The authors thank Mattias Gyllborg for squid facility support and for comments on this manuscript, Michael Hadfield and the Kewalo Marine Laboratory for assistance during field collection, and members of the Ruby and McFall-Ngai Laboratory for contributions to this protocol. Work in the Mandel Laboratory is supported by NSF IOS-0843633.

1. Preparation of Bacterial Inocula
<ol>
	<li>Day 0 
	Two days prior to squid inoculation, plate the relevant bacterial strains on LBS13 agar.</li>
	<li>Incubate bacteria at 25-28&deg;C overnight.</li>
	<li>Day 1 
	Inoculate 3 ml LBS medium in a glass culture tube with one colony of each V. fischeri strain for infection. Prepare duplicate tubes as backup.</li>
	<li>Day 2 
	(Coordinate bacterial steps 1.4-1.6 with squid...

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+normal+bacterial+flora+of+the+oral+cavity.">Defining the normal bacterial flora of the oral cavity.</a> J. Clin. Microbiol. 43, 5721-5732 (2005).</li><li>Mandel, M. J., Wollenberg, M. S., Stabb, E. V., Visick, K. L., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+regulatory+gene+is+sufficient+to+alter+bacterial+host+range.">A single regulatory gene is sufficient to alter bacterial host range.</a> Nature. 458, 215-218 (2009).</li><li>Grice, E. A., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin+microbiome.">The skin microbiome.</a> Nat. Rev. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winnowing:+establishing+the+squid-Vibrio+symbiosis.">The winnowing: establishing the squid-Vibrio symbiosis.</a> Nat. Rev. Microbiol. 2, 632-642 (2004).</li><li>Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+a+cooperative,+bacterial-animal+association:+the+Vibrio+fischeri-Euprymna+scolopes+light+organ+symbiosis.">Lessons from a cooperative, bacterial-animal association: the Vibrio fischeri-Euprymna scolopes light organ symbiosis.</a> Annu. Rev. Microbiol. 50, 591-624 (1996).</li><li>McFall-Ngai, M. J., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiont+recognition+and+subsequent+morphogenesis+as+early+events+in+an+animal-bacterial+mutualism.">Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism.</a> Science. 254, 1491-1494 (1991).</li><li>Jones, B., Nishiguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterillumination+in+the+Hawaiian+bobtail+squid,+Euprymna+scolopes+Berry+(Mollusca:+Cephalopoda).">Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda).</a> Marine Biology. 144, 1151-1155 (2004).</li><li>Graf, J., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+amino+acids+support+the+proliferation+of+symbiotic+bacteria.">Host-derived amino acids support the proliferation of symbiotic bacteria.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 1818-1822 (1998).</li><li>Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+squid+that+glows+in+the+night:+development+of+an+animal-bacterial+mutualism.">A squid that glows in the night: development of an animal-bacterial mutualism.</a> J. Bacteriol. 174, 4865-4870 (1992).</li><li>Lee, P. N., McFall-Ngai, M. J., Callaerts, P., de Couet, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hawaiian+bobtail+squid+(Euprymna+scolopes):+a+model+to+study+the+molecular+basis+of+eukaryote-prokaryote+mutualism+and+the+development+and+evolution+of+morphological+novelties+in+cephalopods.">The Hawaiian bobtail squid (Euprymna scolopes): a model to study the molecular basis of eukaryote-prokaryote mutualism and the development and evolution of morphological novelties in cephalopods.</a> Cold Spring Harbor Protocols. , (2009).</li><li>Stabb, E., Visick, K., Millikan, D., Corcoran, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vibrio+fischeri-Euprymna+scolopes+symbiosis:+a+model+marine+animal-bacteria+interaction.">The Vibrio fischeri-Euprymna scolopes symbiosis: a model marine animal-bacteria interaction.</a> Recent Advances in Marine Science and Technology. , (2001).</li><li>Boettcher, K. 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The colonization assay described allows for analysis of a natural symbiotic process in a controlled laboratory environment. As such, it can be used to assess colonization by mutant strains, by different natural isolates, and under different chemical regimes. Variations on the experiments described are commonly used to assess different aspects of the symbiosis. The kinetics of colonization can be measured by examining luminescence during the first 24 h, which can be detected automatically in a scintillation counter in whi...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Glass Culture Tubes, 16 mm Diameter</td>
