The authors wish to thank Anne Tanner, Bruce Paster, Heike Boisvert, Xuesong He and Batbileg Bor for helpful discussions and for providing bacterial strains. We thank Susan Yost and Jessica Woods for microbial technical assistance. Research reported in this publication was supported by The National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R37 DE016937 (FED), R01 DE024468 (FED) and T32 DE007327 (AJC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

When developing this protocol, IRB approval was sought and approved (#14-10) and informed consent was obtained from all subjects.
1. Preparation
<ol>
	<li>When working with human subjects, obtain necessary IRB approval and informed consent.</li>
	<li>Start cultures of host bacteria with enough time to grow to early stationary phase. For example, inoculate 2-5 mL of tryptic soy broth with 0.1% yeast extract added (TSBY)&#160;with Arachnia propionica and incubate at 37 &#176;C for 24 h.</li>
	<li>Assemble filter holders with track-etched 0.2 &#181;m filter membrane. Wrap the assembly in foil and sterilize by autoclaving.</li>
	<li>Sterilize centrifuge tubes and cap assemblies.</li>
</ol>
2. Obtain sample of oral bacteria
NOTE: While many oral samples contain Saccharibacteria (e.g., saliva, swabs of tonsils, tongue scrapings) dental plaque routinely is the most successful.
<ol>
	<li>Take a plaque scraping using a sterile paper point, Gracey curette, or, if self-sampling, use a sterile toothpick or pipette tip. Transfer plaque to a suitable buffer, such as maximal recovery diluent (MRD, 0.85% NaCl, 0.1% peptone) or PBS. If not processed immediately, keep the sample on ice until ready to proceed.</li>
	<li>Vigorously resuspend the dental plaque in MRD buffer using a combination of vortexing and pipetting with a small pipet tip.</li>
	<li>Add resuspended plaque sample to an additional 9 mL of MRD buffer.</li>
</ol>
3. Prepare filtrate from oral sample
<ol>
	<li>Using aseptic technique, unwrap a sterile filter assembly. Untwist a quarter turn and retighten the filter holder to be sure threads are properly engaged and the apparatus is closed properly. Using a syringe, wash the membrane by passing 10 mL of MRD buffer through the apparatus. Improper assembly is revealed at this step by fluid leaking out of the filter holder. If leakage occurs, obtain another sterile filter assembly and repeat the wash step.</li>
	<li>Apply the sample to the washed filter. Remove the plunger from a syringe and attach it to the filter apparatus. Pour the dispersed dental sample, now in 10 mL of MRD buffer, into a syringe and load it onto the filter.</li>
	<li>Place a sterile centrifuge tube beneath the filter apparatus to catch the filtrate, then apply light pressure to the plunger to push the sample through the filter.</li>
	<li>Repeat the procedure with another 10 mL of MRD buffer to wash the membrane, collecting the flow-through in the same tube as the filtered sample. Aseptically cap the tube.</li>
</ol>
4. Concentrate Saccharibacteria cells by centrifugation
<ol>
	<li>Make an orientation mark on the tube and cap and place the tube in a high-speed centrifuge with the mark on the upper side. The pellet formed from centrifugation is usually invisible. The marking will help determine where the pellet of Saccharibacteria cells is located when centrifugation is done and the tube removed from the centrifuge.</li>
	<li>Centrifuge the samples at 60,000 x g for 1 h at 4 &#176;C. 
