M-350 Procedural Summary

Large Volume Barile Method with DNA Fluorochrome Staining Assay

Direct Culture Method:

  • The direct culture procedure follows the Large Volume Barile method (Reference:  Barile, M.F. and Kern, J. Isolation of Mycoplasma arginini for commercial bovine sera and its implication in contaminated cell cultures.  Proc. Soc. Exp. Biol. 1971; 138: 432-437).  
  • The direct culture procedure utilizes three mycoplasma media:  FC (Fortified Commercial), MH (Modified Hayflick) and HI (Heart Infusion).  FC in both broth and agar formulations. MH and HI in agar based formulation only.
  • A 50 mL sample of serum is inoculated into 200 mL of Fortified Commercial (FC) broth.
  • For non-serum samples, a 50 mL sample is inoculated into 200 mL of FC broth supplemented with 20% horse serum (50 mL).
  • Upon set up (Day 0), two agar plates of each medium formulation are inoculated with 0.1 mL sample/plate.
  • One agar plate of each medium formulation is incubated aerobically and the other microaerophilically.
  • On Day 7, 14 and 21, 0.1 mL aliquots of the FC broth culture are subcultured onto two fresh agar plates of FC and MH media. Plates are incubated under conditions as described above.
  • Appropriate positive (Ë‚ 100 CFU/mL) and negative quality controls are processed with each batch of samples.
  • The broth culture is examined for signs of mycoplasmal growth (i.e./ turbidity and/or pH changes.
  • All agar plates are examined microscopically for mycoplasma at 7 day intervals.

DNA Fluorochrome Staining Assay with Indicator Cell Line:

  • For the indirect DNA fluorochrome staining assay with indicator cell line, a 35 mL sample aliquot is concentrated by high speed centrifugation.
  • The pellet is resuspended in the cell culture media and inoculated onto a mycoplasma-free indicator cell line (Vero).
  • Following a 3 to 5 day period of incubation, a 2.0 mL of the culture is transferred to a CELLshipper® slide for fixation and staining with a DNA - binding fluorochrome (Hoechst 33258). The extranuclear fluorescence staining pattern is then interpreted with UV-fluorescent microscopy up to 600x magnification.
  • Appropriate positive (≤ 104 CFU/mL) and negative quality controls are processed with each batch of samples.
  • As a GMP test, quality assurance reports are provided with the final report packages.
  • Total testing time is 28 days.
Shipping Instructions:

Ship the sample frozen or under usual storage conditions. Bionique Testing Laboratories recommends frozen shipment on dry ice.  A 100 mL sample aliquot should be frozen in a sterile 100 mL PETG Nalgene screw cap bottle. Samples should be shipped overnight in an insulated container with sufficient dry ice to keep frozen for a period of 48 hours. Place the sample vessel inside a plastic bag with crush proof packaging in an appropriate container. Sample can be shipped under ambient conditions or on cold packs in volumes and vessels as described above. However, be aware that shipping ambient may lead to sample compromise due to leakage or exposure to cyclic extremes in temperature. 

Notification of Sample Submission:

We request that you contact us by 12:00 pm ET on the day you ship samples.  Information can be forwarded either by contacting us directly by phone at (518) 891-2356 or faxing your sample submission form to (518) 891-5753.  Please provide details on the number of samples shipped and the requested mycoplasma testing service by catalog #.  This will assist Bionique Testing Laboratories in meeting your needs for quality and timely service.

Please view our general sample submission instructions for more information.