			<td>VWR</td>
			<td>47729-580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Caps for Glass Culture Tubes</td>
			<td>Fisher</td>
			<td>NC9807998</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Visible Spectrophotometer for Determination of OD600</td>
			<td>Biowave</td>
			<td>CO8000</td>
			<td>Any spectrophotometer capable of measuring OD600 will work. This unit can measure the OD600 of liquid directly in the glass culture tubes. Some adjustment of the inoculum calculation may be necessary depending on the instrument used.</td>
		</tr>
		<tr>
			<td>GloMax 20/20 Single-Tube Luminometer</td>
			<td>Promega</td>
			<td>E5311</td>
			<td>Equivalent to the Turner BioSystems 20/20n Luminometer. Includes the microcentrifuge tube holder.</td>
		</tr>
		<tr>
			<td>GloMax 20/20 Light Standard</td>
			<td>Promega</td>
			<td>E5341</td>
			<td>For luminometer calibration.</td>
		</tr>
		<tr>
			<td>Refractometer, Handheld</td>
			<td>Foster and Smith Aquatics</td>
			<td>CD-14035</td>
			<td>Calibrate before each use with deionized water. Rinse after every use with deionized water to prevent salt build-up.</td>
		</tr>
		<tr>
			<td>Instant Ocean (artificial seawater concentrate)</td>
			<td>Foster &amp; Smith Aquatics</td>
			<td>CD-16881</td>
			<td>Prepare at 35 &permil; in deionized water, using the refractometer, then filter through a 0.2 &mu;m SFCA filter.</td>
		</tr>
		<tr>
			<td>Filtration Unit</td>
			<td>Nalgene</td>
			<td>158-0020</td>
			<td>Surfactant-free cellulose acetate (SFCA) membrane, 0.2 &mu;m. We have observed variable results with some surfactant-containing PES filters.</td>
		</tr>
		<tr>
			<td>Transfer Pipettes</td>
			<td>Fisher</td>
			<td>13-711-9AM</td>
			<td>Using scissors or razor blade, cut the tip cleanly above the first ridge to increase the diameter of the pipette tip and avoid squeezing the squid hatchlings.</td>
		</tr>
		<tr>
			<td>Disposable Sample Bowls (plastic tumblers)</td>
			<td>Comet</td>
			<td>T9S (9 oz.)</td>
			<td>Bowls for inoculation, with upper diameter 3 &frac14;", lower diameter 2 &frac14;", height 3". Bowls create a homogenous environment as they have no bottom rim, in which squid can get trapped in a low-oxygen niche. The size is optimized for 40-ml inoculum. Available at webstaurantstore.com, #619PI9.</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>VWR</td>
			<td>89092-720</td>
			<td>Vial diameter matches the opening on the luminometer PMT.</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>ISC Bioexpress</td>
			<td>C-3217-1CS</td>
			<td>Tubes must fit the shape of the pestles.</td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof</td>
			<td>Fisher</td>
			<td>BP2818-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Kimble Chase/Kontes</td>
			<td>749521-1500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plating Beads, 5 mm diameter</td>
			<td>Kimble Chase</td>
			<td>13500 5</td>
			<td>Prepare 5 per tube and autoclave.</td>
		</tr>
	</tbody>
</table>

colonization of euprymna scolopes squid by vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

colonization-of-euprymna-scolopes-squid-by-vibrio-fischeri

Research

JoVE Journal

Immunology and Infection

19.7K visualizações.  Northwestern University. O método descreve o procedimento pelo qual o squid bobtail havaiana, Euprymna scolopes E seu simbionte bacteriano, Vibrio fischeri, São levantadas separadamente e, em seguida, introduzido para permitir a colonização específica do órgão luz lula pelas bactérias. Detecção de colonização por bactérias luminescência e derivados por meio de contagem direta colônia são descritas.