Borrelia carolinensis Rudenko et al.

1. Open thawed vial.
2. Aseptically transfer the entire contents to a 5-6 mL tube of #1914 broth. Additional test tubes can be inoculated by transferring 0.5 mL of the primary broth tube to these secondary broth tubes.
3. Use several drops of the primary broth tube to inoculate a #260 plate and/or agar slant.
4. Or, to obtain a biphasic culture, add several drops of the primary broth tube to an agar slant. Best practice is to incubate these slants at an angle.
5. Incubate at 33°C in the dark in an atmosphere of 5% CO₂ for 7-10 days. Use an anaerobe jar with an active catalyst and a microaerophilic gas generator pack or other acceptable method. All tubes and slants should be incubated with caps loosened.

Growth usually occurs after 5 days, although some strains may take up to 10 days to grow.  Acid formation during growth will change the medium to a light or yellowish-orange color.  Turbidity is not evident.  Cells can be monitored under phase microscopy as long spiral rods and motility by twitching movement.

Borrelia carolinensis is a fragile, sensitive organism that must have the appropriate medium for growth.  Rabbit serum is essential for the growth of this organism.  Fresh medium enhances growth.  Medium older than one month should not be used.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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