CULTIVATION OF BORRELIA

S.E. Phillips, L.H. Mattman, H. Moayad

Introduction
There has long been a medium [Barbour] suitable for isolation of Borrelia burgdorferi from the patient’s spinal fluid or bulls-eye rash tissue. In contrast, it has been difficult to grow the spirochete from the patient’s blood. We describe a medium which is also functional for isolating the pathogen from blood or synovial fluid.

MPM —2001 Medium*
NIH Thioglycollate Broth (Difco #0257)                                          2.9 g
Thioglycollate Medium without indicator (Difco #0430)                     2.9 g
Soluble Stareh (Difco #0178)                                                           0,5 g
Sucrose                                                                                         10g
Tap water                                                                                      100 ml

      The ingredients listed above are dissolved by heating. The broth is then tubed in 9 ml amounts and autoclaved for 15 minutes at 121° -  124°C. A 10% solution of yeast extract (Difco #0127) is made in tap water and autoclaved. Because the yeast extract may contain spores, it is autoclaved for 30-40 minutes. After autoclaving, the separately autoclaved yeast extract is added to each tube to give a final concentration of 1.0%. Aller adding the yeast the tube is shaken only once.

      If the pH of the medium-plus-yeast is too acid, one tube is tested to determine how much sterile 5% sodium bicarbonate must be added to bring the pH to 7.4 — 7.6. This amount of the buffer is then added to each tube. The medium is refrigerated one day before use.
      To make chocolate agar plates or slants, 1.5 g of Nobles agar is incorporated in 100 ml of the broth. Five (5) ml of sheep blood is added to 100 ml of the autoclaved medium. Some sheep blood may contain Borrelia burgdorferi but this contamination is avoided if the blood is added when the medium has been cooled only to 80°C and held at this temperature for five minutes. Yeast extract is also incorporated into the solid medium.
      There is nothing easier than not growing spirochetes. In cultures one will usually not have exclusively the classic spirochete. That is like asking a man to consistently appear in a tuxedo with a name tag.
      The trace of agar and the method of inoculating without aerating the medium allow the pathogen to initiate growth at the desired level of oxygen.

Staining
Heat fixation of a smear is disastrous. Because of it’s high lipid content, any pleomorphic growth appears as an artifact. Although acetone gives firmer fixation, methanol is preferable as causing less distortion of membranes.
      A stained smear is not washed under running tap water, rather with cold tap water from a beaker; otherwise many organisms will go down the dram. As for any study, smears are made part thin, part thick; thus an ideal concentration will be included.
   Acridine Orange may be applied in two ways. A dried smear is stained for 10 minutes. A wet preparation with equal amounts of dye and culture is allowed to stand at room temperature for 10 minutes before examining.
      Kinyoun’s Carbol Fuchsin is an excellent simple stain for revealing spirochetes. Some of the spirochetes will absorb the dye; others will be seen by negative staining.
Sudan Black B stains a few of the spirochetal classic and pleomorphic stages.  
Many of the stains commonly in use are microbiology, such as Wright-Giemsa, do not adequately stain spirochetes.

Inoculum and Incubation
The ideal temperature appears to be 35°C. This must be the shelf temperature; the temperature at another shelf may be too high to permit growth. Incubation is in a sealed jar with BBL Gas Packs to give microaerophllie conditions and CO₂.
      The inoculum is large, e.g. 0.5 ml to a 10 ml tube of medium, disposed from a 3” needle against the side of the tube starting at the bottom. Growth will appear in 24-48 hours, as a line through the medium subsurface, indicating the suitable microaerophilic level.
      Care is taken not to aerate the medium. For example, after adding the yeast, the tube is shaken only once.

Results
Using MPM medium growth of a spirochete which stains with fluorescent antibody to Borrelia burgdorferi has been obtained from the bloods of over 400 Lyme patients and 42 M.S. victims. We believe the agents of these diseases are both in the Borrelia genus, but different species. In an unpublished study, Alva Johnson found that antibody versus the spirochetes from a M.S. case did not react with spirochetes from Lyme cases. The MPM medium has been used in three different laboratories for successfully growing Borrelia.
      The medium, as a broth, has been useful for preparing cultures for Electron Microscopy studies on the Lyme, M.S. and ALS spirochetes. In Agar Plates, the medium has been used to compare the colonies of the spirochete from these three diseases.

Discussion
      Two factors explain why the medium is satisfactory for spirochete growth. The sodium thioglycollate results in the necessary microaerophilic conditions. Secondly, the medium is hypertonic with two agents, Sodium Chioride and Sucrose. Many organisms grow initially in the Cell Wall Deficient stage and show classic growth only after extensive pleomorphic stages.
      Oystein Brorson and co-workers have studied the effect of distilled water on growth of Borrelia burgdorferi. One publication notes that distilled water changes the spirochete to the cystic form. However, the change is reversible. [Brorson paper]
      Growth has been obtained with distilled water. However, most of the studies have utilized tap water from varied municipal sources. A discussion of tap water in culture medium is included in a recent publication. [Lida’s book] We have seen spirochetes in triple distilled water piped to a dispenser. We have not seen spirochetes in tap water. We believe that tap water from any city will work. We used City of Detroit water.
      Sucrose has long been used as a stabilizing agent of Cell Wall Deficient Forms, allowing their reversion to the classic form. Sucrose has been incorporated in concentrations of 10 [Dominque], 15 [Beaman], 20 [Prozorovsky], 30% [Rosner]

PRECAUTIONS
1. Do not use the medium on the day of preparation. Refrigerate 24 hours before use.
2. Do not irradiate the MPM medium with ultra violet light. This could occur when one uses UV to cleanse the inoculating hood.
3. Do not change the brand of yeast extract.
4. Do not shake the tubes to mix in the yeast extract. Place the yeast on top and let it defuse down.
5. Do not shake the tube vigorously when adding the buffer to change the pH, just one shake will suffice.

*Several modifications of the original MPM Medium have been made to simplify preparation.
From: LE. Philips, MD., Greenwich Hospital, 5 Perry Ridge Rd., Greenwich, CT 06830; L.H. Mattman, Ph.D.; H Moayad, D.O., Columbia North Hills Medical Center, 4401 Booth Calloway Rd., North Richland Hills, TX 76180.