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Why do we note 'storage at 4-8°C' instead of room
temperature, when shipping is performed at room temperature?
It is absolutely practicable to ship Mynox® at room temperature
for the following reasons:
Mynox® is sterilized by autoclaving at
125 °C for 20 minutes and is stable at room temperature for at least
6 months even at higher temperatures. Anyhow the shelf life time of the
product is much longer and sometimes customers use products even after the
shelf life time has expired. To provide a product in good condition even
for these circumstances we recommend long term storage as indicated on the
label at +2 to +8°C (Mynox® is stable at these temperatures
for at least 18 months). On the attached quality certificate you can find
the information that shipment is regularly performed at room temperature
to reduce additional costs for the end customer.
Mycoplasma is still
detectable after treatment of cells with Mynox®.
Following theMynox® treatment cells should be examined
for remaining mycoplasma contamination after 4 passages when a sufficiently
high cell density exists. Mynox® lyses the mycoplasma as
an eradication mechanism. Thus, free mycoplasma DNA remains in the supernatant
after treatment. With continuous cultivation and adequate cell density
extracellular DNAses will hydrolyze free DNA.
Different factors might interfere with the efficiency of Mynox®.
A crucial factor is the FCS concentration. FCS contains cholesterol and
other target molecules for Mynox®. Hence, it is pre-requisite
to avoid higher FCS concentrations in the media than suggested (final
concentration of FCS must be 5 %). Another crucial aspect for efficient
mycoplasma elimination is the cell concentration. If elimination of mycoplasma
did not occur with the first treatment we suggest to lower the cell concentration
and/or to increase the incubation time with Mynox®.
Prior to the treatment with Mynox® make sure that you
don't have cell clumps in your suspension. Extended trypsination will
help to avoid the formation of cell clumps clearly.
In case of adherent cells it is highly recommended to use Petri dishes for
the treatment. This will ensure that the cell suspension is not exposed
to aerosols which could be produced when pipetting of the the cell suspension
onto the Mynox® suspension is performed. The aerosols could
stick to the surface of the vessel not being exposed to Mynox®.
These contaminated aerosols could re-contaminate the cell culture later
on. Therefore, it is very important to transfer the cells directly into
the Mynox® suspension and not vice versa. If the
mycoplasma titer at the beginning of a treatment is extremely high it might
be necessary to treat the cells a second time with Mynox®.
In that case it is important to provide enough time for recovery (two days/check
with the microscope) to the cells after the first treatment.
When can I be certain
that Mycoplasma is permanently eliminated?
In case that a few mycoplasma particles survive the treatment with Mynox®
they will grow to detectable titers after four passages. You can detect
mycoplasma at an early stage with the highly sensitive Venor®GeM
Mycoplasma Detection Kit to exclude persisting contamination.
Do I have to remove standard antibiotics for the treatment
with Mynox® ?
No. Standard antibiotics like penicillin/streptomycin could be carried
along during the treatment withMynox®. As a basic principle,
we would not recommend the use of antibiotics. Pen/Strep mainly effects
germs of the mouth and faucal cavity and was introduced in times, when
laboratory staff used their mouth for pipetting. This situation changed
with the introduction of pipetting-aids and security workbenches. Antibiotics
can effect the cellular metabolism and thus the results of experiments.
(compare with: Kuhlmann, Cytotechnology 19:95-105,1996 "The prophylactic
use of antibiotics in cell culture"). Bacterial contaminations can interfere
latently and unrecognized. With Onar®EUB Minerva
Biolabs provides a sensitive PCR-Detection Kit for bacterial contamination
(Cat.-No. 12-1025).
Mynox®
was uneffective in eliminating mycoplasma from virus suspensions.
The cell suspension must be free of cellular debris before treatment.
Before the supernatant can be effectively treated, cellular debris should
be centrifuged (1.000 rpm, 5 min) to form a pellet. Such debris as cell
wall fragments will competively bind Mynox®, thus decreasing
the effective concentration of the elimination reagent.
Mynox®
was harmful to cells during treatment.
