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 steps 3.7-3.10) 
	1 h prior to inoculation, subculture bacteria 1:80 (37.5 &mu;l) into 3 ml LBS in a glass culture tube and grow for 1 h with aeration.</li>
	<li>Measure the OD600 of the sample prior to inoculation. Typical measurements are 0.3-0.6 depending on the strain.</li>
	<li>For a target inoculum of 3-5 x 103 CFU/ml calculate the inoculum volume as follows: Inoculum volume (&mu;l) = 1.25 / OD600 (e.g., For OD600 = 0.5, the calculated inoculum volume = 1.25/0.5 = 2.5 &mu;l).This amount is added directly to the seawater containing squid in Step 4.1. This calculation may need to be adjusted for different strains of V. fischeri or for inoculation at lower or higher levels than those specified here.</li>
</ol>
2. Preparation of Agar Plates for Enumeration of the Inocula
<ol>
	<li>For each treatment, label LBS plates (2 per treatment) to plate samples of the inoculum in Step 4.1.</li>
	<li>Add 5 sterile plating beads per plate.</li>
</ol>
3. Collection of Squid Juveniles
<ol>
	<li>Measure the salinity of Instant Ocean using the refractometer and adjust to 35&permil;.</li>
	<li>Filter 1 L of Instant Ocean using the filtration unit and an attached vacuum line or vacuum pump, to generate filter-sterilized Instant Ocean (FSIO). Oxygenate the water by swirling vigorously prior to each dispensing. The filter unit can be reused for 2 days.</li>
	<li>Aliquot 40-50 ml of FSIO into each of two (2) disposable sample bowls. Label one as earlies and one as timelies.</li>
	<li>Prepare an excess of plastic transfer pipettes for acquiring juvenile squid by cutting the pipette approximately 1 cm from the tip, above the lowest ridges (see Figure 3). This facilitates a wider area through which the squid can pass upon collection. Discard any transfer pipettes in which there is a rough exposed surface.</li>
	<li>Using prepared transfer pipettes, collect E. scolopes that hatched overnight and transfer to the earlies bowl of FSIO. Early hatchlings have been in the egg system for over 1 h and are susceptible to colonization by contaminating V. fischeri in the egg system. Do not use earlies for sensitive colonization experiments.</li>
	<li>Check egg tanks every 30-45 min for new hatchlings. Ensure that all hatchlings are cleared during each check. Remove hatchlings with a transfer pipette, and deposit into the timelies bowl of FSIO. Animals collected in a timely fashion are available for colonization experiments.</li>
	<li>When the collection has finished (~ 45 min after dusk), transfer the squid to the main laboratory. Empirically it is advantageous to colonize the animals under uninterrupted laboratory light conditions for 3 h inoculations.</li>
	<li>For each treatment, prepare a bowl with 40 ml FSIO. Add squid to the bowls for the assay (maximum n=40 per bowl).</li>
	<li>Prepare an additional bowl as an aposymbiotic (negative) control.</li>
	<li>Prepare a dedicated transfer pipette for each treatment.</li>
	<li>Euthanize extra squid in 2% ethanol.</li>
</ol>
4. Squid Colonization
<ol>
	<li>Day 2 - Using a P10 Pipetman, dispense the calculated aliquot of bacteria (Step 1.5) into each squid bowl (Step 3.8) for each treatment. Start a 3 h timer immediately after the first inoculation.</li>
	<li>For each treatment, create a "vortex" in the bowl with the dedicated transfer pipette by placing the pipette near the edge of the bowl and pipetting up and down repeatedly to mix the water and squid for approximately 10 sec. Thorough mixing is critical.</li>
	<li>Plate 50 &mu;l from each bowl onto an LBS agar plate from Step 2.2 (for technical replicates, plate two 50 &mu;l plates per treatment). Incubate at 25-28&deg;C overnight.</li>
	<li>Prepare wash bowls (100 ml FSIO/ea) for each treatment.</li>
	<li>Prepare Drosophila vials (4 ml FSIO/ea) for each squid.</li>
	<li>After exactly 3 h, transfer the squid to their respective wash bowls (complete for all treatment). This stops the inoculation.</li>
	<li>Proceed to transfer each individual squid to its own Drosophila vial with FSIO. Use a designated transfer pipette for each treatment.</li>
	<li>Move trays of Drosophila vials to the squid facility to return to the day/night light cycle the animals experienced during embryogenesis.</li>
	<li>Day 3 - Prepare Drosophila vials (4 ml FSIO/ea) for each squid.</li>
	<li>Prior to dusk at 22-24 h post-inoculation, transfer each squid to a new Drosophila vial. Use a designated transfer pipette for each treatment.</li>
	<li>Day 4 - Prepare labelled 1.5 ml microcentrifuge tubes (1/squid).</li>
	<li>Prior to dusk at 46-48 h post-inoculation, measure and record the luminescence of each squid in the Drosophila vial (luminometer set for 6 s integration and auto-read on lid closure).</li>
	<li>As a negative control for background luminescence, measure a vial with FSIO that does not contain any squid.</li>