	NOTE: This force and time is sufficient to pellet all Saccharibacteria cells. However, Saccharibacteria cells can be at least partially pelleted by centrifuging for as little as 20 min at 20,000 x g.</li>
	<li>Carefully remove the tubes from centrifuge. Pour out the liquid from the tube, keeping the pellet on the upper side of the tube.</li>
	<li>Resuspend the usually invisible pellet in 1-2 mL of MRD buffer by vigorous vortexing.</li>
</ol>
5. Infect host cultures with Saccharibacteria-enriched filtrate
<ol>
	<li>Prepare culture tubes by aliquoting 2 mL of appropriate growth media (e.g., TSBY, BHI, etc.) into tubes.</li>
	<li>Add 200 &#181;L of overnight culture of host organisms to each tube. Add 100-200 &#181;L of resuspended, filtered sample to each tube.</li>
	<li>Incubate combined samples as appropriate for the host organism (e.g., 37 &#176;C, in an aerobic atmosphere for A. propionica).</li>
	<li>Passage the cells every two to three days by transferring 200 &#181;L of binary culture to 2 mL of fresh growth medium in a new tube. If it appears that the passaged cultures show no growth (i.e., the turbidity/optical density of the culture doesn&#39;t increase after passage into fresh media) the Saccharibacteria could be overwhelming or killing all the host organism. To remedy this, add 200 &#181;L of uninfected host culture when passaging the cells. Repeat for at least 5 passages.</li>
</ol>
6. Confirm infection by PCR
<ol>
	<li>After 5 passages, confirm infection with PCR. Five passages will ensure than any non-growing Saccharibacteria cells have been depleted beyond the limit of detection using 25 cycles of PCR.</li>
	<li>Prepare a PCR mastermix. A suggested recipe using is as follows, for each tube needed, prepare 12.5 &#181;L 2x PCR buffer, 0.75 &#181;L 10 &#181;M Forward primer (580F12 - AYT GGG CGT AAA GAG TTG C), 0.75 &#181;L 10 &#181;M Reverse primer (1177R13- GAC CTG ACA TCA TCC CCT CCT TCC), 1 &#181;L 25 mM MgCl2 and 9 &#181;L water. Mix by vortexing.</li>
	<li>Aliquot 24 &#181;L of mastermix into a 0.2 mL PCR tube. Add 1 &#181;L of Saccharibacteria-infected culture to the PCR reaction. Place the tube in a thermocycler and perform the following protocol: Initial denaturation 95 &#176;C for 5 min, 25 cycles of 95 &#176;C for 30 s, 60 &#176;C for 30 s, 72 &#176;C for 30 s, final elongation at 72 &#176;C for 2 min, then final hold at 4 &#176;C.</li>
	<li>Load and run the PCR products in a 1% agarose gel. A band of ~600 bases will indicate the presence of Saccharibacteria.</li>
</ol>
7. Check for purity and remove contaminating organisms
<ol>
	<li>Confirm purity of positive cultures by plating the coculture of Saccharibacteria on nutrient agar sufficient for the growth of the host organism. Perform a 10-fold serial dilution of the culture in sterile buffer (e.g., MRD or PBS) and spread 100 &#181;L on an agar plate. Perform dilutions such that there will be approximately 20-200 colonies growing on the agar plate.</li>
	<li>Incubate the culture in appropriate growth conditions and observe for contaminating organisms. 
	NOTE: Axenic Saccharibacteria colonies have never been observed, so only the host organism is observed growing on these plates. More than one colony type usually indicates contamination. Should contamination occur, the culture should be re-filtered to remove the unwanted contaminants and re-inoculated onto fresh host.</li>
	<li>Occasionally a double infection occurs, where two Saccharibacteria species infect the same host. To separate the Saccharibacteria species, resuspend individual colonies in 20 &#181;L sterile PBS and use 1 &#181;L of suspension in a PCR reaction to detect colonies with Saccharibacteria infections.</li>
	<li>Transfer the suspensions that gave a positive result to growth medium to start a binary culture. The success rate will depend on the titer of each Saccharibacteria species in the culture. It may be necessary to screen more than 50 colonies to find an infected one for propagation.</li>
</ol>
8. Storage of cultures
<ol>
	<li>Grow a binary culture of sufficient volume (10-50 mL) overnight.</li>
	<li>Pellet cells by centrifugation (4,000 x g for 10 min). 
