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

All studies involving samples obtained from human subject were approved by the Institutional Review Board of the relevant University and/or medical facility, and written informed consent was obtained from participants before the samples were collected. All studies involving samples obtained from animals were approved and conducted under the guidelines of the Institutional Animal Ethics Committee. Where relevant, approvals have been obtained for environmental sampling.

NOTE: The success of culture is critically dependent on the quality of ingredients. Commercial BSK medium is available and robustly supports the growth of lab-adapted pure B. burgdorferi and related species where high dose inoculum can be used to seed the culture. For isolates of strains from primary biological material, freshly prepared medium is preferred and often necessary.

Borrelia sp. are known or suspected pathogens, and unscreened human or animal tissues can also be a biohazard. Handling of potentially infectious material requires personal protective equipment and sterile technique for investigator safety. Additionally, the medium is so nutrient-rich that the culture is easily contaminated. Biosafety level 2 containment is generally required for this work and all work with Borrelia sp. or samples should be conducted in a biological safety cabinet.

1. Instructions for making BSK-II medium

1. Preparation of complete BSK-II medium
   1. Obtain reagents to prepare 1 L of the medium, as shown in Table 1.
      NOTE: BSA purity (or lack of purity) is critical for optimal growth. In particular, BSA purity differs between both manufacturer and lot. Please see Table 2 for a list of more versus less successful products. Obtaining several samples from different vendors, testing them for optimal growth, then buying a large quantity of the best lot is an effective strategy to mitigate this problem and produce a good medium.
   2. Start with dissolving the BSA in a beaker by stirring in the water, which will take at least half an hour.
   3. Add all the dry chemicals and let them dissolve. Then add the CMRL.
   4. Adjust the pH of the BSK-II medium to 7.6, if necessary, with 1 M NaOH.
   5. Add rabbit serum to the BSK-II medium to a concentration of 6%-10%. Filter sterilize (0.22 µm pore size) the BSK-II medium. The required amount of serum depends on the Borrelia species. Use 6%-7% for the RF species and strains, and for Borrelia burgdorferi sensu lato. If growth is not strong, add another 1%-2% sterile rabbit serum to the medium.
   6. Freeze the stock of BSK-II medium in 100 mL or 400 mL aliquots.
   7. For RF strains, gelatin is required. Add 100 mL of 7% sterile gelatin (slightly warmed to put it into a solution) to 400 mL BSK-II medium. Warm the BSK-II with gelatin at 37 °C to make the gelatin melt before inoculating the culture.
      NOTE: Medium can be frozen (-20 °C) prior to filter sterilization and the addition of rabbit serum and gelatin (for RF spirochetes) or after filter sterilization and serum (and gelatin) addition. The medium is stable when frozen for a long time. When stored in a fridge, use the medium within 1 month. RF species grow best with fresh medium, usually not more than 1 week old. If not frozen, visually inspect the quality of the medium for turbidity. Discard the medium that appears to be contaminated, precipitating ingredients, or otherwise dubious.
   8. If antibiotics are to be added to the medium, add them to freshly made medium or previously frozen aliquots; the latter is the more flexible approach. Use antibiotic concentrations as described by Oliver et al.³⁹.
   9. While thawing a tube of the medium, weigh antibiotics into sterile (autoclaved) 1.7 mL tubes or sterile 15 mL conical tubes, using a precision balance. Prepare the stock solutions as follows: Dissolve 25 mg of amphotericin B in 10 mL of DMSO, 20 mg of phosphomycin in 1 mL of autoclaved distilled water, and 50 mg of rifampicin in 1 mL of DMSO.
   10. Filter sterilize (0.2 µm syringe filters) the antibiotics and transfer them into a new tube of appropriate size.
   11. Dilute the antibiotics in a ratio of 1:1000 of the medium to reach their final concentration. For 500 mL of BSK, add 0.5 mL of amphotericin B stock solution (2.5 mg/mL in DMSO), 0.5 mL of phosphomycin stock solution (20 mg/mL in sterile H₂O), and 0.5 mL of rifampicin (US: rifampin) stock solution (50 mg/mL in DMSO).
   12. Use the antibiotic-supplemented medium or aliquot and freeze at -20 °C.