	<li>Transfer each squid in a volume of approximately 700 &mu;l to a 1.5 ml microcentrifuge tube from Step 4.12. Move to a cardboard freezer box. Once the lid is placed on the box, do not remove it as the light cues for bacteria expulsion are not well-understood.</li>
	<li>Freeze microcentrifuge tubes at -80&deg;C overnight.</li>
</ol>
5. Determination of Colonization Levels
<ol>
	<li>For each squid, prepare two (2) microcentrifuge tubes, each with 475 &mu;l FSIO (or autoclaved 70% Instant Ocean).</li>
	<li>Prepare pestles by first using a Kimwipe to clean the pestle and remove gross debris and/or tissue.</li>
	<li>Place pestles tip-down in a 50 ml beaker containing 95% ethanol. Ethanol should be added to a height of approximately 3 cm.</li>
	<li>For each pestle, remove from the beaker and wipe the tip with a Kimwipe.</li>
	<li>Dip pestle back into the ethanol bath, remove and insert (tip up) into an microcentrifuge tube rack and allow to air dry completely for approximately 15 minutes.</li>
	<li>Thaw squid in a microcentrifuge tube rack (maximum n=8).</li>
	<li>If necessary, adjust the volume to 700 &mu;l.</li>
	<li>Using a pestle from Step 5.5, disrupt the animal tissue until the ink sac ruptures (the water will turn a murky grey color).</li>
	<li>Remove the pestle and ensure all tissue remains in the tube.</li>
	<li>Vortex the tissue briefly for exactly 10 seconds (use a timer).</li>
	<li>Allow the tissue to rest for 10 min. The tissue will settle and the bacteria and ink remain in solution. For the calculations that follow, the bacteria/ink solution is the [A] dilution (i.e. the E. scolopes light organ homogenate in 700 &mu;l). Serial 1:20 dilutions ([B], [C]) are described below.</li>
	<li>For the [B] dilution, add 25 &mu;l [A] to one of the microcentrifuge tubes prepared in Step 5.1. Vortex.</li>
	<li>For the [C] dilution, add 25 &mu;l [B] to one of the microcentrifuge tubes prepared in Step 5.1. Vortex.</li>
	<li>Plate 50 &mu;l of each dilution onto LBS agar, 2 replicates per treatment.</li>
	<li>Incubate the plates at 25-28&deg;C overnight.</li>
</ol>
6. Data Analysis
<ol>
	<li>To calculate the CFU/light organ (LO), count colonies on the plate for each treatment in which 10-400 colonies are present, and use the appropriate formula: 
	CFU/LO = (colonies on [A] plate) x 14; or 
	CFU/LO = (colonies on [B] plate) x 280; or 
	CFU/LO = (colonies on [C] plate) x 5600.</li>
	<li>Plot individual data points and medians on a logarithmic scale.</li>
	<li>The data are often not normally distributed, with different variances, and the outliers may contain biologically meaningfully information. Therefore, non-parametric tests provide a useful method to determine whether the treatments differ significantly.</li>
	<li>Use GraphPad Prism software for statistical analysis. For two treatments, use the Wilcoxon Rank Sum test. For comparisons among greater than two treatments, use the Kruskal-Wallis test with appropriate post-tests.</li>
</ol>
7. Representative Results
Results from a sample colonization assay are shown in Figure 4. Two strains of V. fischeri that exhibit different relative levels of luminescence were each inoculated into six squid, along with six squid that served as aposymbiotic (uncolonized) controls. The E. scolopes symbiont, ES11414, and the brighter Sepiola robusta symbiont, SR515,16. Similar inoculum levels (Fig. 4A) lead to 100% colonization within 3 h. At 48 h, the luminescence levels (Fig. 4B) and CFU counts (Fig. 4C) were determined to assess the colonization proficiency of the strain. Determination of the specific luminescence (Fig. 4D; per bacterium) allows for determination of the brightness of each bacterial strain during the symbiosis.
<img alt="Figure 1" src="/files/ftp_upload/3758/3758fig1.jpg" /> 
Figure 1. Flow chart of the colonization procedure. Bacteria and squid are harvested separately, then mixed at the specified inoculum. Squid are washed, then transferred to new water at 3 h, 24 h, and 48 h post inoculation. At 48 h the luminescence is measured and the animals are frozen, which serves to surface-sterilize the animals. Light-organ colonized bacteria remain viable at -80&deg;C through one thaw (no additional freeze-thaw cycles).
<img alt="Figure 2" src="/files/ftp_upload/3758/3758fig2.jpg" /> 
Figure 2. Flow chart illustrating homogenization and dilution plating of the bacteria. Serial 20-fold dilutions provide the appropriate dynamic range for enumeration of colonized bacteria.
<img alt="Figure 3" src="/files/ftp_upload/3758/3758fig3.jpg" /> 
Figure 3. Transfer pipettes with an appropriately narrow shaft taper to a narrow bore (A) that would damage juvenile squid. Pretreatment by cutting off the narrowest section with scissors or a razor blade yields an appropriate tool (B) for transferring juvenile squid.
<img alt="Figure 4" src="/files/ftp_upload/3758/3758fig4.jpg" /> 
Figure 4. Sample data for a colonization assay. (A) Levels of the bacteria in the inoculum bowls. (B) Luminescence of individual squid. (C) Colony counts of individual squid. (D) Specific luminescence of individual squid. Apo, Aposymbiotic (uncolonized negative control).