	NOTE: High-speed centrifugation is not necessary here as Saccharibacteria cells will be attached to the larger, heavier cells of the host and will pellet with them.</li>
	<li>Resuspend cells in cryoprotectant. Growth media supplemented with either 5% DMSO or 20% glycerol is usually sufficient. Ensure that the host organism is compatible with cryoprotectant media (e.g., Growth of A. propionica is inhibited by glycerol and frozen stocks may not revive). Test the viability of an uninfected host culture with cryoprotectant to ensure stocks can survive freezing.</li>
	<li>Resuspend cell pellet in the same volume of culture (10-50 mL). Aliquot 0.5 mL into labeled cryovials. Freeze cell cultures at -80 &#176;C.</li>
</ol>

<ol>
	<li>Stewart, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+unculturable+bacteria.">Growing unculturable bacteria.</a> Journal of Bacteriology. 194 (16), 4151-4160 (2012).</li><li>Bomar, L., Maltz, M., Colston, S., Graf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+culturing+of+microorganisms+using+metatranscriptomics.">Directed culturing of microorganisms using metatranscriptomics.</a> mBio. 2 (2), 00012 (2011).</li><li>Derrien, M., Vaughan, E. E., Plugge, C. M., de Vos, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akkermansia+muciniphila+gen.+nov.,+sp.+nov.,+a+human+intestinal+mucin-degrading+bacterium.">Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium.</a> International Journal of Systematic and Evolutionary Microbiology. 54, 1469-1476 (2004).</li><li>Nelson, M. C., Bomar, L., Maltz, M., Graf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucinivorans+hirudinis+gen.+nov.,+sp.+nov.,+an+anaerobic,+mucin-degrading+bacterium+isolated+from+the+digestive+tract+of+the+medicinal+leech+Hirudo+verbana.">Mucinivorans hirudinis gen. nov., sp. nov., an anaerobic, mucin-degrading bacterium isolated from the digestive tract of the medicinal leech Hirudo verbana.</a> International Journal of Systematic and Evolutionary Microbiology. 65, 990-995 (2015).</li><li>Scott, J. C., Klein, B. A., Duran-Pinedo, A., Hu, L., Duncan, 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+two-component+system+regulates+hemin+acquisition+in+porphyromonas+gingivalis.">A two-component system regulates hemin acquisition in porphyromonas gingivalis.</a> PLoS One. 8 (9), 0073351 (2013).</li><li>Martin, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+growth+media+for+oral+bacteria.">New growth media for oral bacteria.</a> Journal of Microbiological Methods. 153 (22), 10-13 (2018).</li><li>Vartoukian, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cultivation+of+'unculturable'+oral+bacteria,+facilitated+by+community+culture+and+media+supplementation+with+siderophores.">In vitro cultivation of 'unculturable' oral bacteria, facilitated by community culture and media supplementation with siderophores.</a> Plos One. 11 (1), 0146926 (2016).</li><li>Strandwitz, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA-modulating+bacteria+of+the+human+gut+microbiota.">GABA-modulating bacteria of the human gut microbiota.</a> Nature Microbiology. 4 (3), 396-403 (2019).</li><li>Hug, L. A., et al. <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+view+of+the+tree+of+life.">A new view of the tree of life.</a> Nature Microbiology. 1, 16048 (2016).</li><li>Castelle, C. J., Banfield, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+new+microbial+groups+expand+diversity+and+alter+our+understanding+of+the+tree+of+life.">Major new microbial groups expand diversity and alter our understanding of the tree of life.</a> Cell. 172 (6), 1181-1197 (2018).</li><li>He, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+a+human-associated+TM7+phylotype+reveals+a+reduced+genome+and+epibiotic+parasitic+lifestyle.">Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (1), 244-249 (2015).</li><li>Hugenholtz, P., Tyson, G., Webb, R. I., Wagner, A. M., Blackall, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+candidate+division+TM7,+a+recently+recognized+major+lineage+of+the+domain+Bacteria+with+no+known+pure-culture+representatives.">Investigation of candidate division TM7, a recently recognized major lineage of the domain Bacteria with no known pure-culture representatives.</a> Applied and Environmental Microbiology. 67 (1), 411-419 (2001).</li><li>Brinig, M., Lepp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+bacteria+of+division+TM7+in+human+subgingival+plaque+and+their+association+with+disease.">Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease.</a> Applied and Environmental Microbiology. 69 (3), 1687-1694 (2003).