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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.2/3.2


TITLE: MYCOPLASMA DECTECTION BY THE GIBCOBRL MYCOTECTâ - INRC -


INTRODUCTION

The Mycotectâ assay is a rapid quantitative method for detecting the presence of mycoplasma contamination in cell cultures; it is based on a defined biochemical difference between mycoplasmas and their host mammalian cells, as to production of adenosine phosphorylase, which is found only in small amounts in mammalian cells, and largely produced by mycoplasma. The adenosine phosphorylase converts the 6-methylpurine deoxyriboside (6-MPDR) in 6-methyl purine and 6-methyl purine riboside, both of which are toxic to mammalian cells.

Materials and solution

  • 24-well cell culture plates
  • Mycotectâ (0.65 ml)
  • Positive control: adenosine phosphorylase (0.65 ml)
  • Indicator cell line (Vero)
  • samples
  • 10% phosphate buffered formalin, pH7 +-0.2
  • 0.2% crystal violet stain in 10% phosphate buffered formalin, pH7 +-0.2
  • distilled water

Reagent storage and preparation

Mycotectâ

  • The stock solution must be stored at -60C to -80C
  • Dilute the stock solution 1:10 with sterile PBS to obtain a 1mM working solution
  • Prepare ten 0.65 ml aliquots and store at -60C to -80C

Positive control

  • The stock solution of adenosine phosphorylase must be stored at -60C to -80C
  • Dilute the stock solution 1:10 with sterile PBS to obtain a working solution
  • Prepare ten 0.65 ml aliquots and store at -60C to -80C

10% phosphate buffered formalin, pH7.0 +-0.2

  • 100ml 37% formaldehyde
  • 900 ml distilled water
  • g dibasic sodium phosphate, anhydrous
  • 4 g monobasic sodium phosphate, monohydrate

Stain solution

  • Dissolve 1 g crystal violet in 500 ml 10% phosphate buffered formalin pH7 +-0.2
  • Filter through paper

N.B. the crystal violet is toxic, and should be managed with extreme care

Specimen collection

  • Collect 1 ml supernatant containing a small number of cells, for adherent cells use a rubber policeman. Cell lines to be screened should be grown in antibiotic free media for at least 2 passages prior to testing.

Procedure

  • Seed a 24-well cell culture plate with mycoplasma free Vero cells grown in antibiotic free medium: 1.5 ml suspension in complete DMEM medium at a concentration of 2 x 104 cells/ml (3 x 104 cells/well)
  • Incubate at 37C 5%CO2
  • Collect samples of cell lines to be tested (to five cell lines per plate)
  • Thaw aliquot of positive control working solution, keep at +2C to +8C until ready to use
  • After Vero cells have attached add 0.2 ml of test and control samples to wells, use seeding medium as negative control
  • Incubate for 24 hrs at 37C 5%CO2
  • Thaw aliquot of Mycotectâ working solution and keep at +2C to +8C until ready to use
  • Add 50l Mycotectâ (final concentration 30M) to the relevant wells
  • Incubate for 72 - 96 hrs at 37C 5%CO2 until cells in negative cell control wells are confluent
  • Observe the plate microscopically for confluence
  • Aspirate medium from each well
  • Fill wells with 0.5 ml 0.2% crystal violet in 10% phosphate buffered formalin, pH 7.0 +-0.2
  • Incubate for 20 min. at room temperature
  • Aspirate stain and wash wells with distilled water, until rinse water is clear

Interpretation of result

  • Negative control wells (rows 1 column 1) and row 4 should show a confluent layer of cells
  • If samples are positive for mycoplasmas, the layer will not be confluent
  • If infection is extensive, the monolayer may be non existent
  • Positive control wells, which contain an exogenous source of adenosine phosphorylase, will exhibit staining pattern which mimic the staining pattern of typical mycoplasma infections.

 

 

 


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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