M-800 Procedural Summary

Mycoplasma Test for Human Vaccines and Biologicals Compliance with 21 CFR 610.30

Direct Culture Method:

• The procedure requires the submission of both test article and control fluid.
• Two sets of 4 x 10 mL cultures of Fortified Commercial (FC) semi-solid broth are inoculated with 1 mL of sample.  One set is designated for the product harvest and the other for the control fluid.
• At set-up, a minimum 2 mL volume of product sample and control fluid must each be inoculated onto two different media formulations of agar plates. 8 plates of Fortified Commercial (FC) and 8 plates of Heart Infusion (HI) agar media are inoculated with 0.15 mL per plate.
• The semi-solid broth and agar cultures are incubated under appropriate aerobic and microaerophilic conditions as specified.
• Subcultures of the semi-solid broth cultures for the test article and control fluid are performed on Days 4 and 14.
• Each subculture includes the inoculation of eight (8) fresh FC agar plates with 0.1 mL/plate of pooled material from the two aerobic broth cultures (0.5 mL from each tube).  This process is repeated with the two microaerophilic broth cultures for the product test article and control fluid.
• All agar plates are examined microscopically for mycoplasma at 7 day intervals.
• Broth cultures are observed for signs of mycoplasmal growth (i.e. turbidity and/or pH changes.
• 21 CFR 610.30 test specifies the use of two mycoplasma species as positive controls, one of which must be M. pneumoniae.
• Positive controls (M. pneumoniae and M. salivarium at ≤ 100 CFU) and a negative control are processed for each sample batch.

DNA Fluorochrome Staining Assay with Indicator Cell Line:

• The 21 CFR 610.30 test does not specify the inclusions of an indicator cell culture procedure. However, Bionique Testing Laboratories enhances the M-800 assay by the inclusion of an indicator cell culture with DNA fluorochrome staining assay for both the test article and the control fluid to broaden the detection of fastidious mycoplasma species or strains.
• The indicator cell culture procedure involves the inoculation of 1.0 mL of sample volume onto a mycoplasma free indicator cell line (Vero).  This procedure is performed for both the product test article and control fluid. 
• Following a 3 to 5 day period of incubation, 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 (≤ 100 CFU) 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.

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.

Per 21 CFR 610.30, samples should be stored at ≤ -20°C for 24 hours or longer. Bionique Testing Laboratories requires that samples for the M-800 Mycoplasma Test be shipped and received frozen with dry ice remaining upon receipt at our facility. 10 mL sample aliquots of both the product and control fluid should be frozen in a 15 mL sterile polypropylene centrifuge tube or a sterile 20 mL Nalgene PETG screw cap bottle. Sample aliquots greater than 10 mL (>10.5 mL) should be submitted in a sterile 50 mL polypropylene centrifuge tube or a sterile 40 mL Nalgene PETG screw cap bottle.  Place the sample vessels inside a plastic bag with crush proof packaging in an appropriate container. The frozen samples should be shipped overnight in an insulated container with sufficient dry ice to keep frozen for a period of 48 hours.