</li><li>Bor, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+obtained+by+culturing+saccharibacteria+with+their+bacterial+hosts.">Insights obtained by culturing saccharibacteria with their bacterial hosts.</a> Journal of Dental Research. , 689-694 (2020).</li><li>Bor, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+physiological+characterization+of+the+epibiotic+interaction+between+TM7x+and+its+basibiont+actinomyces.">Phenotypic and physiological characterization of the epibiotic interaction between TM7x and its basibiont actinomyces.</a> Microbial Ecology. 71 (1), 243-255 (2016).</li><li>Skopek, R. J., Liljemark, W. F., Bloomquist, C. G., Rudney, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+plaque+development+on+defined+streptococcal+surfaces.">Dental plaque development on defined streptococcal surfaces.</a> Oral Microbiology and Immunology. 8 (1), 16-23 (1993).</li><li>Murugkar, P. P., Collins, A. J., Chen, T., 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=Isolation+and+cultivation+of+candidate+phyla+radiation+Saccharibacteria+(TM7)+bacteria+in+coculture+with+bacterial+hosts.">Isolation and cultivation of candidate phyla radiation Saccharibacteria (TM7) bacteria in coculture with bacterial hosts.</a> Journal of Oral Microbiology. 12 (1), 1814666 (2020).</li><li>Cross, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+isolation+and+cultivation+of+uncultivated+bacteria+by+reverse+genomics.">Targeted isolation and cultivation of uncultivated bacteria by reverse genomics.</a> Nature Biotechnology. 37 (11), 1314-1321 (2019).</li><li>Kantor, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+genomes+and+sparse+metabolisms+of+sediment-associated+bacteria+from+four+candidate+phyla.">Small genomes and sparse metabolisms of sediment-associated bacteria from four candidate phyla.</a> MBio. 4 (5), 00708-00713 (2013).</li></ol>

The authors have nothing to disclose.

Our method of filtering plaque and applying it to pure cultures of host organisms is largely based on earlier observations on the first cultured Saccharibacteria, &#39;Nanosynbacter lyticus&#39; strain TM7x11,14,15. Given the small cell size, we deduced that they could be separated from dental plaque using a filter and concentrated with centrifugation. Second, as these organisms live as parasites, providing these cells pure cultures of hosts would allow them to enter into a symbiosis and grow as binary cultures.
One advantage of this method is that it does not require an enrichment culture or selective pressure. &#39;Nanosynbacter lyticus&#39; strain TM7x was cultured from an enrichment culture using streptomycin as a selective agent, which sequencing had suggested would be effective at enriching for Saccharibacteria. Fortuitously, the host for &#39;Nanosynbacter lyticus&#39; Schaalia odontolytica, is known to be resistant to streptomycin16. Using antibiotics as a selective agent could also prevent the host organism from growing, which in turn would preclude the growth of Saccharibacteria.
A larger issue of using enrichment cultures is that fast-growing organisms will quickly displace organisms of interest. In the oral cavity, for example, Streptococcus species can grow quickly and, if sugar is present in the growth medium, produce enough acid to acidify the medium, further selecting against organisms of interest. By avoiding an enrichment culture and selective antibiotics, our method provides a general approach that could be applied to a wider range of Saccharibacteria and potential hosts without the complications of these other methods.
There are some obstacles to the method presented here. First, this method assumes that Saccharibacteria live in a binary culture. We have not tested combinations of trinary or ternary cultures to gauge their effectiveness, but there are likely Saccharibacteria that require growth factors that a single host organism cannot supply. Testing the vast combinations of oral bacteria that could support the growth of Saccharibacteria would be a daunting task. Second, the method assumes all Saccharibacteria are small enough to pass through a 0.2 &#181;m filter. It could be that other Saccharibacteria are larger than believed and the filter is selecting against these organisms. A filter with a larger pore size could be used, but this runs the risk of allowing more unwanted oral bacteria into the infected co-culture. Lastly, it is very difficult to find host species outside of those that have already been published. Thus far, the only successful hosts are species from the genera Actinomyces, Schaalia, Arachnia and Cellulosimicrobium, all members of the phylum Actinobacteria15,17,18. However, these hosts only support the growth of specific Saccharibacteria. To culture more species of Saccharibacteria, many more hosts must be explored.