Table 1: Composition of 1 L of BSK-II medium.

Table 2: Sources of BSA and suitability for BSK-II medium.

2. Instructions for making MKP

1. Preparation of basic medium
   1. Carefully weigh and dissolve all powdered components as listed below in Table 3 in 3/4 of the final volume of ddH₂O (ca. 750 mL) by stirring until complete dissolving, which takes ~1 h.
   2. After complete dissolving, adjust the pH with NaOH to 7.6 and the volume to 1000 mL, and then sterilize by filtration (0.22 µm filter).
      NOTE: Basic medium can be stored at -20 °C for up to 3 months.
2. Preparation of MKP complete medium
   1. Mix all components as listed in Table 4 in a sterile manner; sterilize the gelatin by autoclaving and handle in a sterile manner. Work in a sterile biological safety cabinet and use a sterile filtered serum.
   2. Aliquot MKP in 50 mL tubes. Put one small aliquot at 33 °C for several days, then check under a darkfield microscope for the absence of contamination.
      NOTE: Keep MKP complete medium at +4 °C for no more than 1 month, and do not freeze. The complete medium can be additionally sterilized by filtration using a 0.22 µm filter.

Table 3: Composition of 1 L of basic MKP (Modified Kelly-Pettenkofer) medium.

Table 4: Composition of MKP (Modified Kelly-Pettenkofer) complete medium.

3. Culture of Borrelia, with or without antibiotics

1. Perform all experimental steps under sterile working conditions in a biological safety cabinet. Disinfect the surface and all materials with 70% ethanol, and transfer them immediately to the biological safety cabinet. UV sterilize all non-biological materials in the biological safety cabinet for 5 min before the start of the work.
2. Use the freshest medium possible.
3. To start a culture, pre-warm the medium (33 °C – 37 °C) in an incubator. Apply shaking if needed.
   NOTE: Depending on the volume of medium, this step may take several hours.
4. Add ~1 mL of thawed Borrelia culture from a glycerol or similar stock, previously stored at -80 °C and thawed at room temperature (RT), as soon as it is liquid, to 5-15 mL of pre-warmed BSK medium. Inoculate the culture into a small volume (5-15 mL of BSK) using commercially available glass or plastic vials with screw caps. Fill the tubes almost full to make a micro- or anaerobic atmosphere. Close the tubes tightly; if cultured in this manner, CO₂ is not needed.
5. Grow RF species (and B. persica) at 37 °C in an incubator without stirring or agitation. Grow B. burgdorferi sensu lato species at 33-34 °C.
6. Monitor the growth of the Borrelia culture using phase contrast or darkfield microscopy at 200x-400x magnification (Figure 3). To ensure even distribution of cells, mix the culture with a serological pipette or for more mixing with a 3 mL transfer pipette while pipetting up and down.
   NOTE: Borrelia are most visible at higher magnification (200X-400X), but are best countable at 200X magnification on a hemocytometer⁴⁰. Borrelia movement and morphology are indicative of the presence of these bacteria.
7. To quantify bacterial growth, count several (10) individual small squares in the sixteen-field grid pattern of counting area on both sides of the hemocytometer and average. Multiply the average cell number per small square with the conversion factor appropriate for the hemocytometer being used to determine the cell count per mL.
8. Monitor the bacterial growth at 1-2 day intervals. The exponential growth of Borrelia is dependent on the viability and cell density and may take 1 week or longer at 34 °C. Dilute the exponential phase cultures at a ratio of 1:2 or 1:4 with the medium in a 1.7 mL tube before loading on the hemocytometer. Include appropriate consideration of the dilution factor when determining the cell count. After use, spray the surfaces and hemocytometer with diluted bleach (10%) and/or 70% ethanol.
   ​NOTE: Borrelia are in exponential growth when cell concentrations are 1-5 x 10⁷ cells/mL. Within this range, the cells grow well.
9. Extended culture will deplete nutrients and change medium pH so subculturing is required. Under sterile conditions in a biological safety cabinet, transfer 1/10 of the culture volume into a new tube and fill it with the new medium (a fresh aliquot of the same medium used to grow the previous passage). A 1:10 dilution is optimal. If culture volume is to be changed, adjust the inoculum amount accordingly, and continue incubation. The number of passages increases with each medium change.
10. For extraction of DNA or isolation of Borrelia cells, centrifuge the exponential phase Borrelia for 10 min at 4000 x g to pellet the bacteria. Discard the supernatant in suitable biohazard waste. Process the pelleted bacteria with a commercial DNA extraction kit or resuspend the bacteria in an appropriate medium for the intended experiment.
11. At the end of each manipulation, sterilize all equipment with ethanol and UV radiation, as appropriate for the equipment. Collect the liquid waste, including excess culture, in a waste bottle and add bleach to a final concentration of 10%. Place this bottle at -80 °C before discarding it. Alternatively, sterilize the liquid waste and culture without bleach or alcohol by autoclaving.

