1. health and fitness
2. disease

Mycoplasma Detection Kit-QuickTest
Description
Perform all steps at room temperature: 22- 28°C.
Should the cell culture system be contaminated by mycoplasma,
typical metabolic enzymes from mycoplasma will degrade the
culture medium components, with typical metabolite products
secreted into the cell culture supernatant. The specificity of the
metabolites produced by the mycoplasma are very high. Any other
type of eukaryotic cells or bacteria will not produce the metabolites.
If the test sample contains the metabolites, the reaction system
(including the test sample, the reaction sample well and the
reaction buffer) will turn greenish-blue in color. The concentration
of metabolites produced is proportional to how dark the color is,
which directly indicates the amount of mycoplasma in each
sample.
1. Open the test plates.
2. Add 40 uL Reaction Buffer A to the sample wells.
3. In the first well, add 10 uL of the negative control. In subsequent
wells, add 10 uL of the test samples or positive controls.
4. Shake gently, then let stand at room temperature for 5 min.
5. Add 40 uL Reaction Buffer B to all sample wells.
6. Shake gently, then let stand at room temperature for 4 min.
7. Immediately monitor sample color changes.
a) If the color of the test well is darker than the negative control
well, the sample is positive for mycoplasma.
Components
Contents
Cat#:B39032
Cat#:B39035
Cat#:B39038
Plates
20
100
1000
Reaction BufferA
3.2 mL
16 mL
16 mL x 10
Reaction BufferB
3.2 mL
16 mL
16 mL x 10
b) If the color of the test well is the same as the negative control
well, the sample is negative for mycoplasma.
Important Notes
1. As the method is based on detecting metabolic products of
mycoplasma, we recommend performing the test after 36 - 48 h
continuous cell culture.
Storage
1. Kit is delivered with all component sealed against contamination.
2. Stored at 4 ~ 25°C for optimal results.
3. The product is stable for up to 12 months.
Notice
• Perform all assay steps at room temperature (22- 28°C).
• Do not use H2O as the negative control.
• Be sure to fully mix samples after addition of reaction buffer.
1. Negative control: the same unused cell culture medium (without
mycoplasma contamination).
2. Test samples: cell culture supernatant.
Protocol
① add 40 ul reation buffer A
② add 10 ul sample
③ oscillate gently
stand at room
temperature for
5 min
④ add 40 ul reation buffer B
⑤ oscillate gently
stand at room
temperature for
4 min
9 min
5 min
⑥ analyze
the color
change
2. The optimal reaction conditions are at room temperature: 22 - 28°C.
The reaction temperature must be over 18°C. We recommend
warming the kit to room temperature before starting the
experiment, especially in cold environment.
3. For most samples, we recommend batch testing. Please set up
independent negative control samples for each batch to ensure
that color reaction timing is controlled for different wells.
4. We recommend analyzing results immediately following reaction
mixing, for the following reasons:
a) After adding Reaction Buffer B, the color reaction will continue
to deepen. As the there is a limited amount of chromogenic
substrate, when all chromogenic substrate is consumed, the
color of the positive test well will plateau. The color of the
negative or weak positive test well will also continue to darken,
and the color difference of the test wells will get increasingly
small, and finally converge.
b) Product studies show that the color difference between
negative, weak positive, and positive samples is most obvious
within the 4 min after adding Reaction Buffer B. This period
represents the linear relationship between color depth and the
concentration of analyte.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: order@biotool.com
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: eu.order@biotool.com
5. The material used to coat the test plate may change color when
exposed to air. Only open the test plate immediately prior to use.
6. For research use only. Cannot be used for clinical purpose.
Assay Principle
When a cell culture system is contaminated by mycoplasma, its
metabolic enzymes will degrade culture medium components and
produce metabolic products which are secreted into the cell culture
supernatant. The presence of these metabolites indicates the
presence of mycoplasma, as eukaryotic cells or bacteria will not
produce these metabolites.
If the test sample contains the metabolites, the reaction system
(including the test sample, the reaction sample well, and the reaction
buffer) will turn green in color. The concentration of metabolites
produced is proportional to how dark the color is, directly indicating
the amount of mycoplasma in each sample. If there is no metabolite
present, the reaction result (color) will be the same as the negative
control, showing no mycoplasma contamination in the cell culture
system.