We hope the method presented here will aid future research of the Saccharibacteria and other CPR organisms. Metagenomic sequencing suggests that these organisms also have small genomes and are suspected to be symbionts or rely on the local microbial community to supply metabolites and other factors critical to their survival19. A similar filtering strategy could be used to isolate these organisms, provided they are small enough and that their host organisms(s) can be cultured. The methods described here are a first step to bringing the powerful tools of laboratory culture to this large and diverse group of bacteria.

Culturing novel species of bacteria and bringing them into the laboratory allows for powerful experiments to better understand their physiology and broader interactions within their microbial community. While there are culture-free methods of interrogating these questions (e.g., &#34;meta-omics&#34;), the complex interactions of diverse microbial populations make it difficult to tease apart single variables and reach meaningful conclusions. While culturing bacteria has many benefits, there are many potential barriers to isolating a bacterium and growing it in pure culture. Potential specific growth requirements include pH, oxygen tension, vitamins, growth factors, signaling molecules or even direct cell contact to elicit growth1. However, it is believed that specific auxotrophies are the primary deterrent to culturing new species of bacteria. Standard media formulations lack many nutrients required by uncultivated bacteria, such as specific vitamins or carbons sources. These missing molecules can be key to the physiology of the uncultured bacteria and are usually provided by either another organism in the microbial community or a host organism. For example, complex carbohydrates such as mucins can be provided by animal hosts. Adding these to media has allowed cultivation of several bacteria from animal guts, including Akkermansia muciniphila and Mucinivorans hirudinis2,3,4. Many pathogenic bacteria have evolved the ability to use iron bound to hemin in animal cells, including the oral pathogen Porphyromonas gingivalis5. In the laboratory, the growth of Porphyromonas and other organisms, can be stimulated by the addition of hemin6.
Recently, many breakthroughs in culturing novel isolates of bacteria have come through co-culturing, using a &#34;feeder&#34; organism to provide specific factors to uncultured bacteria necessary for their growth. An elegant study by Vartoukian and colleagues showed that siderophores, iron binding molecules produced by bacteria, stimulated the growth of several novel oral isolates. Pyoverdines, a type of siderophore produced by pseudomonad species, were shown to significantly facilitate the growth of a novel Prevotella species7. In the same study, the first oral isolate for the phylum Chloroflexi was cultivated, also using F. nucleatum as a helper for providing some as yet unknown compound7. More recently, a bacterium from the genus Ruminococcaceae was isolated using Bacteroides fragilis as helper organism8. It was later shown that gamma aminobutyric acid (GABA), an inhibitory neurotransmitter, was required for growth on laboratory media. Using feeder organisms has proved to be a key strategy to mimicking specific microenvironments where uncultured bacteria grow, being more efficient than continuously reformulating growth media with different additives in varying concentrations.
One of the largest groups of uncultured bacteria are in the &#34;Candidate Phyla Radiation&#34; (CPR), a monophyletic group of several candidate bacterial phyla9,10. As of this writing, only members of the Saccharibacteria phylum within the CPR have been successfully cultured in the laboratory. The first isolate, &#39;Nanosynbacter lyticus&#39; strain TM7x, was isolated using the antibiotic streptomycin, which had been predicted to enrich for the uncultured TM711,12. A key discovery of this work was that the new isolate grew as a parasite growing in direct contact with a bacterial host, Schaalia odontolytica, and microscopy showed these parasites were ultrasmall bacteria.