4. Long-term storage of Borrelia cultures

1. Preparation of glycerol stock of Borrelia culture
   1. Take ~1.8 mL aliquots of exponential phase cultures, cell concentrations ~10⁷-10⁸ cells/mL, and add sterile glycerol to a final concentration of 10% (concentrations of 5%-20 % work).
   2. ​Place in 2 mL screw cap cryogenic vials. Store at -80 °C.
   3. Maintain a stock with at least 10 tubes of each strain in the freezer.
2. An alternate storage recipe to produce a glycerol stock for permanent storage
   1. Mix 2/3 of Borrelia culture with 1/3 of 15% glycerol in Brucella broth. Use 2.8 g of Brucella broth dissolved in 100 mL of ddH₂O, which was sterile filtered (0.22 µm).
   2. Keep the glycerol stock of samples at -70 °C to -80 °C.

5. Isolation of spirochetes from environmental or clinical sources

NOTE: If isolating material from human, animal, or environmental sources, research ethics, animal care, or ecological certification, as appropriate, must be obtained.

1. Cultivation of Borrelia from hard ticks
   1. Collect adult females, males, and/or nymphal ticks.
   2. Surface sterilize all tick samples by dipping them into 70% ethanol for 2 min and air-dry in a biological safety cabinet. Dissect individually into several pieces with a sterile scalpel. Place the pieces into a 2 mL tube containing 1.5 mL of medium, supplemented with antibiotics.
   3. Incubate the cultures at 34 °C and check for spirochetes by darkfield or phase contrast microscopy twice weekly for the first 2 weeks and weekly thereafter for 6 weeks (more often for RF spirochetes). Subculture culture-positive samples in 5 mL of the same medium.
      NOTE: Contaminated cultures can be purified to some extent by filtration using a 0.2 µm filter; antibiotics may be required to fully resolve contamination.
2. Cultivation of Borrelia from blood samples
   NOTE: The blood sample should be taken by a qualified medical, veterinary, or research professional, depending on the source of the material. Less than 24 h at RT should elapse between sample
   removal and arrival in the lab.
   1. Obtain 10 mL of blood, collected into glass blood collection tubes with acid citrate dextrose (ACD; "yellow top") as an anticoagulant. Leave at room temperature. It is recommended that less than 24 h pass between blood collection and inoculation.
   2. Centrifuge the blood with anticoagulant for 10 min at 200-400 x g to separate plasma from blood cells.
   3. Gently remove the plasma from the tube and inoculate 1 mL of plasma into 5 mL of pre-heated MKP complete medium. MKP can be supplemented with antibiotics. Incubate cultures at 33 °C with daily visual inspection and weekly darkfield or phase contrast microscopy until borrelial growth is noticed (more often for RF Borrelia), which can take up to 9 weeks.
      NOTE: If using larger or smaller inoculation volumes, maintain the 1:5 ratio of inoculant to medium. Use sera or plasma rather than whole blood.
3. Cultivation of Borrelia from skin biopsy
   1. Surface sterilize the skin biopsy (ideally a 3 mm x 3 mm x 3 mm cube; i.e., with 70% ethanol and hydrogen peroxide). Place the biopsy sample in physiological saline.
