Cell Mycoplasma Testing

MYCO001 PCR-based test

MYCO002 Direct Culture test

Background

The presence of mycoplasma in cell cultures directly impacts your work. Mycoplasma contamination in cell culture is wide-spread and difficult to notice. Usually there is an absence of visible morphological changes but the invisible effects on the cell cultures are what make mycoplasma contamination so serious a problem. Mycoplasmas have been shown to alter virtually every cell culture property and characteristic measured.

Screening for mycoplasma contamination is recommended on a regular basis, and before cell banking and cryopreservation. AddexBio offers mycoplasma testing using a PCR-based and direct culture methods.

The PCR-based assay identifies Mycoplasma positive samples using primers developed in-house for screening purposes, and the PCR test is useful for detecting contamination with most species of mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma including species most likely to afflict cell cultures: Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma arthritidis, Mycoplasma bovis, Mycoplasma fermentans, Mycoplasma gallisepticum, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma neurolyticum, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma pulmonis, Mycoplasma salivarium, Mycoplasma synoviae, Spiroplasma citri, Ureaplasma urealyticum .

Ordering

We require that customers contact our customer support to submit an order form to use our Mycoplasma Testing Service before sending samples to us.

Procedures for Submitting Cell Cultures for Mycoplasma Testing

1. Samples should be submitted from growing culture (please see below for detailed cell culture preparation). We accept only BSL-1 and BSL-2 samples for mycoplasma testing.

2. Prior to sending the cells for testing, please contact customer support and submit an order form either by email at customersupport@addexbio.com or by fax at 858-538-8847, so that we will be receiving the cells.

3. If you are submitting more than one cell culture sample, place samples for each culture in separate zipper-locked or sealed bags. This will protect the integrity of the other sample(s) if one of them leaks.

4. Fill out the order form that you received from customer support; make sure you include your reference number. Pack cell culture sample(s) at room temperature in a sturdy box to prevent breakage. Each flask/tube should be wrapped in plastic bubble wrap or comparable packing material to prevent breakage or leakage. Additional packing material should be used for protection in shipping. Do not use vented caps as they may leak during shipping.

5. Send materials at room temperature by courier, such as FEDEX or UPS (maximum 2-day delivery). Materials must arrive at AddexBio by noon on Monday through Thursday of the week. Please include signed order form with sample(s).

Cell Culture Preparation for Mycoplasma Testing

1. Passage cells in antibiotic-free media twice. Do not change the medium during the last 72 hours before testing. Cells should be 50-80% confluent.

2. Samples grown in antibiotics or given fresh medium prior to testing may have artificially low mycoplasma levels if present or may give a false negative result.

3. Prepare samples(s) following one of the procedures based on cell type:

For Preparation with Adherent Cells

1. Aseptically seed a sterile T-25 with approximately 5x10⁴ cells in 10 mL of medium and grow for 2 population doublings (about 48 hours).

2. Without replacing the current culture medium, aseptically fill the flask completely with fresh culture medium. Seal the lid of the closed flask with Parafilm sealer.

For Preparation with Suspension Cells

Aseptically place approximately 2x10⁵ cells in a sterile container topped up with medium, container can be either a small plastic tube with a screw-cap closure or a T-25 flask. Seal the lid of the closed container with Parafilm sealer.

Samples received that show signs of leaking or obvious bacterial/fungal contamination will not be tested.

We do not accept culture samples(s) containing HIV, virulent microbials (fungi, bacteria or viruses), or other BSL 3 or 4 agents for mycoplasma testing.

We offer Mycoplasma Testing for research purposes only. This service has not been validated to support any clinical diagnosis. Testing is not carried out under GLP facilities.