Using these clues, we devised a method to quickly establish binary cocultures of Saccharibacteria with their partners by filtering dental plaque and other oral samples through a 0.2 &#181;m filter, collecting cells in the filtrate by centrifugation and using them to infect cultures of candidate host bacteria. This method has the advantage of avoiding enrichment cultures, which can be overwhelmed with fast-growing organisms. It also avoids the use of antibiotics, which could stop the growth of either the targeted Saccharibacteria species or their hosts. Using the method demonstrated here, we have successfully cultured 32 isolates from the Saccharibacteria phylum.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alphaimager</td>
			<td>Cell Biosciences</td>
			<td>FluorChem HD2</td>
			<td>Or equivalent UV gel imaging system</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion Broth (dehydrated powder)</td>
			<td>Becton-Dickinson</td>
			<td>211059</td>
			<td>Or other growth media suitable for target organisms</td>
		</tr>
		<tr>
			<td>Centrifuge Rotor 70-Ti</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Fisher Scientific</td>
			<td>12-567-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645052</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis Rig</td>
			<td>Bio-Rad</td>
			<td>1704467</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Forceps</td>
			<td>Millipore Sigma</td>
			<td>XX6200006P</td>
			<td>Not essential, helps ensure filters are not punctured during handling</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>G33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green Mastermix</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler Pro Thermocycler</td>
			<td>Eppendorf</td>
			<td>950040025</td>
			<td>Or equivalent thermocycler for PCR</td>
		</tr>
		<tr>
			<td>MgCl2 solution 25mM</td>
			<td>Promega</td>
			<td>A3513</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology grade water</td>
			<td>Fisher Scientific</td>
			<td>BP2819100</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Control InVitro Glove Box</td>
			<td>Coy Laoratories</td>
			<td>031615</td>
			<td>If needed for microaerobic organisms</td>
		</tr>
		<tr>
			<td>Optima L-100 XP High Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>8043-30-1124</td>
			<td></td>
		</tr>
		<tr>
			<td>P-10 micro pipette</td>
			<td>Gilson</td>
			<td>F144802</td>
			<td></td>
		</tr>
		<tr>
			<td>P-1000 micro pipette</td>
			<td>Gilson</td>
			<td>F123601G</td>
			<td></td>
		</tr>
		<tr>
			<td>P-2 micro pipette</td>
			<td>Gilson</td>
			<td>F144801</td>
			<td></td>
		</tr>
		<tr>
			<td>P-20 micro pipette</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		</tr>
		<tr>
			<td>P-200 micro pipette</td>
			<td>Gilson</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>BP399500</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes 0.2 mL</td>
			<td>Fisher Scientific</td>
			<td>14-230-205</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips - 10 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>02-717-157</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips - 1000 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>02-717-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips - 20 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>02-717-161</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips - 200 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>02-717-165</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filters - 47mm, 0.2 &#956;m pore size</td>
			<td>Millipore</td>
			<td>GTTP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-cap conical centrifuge tubes 15 mL</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>Or other tube suitable for bacterial culture</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Swin-Lok Filter&#160; - 47mm</td>
			<td>Whatman</td>
			<td>4200400</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes - 20 mL</td>
			<td>Fisher Scientific</td>
			<td>14-955-460</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer (50x) concentrate</td>
			<td>Fisher Scientific</td>
			<td>P1332500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thickwall Polycarbonate 25 x 89 mm (26.3mL capacity) centrifuge tubes with caps</td>
			<td>Beckman Coulter</td>
			<td>355618</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Blood Agar Plates</td>
			<td>Northeast Laboratory Services</td>
			<td>P1100</td>
			<td>Or other agar plate sufficient for growth of host organisms</td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth (dehydrated powder)</td>
			<td>Becton-Dickinson</td>
			<td>211825</td>
			<td>Or other growth media suitable for target organisms</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>Coy Laboratories</td>
			<td>032714</td>
			<td>If needed for anaerobic organisms</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing stable binary cultures of symbiotic saccharibacteria from the oral cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

PCR for detection of Saccharibacteria may appear negative (i.e., no product seen) in initial infection cultures due to a low number of Saccharibacteria symbionts. However, after a few passages a strong PCR product should appear showing that a stable infection has occurred (Figure 1A). Conversely, some infections will initially appear positive by PCR, but diminish to undetectable after 1-4 passages (not shown). This indicates that a large amount of Saccharibacteria cells were present in the initial filtrate but were diluted out by passage and none were able to enter into a stable symbiosis with the host culture. Testing several host species with the same Saccharibacteria filtrate from the oral cavity will usually have a low success rate (Figure 1B) as the symbiont-host interaction is very specific. A researcher may also test the same host with filtrates from several different subjects. If a good host organism (such as Arachnia propionica or Schaalia odontolytica) is used, a success rate of 50% can be expected.
