Establishing a Binary Culture of Saccharibacteria with Host Bacteria

Abstract

Source: Collins, A. J., et al., Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity. J. Vis. Exp. (2021)

This video demonstrates the establishment and maintenance of a binary culture of Saccharibacteria and its host bacterium by sequentially co-incubating them in host-specific growth medium.

Protocol

1. Concentrate Saccharibacteria cells by centrifugation
   1. 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 is removed from the centrifuge.
   2. Centrifuge the samples at 60,000 x g for 1 h at 4 °C.
      NOTE: This force and time are 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.
   3. Carefully remove the tubes from the centrifuge. Pour out the liquid from the tube, keeping the pellet on the upper side of the tube.
   4. Resuspend the usually invisible pellet in 1-2 mL of MRD (Maximal recovery diluent) buffer by vigorous vortexing.

2. Infect host cultures with Saccharibacteria-enriched filtrate

1. Prepare culture tubes by aliquoting 2 mL of appropriate growth media (e.g., TSBY (Tryptic soy broth with 0.1% yeast extract), BHI (Brain Heart Infusion), etc.) into tubes.
2. Add 200 µL of the overnight culture of host organisms to each tube. Add 100-200 µL of resuspended, filtered sample to each tube.
3. Incubate combined samples as appropriate for the host organism (e.g., 37 °C, in an aerobic atmosphere for Arachnia propionica).
4. Passage the cells every two to three days by transferring 200 µL 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't increase after passage into fresh media), the Saccharibacteria could be overwhelming or killing all the host organisms. To remedy this, add 200 µL of uninfected host culture when passaging the cells. Repeat for at least 5 passages.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Brain Heart Infusion Broth (dehydrated powder)	Becton-Dickinson	211059	Or other growth media suitable for target organisms
Centrifuge Rotor 70-Ti	Beckman Coulter	337922
2 Control InVitro Glove Box	Coy Laoratories	31615	If needed for microaerobic organisms
Optima L-100 XP High Speed Centrifuge	Beckman Coulter	8043-30-1124
P-1000 micro pipette	Gilson	F123601G
P-200 micro pipette	Gilson	F123602G
PBS (Phosphate buffered saline)	Fisher Scientific	BP399500
Peptone	Fisher Scientific	BP1420-500
Pipette tips - 1000 μL	Fisher Scientific	02-717-166
Pipette tips - 200 μL	Fisher Scientific	02-717-165
Screw-cap conical centrifuge tubes 15 mL	Falcon	352096	Or other tube suitable for bacterial culture
Thickwall Polycarbonate 25 x 89 mm (26.3mL capacity) centrifuge tubes with caps	Beckman Coulter	355618
Tryptic Soy Blood Agar Plates	Northeast Laboratory Services	P1100	Or other agar plate sufficient for growth of host organisms
Tryptic Soy Broth (dehydrated powder)	Becton-Dickinson	211825	Or other growth media suitable for target organisms
Yeast Extract	Fisher Scientific	BP1422-500