Saccharibacteria are obligate parasites that require co-culture with appropriate host bacteria. As human oral saccharibacteria can be easily isolated for most individuals using known host bacterial species. This protocol will allow any investigator or laboratory to isolate their own strains in binary co-culture.

This technique uses simple devices and equipment present in most microbiological laboratories and has been successfully used to culture over 30 isolates representing six species of saccharibacteria. This protocol may be useful for the isolation of other candidate-followed radiation species, as well as saccharibacteria from different environments and other mammals if appropriate actinobacterial hosts can be identified and isolated. Demonstrating the procedure will be Andrew Collins, a former postdoctoral fellow in my laboratory.

To begin, start cultures of host bacteria such as arachnia propionica, with enough time for them to grow to early stationary phase. To initiate the culture, inoculate two to five milliliters of tryptic soy broth containing 0.1%yeast extract with arachnia propionica, and incubate it at 37 degrees Celsius for 24 hours. Next, assemble filter holders with track etched 0.2 micrometer filter membranes.

Wrap them in foil and sterilize by autoclaving, along with centrifuge tubes and cap assemblies. Sterilize extra supplies in case of accidental contact with non-sterile surfaces. Collect dental plaque by scraping along the gum line, using a sterile paper point, gracey curette, or a sterile toothpick or pipette tip, and transfer it to a suitable buffer such as MRD or PBS.

Keep the sample on ice until ready to proceed further. Resuspend the dental plaque vigorously using a combination of vortexing and pipetting with a small pipette tip, then add the resuspended plaque sample to a tube containing nine milliliters of MRD buffer. Carefully unwrap a sterile filter assembly using aseptic technique.

Untwist a quarter turn, and re-tighten the filter holder to ensure that the threads are properly engaged, and the apparatus is properly closed. Wash the membrane by passing 10 milliliters of MRD buffer through the apparatus, using a syringe. To apply the sample to the washed filter, remove the plunger from a syringe and attach it to the filter apparatus.

Place a sterile centrifuge tube beneath that filter apparatus to catch the filtrate, then pour the dispersed dental sample into the syringe and load it onto the filter. Then apply light pressure to the plunger to push the sample through the filter. Wash the membrane with another 10 milliliters of MRD buffer, and collect the flow through in the same tube as the filtered sample.

Then cap the tube aseptically, and keep on ice. Make an orientation mark on the tube and cap to identify the location of the cell pellet after centrifugation. Place the tube in a high-speed centrifuge with the mark on the upper side and centrifuge the samples at 60, 000 G for one hour at four degrees Celsius.

After centrifugation, carefully pour out the supernatant, keeping the pellet on the upper side of the tube. Then resuspend the pellet in one to two milliliters of MRD buffer by vigorously vortexing. Prepare culture tubes by aliquoting two milliliters of appropriate growth media into the tubes.

Then, add 200 microliters of overnight culture of host organisms and 100 to 200 microliters of resuspended filtered sample to each tube. Incubate the combined samples under conditions appropriate for the host organism. Passage the cells every two to three days by transferring 200 microliters of the binary culture to two milliliters of fresh growth medium in a new tube.

If the passaged cultures show no growth at 200 microliters of uninfected host culture, when passaging the cells, return inoculated tubes to the incubator. Aliquote 24 microliters of the PCR master mix into a 0.2 milliliter PCR tube. At one microliter of saccharibacteria infected culture, place the tube in a Thermo cycler.

Set the PCR program as described in the text manuscript. Load and run the PCR products on a 1%Agros gel. A band of approximately 600 bases will confirm the presence of saccharibacteria.

To confirm the purity of positive cultures, perform a 10 fold serial dilution of the co-culture of saccharibacteria in a sterile buffer, and spread 100 microliters on an agar plate. Incubate the culture under appropriate growth conditions and observe for contaminating organisms. A band of approximately 600 bases will confirm the presence of saccharibacteria.

PCR detection may appear negative in initial infection cultures due to a low number of saccharibacteria symbionts. However, after a few passages, a strong PCR product should appear, indicating that a stable infection has occurred. Testing several hosts species with the same saccharibacteria filtrate will usually have a low success rate.

As the symbiont host interaction is very specific. 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.

A contaminated culture can be purified by picking infected colonies after plating. Infected colonies can sometimes be identified by an irregular colony shape compared to uninfected colonies. The proportion of a regular colonies depends on the titer of saccharibacteria in the binary culture.

If the titer is low, fewer colonies will appear irregular, making it easy to pick the infected colonies and start a pure binary culture. Cultures may not appear positive for several passages. So some patients will be needed.

It is also necessary to regularly check for contamination of binary cultures, as this can destabilize the culture and lead to the death of the saccharibacteria. The binary culture can be used for all sorts of laboratory experiments. DNA and RNA can be extracted for genome sequencing and transcriptome analysis, respectively.

Pathogenicity can also be investigated using animal models.

Summary

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Microbiological Techniques