<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. Microbiol. 9, 244-253 (2011).</li><li>Malic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+specific+bacteria+in+wound+biofilms+using+peptide+nucleic+acid+fluorescent+in+situ+hybridization+(PNA+FISH).">Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).</a> Microbiology. 155, 2603-2611 (2009).</li><li>Turnbaugh, P. 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+human+microbiome+project.">The human microbiome project.</a> Nature. 449, 804-810 (2007).</li><li>Nyholm, S. V., 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=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. 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=Depressed+light+emission+by+symbiotic+Vibrio+fischeri+of+the+sepiolid+squid+Euprymna+scolopes.">Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes.</a> J. Bacteriol. 172, 3701-3706 (1990).</li><li>Fidopiastis, P. M., von Boletzky, S., 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+new+niche+for+Vibrio+logei,+the+predominant+light+organ+symbiont+of+squids+in+the+genus+Sepiola.">A new niche for Vibrio logei, the predominant light organ symbiont of squids in the genus Sepiola.</a> J. Bacteriol. 180, 59-64 (1998).</li><li>Bose, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+rapid+evolution+of+the+luxR-luxI+intergenic+region+to+the+diverse+bioluminescence+outputs+of+Vibrio+fischeri+strains+isolated+from+different+environments.">Contribution of rapid evolution of the luxR-luxI intergenic region to the diverse bioluminescence outputs of Vibrio fischeri strains isolated from different environments.</a> Appl. Environ. Microbiol. 77, 2445-2457 (2011).</li></ol>

The authors have nothing to disclose.

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 ...

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Immunology and Infection

19.8K Views.  Northwestern University. 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.

Video: Colonization of Euprymna scolopes Squid by Vibrio fischeri

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

TransFLP &amp;#x2014; A Method to Genetically Modify &lt;em&gt;Vibrio cholerae&lt;/em&gt; Based on Natural Transformation and FLP-recombination

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...