothesis of this experiment is that there are V. harveyi present in oysters that can be isolated and characterized.
V. harveyi produces an interspecial autoinducer AI-2, which could have a major role in biotechnology. This autoinducer could be used to produce synthetic inhibitors and lead to the development of novel antimicrobials (4). This is the topic of many research projects and is being looked into today to help make the medicine of tomorrow.
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Materials and Methods
Media
Marine Broth 2216 by Difco was used to make large cultures of bacteria from the oysters, that we could get isolates from. Marine agar of Fermtech Agar was used for plating cultures and getting single colonies.
Isolation of Vibrio harveyi
Method 1
Two sets of marine broth in 250ml quantities was prepared to grow bacteria in. 2 Oysters were collected and shucked using aseptic technique and chopped using sterile scalpels. The chopped oysters were then mixed with 50 ml of marine broth each. Each was mixed and the supernatants were decanted into the separate larger volumes of broth adding up to 250ml per oyster. The 2 samples were then incubated at 26oC for 48 hours. Each oyster broth was plated on
4 plates of marine agar via streaking method. The two sets of 4 plates were incubated at 26oC for
24 hours to get single colonies. 2 more sets of marine broth were prepared, 1 liter per flask. Each flask was inoculated with a single colony from the plates and left to incubate for 24 hours at
26oC. They were checked for growth and luminescence, and if none occurs allow to incubate for an additional 24 hours at 26oC.
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Method 2
Prepared 1 liter of marine broth split into 4 separate flasks of 250ml. 4 Oysters were collected and shucked using aseptic technique and chopped using sterile scalpels. The chopped oysters were each mixed with 50 ml of marine broth and then mixed. Each was then decanted into their own separate flasks of marine broth adding up to 250ml each. The broths were incubated at 26oC for 48 hours. Samples from each broth were streaked onto marine agar plates. 4 plates were used per oyster, and they were incubated for 48 hours at 26oC checking for luminescence every 24 hours.
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Results
Method 1 was used with 2 oysters that were obtained by Dr. Broussard after an in class experiment involving oysters. After the inoculation of the oysters the broth became slightly turbid. After the incubation period the solutions were extremely cloudy and clear growth was seen. However, when put in a dark room, no luminescence was seen. The streaked plates that came from the broths had normal growth of a white bacteria in clear colonies. After the 24 hour incubation of the plates, 2 colonies were seen on the plate; one white and one yellow. Each species of bacteria was inoculated into one liter of marine broth each. After 24 hours of incubation there was no light emitted and after an additional 24 hours of incubation still no luminescence. Had luminescence occurred in the broths one would have seen a similar phenomenon as seen in figure 1.
Method 2 was used on 4 oysters obtained from Wegmans fresh fish market. One oyster still had a baby crab partially digested inside it. After shucking and inoculating the broths with the oysters, the solution became turbid. Due to time restraints the broth solutions had to be kept at 10oC over the weekend before being able to incubate at 26oC. After incubation the oyster broth that contained the baby crab had turned black when swirled around. The other 3 remained the turbid yellow/grey color as usual. After streaking plates the flasks of broth were kept at 4oC. The streaked plates bore the same white colonies as the previous method but no yellow colonies formed this time. Also when placed in a dark room, no luminescence was seen either. Had the colonies possessed the capabilities to luminesce, one would have observed something similar to what can be seen in figure 2.
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Discussion
The purpose of this experiment was to isolate and characterize bioluminescent bacteria.
This purpose was not accomplished within the confines of this experiment. The results were all negative when looking for signs of bioluminescence from the bacteria that were isolated and grown from the oyster samples.
A good reason that we did not find any signs of vibrio harveyi is that they pose a serious threat to shrimp and marine life farmers. Vibrio harveyi can be pathogenic depending on their concentration, they can cause luminous vibriosis. “Luminous vibriosis is a leading cause of death among commercially farmed shrimp and other aquaculture” (5). Due to this it can be reasoned that farmers who sell to commercial businesses would want to limit this and work to eradicate a
V. harveyi colonies in their fish farms.
What should have been seen was a slight blue glow from the collection of plates or from the flasks of broth that were inoculated. It should be stated that the broths may have been incubated too long and so the cells began to die out before we could see the luminescence. If we had been able to see the glow the characterization would have relied on a microscopy where the bacteria would be examined to see if it followed the known characteristics of V. harveyi. One would look for gram negative rod shaped bacteria with flagellum about 1 micron long. That would have been enough evidence to support the hypothesis that V. harveyi, a bioluminescent bacteria, could be found and characterized in oysters.
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In conclusion a better way to go about this experiment would be to get samples from the bacteria’s main habitat, tropical waters, and marine wildlife. And to take fresher samples not from a farm but from the wild. Also it would be a better possibility to find it when looking into some of its symbiotic partners in the wild, such as squid. Although the experiment did not yield any positive results you cannot conclude that V. harveyi do not live in oysters. There are many factors that were not able to be controlled and obtaining a good sample of possible hosts proved to be the most difficult aspect of the experiment. Given better accessibility to probable hosts or habitats of bioluminescent bacteria this experiment might have different results.
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Legends of Figures
Figure 1
Figure 1 shows a side by side comparison of the same marine broth in the light or in a dark room. In the first picture here is just a broth of a brown color in a flask, while the second picture shows the same flask but the broth glows in a green blue color in the flask surrounded by darkness. This shows the luminescent properties of the Vibrio harveyi that were growing in the broth.
http://www.biology.pl/bakterie_sw/bac_hp_en.html
Figure 2
The figure shows a side by side comparison of the same plate of Vibrio harveyi in the light or in a dark room. The bug is grown on marine agar, and presents itself in the first picture as single white colored colonies. In the second picture the same plate is in a dark room and the single colonies are glowing with a green blue color.
http://www2.fiu.edu/~makemson/
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Figures
Figure 1
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Figure 2
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References
1. Showalter, R.E., Martin, M.O., and Silverman, M.R. Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi. J. Bacteriol. 1990. 172: 2946–2954. 2. "Genome: Vibrio Harveyi." The Genome Institute at Washington University. Washington University in St. Louis School of Medicine, n.d. Web. 20 Nov. 2014.
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