M-250 Procedural Summary

Multi- Media Direct Culture Method with DNA Fluorochrome Staining Assay

Direct Culture Method:

  • The direct culture procedure utilizes three mycoplasma media formulations: Fortified Commercial (FC), Modified Hayflick (MH), and Heart infusion (HI) in both broth and agar formulations.
  • 0.5 mL of sample is inoculated into 6 mL of each broth medium (FC, MH, & HI).
  • 0.1 mL of sample is inoculated onto duplicate plates for each agar formulation.
  • Each broth culture is subcultured onto like agar plates on Day 7 post setup.
  • The agar plates are incubated under both aerobic and microaerophilic conditions. Plates are examined microscopically for mycoplasma at 7 day intervals.
  • Appropriate positive (Ë‚ 100 CFU) and negative controls are processed with each sample.

DNA Fluorochrome Staining Assay with Indicator Cell Line:

  • The DNA fluorochrome procedure is an indirect assay which involves the inoculation of 1.0 mL of sample volume into a mycoplasma free indicator cell culture (Vero). 
  • Following a 3 to 5 day period of incubation, 2.0 mL of the culture is transferred to a CELLshipper® slides for fixation and DNA staining with Hoechst 33258.   The extranuclear fluorescence staining pattern is then interpreted with UV-fluorescent microscopy up to 600x magnification.
  • Advantages of this assay include enhancement of sensitivity and specificity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.), amplification of mycoplasma titers and detection of "non-cultivable" organisms.
  • Appropriate positive (Ë‚100 CFU) and negative controls are processed with each sample.
  • As a GMP test, quality assurance reports are provided with the final report packages.
  • Total testing time is 28 days.

ADHERENT Cells:

Grow adherent cell culture in a T-25 flask with antibiotic free media. When the cell culture is nearly confluent, scrape and aliquot 5 mL of suspension.  Dispense sample in sterile screw cap 15 mL polypropylene centrifuge tube for shipment under frozen or ambient conditions. Alternatively, sample may be submitted in a T-25 flask ambient. In this situation, simply add fresh antibiotic free media to the vessel prior to shipping. Leave sufficient room in the neck of the flask to allow for expansion and to prevent leakage. Samples should be cryopreservative free and have undergone at least 3 passages in antibiotic free media prior to submission for mycoplasma testing to achieve the highest level of assay sensitivity.  It is recommended that a back-up sample be retained and stored at -60oC or colder.  

NONADHERENT Cells:

Take 5 mL of non-adherent cell suspension and place in a sterile screw cap 15 mL polypropylene centrifuge tube. Cell culture samples can also be provided in a T-25 flask as above.  Samples should be cryopreservative free and have undergone at least 3 passages in antibiotic free media prior to submission for mycoplasma testing.  It is recommended that a back-up sample be retained and stored at ≤ -60oC.

Notification of Sample Submission:

We request that you contact us by 12:00pm 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.

View our general sample submission instructions for more information.

Shipping Instructions:

Ship Frozen on dry ice or ship under ambient conditions. Bionique Testing Laboratories recommends frozen shipment on dry ice.  A 5 mL sample aliquot should be frozen in a sterile 15 mL polypropylene screw cap centrifuge tube and shipped overnight in an insulated container with sufficient dry ice to keep frozen for a period of 48 hours. If shipping sample under ambient conditions, a nearly confluent T-25 flask or 5 mL sample aliquot in a 15 mL polypropylene screw cap centrifuge tube should be used.  Place the sample vessel inside a plastic bag in a crush proof package and ship overnight.  Be aware that shipping under ambient conditions may lead to sample compromise due to leakage, exposure to cyclic extremes in temperature, etc.