Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

Borrelia is a genus of spirochete bacteria that encompasses three major kinds. The Lyme Borreliosis group, the Relapsing Fever group, and the less well-characterized group found in reptiles. Many Borrelia species are pathogenic.

The culture of bacteria is the gold standard for the detection of infection. This is true for both research and clinical work. The culture of bacteria from body fluid and tissues confirms the the bacteria are both viable and replicating.

This protocol demonstrates the methodology and recipes required to successfully culture and preserve the Lyme Borreliosis group of spirochetes, Relapsing Fever Borrelia, and Borrelia miyamotoi. Methods are provided for the culture of previously isolated species and strains, as well as new isolates and monoclonal isolates from environmental or mammalian sources. Demonstrating the procedure will be Marie-Line Faucillion, postdoctoral researcher, and Ingela Nilsson, technician from the Bergstrom Lab, Anne Berthold, research associate from the Lloyd Lab, and Maryna Golovchenko, research associate from the Rudenko Lab.

To begin, obtain the reagents required to prepare one liter of Barbara-Stoner-Kelly, or BSK-II medium. Start with dissolving the BSA in a beaker by stirring the water. Then add all the dry chemicals and let them dissolve before adding CMRL.

Adjust the pH of the medium to 7.6. If necessary, use one molar sodium hydroxide. Next, add rabbit serum to the medium to a concentration of six to 10%If the bacterial growth is not strong, add another one to 2%serum to the medium.

Then sterilize the medium using a 0.22 micron pore filter. Freeze the stock of medium in 100 or 400 milliliter aliquots. For Relapsing Fever strains, add 100 milliliters of 7%sterile gelatin to 400 milliliters of BSK-II medium.

Disinfect the surface and all materials with 7%ethanol and transfer them immediately to the biological safety cabinet. UV sterilize all non-biological materials for five minutes before the start of the work. To start a culture, pre-warm the medium in an incubator.

Then add one milliliter of thawed Borrelia culture from a glycerol stock to five to 15 milliliters of pre-warmed BSK medium. Fill the tubes almost completely to make a micro or anaerobic atmosphere before closing them. Grow Relapsing Fever species at 37 degrees Celsius and Borrelia burgdorferi sensu lato species at 33 to 34 degrees Celsius.

Monitor the growth of the Borrelia culture using phase contrast or dark field microscopy at 200 to 400 times magnification. To ensure the even distribution of cells, mix the culture with a serological pipette or use a three milliliter transfer pipette while pipetting up and down. Then load to 10-microliter culture aliquot on each side of a hemocytometer for counting.

Next, dilute the exponential phase Borrelia culture at a ratio of one-to-two or one-to-four with a medium in a 1.7 milliliter tube. When determining the cell count, consider the dilution factor appropriately. After use, spray the surfaces and the hemocytometer with 10%diluted bleach and/or 70%ethanol.

For extraction of DNA or isolation of Borrelia cells, centrifuge the exponential phase culture for 10 minutes at 4, 000 G and discard the supernatant in a suitable biohazard waste. Process the pelleted bacteria with a commercial DNA extraction kit or resuspend it in an appropriate medium for the intended experiment. At the end of each experiment, sterilize all equipment with ethanol and UV radiation.

Collect the liquid waste, including excess culture, in a waste bottle and add 10%bleach. Place the bottle at 80 degrees Celsius before discarding it. For long-term storage of Borrelia cultures, take the desired volume of an exponential phase culture at 10 million to 100 million cells per milliliter, add 10%sterile glycerol, and mix by pipetting up and down.

Transfer 1.5 milliliter aliquots of the mixture into two milliliter screw cap cryogenic vials. Store the stock vials at 80 degrees Celsius. Maintain a stock with at least 10 tubes of each strain in the freezer, To cultivate Borrelia from hard ticks, collect adult females, males, and nymphal ticks.

Surface sterilize ticks by dipping them into 70%ethanol for two minutes and air dry in a biosafety cabinet. Next, dissect the ticks individually into several pieces using a sterile scalpel. Then place the pieces into two milliliter tube containing 1.5 milliliters of the medium supplemented with antibiotics.

Incubate the cultures at 34 degrees Celsius and check for spirochetes by dark field or phase contrast microscopy twice weekly for the first two weeks and weekly after that for six weeks. To cultivate Borrelia from blood samples, gently remove the plasma from the tube and inoculate one milliliter of plasma into five milliliters of preheated, Modified Kelly-Pettenkofer or MKP Complete medium. For the cultivation of Borrelia from a skin biopsy, surface sterilize the skin biopsy with 70%ethanol and hydrogen peroxide.

Then place the biopsy sample in physiological saline. Dice the sample into two to six smaller pieces using a sterile razor blade in a glass Petri dish while immersed in the physiological saline. Transfer the diced sample and one milliliter of saline in which the skin biopsy was stored into five milliliters of pre-warmed medium supplemented with antibiotics, and incubate at 33 degrees Celsius.

For making plates, warm 300 milliliters of 1.5x BSK or MKP Complete medium without gelatin. Also warm 200 milliliters of 1.7%agarose to 55 degrees Celsius. Mix the liquids and pipette 15 milliliters each onto 16 deep Petri dishes to make the bottom agar layer.

Maintain the rest of the medium in a water bath. Quantify Borrelia culture density and dilute to the desired density in a liquid medium. After the bottom plates have solidified, add 10 milliliters of the remaining agar mixture as top agar to a 15 milliliter tube containing the appropriate number of spirochetes.

Pour the suspension onto the bottom agar plate into the labeled Petri dish. After the plates are solidified, close them and place them upside down at 34 to 35 degrees Celsius in 2.5%carbon dioxide. When prepared correctly, the BSK medium appeared red-orange and clear.

For comparison, MKP uninoculated medium is also shown here. The overgrowth by competing bacteria generates turbidity and clumped materials in the medium. The bacterial growth was monitored by visual inspection by phase contrast or dark field microscopy.

In the exponential phase, Borrelia cells are typically slender, kinked, and modal to some extent. As the cultures enter at the stationary phase round bodies are increasingly evident. Borrelia cells are fastidious and each species and strain and even isolate differs both genetically and through adaptation to the host from which they have been isolated.

When making media, appropriate ingredients make an enormous difference in the vigor of Borrelia culture or even the viability of the culture. Borrelia spirochetes are challenging to culture, but culture is possible. Culture in the laboratory is the springboard for research on the biology of these bacteria.

Culture provides the material to understand the genome, the proteome, the evolution, and host pathogen interactions of the Borrelia species. Borrelia culture can take several weeks before spirochetes are sufficiently abundant for detection. This limits diagnostics applications.

But culture can show if Borrelia species are present, viable, and replicating, and it can help access treatments.