Most cases in which Mynox® was believed to be detrimental
to cells, the mixture as prescribed in the Instruction Manual was not
properly followed, thus resulting in a cytotoxic Mynox®
concentration. Cells should be observed frequently during treatment. If
cytotoxic effects are clearly evident, the treatment should be immediately
stopped by medium change. For cells known to be sensitive to Mynox®,
the treatment time should be reduced by passage of cells after 30 minutes
for adherent cell lines and 15 minutes for suspension cell lines.
Can the cellular concentration
be increased for treatment with Mynox® ?
The cell concentration may be increased by 10-fold, however an overall
decrease in the elimination efficiency of Mynox® should
subsequently be expected.
Can Mynox®
eliminate intracellular contaminants?
Mynox® does not integrate into the cellular membrane.
Therefore it cannot eliminate intracellular contamination. However, mycoplasma
is an extracellular contaminate. Mycoplasma penetrans and Mycoplasma
gallisepticum are the only species described intracellulary. Both
M. penetrans and M. gallisepticum have not been reported
as cell contaminants.
Is Mynox®
effective against bacteria, fungi, or chlamydia?
No, Mynox® is only effective against mycoplasma.Mynox®
is especially useful against mycoplasma contamination in chlamydia cultures,
as standard antibiotic treatments are damaging to chlamydia in cell lines.
Is Mynox suitable for removing mycoplasma from murine
ES cells?
Murine ES cells can be treated with Mynox. Mynox generally shows a low
cytotoxicity in contrast to other anti-mycoplasma reagents, but still
is not completely free of such complications. We usually like to recommend
Mynox Gold which shows basically no cytotoxicity anymore, but treatment
takes longer.
Can primary cells be treated with Mynox®
?
Yes, primary cells can be treated with Mynox®. However,
we recommend a 10-fold increase in the cell concentration. (Note that
an overall decrease in the elimination efficiency of Mynox®
should subsequently be expected).
What are
recommended methods of eliminating mycoplasma contamination on surfaces
or other laboratory apparatus?
Mycoplasma-Off® Surface Disinfection Spray is ideal for
cleansing and disinfection of all laboratory surfaces and apparatus including
clean benches, incubators, work benches, cell storage boxes and liquid
nitrogen containers. Mycoplasma-Off® is also effective
against a broad range of other pathogenic contaminants.
Is Mynox®
effective against viruses?
1. Mynox® is active against some enveloped viruses only.
If you work with a Mynox®-sensitive virus, the titer might
be reduced. We recommend to follow the instructions in the handbook carefully.
Do not exceed the incubation time. Propagation of the virus on host cells
is recommended after treatment for highest titers.
2. Mynox® is not active against non-enveloped viruses due
to the biophysical mode of action. Mynox® integrates into
the phosholipid/cholesterol layer of the membrane/envelope forming pores
and finally bursting the mycoplasma. Non-enveloped viruses do not have target
regions in their structure.
Does
Mynox® interfere with ELISA applications?
No, Mynox®
does not interfere with any type of ELISA parameter due to the
low molecular weight of the active component. It's the mycoplasma and
their effects on the cells providing, after their removal, real results
with the ELISA.
Is
it possible to do a second treatment with Mynox®
?
Yes, it is possible to run a second treatment after the first passage.
The first treatment reduces the mycoplasma load drastically. The second
treatment would kill remaining mycoplasma.
What is the principle of Mycoplasma-Off®
? How does it kill mycoplasma and how long will mycoplasma survive in
general?
Mycoplasma-Off® contains membrane active
components acting in combination with the alcohols included. It also includes
aldehydes mainly for the inactivation of non-enveloped viruses and spores.
Mycoplasma-Off® is also active against
bacteria and other microorganisms. It is not quite understood how long
mycoplasma can survive outside a culture or their natural habitat. By
our knowledge no study is available. It is only known that cross contamination
is very prominent for cell cultures. That can only be the case with quite
stable mycoplasma.
Is the DNA-RemoverT
active against RNA?
The DNA-RemoverT is highly active against DNA and RNA and is intended
for use at PCR workstations and other workareas.
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