STANDARDIZATION OF MEDIUM FOR CULTURING LYME-DISEASE SPIROCHETES

To standardize the procedure for isolating and culturing Lyme disease spirochetes, we modified the composition of the medium generally used for this purpose (BSK-II) and developed a system for its distribution. This medium contains no gelatin or agarose, and various components ar e used in proportions that differ from those in BSK-H. Each of the maj or proteinacious components was screened by substitution in samples of the complete product. The final medium was evaluated for the capacity to grow related spirochetes including Borrelia burgdorferi N40, Guilf ord, and JD-1 as well as strains of Borrelia hermsii (HS-1) and of Bor relia coriaceae (CO53). Each isolate developed from inocula containing as few as one to five organisms. Doubling time of B. burgdorferi duri ng log-phase growth at 37-degrees-C was 10 to 12 h. Lyme disease spiro chetes were isolated in this medium from ear punch biopsies and dermal aspirates from naturally infected mice and rabbits, from dermal biops ies from a human patient, and by sampling field-collected deer ticks ( Ixodes dammini). Cultured spirochetes remained infective to mice and t o ticks. The medium can be stored at -20-degrees-C or lower temperatur es for at least 8 months without effect on its ability to support grow th of small inocula to densities exceeding 10(8) spirochetes per ml. L yme disease spirochetes remained infective to mice after being stored at -80-degrees-C in this medium for at least 8 months. We anticipate t hat the availability of this standardized medium (Sigma Chemical Co.), supplemented with prescreened rabbit serum, will facilitate compariso n of research results between laboratories and may eventually permit d efinitive clinical diagnosis of Lyme disease based on demonstration of the pathogen. The standardized medium is designated BSK-H.