By sampling cell culture medium without mycoplasma contamination
as a negative control and comparing the reaction color between the
control sample and the test sample, this kit can indicate whether there
is mycoplasma contamination in the cell culture system.
Problem
Suggestion
Even if the mycoplasma is dead, the results of
PCR method will come out as positive, while
the results of this detection kit are negative,
for the following reasons: PCR methods
For the same
sample, why are the detect the existence of mycoplasma via
amplify 16S rRNA sequences. Whether the
results of PCR
methods positive, mycoplasma is dead or alive, once there is
while the results of the presence of mycoplasma 16S rRNA, the
this detection kit are result will be positive. This kit detects
mycoplasma contamination based on
negative?
detecting the metabolites produced by the
mycoplasma, so this kit will only detect the
viable mycoplasma.
For the same
sample, why are the
results of PCR
methods negative,
while the results of
this detection kit are
positive?
PCR method detects the existence of
mycoplasma via amplification of 16S rRNA
sequences. The mycoplasma species can be
detected by PCR reactions closely related
with the primer sequence. This rationale
explains how the PCR methods can only
detect a limited variety of mycoplasma. This
kit detects mycoplasma contamination based
on metabolites produced by many types of
mycoplasma. When the primers used in the
PCR method cannot amplify the target
sequence, the result of PCR method is
negative, even if there is viable mycoplasma
in the sample.
Can this
mycoplasma
detection kit be
used to detect
contamination of
cell culture
supernatants stored
at 4°C which were
collected in batch?
Yes. The metabolites produced by the
mycoplasma are stable when stored at 4°C.
This kit can be used to reliably detect
metabolites found in the cell supernatant
stored at 4°C for up to 5 days post-collection.
During the process
of adding sample, if
the pipette tip
touched the filter
paper of the test
well, will it influence
the test result?
No.
Can this
mycoplasma
detection kit be
used to detect
whether a cell
suspension is
contaminated by
mycoplasma?
Yes.
Limitations of This Method
1. This kit cannot distinguish between species of mycoplasma, but
can effectively detect all types of mycoplasma.
2. If the cell culture system is contaminated by trace mycoplasma
(less than 10 mycoplasma copies / uL cell culture supernatant), the
result may show weakly positive. We suggest re-testing the
mycoplasma contamination after appropriate extension of cell
culture time (24-48 h).
Troubleshooting
Problem
Suggestion
Culture the fresh cell culture medium by cell
culture flask in a CO2 incubator for 48
hours (sample A). Take fresh cell culture
medium stored in a refrigerator as the control,
and then test these samples with our kit.
How can I test if the Compared to the control test well, if the color
fresh cell culture
of sample A is deeper, the cell culture
medium is
medium is contaminated by mycoplasma.
contaminated by
If the colors are similar, the cell culture
mycoplasma?
medium is not contaminated by mycoplasma.
We recommend culturing the fresh cell
culture medium by cell culture flask in a CO2
incubator 48 hours before testing the cell
culture supernatant, in order to exclude the
existence of mycoplasma contamination of
the fresh cell culture medium.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: order@biotool.com
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: eu.order@biotool.com
Problem
Why is it not
recommended to
use H2O as
negative control?
Suggestion
The cell culture medium contains serum.
During the serum preparation process,
metabolites of mycoplasma maybe still
retained, even if the mycoplasma has been
removed. Therefore, when using this
mycoplasma detection kit, it's highly
recommend to use the same cell culture
medium as a negative control, with the same
batch of cell culture medium as the best
negative control.
Customer Reviews
Fig A
Fig B
Fig C
Fig A. Test results from Lab 1, the sample A and B were not
contaminated by mycoplasma.
Fig B. Test results from Lab 2, the sample C and D were
contaminated by mycoplasma.
Fig C. Test results from Lab 3, the sample E and F were contaminated
by mycoplasma. The mycoplasma contamination of Sample E was
more severe than Sample F.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: order@biotool.com
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: eu.order@biotool.com