Contributor: Suprya Jayadev
Date: August 11, 1996
Reagents:
1) 1 mM MycoTect reagent working solution (Gibco # 189-5672-1)
- Dilute the 0.65 ml of 10 mM 6-methylpurine deoxyriboside (6MPD) solution
1:10.
- Aliquot the 1 mM working solution of 6-MPDR into 10 cryovials,0.65 ml/vial.
(Each aliquot is sufficient to test five samples.)
- Store in -80°C until necessary, and try to avoid repeated freezing
and thawing.
2) Adenosine phosphorylase working solution (Gibco # 189-5672-2)
- Dilute the stock solution 1:10.
- Distribute the working solution into ten 0.65 ml aliquots. (Each aliquot
is sufficient for one positive control.)
- Store in -80°C freezer, and try to avoid repeated freezing and thawing.
3) 10% Phosphate-buffered formalin, pH 7.0 + 0.2 (PBF)
100 ml 37-40% formaldehyde
900 ml distilled water
6.5 g dibasic sodium phosphate, anhydrous
4.0 g monobasic sodium phosphate, monohydrate
4) 0.2% crystal violet stain in 10% PBF
- Dissolve 1 g of crystal violet in 500 ml of 10% PBF.
- Filter through coarse paper.
5) Test cultures
a) Cultures should be assayed 3-4 days after media is changed, and the cultures
should be at or near maximum density.
b) Cells are important since some mycoplasmas cytadsorb to the cell line
they are infecting.
c) For monolayer cultures, remove cells by scraping and then collect 1.0
ml of media plus cells. DO NOT USE TRYPSIN!! (Trypsin can be detrimental
to the growth of mycoplasma.)
d) Maximum results are obtained if the cells are tested on the same day
they are collected; however, cells with media can be stored for one or two
days at 4°C or frozen at -20°C
Procedure:
Day 0
1) Collect 0.6 mls of media and cells from each cell line to be tested.
2) Thaw an aliquot of positive control working solution and keep on ice
until ready to use.
--> Always use the same day.
3) Seed a 24-well cell culture dish with 1.5 ml of 2 X 104 cells/ml mycoplasma-free
Vero cells.
--> Allow for positive and negative controls as well as for duplicates
of each test culture.
--> These cells must be grown in antibiotic-free media.
--> The seeding density should insure that the cells will form a confluent
monolayer in approximately four to five days.
4) Allow cells to attach to wells by incubating ~2 hours.
5) Add test and control samples to wells and use seeding media as the negative
control.
--> ALWAYS, keep a template of test and control samples!
--> Final volume/well should be ~1.7 mls.
6) Incubate cultures for 24 hours.
Day 1
7) Thaw an aliquot of 1 mM MycoTect working solution and keep on ice until
ready to use.
--> Always use the same day.
8) Add 50 µl of MycoTect/well - to get a final concentration of 30
µM 6MPD.
9) Incubate a further 72-96 hours.
--> Incubation should be such that negative cell control wells are confluent
and cells are not sloughing off of the dish.
Day 4/5 - No longer require sterile conditions!
10) Determine relative confluency of each well then aspirate media from
each well, without disturbing the monolayers.
11) Fill wells with 0.2% crystal violet in 10% PBF and incubate at room
temperature for 20 minutes.
12) Aspirate satin and wash wells until the rinse water is clear and allow
plates to air dry.
Interpretation:
NOTE: Stained plates may be kept indefinitely at room temperature after
drying.
13) The MycoTect control wells and any cell control wells should show a
confluent layer of cells.
14) If cells are positive for mycoplasma, the cell monolayers should be
diminished. If infection is extensive, the monolayer may be nonexistent.
15) Positive control wells should exhibit staining patterns which mimic
the staining pattern of typical mycoplasma infections.