Testing by PCR is crucial. Experienced researchers have attempted to confirm infection by microscopy, only to report false positives. The Saccharibacteria cells are small and difficult to distinguish between vesicles or irregular cell envelope bulges. A PCR signal stable through several passages is key to confirm a successful infection.
As the infected cultures grow, normal turbid growth should appear. As the symbiosis establishes itself, infected cultures will appear less turbid than cultures of uninfected host organisms (Figure 2A). In some cases, infected cultures may appear to stop growing completely and not become turbid at all. This may be due to an infection where the Saccharibacteria cells are overwhelming the host organism. Adding &#34;fresh&#34; (uninfected) host to these cultures should provide a sufficient population of host to support the continued growth of the Saccharibacteria parasites.
Overgrowth, or excessively turbid cultures, may indicate contamination by either a laboratory contaminant or another small oral bacterium that was able to pass through the 0.2 &#181;m filter. Campylobacter and Capnocytophaga spp. are typical oral contaminants of these experiments. This can be confirmed by plating the culture and looking for colonies that are atypical of the host organism followed by 16S rRNA sequencing. If contamination is seen, filtering these cultures through a 0.2 &#181;m filter is usually sufficient to remove the contaminants. Saccharibacteria cells in the filtrate can be concentrated by centrifugation and used to re-infect a pure host culture.
Another way to purify a contaminated culture is by picking infected colonies from plating an infected culture. Colonies of infected hosts can sometimes be identified by irregular colony shape compared to uninfected colonies (Figure 2B-D). These irregular colonies are dependent on the titer of Saccharibacteria in the binary culture and a smaller proportion of colonies will appear irregular if the titer is low. This can make it easy to identify infected colonies, which can be picked and used to start a pure binary culture. If no irregular colonies are seen on plating from a Saccharibacteria infected culture, it is possible that the symbiont is at a low titer or does not cause irregularly shaped colonies. In such a case, PCR screening colonies with a normal appearance can show infection by Saccharibacteria, but at a low rate (2-10% of all colonies).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62484/62484fig1v2.jpg" /> 
Figure 1: Typical PCR results of Saccharibacteria infections of host cultures. (A) A PCR product indicating presence of Saccharibacteria may not appear with the initial infection but can appear and become stronger in subsequent passages as the coculture establishes itself. (B) Most hosts infected with Saccharibacteria-enriched filtrate will not support their growth due to the specificity of the symbiosis. In this example, only A. propionica was successfully infected. <a href="https://www.jove.com/files/ftp_upload/62484/62484fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62484/62484fig02.jpg" /> 
Figure 2: Growth characteristics of Saccharibacteria infected cultures. (A) Growth curves of infected and uninfected cultures of A. propionica showing infected culture will not grow to the same density as the uninfected control and appear less turbid. (B-D) Plating of cocultures can produce irregular colonies, caused by Saccharibateria infections. Irregular colonies will decrease in proportion relative to the titer of Saccharibacteria in the coculture. (B) Host with a high level of Saccharibacteria. (C) 10-fold diluted Saccharibacteria (D) Uninfected host culture. White arrows indicate examples of irregular colonies. Scale bar= 1 cm.&#160;<a href="https://www.jove.com/files/ftp_upload/62484/62484fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity at JoVE.com

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

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3.0K Views.  The Forsyth Institute. We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Video: Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Extraction of the EPP Component from the Surface EMG

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.

The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...