      NOTE: A biopsy sample should be taken by a qualified medical, veterinary, or research professional, depending on the source of the material. Less than 24 h at RT should elapse between sample removal and arrival in the lab.
   2. Dice the sample into two to six smaller pieces using a sterile razor blade in a sterile glass Petri dish while immersed in the physiological saline. Transfer all of the samples and ~1 mL of the physiological saline in which the skin biopsy was stored into 5 mL of prewarmed medium supplemented with antibiotics and incubate at 33 °C.
   3. In case of overgrowth of contaminating bacteria, add antibiotics as in step 1.1.10 above, or by adding two tablets of sulfamethoxazole (23.75 mg; final concentration 5 mg/mL) + trimethoprim (1.25 mg; 0.25 mg/mL) or Bactrim (minimum inhibitory concentration >128 µg/mL), two tablets of Amikacin (2 x 30 µg; final concentration 12 µg/mL), and two tablets of Phosfomycine (2 x 200 µg; final concentration 80 µg/mL).
      NOTE: In all cases, be sure to remain below the minimum inhibitory concentration of that antibiotic for the Borrelia species for which culture is being attempted^41,42,43.
   4. If the culture is positive for Borrelia, keep the culture for an additional 3-4 days or until the amount of Borrelia reaches 10+ spirochetes in a microscope field using a 40X objective. Create a glycerol stock for permanent storage.

6. Isolation of monoclonal populations of  B. burgdorferi  sensu lato spirochetes and  B. miyamotoi  from liquid cultures by cultivation on solid media

1. For making plates, warm 300 mL of 1.5X MKP complete medium (without gelatin). Also, warm 200 mL of 1.7% agarose (autoclaved in deionized water, stored at RT, and microwaved before use) to 55 °C. Mix both liquids.
2. Pipette 15 mL of this mixture into each of 16 deep Petri dishes to make the bottom agar oselayer.
3. Retain the rest of the medium at 55 °C in a water bath.
   NOTE: The Borrelia are mixed into the top agarose layer.
4. First, quantify Borrelia culture density using a hemocytometer and dilute to the desired density in a liquid medium.
   NOTE: 500, 200, 100, and 50 spirochetes per plate work well.
5. After the bottom plates have solidified, add 10 mL of the remaining agarose mixture to a 15 mL tube containing the appropriate number of spirochetes.
6. Pour the suspension onto the bottom agarose in the labeled Petri dish.
   NOTE: Since Borrelia spirochetes are sensitive to high temperature, care should be taken to ensure that the bacteria are not exposed to 55 °C for an extended period of time; pour the top agar immediately upon adding it to the bacterial cells in the medium.
7. After the plates are completely solidified, close the Petri dishes and place them upside down at 34-35 °C in 2.5% CO₂.
   NOTE: The colonies will appear 1 week after plating if the procedure is successful.
8. To screen and expand the individual colonies, select single colonies from the plates and put them into 1.5 mL of modified liquid MKP, or other suitable media, in sterile 2 mL culture tubes.
9. Incubate the cultures at 34 °C and examine for spirochetes by darkfield or phase microscopy. Use these cultures for analysis or stocks, made as described above.